MBB6346 Genetic pathways from zygote to organism Flashcards

Question

How do PAR proteins become polarised?

Answer 1

Goldtsein and Hird, 1996 - The sperm entry point defines the posterior pole - Manipulated sperm entry so it occurs anteriorly. This reverses the polarity of the zygote - Experiments were also done examining what happens if the sperm enters at a lateral position and in these instances, the sperm pronucleus quickly migrated to the nearest pole and this became the posterior. Cytochalasin D treatment inhibited this movement indicating that intact actin microfilaments are needed for this process. - Sperm donates a centrosome to the entry point (contains actin) affecting actin within the oocyte

Answer 2

- The mesh of actin is contracting and relaxing and sperm entry results in relaxation at the posterior pole meaning actin only contracts at the anterior. This forces cytoplasm to move to the other end. This is known as a fountain head streaming affect. - This is critical for the localisation of PAR proteins

Answer 3

Before fertilisation, PAR3, PAR6 and PKC-3 are present around the entire cortex. These proteins inhibit PAR1 and PAR2 from the cortex

Answer 4

- After fertilisation, and fountain head streaming occurs, PAR3 and 6 are driven into the anterior pole allowing PAR1 and PAR2 from the cytoplasm to bind to the membrane in the posterior pole. - This results in PAR3 and 6 localised at the anterior cortex and PAR1 and 2 and the posterior cortex. The PAR proteins are mutually exclusive.

Answer 5

PKC-3 phosphorylation inhibits their ability to bind the anterior region by phosphorylating PAR-1 and PAR-2. In turn, PAR-1 inhibits PAR-6/6/PKC-3 from binding the posterior region by phosphorylating PAR-3, whilst PAR-2 can inhibit PKC-3.

Answer 6

- PAR1 acts on MEX5 causing its dissociation from proteins and RNA. Usually it is associated with protein and RNA making it slow moving. - Once PAR1 causes its dissociation, it produced two pools of MEX5 – one bound to proteins which is slow moving and another which is free from proteins and fast moving. - MEX5 is unable to bind proteins in the posterior due to the presence of PAR1 and its subsequent phosphorylation meaning MEX5 moves anteriorly where it can bind proteins and become slow moving again. - This results in MEX5 to gradually localise anteriorly

Answer 7

- A stomata is formed of two specialised guard cells that flank the stomatal pore. - CO2 exchange and fixing via the Calvin Cycle into glyceraldehyde-3-phosphate. The cuticle of most plants is reasonably impermeable to gas exchange, so these pores allow CO2 to enter the plant. - It is required to balance CO2 and H20 levels

Answer 8

The stomatal lineage consists of a series of defined cell divisions that ultimately result in the stomatal complex, consisting of two guard cells flanking the pore.

Answer 9

- A protodermal cell takes a meristemoid mother cell fate (MMC). - The MMC is a cell that is competent to enter the stomatal lineage. Asymmetric division occurs and gives rise to a smaller meristemoid (M) cell and a larger stomatal lineage ground cell (SLGC). - Eventually the M differentiates to form a guard mother cell (GMC), which is the immediate precursor of a stomata. The transition from GMC to guard cells involves a symmetrical division, which sees the production of two guard cells and the formation of the pore.

Answer 10

The cell fate determinant is a basic helix-loop-helix (bhLh) transcription factor called speechless and is found in the meristermoid cell

Answer 11

- It is present in the MMC before the asymmetric division and is present in both daughter cells immediately after division – equal segregation. - There is then a mechanism that activates the degradation of speechless in the SLGC. This is different to what occurs in the C. elegans zygote asymmetric division

Answer 12

- A mechanism activates the degradation of speechless in the SLGC. - This mechanism involved MAPK and BASL, a polarity determining factor which acts as a scaffold for the MAPK module. This scaffold makes sure that this MAPK is only found in the SLGC by sequestering it away to the membrane (polarity) before division and allowing it to degrade speechless in the SLGC after division

Answer 13

This mutant is characterised by a massive overproliferation of stomata, which are severely clustered together. This clustering is rarely seen in WT plants as they always have at least one cell between stomata.

Answer 14

Yoda is therefore a negative regulator of stomatal cell fate and their spacing.

Answer 15

- It is a MAPKKK (top of the MAPK cascade). It has a regulatory N terminal domain and deletion of this results in constitutively active yoda (turns on the pathway always). This results in an epidermis that has no stomata. - This agrees with Yoda being a negative regulator of stomatal development. - This also implies the involvement of MAPK and MAPKK.

Answer 16

MAPK signalling is difficult to analyse due to large numbers of MAPK in Arabidopsis and its wide use. This lab therefore focused on MAPKs that had been shown to have a role in environmental stress signalling.

Answer 17

- Determined this using double mutant analysis. If knockout MKK4 and MKK5, similar phenotype to yoda knockout. If knockout MPK3 and MPK6 then there is a phenotype of stomata everywhere (yoda knockout). - This shows that these kinases act downstream of yoda

Answer 18

Used microarrays to look for sets of genes whose activity was altered in constitutively active yoda and mutant yoda.

Answer 19

Identified a basic helix loop helix (bHLH) transcription factor FAMA which is expressed in guard cells. FAMA mutants have defects in specification of guard cells (final step)

Answer 20

A phylogenetic analysis of the bHLH family in Arabidopsis revealed that FAMA is closely related to two other bHLH Transcription factors. One of these was speechless and the other was mute.

Answer 21

There are no asymmetric divisions in a speechless mutant and produces no stomata – lethal mutation

Answer 22

Over expression of speechless leads to ectopic asymmetric divisions

Answer 23

- Can see using SPCH-GFP that speechless is localised to meristermoid cells. - Its overexpression leads to ectopic asymmetric divisions and its loss leads to no asymmetric divisions. - This is evidence that speechless is the cell fate determinant

Answer 24

- Created a double mutant. - The double mutant resembles a speechless mutant despite yoda usually causing lots of stomata. - This is evidence for epistasis and that yoda acts on speechless.

Answer 25

Lampard et al (2008) - Speechless has a domain that is not conserved in mute and fama. - This region has a number of residues that conforms with a MAPK target sequence. MAPK usually acts on serine or threonine that are surrounded by proline - Mutated these known residues to alanine to prevent their phosphorylation – created a phospho-mutant. - The result is speechless variants with increased stability, such that they persist and induce ectopic asymmetric divisions. If mutate more serine residues (more representative of what you see if you overexpress MUTE and FAMA)

Answer 26

Breaking of Asymmetry in the Stomatal Lineage. These mutants have lost their ability to orient their divisions and results in asymmetric divisions occurring next to each other so that meristemoids are touching

Answer 27

- In meristemoid, BASL was always in the nucleus. - In the larger SLGC – immediately after an ACD, BASL is expressed both in the nucleus AND the cell periphery. - The BASL in the periphery is opposite the division plane.

Answer 28

- Analysed the sequence of BASL. There are three kinase interrupting motifs. There is also a series of serine’s that conform to MAPK targeting. This suggests that BASL has the capability to bind MAPK and is phosphorylated by them - In a kinase assay, BASL was shown to be phosphorylated by MPK6. - In a pull down assay, they were investigating with BASL interacts with yoda. They found that it does and the kinase domains are required for this interaction and deletion of these domains results in failure to interact with yoda.

Answer 29

- BASL is acting as a MAPK scaffold protein – a protein that interacts with MAPK signalling modules and brings them together to enable them to signal in response to specific stimuli – that cycles between different places within the cell. - In the nucleus, it is inactive. It then becomes phosphorylated by MPK6 which causes BASL to move out to the membrane and act as a scaffold to bring together the MAPK components (yoda) together at the periphery of the cell.

Answer 30

Zhang et al, 2016 - In the meristermoid speechless is in the nucleus but in the SGLC it is in the periphery and nucleus - After division, there is an equal amount of speechless in both daughter cells meaning that there is no polarisation before the asymmetric division. - |t is the BASL that has the integral role in degradation of speechless - BASL is only found in the SLGC meaning that is where the MAPK module is localised resulting in speechless degradation in that daughter cell.

Answer 31

- This occurs in a sheet of cells (the epidermis) so there are many cell interactions. - Stomatal lineage cells secrete an extracellular peptide called Epidermal patterning factor 2 (EPF2). EPF2 is the ligand for the erecta receptor like kinases. Binding of EPF2 leads to activation of the Yoda-MAPK cascade. - Both intrinsic and extrinsic signals are involved but due to this happening in a field of cells it is almost impossible to completely dissociate the two components to answer unequivocally the role of intrinsic v extrinsic factors.

Answer 32

short-root (SHR) and scarecrow (SCR).

Answer 33

Levesque et al (2006) - Isolated two mutants called Scr and Shr which only had a single ground meristem layer and this was due to their being no asymmetric division of the CEI (Cortex-Endodermis Initial).

Answer 34

- Transcriptional constructs were generated in which either the SHR or SCR promoters was linked to GFP. - Using confocal microscopy it was then clear that SHR mRNA is found in the stele (the vascular tissue) whilst SCR expression occurs in the meristem and CEI as well as the endodermis. - SCR expression is significantly reduced in a Shr mutant suggesting the Shr is required for SCR expression. - SHR is having an impact on CEI asymmetric division and SCR expression

Answer 35

- SHR interacts with SCR and together, this results in a feedforward loop resulting in more SCR expression. - This ensures all the SHR is bound by SCR and remains in the nucleus, where the SHR-SCR complex induce expression of CYCD6 and you get asymmetrical division of the CEI.

Answer 36

Carried out ß-galactosidase assay and yeast-2-hybrid assays

Answer 37

- In the CEI, SHR is expressed in the nucleus but is cytoplasmic in the stele but in a SCR mutant, SHR is cytoplasmic in the CEI, endodermis and stele meaning that SCR is required for the nuclear localisation of SHR in the endodermis and CEI. - In SCR-RNAi roots, SHR has moved more than one cell layer causing ectopic divisions meaning that SCR is required to limit SHR movement.

Answer 38

- In the SCR mutant, there is reduced SCRproGFP meaning SCR upregulates its own expression

Answer 39

- This is not seen in the SCR mutant because SCR is vital for these cell divisions – you need a little bit of SCR in order to promote the division. No SCR results in no division, a little bit means that some SHR protein escapes the endodermis and moves another cell layer, where it interacts again with SCR and promotes division.

Answer 40

Cyclin-D6 | - By inducing this in the CEI, an asymmetrical division is promoted.

Answer 41

- SHR is expressed in the stele - It’s protein moves into the next cell layer, this includes the CEI. - It interacts with SCR and together, this results in a feedforward loop resulting in more SCR expression. - This ensures all the SHR is bound by SCR and remains in the nucleus, where the SHR-SCR complex induce expression of CYCD6 and you get asymmetrical division of the CEI. - There is now an endodermis and cortex. SCR is found in both cell types but it is its presence in the endodermis that prevents SHR moving into the cortex and causing further cell divisions

Answer 42

Wing development is something that is initiated late in development and progresses as the larva pupates and undergoes metamorphosis.

Answer 43

- Imaginal discs are groups of cells that form later in development and divide and exit through the body wall and form the external organs of the body e.g. head, wings, legs. - There are 19 in the late stage pupae each consisting of as few as 10-15 cells. After proliferation, the wings consist of 50000 cells – lots of proliferation

Answer 44

During metamorphosis, these cells have a very high rate of proliferation and they move from inside the larva, through the larval wall and into the space under the pupal case. There, they unfold and elongate and complete the final differentiation steps to form the structures of the adult fly – they also fuse together to form a continuous epithelium.

Answer 45

The wing consists of: - the notum – forms part of the dorsal thorax to which the wing attaches - the hinge – allows wing movement - the wing – this is the set of cells that the wing as you envisage it, is derived.

Answer 46

- Segment polarity is defined by the transcription factor engrailed which regulates the transcription of Hh. Hh is secreted from the cells that produce it and diffuses away. - This then activates Hh signalling in adjacent cells and activates target genes including wingless. - This system is not relevant in the formation of the drosophila wing.

Answer 47

- Hedgehog binds to its receptor patched and inhibits its action. - Patched usually inhibits these actions of the signal transducing component smoothened. The relieving of this inhibition allows the transcription factor Ci to stabilise and regulate the transcription of target genes. - In the absence of Ci acts as transcriptional repressor - When there is Hh, Ci is not cleaved and it activates gene expression - The balance of these forms of Ci defines the target gene expression

Answer 48

- The cells in the anterior and the posterior of the wing come from different linages - This results in a defined boundary between the two lineages (between AP)

Answer 49

- Hh | - Dpp

Answer 50

In situ hybridisation of wing disc - In situ for engrailed protein. En is only found in posterior cells. En is the transcription factor which is limited to the cells in which it is expressed and activates Hh - Can also see Hh in the posterior cells of the wing disc showing that En is regulating Hh in the posterior cells

Answer 51

- Although Hh is expressed in these cells, they are incapable of responding to it (refractory to Hh). - It is En that prevents these cells from responding to Hh. En regulates Hh and inhibits its action in the same cells by inhibiting the expression pf patched

Answer 52

Anterior - En is limited to the posterior cells and prevents them repsonding to Hh. - Hh can diffuse extracellularly and move away to the anterior compartment to cells with no En and competent to respond

Answer 53

Ci is expressed in the anterior but not the posterior due to the presence of En inhibiting the expression of patched and Ci these cells. Once this inhibition is removed, these genes can be expressed and receive Hh signalling and initiate the signalling cascade

Answer 54

Hh is being expressed at the posterior side so the highest concentration of Hh will be at the AP boundary and reduce as it moves across the anterior domain.

Answer 55

- En regulates Hh expression in the posterior cells but Engrailed also blocks the expression of Patched (Hh receptor) and Ci (transcriptional regulator) in these posterior cells. - Even though HH is there, these posterior cells cannot perceive it and hence do not respond (are refractory)

Answer 56

- These anterior cells do express Patched and Ci – so they do respond to this HH gradient - You get active Ci when Hh is present and repressive Ci when Hh is absent. - Across the Hh gradient you get a similar gradient of Ci(active):Ci(repressor) with the active Ci being at the AP boundary and the Ci repressor being at the other side of the anterior compartment

Answer 57

Can be used to determine whether factors impact on wing development e.g. how these factors interact to regulate fate.

Answer 58

The anterior compartment, it has veins I, II and III. The posterior has veins IV and V. So, consider each vein as a marker of particular cell fate.

Answer 59

Tabata and Takei, 2004 - Cells in the anterior region of the wing were made to express Hh- ectopic expression. These cells are competent to respond to Hh and Hh will diffuse out - Result in a mirror image duplication event centred around at vein 3. This vein is found just into the anterior compartment in the Wt wing - The concentration of Hh is influencing cell fate and where Hh is at its highest concentration (highest Ci activation to repressor), vein 3 forms. - Veins 4 and 5 are not affected as they are in the posterior compartment and these cells are not competent to respond to Hh.

Answer 60

Homologue of BMP and is a secreted peptide

Answer 61

It is regulated by Hh and can diffuse into both the anterior and the posterior compartments

Answer 62

Ectopically expressed Dpp anteriorly - This resulted in mirror image duplication centred around vein 2. Highest concentration of Dpp defines vein 2 identity Ectopically expressed Dpp posteriorly - Resulted in mirror image duplication centred around vein 4 showing that posterior cells are competent to respond to Dpp and highest concentrations result in vein 4 identity - Different to rctopic Hh expression in posterior as cells are not competent to respond to Hh

Answer 63

- Receptor pair at the cell surface Tkv and Punt. The ligand binds to the extracellular domain and this in results in the cytoplasmic serine/threonine kinase domain of the receptors being activated - This then phosphorylates MAD forming p-MAD. - This activated p-MAD translocates into the nucleus where, together with two other factors called Medea and Schnurri, it regulates the transcriptional repressor Brinker. - Brinker negatively regulates the expression of DPP target genes (vein 4 cell fate)

Answer 64

optomotor-blind (omb) and salm (sal)

Answer 65

- DPP is expressed in a strip at the A-P boundary and is regulated by Hh. As a morphogen, it can diffuse in both directions – so into both the Anterior and Posterior compartments. - As DPP represses Brinker transcription via p-MAD, Brinker expression is seen most strongly at the extreme Anterior and posterior poles. - The localisation of p-MAD and Brinker, they are relatively exclusive

Answer 66

- Sal requires higher concentrations of DPP (p-MAD) signalling than omb, hence the broader expression pattern of OMB. The expression of both Sal and Omb is repressed by Brinker

Answer 67

- This can be experimentally proven by knocking out Brinker expression in the Brinker expressing cells (most anterior or posterior cells) therefore allowing Omb expression (as Dpp diffuses in) and creating a secondary Omb:Brinker boundary. - This distinct boundary results in the formation of a secondary vein 5 showing that this boundary is what drives vein 5 cell fate.

Answer 68

- Genetically alter the expression of one of your key genes, using a DNA recombination based system to excise the relevant gene from the genomic DNA in a random cell, so that the cell becomes mutant for that gene. - You then allow development to occur and that cell will divide and you then look at where the clone of cells derived from this original cell is now found.

Answer 69

- A wing disc expressing GFP in both anterior and posterior compartments was engineered so that the expression of EN, Ci or both could be switched off in cells using a heat shock recombination based system (also removed GFP). - Researchers then focused on cells where the knockout event was close to the AP boundary. - Cells were then allowed to divide - In some cells, the excision event occurred in a cell in the anterior compartment (knocked out Ci, Eg or Hh). - After it was allowed to divide, we see that the clone of cells derived from this have now expanded into the posterior compartment. - Anterior-posterior cells are derived from different lineages and would not normally be able to do this.

Answer 70

- FLP recombinase expressed from a heat shock promoter. - Construct with FRT sites flanking the region you want to excise and once FLP expression is induced by a heat shock, you will get excision of that WT gene.

Answer 71

- In an anterior cell near the A-P boundary, a cell was manipulated to mutate Ci and to turn on En. - The clone of cells expanded into the posterior compartment and took on posterior fate. So, this is suggesting that En alone is sufficient for cells to have posterior cell identity and it can do this in a HH independent manner

Answer 72

- A cell in the posterior compartment has been mutated so it can’t express En. The cells are still able to express Ci. - The cells divide and move into the anterior compartment, indicating that Ci is sufficient to specify Anterior cell segregation. - As Hh is required for active Ci, this would suggest that this is Hh-dependent as Hh can diffuse so even though it is expressed in the posterior compartment it can diffuse into the anterior one

Answer 73

HH-independent mechanism operating to sort Posterior cells and HH-dependent mechanism to sort Anterior cells. - When a posterior cell expressed both Ci and EN it sorts into the anterior compartment. - This suggests that the HH-dependent pathway is dominant to the HH-independent pathway.

Answer 74

- FLP-FRT recombinase system has been used to generate clones of cells mutant in either Omb or Brinker.

Answer 75

- Produced two Omb mutant clones. The top one, is not in contact with the vein area and has no impact. However, the second Omb mutant clone overlaps where vein V is and results in loss of vein development in that mutant clone. - This means Omb is required for the positioning of vein V. To make vein V, cells must express Omb.

Answer 76

- A clone of cells posterior to the normal vein V have had brinker expression knocked out causing an ectopic vein now forms along the boundary of this clone. - Where Brinker has been lost, Omb expression now occurs and the vein forms at the boundary between the two –Brinker repression of Omb sets a tight boundary in which vein V forms.

Answer 77

An embryonic structure and the precursor to the central nervous system

Answer 78

- The dorsal plane results in sensory neurones while the ventral plane results in motor neurones. There are interneurons that connect the two - Formation of the neural tube

Answer 79

Neurulation - The ectoderm differentiates either into the epidermis or the neuroepithelium - BMP drives epidermal fate and the organiser (mesoderm derived) secretes BMP antagonists such as chords and noggin which block BMP and induce neuroepithelium - The neuroepithelium grows and undergo columnisation to form the neural plate which is found at the bottom of the neural tube - Then elongates along the AP axis and rolls into the neural tube - Simple cell movements that underlie these morphological changes

Answer 80

- Found in all chordates | - Found below the ventral surface of the neural tube

Answer 81

- As soon as the organiser induces the neural plate, the organiser self-differentiates into the axial mesoderm. The axial mesoderm then involutes and undergoes convergent extension - The axial mesoderm consists of the prechordal mesoderm and the anterior endoderm which mark the head of the organism (anterior) and the notochord which marks the body of the organism (posterior)

Answer 82

Acts as a structural support and plays a crucial role in dorsoventral patterning of the neural tube.

Answer 83

Beddington et al, 1994 - The node/organiser gives rise to the notochord and they knew that the notochord was involved in neural tube patterning - They excised the node/organiser from one embryo and transplanted onto the lateral surface of another embryo. - A control was also done using anterior endoderm transplantation - Developed a secondary neural tube at the graft site showing that the node itself induced and developed into the neural tube

Answer 84

Examined the ability of the notochord to regulate motor neuron development

Answer 85

- Used Islet-1 as a marker for motor neuron fate and stained a chick spinal cord cross section - Excised neural plate from embryo and incubated in collagen gel (control) and stain for islet-1. There were no motor neurons formed showing that the formation of motor neurons in not inherent to that tissue – not autonomous - Repeated with cells incubated on an isolated notochord – resulted in motor neurones. - Notochord is capable of inducing motor neuron cell fate from neural plate cells

Answer 86

- They took notochord and incubated in buffer over night and removed all the cells from the buffer. - They then repeated the experiment with the neural plate cells incubates in the buffer. This too resulted in the formation of motor neurones. - This shows that the notochord must be producing a diffusible signal that is capable of inducing this developmental change in the neural plate cells.

Answer 87

Echerlard et al, 1998 - Isolated Shh in zebrafish and mice – homologue of Hh in drosophila - In situ hybridisation of Shh in chick notochord. Can see that Shh is present in the notochord and in the neural plate cells in late development - This shows that Shh is expressed in the right place at the right time to be the diffusible molecule expressed by the notochord. - Only circumstantial evidence

Answer 88

- Shh is a peptide and produced as a pro-protein and undergoes autoproteolysis to cleave to itself into the mature form - They looked to see which part of the Shh is involved in regulating these processes and is there a concentration dependant manor to this control and used full length Shh, mutated Shh which cannot undergo autoproteolysis, C terminal Shh and N terminal Shh. - They expressed these proteins in Cos cells and incubated with neural plate explants

Answer 89

- Incubation with full length Shh, both islet1 and HNF3b (floor plate identity marker) is expressed. - This is also seen in N terminus but not the C terminal protein showing that the c terminus is not biological active. - If Shh cannot be cleaved (not N and C terminus) there is also no markers. - This shows that it is the N terminus that is biologically active.

Answer 90

As a morphogen - Then repeated the experiments but took dilutions of Cos cells to have different concentrations of Shh. - The different concentrations resulted in different outcomes. - High concentration results in floor plate cells but lower concentrations result in motor neuron formation. - This shows that Shh has concentration specific effects on cell identity

Answer 91

- Different precursor cells develop at specific points in the ventral neural tube due to the gradient of Shh, starting with the floor plate cells and progressing to the p0 pool. - pMN is the progenitor pool for motor neurons whereas the other progenitors give rise to a distinct type of interneuron – which enable communication between sensory and motor neurons.

Answer 92

- Balance between active and repressive forms of Ci (Gli in mammals) determines the cell fate of targets. This results in two classes of genes: dorsal and ventral expressing genes.

Answer 93

- Pax7 is highly expressed when there is no Shh. When Shh increases, the expression is repressed - Pax6 is more widely expressed then pax7 but is primarily expressed in the mid and dorsal domain - still expressed when Shh is present but high Shh represses it. - Nkx2.2 is expressed in the ventral domain - high Shh is required for its expression - The concentration of Shh therefore regulates these transcription factors

Answer 94

- Incubated floor plate cells with anti-Shh to bind Shh and look what happens in a Shh mutant. - This results in an increase in Pax6 expression in these cells and a reduction of Nkx2.2 and causes a change from motor neuron formation to interneurons V1 and V2. - Pax6 expression is defines by Shh which in turn defines cell identity - Shh therefore represses (pax6) and induces (nkx2.2) various transcription factors

Answer 95

- Shows that there are distinct boundaries between their expression domains - The location of the progenitor cells within in the neural tube relates to the boundaries formed between these transcription factors and tells us which transcription factors define which progenitor cells. - It is the boundaries that defines where the progenitor cells form - If the boundaries move, then that results in the reprogramming of the dorsoventral axis.

Answer 96

- A morphogen can rarely define these tight boundaries alone. Interactions refine these boundaries - Negative interactions between the transcription factors are required for the formation of the tight boundaries

Answer 97

Nkx6.1 usually negatively regulates Dbx2. Mutant Nkx6.1 therefore has an expanded Dbx2 expression domain due to the removal of the negative repression

Answer 98

- The homeodomain transcription factors in the neural tube are primarily transcriptional repressors – so they switch target genes off, meaning transcriptional activators are required, usually the widely expressed transcription factors Pou and Sox

Answer 99

Shh has many uses in development so studying its role in a specific system is difficult because mutant Shh will have large effects

Answer 100

What role does Patched regulation have in Shh patterning?

Answer 101

- Shh is required for patch expression so they uncoupled this relationship - They did this by generating patched knockout mice but introduced a construct whereby Patched was expressed at low level from a widely expressed metallothionein promoter (MtPtch1) which doesn’t respond to Shh. - This means that the cells in the neural tube can respond to Shh but they cannot induce Patched - In the wild type mice, Shh is in a concentration gradient which induced patched expression in the same gradient. In these mutant mice, the patched expression is the same across the neural tube

Answer 102

They looked at the expression of some ventrally expressed genes and found that in the mutant mice their expression domain had expanded showing that Shh regulation of patched usually acts to limit Shh signalling and its own action

Answer 103

- Shh binds to its receptor patched which in turn upregulates patched receptors in that cell meaning more Shh can bind. - This binding and signal activation causes the degradation of Shh molecules meaning they cannot diffuse further away. - As there is more Shh towards its origin, it will activate more patched receptors on adjacent cells and therefore initiate more degradation. - This creates a steep gradient for Shh as the cells further away will only be exposed to the Shh molecules that have not already been degraded by the adjacent cells expressing high patched receptors.

Answer 104

- The expression of the transcription factors is dependent on both the number and affinity of Gli binding sites within the promoter of a target gene. - So, Gene A (see diagram) has only 1 weak affinity site and so needs a high concentration of Shh to drive its expression. - Gene c however, has 3 high affinity sites so doesn’t require as high of Shh and so is expressed broadly.

Answer 105

- Broadly expressed regulatory transcription factors that either positively or negatively regulate targets. - There are also interactions between the target genes themselves to refine the boundaries - Meaning there are three mechanisms involved in the expression pattern domains: It is the combination of Gli binding sites, the binding sites of broadly expressed positive or negative regulators and target gene interactions that refines pattern.

Answer 106

- Meristems are found in most plants organs and are stem cell niches. For example, in the leaf, meristematic tissue is associated with the vascular tissue in the stems and contribute to radial growth - E.g. root and shoot meristems

Answer 107

- Shoot apical meristem (SAM) – formed during embryogenesis and responsible for all above ground tissue (so ultimately, other meristems will be derived from this meristem). - Root apical meristem – root cells are all derived from this, though lateral roots then derive from lateral root meristems, which are associated with vascular tissue. - Floral meristem – gives rise to the reproductive organs of the plant - Procambium – gives the xylem and phloem -

Answer 108

- They are a collection of stem cells and are held in an undifferentiated state but when a stem cell divides and leaves the meristem they divide into a specific cell type. - The meristem cells will persist only if the cells lost to organ recruitment are in turn replaced – maintain the stem cell pool. If not then left with the resulting organ and a terminal fate

Answer 109

- Normal plant growth is known as the vegetative phase. In this phase, the apical meristem forms organs such as leaves from flanking cells - Depending on the type of plant, there becomes a stage, usually triggered by environmental signals, where a developmental phase transition is induced and results in reproductive growth. This forms flowers instead of leaves. This is the same as puberty in humans

Answer 110

- The SAM could either convert to inflorescence meristem which is a meristem that forms floral meristems rather than leaves and the floral meristems on its flanks then develop into flowers. This produces lots of flowers while maintaining the inflorescence meristem population - Or the SAM convert straight to the floral meristem – a terminal differentiation and the stem cell pool is lost

Answer 111

- SAM converts to inflorescence meristem (pool of stem cells maintained and therefore indeterminate) which converts to floral meristems (stem cells used up and therefore determinant)

Answer 112

- Determinant includes when each branch point ends in a terminal flower - Indeterminists also flower but the meristem is not being consumes in the process. It continues until senescence occurs. During normal growth, the meristem is not consuming and it continues to form floral meristems. If it branches, they will also remain indeterminate.

Answer 113

- Terminal flower - Leafy - Apetala 1

Answer 114

In these mutants, the inflorescence meristem is converted to a floral meristem and forms terminal flowers at the end of the branches - determinant

Answer 115

Produce more vegetative like organs instead of flowers - indeterminate

Answer 116

same as leafy - floral meristem converts to a vegetative meristem

Answer 117

When both knocked out, there are no floral organs forming at all and the meristems are vegetative

Answer 118

- Results in the same phenotype as the leafy Apetala 1 double mutant - This suggests that there is an interaction between these genes and that terminal flower (TFL) is inhibiting the action of leafy and Apetala 1 – overexpression turns them off and therefore resembles the double mutant

Answer 119

- Same phenotype as the terminal flower mutant so therefore leafy switches off TFL - This tells us that the interactions are going in both directions

Answer 120

- TFL1 and leafy/Apetala 1 mutually repress each other - Leafy and Apetala 1 promote vegetative to floral transition and terminal flower inhibits this transition and therefore promotes indeterminacy

Answer 121

Expression in vegetative meristem before flowering - Where there is TFL there isn’t the other two genes – negative interactions - Inflorescence meristem (middle) has strong TFL. In the floral meristems (on the flank), leafy and Apetala 1 is highly expressed on the meristems - TNF and leafy/Apetala 1 are mutually exclusive

Answer 122

Leafy and AP1 moves into the domain where TfL would be expressed and converts it to a determinant floral meristem

Answer 123

TFL1 expression expands into the organs forming on the flank and maintains in vegetative fate – indeterminate

Answer 124

- Used comparative genomics and looked at the terminal flower locus. They asked what regulates its expression and what impacts the expression has on determinacy - Sequenced the entire locus including regulatory regions in Arabidopsis and compared with four plant species

Answer 125

- The TFL1 exons are highly conserved between all four species - There are regulatory regions that are shared in some species but not others.

Answer 126

- Producing transgenic plants that had deletions of these regions e.g. reporter gene instead of TFL gene and removed a regulatory region

Answer 127

- Wt - TFL had strong expression in the middle of the meristem and rib meristem - Delete box B conserved regulatory region – drop in TFL expression. Then expressed TFL in the TFL mutant to ask if they can rescue the mutant phenotype – still get a terminal flower mutant showing that the regulatory region is important in how much TFL is expressed - If removed E and F regulatory regions, you lose expression of TFL in the middle of the meristem resulting in a terminal flower

Answer 128

- Leafy binds to box f - Apetala 1 binds to box E - These genes can regulate each other and determines when where and how much is expressed - One of the genes involved needs to be altered expression and the whole system is changed

Answer 129

MADS domain transcription factor

Answer 130

plant specific transcription factor

Answer 131

phosphatidylethanolamine-binding (PPB) protein

Answer 132

- Flowering locus T (56% amino acid identity) but have exact opposite phenotypes - If overexpress FT then it resembles a terminal flower mutant

Answer 133

Appear to have negative interactions between them

Answer 134

- TFL1 expression is excluded from the centre of the meristem but is still found in the axillary meristems - AP1 and leafy is increased compared to Wt – excludes TFL1

Answer 135

- Suppressor screens – mutagenize a mutant to see if the phenotype is reverted - Yeast -2- hybrid – proteins interact

Answer 136

- Found a transcription factor FD which interacts with both TFL1 and FT - FT has a positive impact on leafy and AP1. - Used RT-PCR to look at the outcome of a FD and FT interaction. Overexpression of FD, AP1 is expressed. - FD and FT expression drives AP1 and leafy but TFL1 and FD blocks leady and AP1 expression

Answer 137

This ability is dependent on FT because if overexpress FD in a FT mutant, there is no AP1 expression

Answer 138

- In inflorescent meristem FD interacts with TFL1 and inhibits leafy and AP1 – indeterminate. FT is directed to the flank and becomes floral meristem - In floral meristem, FD interacts with FT and drives leafy and AP1 – determinant – TFL1 and FD are directed to the flank are drive branching

Answer 139

If any part of this system changes its expression (quantitative or temporal – anything to change the equilibrium), then the interactions change and so does the determinacy phenotype

Answer 140

SHH is vital to hind limb development and is found in the zone of polarising activity (ZPA). There is a reciprocal signalling between the ZPA and apical ectodermal ridge (AER) which produces FGF and reinforces patter. FGF and SHH maintain each other’s expression

Answer 141

Advanced snakes have no limbs but pythons and boas have hind limb buds but development stops

Answer 142

- After egg laying (oviposition), there is no SHH in python but SHH expression could be induced in relevant python cells transplanted into a chick wing bud - This suggests that the cells can make SHH but is prevented by regulation

Answer 143

In situ hybridisations of SHH and other components SHH signalling in a python (no legs) and in anole hind limb (forms hind leg) and compared samples at similar times of development

Answer 144

- Previously, SHH has not been detected in the developing limb buds of snakes. - They looked earlier in development and saw that SHH is expressed at low levels and then is lost as development continues. - This is compared to the stages in Anole which expresses SHH at higher levels and persists through development.

Answer 145

- They also examined transcriptional readouts of SHH signalling including PTCH1 and GLI1. - These are markers of SHH activity. In the python, their expression drops trough development and is maintained in the Anole. - This consolidates the idea that SHH is expressed in early development in the python and decreases during development

Answer 146

GLI3 is a repressor of SHH and is found to increase its expression domain in the python hind limb during development. This increase is not seen in the anole hind limb

Answer 147

- They looked at the expression of FGF8 and found that in early python development, FGF8 is expressed at the AER. As SHH is repressed in development, so does the presence of FGF in the AER – breakdown of the AER. In the anole hind limb, the FGF8 expression in the AER is maintained through development - This evidence shows that the limb formation signalling is present in early development in snake but breaks down during development

Answer 148

regulatory genes of SHH

Answer 149

The expression patterns of HAND2, HOXD13, HOXA13 are similar between python and Anole hind limbs – species specific differences showing that SHH and its regulatory genes are still present in pythons

Answer 150

The ZRS is an enhancer region found in the intron of another gene and is essential for SHH expression in hind limbs. Regulatory sequences bind to the ZRS

Answer 151

- They expanded out this 2kb region and compared this region across species capable of forming limb buds. - There are high conservation sequences in the ZRS locus but there is a missing conservation sequence apparent in snakes that is present in other limb forming organisms. - The region is partially deleted in pythons and boas and is almost completely deleted in advanced snakes that cannot produce hind limbs - This suggests that the change in SHH expression is due to regulatory changes

Answer 152

The regulatory genes are still present and expressed (HAND2, HOXD13, HOXA13) at sufficient levels but key regions in the SHH enhancer, ZRS, that are missing which results in its loss of expression in snakes. The binding sites of the SHH regulatory regions are lost

Answer 153

Limbs would form

Answer 154

- Every somatic cell nucleus contains identical DNA - Unused genes in differentiated cells are maintained and retain the potential for being expressed - Only a small proportion of the genome is expressed in each cell type and a proportion of RNA expression is cell type specific

Answer 155

- Totipotent: all cell types of the human body including trophoblasts (zygote) - Pluripotent: derivatives from the three germ layers (embryonic stem cells) - Multipotent: different cell types from a tissue or organ - Progenitor cells: Can divide a limited number of times before differentiation

Answer 156

from the inner cell mass of the blastocyst

Answer 157

- A feeder layer is a carpet of fibroblasts that are put in a tissue culture dish and treated in a way to stop the fibroblasts from dividing. - They produce LIF and BMP which condition the media and produce an environment so the embryonic stem cells can maintain pluripotency indefinitely

Answer 158

Oct4, Nanog, Sox2 | - They are homeodomain transcription factors and are master regulators of cell fate

Answer 159

Takahashi and Yamanaka, 2006 | - Reprogrammed somatic cells using these factors to produce IPS cells

Answer 160

Transcription factors that can directly bind to condensed chromatin and activate gene expression

Answer 161

They maintain ESCs in pluripotent state by autoregulation of their own expression, co-binding and co-ordinated regulation of target genes and collectively target lineage determining transcription factors. They work within an open chromatin state

Answer 162

- Induction of Sox2 in mouse ES cells where Sox2 is knocked out, Sox2 and other pluripotency factors are upregulated - Co-immunoprecipitation of these factors, shows the other factors showing that they interact and regulate each other’s expression

Answer 163

They carry out two opposing functions: they work to promote activation of genes required for pluripotency and silence genes required for differentiation

Answer 164

The transcription factors occupy pluripotency target genes promotors and recruit RNA polymerase II and activate their transcription

Answer 165

ChiP sequencing showed that the repressed genes are bound by Polycomb complexes which modify the histones of lineage specific genes

Answer 166

Closed chromatin or heterochromatin represses transcription while open chromatin or euchromatic activates transcription. The dark regions of heterochromatin are usually at the periphery of the cell while euchromatic is at the centre

Answer 167

They aim for an open chromatin stateand as differentiation occurs, cells become more tightly packed. The open chromatin state allows the rapid swapping to different cell fates. This allows him to respond to signals quickly along differentiation pathways

Answer 168

SWI/SNF - E.g. BAF - Ejection – removes a nucleosome and opens chromatin CHD - NuRD - Ejection – removes a nucleosome and opens chromatin ISWI - NuFR - Sliding – moves nucleosomes over to access sequence INO80 - TIP60 - Dimer exchange - Changes the histone modification

Answer 169

- Chromatin remodelling proteins use energy released form ATP hydrolysis to disrupt the interactions between histones and DNA. E.g. INO80, SWI/SNF. - Expression of these chromatin remodellers is elevated in stem cells

Answer 170

DNA methylation is a repressive epigenetic modification that occurs on Cytosine residues of CpG nucleotides

Answer 171

DNA methyltransferases, DNMT3A and DNMT3B are responsible for this de novo methylation

Answer 172

DNA modifications are considered true epigenetic mechanisms because they can be inherited by cells This maintenance methylation is carried out by the enzyme DNMT1 which recognises hemi-methylated DNA (only one strand is methylated from the parent cell).

Answer 173

DNA methylation of CpG islands are high at promotors of inactive genes in pluripotent ES cells

Answer 174

Totipotent zygotes are essentially devoid of DNA methylation - As preimplantation embryo is created and develops, methylation is decreasing. Once is becomes the epiblast (post implantation), remethylation occurs. This demethylation wave in the preimplantation embryo is erasing the epigenetic imprints that the sperm and egg has - 30% of genes in ES cells are methylated at promotors

Answer 175

Lysine acetylation on the NH3+ side chain is associated with active chromatin

Answer 176

Carried out by histone acetyltransferases HATs and removed by HDACs

Answer 177

- Usually repressive – if the methylation occurs on H3K9 or H3K27 - H3K4 methylation is active

Answer 178

Carried out by histone methyltransferases (HMTs) and removed by histone demethylases

Answer 179

- Most developmental genes that are silenced by Oct4, Sox2 and Nanog in pluripotent stem cells are co-occupied by Polycomb group proteins. They add the repressive mark H3K27me3 and the activating mark H3K4me3 on lineage specific gene - This is known as bivalent modifications and allow the quick removal of one of these marks once the cell receives a signal to promote differentiation

Answer 180

- Oct4 is rapidly silenced and repressive H3K27 and H3K9me3 are added and activating modifications are removed. - H3K9me3 is catalysed by the HMT G9a and this then promotes DNA methylation at OCT4s promotor also occurs

Answer 181

Asked how many genes controlled pluripotency, are there a few genes required or just one?

Answer 182

- Created a new system where he could screen for pluripotency. - He knew that Fbx-15 gene is highly expressed in ES cells but it is not important in pluripotency meaning it could be manipulated without damaging pluripotency. - He knocked in Bgeo or G418 (neomycin selection gene) instead of Fbx-15. If this gene is active then neomycin resistance gene will therefore be active. - Somatic cells can be killed with low levels of neomycin and if turn on this gene (only turned on in pluripotent ES cells) then they become resistant to this and the cells will survive

Answer 183

- Used fibroblasts and with the G418/Fbx-15 system - Used viral infection to add 24 genes (hypothesised to be involved in pluripotency) one by one. No cells survived meaning they hadn’t acquired ES cell properties. - He then added them all together and this resulted in 22 clones and 5 of them looked very like ES cells. - He then purified this further and found that they expressed pluripotent factors

Answer 184

Oct4, Sox2, C-Myc and Klf4 | - Nanog was dispensable

Answer 185

- They are genetic elements consisting of 100-1000bp of non-coding DNA and positively active transcript - They act independent to location, orientation and distance and are crucial determinates of lineage specific gene expression

Answer 186

- cis-regulatory (located on the same chromosome) and reside away from the promotors they act upon

Answer 187

They contain clusters of different transcription factor binding motifs which allows them to integrate information from multiple different signalling events into one regulatory locus to drive gene expression

Answer 188

Controls shh expression

Answer 189

- Located away from the shh promotor site and when mutations occur, there is ectopic expression of the shh gene - Can result in polydactyly (extra digit formation)

Answer 190

Look if there are clusters of transcription binding

Answer 191

- H3K4me1 - H3K27ac - Higher levels of DNAse1 hypersensitivity - CpG low enrichment

Answer 192

- H3K4me3 is found at promotors but only mono-methylation at enhancers. The more methylation is a mark of transcriptional activity which is higher at promotors than enhancers

Answer 193

- Enhancers are highly dependent on transitional coactivators that bind and modify the chromatin at that region: CBP and p300. - These are histone acetyltransferases which give the activating mark. This recruitment results in increased histone acetylation of H3K27 and increased transcription

Answer 194

The chromatin are more open and available for incoming transcriptional activators

Answer 195

Prevents DNA methylation which reduces DNA transcription

Answer 196

Overall levels of transcription are lower in enhancers than promotor but can see small transcription.

Answer 197

TAL1 is a lineage determining transcription factor - In cell lineages where TAL1 is active, there is elevated H3K4me2 and acetylated H3K27 at the enhancer regions of TAL1. - In cells where the gene is turned off, there are none of these modifications at the enhancer sites showing that the enhancers work in a cell type specific manor

Answer 198

The transcription factors can bind to DNA sequences even in closed chromatin - can bind to DNA and nucleosomes and loosen the tightly packed chromatin and makes some of the other TF binding sites more accessible for binding by chromatin remodellers and other TFs

Answer 199

- Lineage determining transcription factors have pioneer activity and bind to the enhancer - This makes the enhancer site more open and active – recruits coactivators and results in modifications at the chromatin creates a primed enhancer

Answer 200

- After pioneer activity has opened chromatin at the enhancer element, the signal dependant transcription factors can then bind allowing integration of the genetic response with signalling factors received by the cell - This is highly cell type specific because the ability to respond to these signals is due to the pioneer activity of the lineage specific transcription factors

Answer 201

- Enhanceosome model | - Billboard model

Answer 202

- The position and order of the sequences in the enhancers that’s important in defining TF binding - Multiple transcription factors bind to the enhancer and forms a highly structured complex called the Enhanceosome. The targets genes would only be activated by the enhancer upon precise assembly of this complex

Answer 203

- More flexible than enhanceosome model - The identity of the TFs that bind to the enhancer is important for the function of the enhancer site - Multiple transcription factors of different purposes bind to the enhancer and discrete regions of the enhancer containing a few of these binding sites is sampled by the basal machinery and determines the overall output - A rigid complex is not required

Answer 204

- Histone methyltransferases (MLL) - Histone acetyltransferases (CBP) - Chromatin remodellers (BAF) - Mediator complex

Answer 205

Recruited to loci in the genome by sequence dependant transcription factors (lineage specific) binding to enhancers which in turn recruit the co-activators. The co-activators act independently to the underlying DNA sequences

Answer 206

After pioneer transcription factors bind to enhancers, co-activators are recruited which modify histones and allow the enhance to enter an open state - primed - These coactivators are required for the function of DNA binding activators

Answer 207

The same co-activators are used - Lineage specific transcription factors bind to the enhancer regions and open chromatin in different cell lineages. - The same coactivators are then recruited to the newly opened enhance regions by these lineage specific transcription factors

Answer 208

- After linage determining factor binding, transcription factors binds to UTX, a histone demethylase, which removes repressive histone modifications H3K27me3 made by EzH2. This is then replaced with H3K4me by the MLL/COMPASS complex - p300 is recruited which with adds H3K27ac to open the chromatin making the enhancer poised for activation - When in the poised state and the enhancer is off, there is high level of H3K27me3 and low/no H3K4me and H3K27ac. When activated, the repressive marks are removed and p300 binds to add H3K27ac. The differences are on the level of chromatin modifications

Answer 209

Embryonic stem cells are enriched in the poised enhancers and upon differentiation these enhancers transfer to an active state - poised enhancers are later in activation stage and characterised by epigenetic marks (primed only TF binding)

Answer 210

RNA pol II transcribes when the enhancer is active to produce eRNAs which promote the activity of the enhancer to drive gene expression

Answer 211

- They can promote looping between the promotor and the enhancer using cohesin and the mediator. - They can release Poll II from the gene promotor to the gene body and stabilise TF binding to enhancers. - They can also stimulate the CBP activity to increase H3K27ac

Answer 212

Higher order organisational units of chromatin folding and function within the nucleus - TADs identify the extent of a chromatin domain containing transcriptionally co-regulated gene

Answer 213

Enhancers interact with genes within the same topologically associated domains due to the presence of TAD boundaries which are defined by the insulator protein CTCF.

Answer 214

Hand-cuff model - Two ends of a TAD are brought together in 3D space by CTCF proteins which bind to each boundary via the DNA motifs ad recruit cohesin. - This is supported as the cohesin complex generally co-localises with CTCF throughout the mammalian genome

Answer 215

A problem is that the number of CTCF binding sites are much more abundant than the number of TAD boundaries. Therefore, why are TADs not more abundant?

Answer 216

Hi-C | - Based upon chromatin capture e.g 3C

Answer 217

- Close chromatin are cross linked using formaldehyde - DNA is digested - Before DNA ligation, the newly made restriction ends are filled in with biotin-labelled nucleotides which tag the DNA at different positions - Then ligated to produce a circular DNA and then sequenced - A Biotin pull-down is the performed to ensure that only ligation junctions are selected. - Reads are then mapped back to the genome and when a pair of tags are found on two different restriction fragments then these two fragments are known to interact in the 3D nucleus.

Answer 218

When chromatin are interacting, they are near each other. This process works by crosslinking chromatin in the nucleus and joins together regions of the chromatin that are in proximity to each other by treating the chromatin with formaldehyde

Answer 219

- You can use the results of Hi-C interactions to map the genome and investigate contact frequency between different loci - Can then find domains within in the chromosome that are in contact with each other in 3D

Answer 220

Interactions that are no further than 5kb a part. Can see gene and enhancer loops, Polycomb mediated

Answer 221

Can see TADs. Dark triangles show the TADs regions that interact with each other in 3D space. In the nucleus, each domain is more closely interacting with each other in 3 dimensions

Answer 222

Can see that individual TAD domains start to group together to form multi TAD domains and form chromatin compartments and can be defined more by histone modifications

Answer 223

The hi-C map indicates individual chromosomes. Can see that there are very few interactions in trans between different chromosomes

Answer 224

In vertebrates, there is a high level of conservation of the location of the TADs between cell differences. The differences seen between cell types are at the level of structure within a TAD, such as enhancers active

Answer 225

- TADs form between the binding sites of the insulator protein CTCF. - Housekeeping genes are also found at the boundaries of TADs as they do not require enhancer for their activity - Repetitive parts of the genome

Answer 226

TADs are crucial for development and disease. If disrupt the TAD boundaries, enhancers will begin to act in parts of the genome that it should not and cause ectopic gene expression

Answer 227

- Driven by the cohesin complex - Forms ring around two strands of DNA and uses ATP hydrolysis to push DNA through the ring structure forming a loop region - Two CTCF binding sites sit at the neck of the loop that are in the same direction. - When the cohesin complex meets these sites during loop formation, the looping stops at his point and the CTCF binds to its two opposite binding sites and dorms a TAD domain

Answer 228

TAD structure is important for enhancer activity as it limits the range of genes that an enhancer can act upon

Answer 229

CTCF sites are crucial for maintaining TAD structure. Disruption of CTCF binding allows enhances to regulate gene expression in a different manor deviating from it’s normal regulation.

Answer 230

Cohesin complex and CTCF can also interact with cell type specific factors which can give TADs a cell type specific function

Answer 231

- Experimental deletion of CTCF domains - Resulted in the formation of larger TADs by the joining of smaller TAD regions where the CTCF binding site had been lost - This changes the activity of enhancers

Answer 232

Compartments are regions of chromatin formed of multiple TADs over large distances

Answer 233

A active and B inactive

Answer 234

- TADs within these regions preferentially interact with each other and is defined by chromatin modifications - e.g. An A compartments TAD has more activing histone modifications and chromatin within B compartments have more repressive modifications

Answer 235

The overall TAD structure does not significantly change during differentiation but they can shift from an active to an inactive compartment depending if they are going to be expressed in that cell lineage.

Answer 236

This is driven by chromatin regulatory complexes. This shown by artificially recruiting dCas9 to an artificial region which resulting in shifting the TAD domain between compartments

Answer 237

Post translational modifications of histone tails which change the chromatin state

Answer 238

Lysine acetylation or methylation

Answer 239

- Writers: introduces the post-translational modification - Erasers: removes the modification - Readers: binds the modification and acts upon this modification

Answer 240

These complexes are involved in the establishment of transcriptionally repressed chromatin. When these complexes can’t work (due to one gene mutation) then the system falls apart and it results in derepression of the Hox gene cluster

Answer 241

The core contains histone ubiquitinates: RING1A and RING1B which are mutually exclusive. They require a stable association with one of the polycomb group ring finger (PCGF) proteins. There are 6 different PCGF proteins. - There are at least 100 different forms of PRC1 could functionally exist in each cell at a given developmental stage. The specific functional properties and regulation of these complexes are poorly understood

Answer 242

- EZH1 and EZH2 are mutually exclusive (PCR2 only has one type) - They methylate H3K27 and require stable associative with Eed and Suz12 and recruit RBBP4 or RBBP7.

Answer 243

- The enhancer of zeste is the catalytic core of the complex to create the trimethylation and Esc recognises the mark. This pair of activity allows chromatin to be progressively methylated. - Enhancer of zeste methylates histone, the Esc then binds to this methylation mark and positions enhancer of zeste in the correct location to methylate the next histone H3 in the next nucleosome - The enhancer of zeste protein is inactive without ESC showing the need for the whole PRC2 complex

Answer 244

- Recognises the trimethyl mark through the chromodomain - Methylation on lysine 27 from PRC2, the Polycomb protein in PCR1 then sits in between these methylations and creates condensed chromatin

Answer 245

Functionally antagonistic

Answer 246

methylates lysine 4 - activating mark

Answer 247

PHD fingers

Answer 248

- ES have a bivalent pattern of modification. Contains both methylated lysine 4 and lysine 27 which have distinct functional characteristics. Lysine 4 is activating and lysine 27 is repressive - In differentiated cells some retain the lysine 4 or the lysine 27 methylation. They make the decision to remove one of the methylation marks allowing either transcription or repression. It loses bivalency. - Genes required for a specific lineage maintain their lysine 4 mark and remove their lysine 27 mark.

Answer 249

- The vast majority of promoters are enriched in H3K27 and H3K4 methylation. They are bivalent and are all inactive genes but have an association with RNA polymerase II so are ready to differentiate (poised)

Answer 250

H3K4 methylation requires the activity of the Trithorax protein Mll2 - In Wt ES cells there is a strong peak of H3K4 methylation in various genes - Knockout of Mll2 then the level of methylation has been completely abolished - However, H3K27 methylation is unaffected by Mll2 knockouts

Answer 251

H3K37 require polycomb function - Knockout components of the Polycomb complexes - Knockout of EED (binds to EzH2), or EzH2 then lose tri and di methylation of H3K27 - Expression of pioneer transcription factors (nanog ect) in knockouts is gone. Polycomb function is required for pluripotency - Loss of PRC2 function causes spontaneous differentiation of ESCs into meso-endodermal tissue and lose pluripotency

Answer 252

Polycomb and Trithorax Group proteins are functionally antagonistic - Come together on bivalent genes to create the state of poised transcriptional inactivity - Induction of differentiation disturbs this balance and breaks the transcriptional silence to allow for the removal of H3K27 marks and differentiation into a specific lineage.

Answer 253

Every somatic cell nucleus contains identical DNA. Unused genes in differentiated cells are maintained and retain the potential for being expressed. Only a small proportion of the genome is expressed in each cell type and a proportion of RNA expression is cell type specific

Answer 254

- How do reprogramming transcription factors initially interact with the genome during reprogramming? - How different transcriptional networks are regulated during reprogramming? - How do OSKM factors work together to drive re-programming?

Answer 255

Oct4 Sox2 Klf4 C-Myc

Answer 256

- Induced expression of OSKM in human fibroblasts using Dox inducible expression. OSKM are maximally expressed at 48 hours. If then remove the exogenous expression of the OSKM by removing dox, the cells continue to grow and express pluripotency markers – induced pluripotent cells - After 48 hours, ChIP-Seq for OSKM following 48 hours of Dox to look across the genome to see where the OSKM factors are binding.

Answer 257

- Oct4 Chip-Seq also showed enrichment of the other factors at the same point. - This shows that the factors all bind together and the same sites in the genome suggesting in early stages of reprogramming the factors act in the same transcriptional network

Answer 258

- OSKM factors bind and regulate genes required in reprogramming - turn off fibroblast specific and turn on pluripotent. - Many of these bindings occur at enhancer regions rather than promotors

Answer 259

- C-Myc binds open chromatin while OSK factors bind closed chromatin. - OSK factors therefore have pioneer factor ability. 70% of all OSK factors bind at closed chromatin regions using this pioneer factor activity- they bind to genes involved in pluripotency which are closed in differentiated cells - This pioneer ability is due to the varying crystal structure of the factors

Answer 260

C- myc binding at enhancers and promotors in DNAse resistant areas requires the presence of the other factors – requires the pioneer activity of oct4 and sox2

Answer 261

Whole genome sequence found large domains where OSKM factors did not bound

Answer 262

DBRs have large enrichened regions of repressive H3K9me3 | - Once reprogrammed these regions lost methylation and OSKM could bind

Answer 263

They depleted the methyltransferase responsible for this repressive mark (Suv39H1/2) by siRNAs. This resulted in a recovery of Oct4 and sox2 binding in the DBRs showing that it is the H3K9me3 that is blocking OSKM binding. This recovery is not seen in regions that aren’t affected by methylation

Answer 264

The vulva is the egg laying organ in the hermaphrodite worm

Answer 265

Worms that have developed without a vulva exhibit egg laying defect phenotype

Answer 266

They are good model organism because they are small, rapid life-cycle, free living, few cell types, compact genome, transparent cuticle (look in a live worm) and there are hermaphrodites and males, every cell linage is mapped

Answer 267

Hermaphrodites start their life as a male and produce sperm and then later in development produce eggs which can be self-fertilised

Answer 268

The C. Elegans hermaphrodite vulva is one of the best studied models for signal transduction and cell fate determination during organogenesis

Answer 269

These pathways were discovered by forward genetic screens and laser ablation. Laser ablation is when they kill one of the cells in the worm and then see how this effects development of the worm

Answer 270

Vulval precursor cells

Answer 271

Begins with 6 vulval precursor cells (VPC) which overlay an anchor cell in the L2 stage of larval development

Answer 272

The process is always the same | - One exception: P3.p sometimes becomes a VPC and sometimes fuses with Hys7

Answer 273

EGFR, NOTCH and Wnt

Answer 274

The combined actin of the three pathways result in a single primary VPC (P6P) that Is always flanked by two secondary VPCs (P5p and P7p). If there is no signal at all from the signalling then they become tertiary VPC

Answer 275

Wnt and EGFR are an example of paracrine signalling while NOTCH is Juxtacrine signalling

Answer 276

- NOTCH signalling occurs between secondary and primary VPCs. Wnt and EGFR occurs between anchor cells and VPCs - These pathways cooperate acting with partial redundancy to specify cell fate.

Answer 277

- Beta catenin is bound to a destruction complex in the cell cytoplasm, formed by the kinases CK1 and GSK3 and proteins APC, Axin and Slimb - CK1 and GSK3 phosphorylate beta catenin causing the slimb protein to ubiquinate it. These targets the beta catenin for degradation by the proteasome - Without the presence of beta catenin in the nucleus, repressor proteins such as Groucho block activation of the target genes

Answer 278

- Groucho represses transcription by recruiting histone deacetylases which causes acetyl groups to be removed from histones so that they are packed tighter. - Therefore, transcription cannot occur because the genes are not accessible

Answer 279

- Wnt binds to the transmembrane receptor frizzled and co-receptor arrow/LRP - Destruction complex proteins CK1 and GSK3 phosphorylate the cytoplasmic tail of the activated arrow/LRP and this leads to the phosphorylation of Dsh. This stops the formation of the destruction complex and the loss of slimb protein - Beta catenin is still phosphorylated but it cannot be degraded causing the release of beta catenin which enters the nucleus and removes the repression protein Groucho and activate transcription of the selected target genes

Answer 280

- Beta catenin removes repression by binding to the promotor region and activating histone acetyl transferases and BRC1 (a chromatin remodelling factor). - This causes the histones to be less densely packed so transcription can occur.

Answer 281

- EGF activates a receptor tyrosine kinase (RTK) pathway - Binding of the ligand LIN-3 triggers dimerization of the receptor causing transphosphorylation and triggers a kinase cascade Eventually the activated ERK kinases alters gene expression by phosphorylating transcriptional factors

Answer 282

- Notch (transmembrane receptor) binds to Delta (transmembrane receptor on adjacent cell). - This occurs at two receptors at the cell surface which then interact and causes proteolysis of the Notch receptor. - The intracellular domain of NOTCH to enter the nucleus and activates target genes by binding to CSL which displaces transcriptional repressors and recruits transactional activators e.g. histone acetyl transferase p300 This is how the primary and the secondary VPCs communicate

Answer 283

The syncytium

Answer 284

- The primary cell fate is the primary VPC (P6.p) and forms the central vulval cells - The secondary cell fate (P5.p and P7.p) and forms lateral vulval cells - The tertiary cell fate (p3.p p3.p and p8.p) fuse with hyp7 and forms the hypodermal cells

Answer 285

- In L1 stage of development P cells divide into Pn.a and pn.p. (where n is any number of cells) - The pn.p goes onto form the epidermis but wnt signalling selects six of the Pn.p cells to become the vulval precursor cells. - It does this by inducing the Hox gene Lin-39. - The other Pn.p cells lose this potential and become the hypodermis

Answer 286

- The Wnt signalling comes from the anchor cell and without it, no pn.p cells are driven to vulval competence and the vulva does not form - Activates Hox gene Lin-39

Answer 287

- P3.p can either become a VPC or fuse with hyp7 - Animals over expressing Lin-3 (EGF like ligand), the fusion of P3.p is not observed. - This showed that the vulval equivalence is decided by Wnt and EGFR signalling. If it was only decided by Wnt, the amount of Lin-3 would not affect the outcome of p3.p

Answer 288

The anchor cell

Answer 289

- LIN-3 is secreted from the anchor cell in a gradient and activates LET-23 (EGFR) in all VPCs - P7.p receives the most of LIN-3 and becomes the primary VPC – EGFR tyrosine kinase pathway is activated. This pathway ends with phosphorylation of the transcription factors LIN-1 and LIN-31. - The phosphorylated version of these factors cannot repress transcription and activate genes associated with a primary cell fate. - One targets of these genes is the lin-39 hox gene which promotes the primary cell fate - Wnt also activates Lin-39, showing that both Wnt and EGFR contribute to the primary cell fate

Answer 290

NOTCH signalling - the primary VPC expresses transmembrane receptor ligands LAG-2, DSL-1 and APX - other VPCs express NOTCH receptor Lin-12

Answer 291

- The secondary cells already had less of the lin-3 signal from the anchor cell - Notch signalling inhibits EGFR signalling in the flanking cell by lateral inhibition. - It triggers the endocytosis of the EGFR to prevent further activation of EGFR signalling and it also triggers activation of MAPK phosphatases which is undoing the kinase cascade activated by EGFR signalling already

Answer 292

- This was evidenced in a constitutive LIN-12 mutant as all the 6 VPCs adopt a secondary cell fate. - This shows that NOTCH must have an inductive role even if LIN-3 signalling is absent. - NOTCH signalling is sufficient to drive secondary VPC fate

Answer 293

The distal VPCs that receive neither lin-3 nor lateral Lin-12 signal adopt the tertiary non-vulval cell fate

Answer 294

Sternberg et al, 1986

Answer 295

- The pluripotency factors all drive expression of different tissues so the balance of them results in no differentiation and the cells remaining in a self-renewing state - This is achieved by the factors mutually repressing each other. Oct4 drives mesoderm differentiation but represses trophoectoderm formation. Sox2 drives ectoderm but repressed trophoectoderm and mesoderm

Answer 296

DNA methylation can also be removed by TET enzymes in a multi-step process - Forms hydroxyl methylcytosine (sign of active demethylation) - Leads to full removal of the nucleotide to create a gap in the DNA - Repaired by the base excision repair mechanism - Is then repaired to unmethylated cytosine

Answer 297

Poised enhancers are commonly found in ES cells

Answer 298

- Poised are characterised by the presence of epigenetic modifications and require longer to activate then primed - Primed state is characterised by transcription factor binding but no epigenetic modification

Answer 299

Pattern formation during vulval development in C. elegans

Answer 300

- They looked at the pattern of cell division to identify each cell type using Normatski optics - Ablated individual cells with a laser

Answer 301

- Determination occurs in the Pn.p (P(3-8).p cells and not their progeny (pn.px). After cell division, the cells are committed to their fate - The unindicted ground state of the Pn.p cells is to adopt a tertiary fate – fuses with the syncytium - A graded signal from the anchor cell specifies which of the three fates are adapted

Answer 302

- They ablated different of the VPCs in 9 different C. elegans before the first cell division - When no cells are ablated, P6.p is primary, P5 and P7 are secondary and the others are tertiary - When ablated P5.p cells, everything to the right stayed the same: the primary secondary and tertiary but to the left the p4.p moved and adopted the secondary cell fate on the left and the P3.p remained tertiary - If ablate a cell before division, cells can be replaced. If occurs after cell division, replacement cannot occur

Answer 303

- Used Ion-1 mutant which are 50% longer than wild type. - Because of this, the anchor cell is not always in the correct place giving a shifted vulvae phenotype. - This could therefore change the fate of Pn.ps and the anchor cell would be between the P5.p and the P6.p and expect to see a half shit in cell identity. - However, this never occurred suggesting that the cell identity cannot be changed after division

Answer 304

The anchor cell is not required after the first division. If ablate, development is normal

Answer 305

- The anchor cell could stimulate P(5-7) to adopt a primary or tertiary cell. This would mean that the ground state of the pn.p cell is to adopt a 3’ fat. - Pn.p cells could inhibit each other from adapting primary or secondary fates and that the anchor cell could release P(5-7) from inhibition by their neighbours

Answer 306

- If remove the gonad (row B), this removes the anchor cell, everything becomes tertiary - Ablated P5-7 (row C) and kept the gonad (and therefore anchor cell), this resulted in cells moving in o replace the lost primary and secondary cells - Ablated the gonad and P5-P7, all cells adopt a tertiary cell fate - Therefore, the ground state is tertiary and the anchor cell induces the primary and secondary cell fate

Answer 307

- This model suggests that an isolated Pn.p cell could not adopt a secondary cell fate. - They therefore used an unc-84 mutant where cells cannot move to generate isolated pn.p cells. - The isolated pn.p cells could adopt any fate depending on its distance from the anchor cell – it doesn’t need to talk to other pn.p cells, proving model B wrong

Answer 308

Distance from the anchor cell dictates pn.p cell - When a p8.p is under the anchor cell it forms the primary VPC. - When p8.p is further away it becomes a secondary cell and when it is at its furthest away it adopts a tertiary cell fate.

Answer 309

- The anchor cell releases EGF ligand Lin-3 where most is received by primary and least by tertiary - The primary (P6.p) and secondary cells (p5.p, p7.p) which becomes the vulva. While the tertiary cells become the hypodermis syncytium - There is also notch signalling between the primary and secondary cells - After specification, the cells divide - For morphogenesis to occur, interaction between primary and secondary cells is required (notch signalling)

Answer 310

Notch signalling in bone development (Yamada et al, 2003)

Answer 311

- Early in vitro studies have found the Notch signaling pathway functions as down-regulator in osteoclastogenesis and osteoblastogenesis - Constitutively active Notch1-transfected stromal cells showed increased expression osteoclast repressive genes, resulting in reduction of their ability to support osteoclast development - Overexpression of Notch signaling inhibits BMP2-induced osteoblast differentiation.

Answer 312

Overall, Notch signaling has a major role in the commitment of mesenchymal cells to the osteoblastic lineage and provides a possible therapeutic approach to bone regeneration

Answer 313

Flowering decision in response to cold weather

Answer 314

Vernalisation in plants Metabolic disease risk or behavioural traits in mammals

Answer 315

Flowering Locus C (FLC)

Answer 316

- FLC is floral repressor - If on, there is late or no flowering - If off, early flowering

Answer 317

- Because this is so well characterised, it is an experimentally robust way to study gene regulation. Refers to other organisms

Answer 318

The length of time and when flowering occurs

Answer 319

The prolonged exposure to cold leads to a mitotically stable state that survives vegetative propagation – gene stays off even when reprogrammed to a stem cell

Answer 320

- The repression is limited to one locus and the genes adjacent to FLC are not affected by cold temperature. - This is quite rare in epigenetics – usually whole regions affected

Answer 321

- There is a clear temporal separation of the establishment (cold winter) and the maintenance (spring) - The silencing is quantitative – the degree of stable FLC silencing after vernalisation reflects the length of cold exposure – this occur is a cell autonomous way

Answer 322

The epigenetic state is re-set at every generation to ensure that the vernalisation requirement is inherited in the offspring

Answer 323

- 6 weeks- seed to seed - Germination, seeding establishment, vegetative growth (continues until cold), floral transition, senescence - Embryo development occurs after the flower had been fertilised - There is a transition from vegetative meristem to reproductive meristem when the FCL gene is involved

Answer 324

- If grow a plant at 20 degrees, it won’t flower because high expression of FLC. If put plant in fridge with light, FCL expression deceases. When return the flower to 20 degrees, the plant flowers - A longer exposure to the cold results in a quicker flowering when removed from the cold

Answer 325

It temperate climates, vernalisation is advantageous to keep plants in a vegetative state in winter

Answer 326

- Light quality, light time and temperature is about the same - Doesn't know when to flower

Answer 327

- It does this by measuring the prolonged period of cold – has it been winter before

Answer 328

- The repression of FCL is quantitative and increases with accumulating exposure to cold. This leads to the progressively earlier flowering - Can see this affects the evolution in the genomes of plants in different countries – require different amounts of cold to flower

Answer 329

Vernalisation involved the down regulation and epigenetic silencing of a key floral repressor gene called FLC

Answer 330

- During the cold, silencing occurs at small patches of the gene called nucleation sites which then spreads across the while locus when returned to the warm – known this because can separate the exposure and memory sections in the genome

Answer 331

- PRC2 is already at the FLC gene in the cold. - Cold activates anti-sense transcript to FLC called COOLAIR (a ncRNA) which transcriptionally shuts down FLC - After longer exposure to cold, VIN3 upregulates which recruits a PHD protein to the PRC at the FLC locus. - The PHD protein adds repressive marks to the locus – e.g. H3K27me3 - VIN3 also nucleates the PRC to small nucleation regions in the FLC - Nucleation increases with time in the cold

Answer 332

- In the warm, VIN3 turns off and the silencing marks in the small nucleation regions of the FLC locus spread across the whole locus and FLC is turned off

Answer 333

After fertilisation, resetting occurs and ELF6 removes the H3K27me3 marks from the FLC locus. LEC1 is also involved in this process

Answer 334

- When warm and FLC is transcribed, there is expression of distally polyadeylated COOLAIR – long anti-sense - FRI-C is associated to the FLC locus and adds activating histone modification such as H3K36me3

Answer 335

- When cold and transcription of FLC decreases, COOLAIR is polyadenylated proximally forming a small transcript which binds to flowering genes such as FCA and FY. - They then recruit FVE and FLD to the FLC locus. - These acts independently. FLD removes the active histone marks (it’s a H3K4 demethylase) meaning that site is available for a repressive mark. - FVE is histone deacetylates 6 and adds a different repressive mark

Answer 336

- Rapid cyclers are from hot places such as Columbia and have no vernalisation requirement - Rapid vernalisers e.g. UK – low vernalisation requirement but requires small cold for flowering - Slow vernalisers e.g. Sweden – requires a long winter for FLC to be repressed. If taken to England, the plant will never flower as FLC is not repressed for long enough to shut it off

Answer 337

The wild type Arabidopsis, Columbia, is a rapid cycler and has lost FRIGIDA gene

Answer 338

- A strong up regulator of the FLC | - Absolutely required for active FLC transcription and delay flowering

Answer 339

- Directly binds to the nuclear cap-binding complex - Recruits a WD40-domain protein that promotes H3Kt6 trimethylation - Forms transcriptional activator complex

Answer 340

- FLC is silenced after winter and the silencing is maintained through mitosis. However, this gene needs to be turned back on before it is passed on to the next generation - Plants DO NOT have a germ line

Answer 341

LEC encodes a subunit of NF-Y and is master regulator of embryogenesis

Answer 342

- A plant grows in the warm in the seedling stage, FLC is on and LEC1 is off. - Vernalisation occurs and FLC is turned off. - Return plant to the warm and the plant flowers and undergoes meiosis and gametogenesis – FLC is still off. - In early embryo development, LEC1 turns on and exchanges the silencing marks to the active marks on the FLC locus – FLC is turned on. - The cycle repeats

Answer 343

- Pioneer transcription factors are special types of transcription factors that can access their target sequences in condensed chromatin - They are often associated with changes in cell fate and developmental switching.

Answer 344

- The seed-specific transcription factor LEAFY COTYLEDON1 (LEC1) acts as a pioneer transcription factor.

Answer 345

- Is necessary for FLC resetting during embryo formation | - LEC1 is necessary for the increase in activating histone marks at the FLC locus

Answer 346

The role of LEC1 in the reprogramming of embryonic FLC expression

Answer 347

- Vernalised and non-vernalised plants 1-5 days after pollination and the expression of FLC with the reporter gene GUS - Difference in expression disappears by day 3 and FLC is turned back on in the embryo as there is no visual difference between FLC expression in vernalised and non-vernalised parental plants

Answer 348

- Wild type Columbia flowers early (17-18 leaves) - Columbia with FRIDGIDA (FRI) put in (70 leaves) flowers late - Knockout of Lec1 in FRI background results in intermediate flowering time (partly turns FLC back on)

Answer 349

- Hypothesised that LEC1 would bind to the cis element CCAT in the FLC promotor - There are 4 of these sites in the FLC promotor - Used antibody against LEC1 and pulled DNA down and then PCR to see what the binding sequence is - LEC1 binds partly to the first site, mostly at position 2, partly at 3 and none at 4 - LEC1 directly binds to the promotor of FLC

Answer 350

DEX inducible system for turning on LEC1 quickly. - Express transgene with glucocorticoid receptor bound to LEC1. - This binds to HSP90 and restricted to the cytoplasm. - Then treat with DEX which binds to GR, dissociates HSP90 and allow translocation of LEC1 to the nucleus where it can activate target genes

Answer 351

- Active marks are reduced in lec1 mutants (H3K36me3) and an increase in repressive H3K27me3. This suggests that LEC1 is needed to recruit active marks on FLC - EFS complex is recruited to the FLC locus by LEC1

Answer 352

- LEC1 acts in 15-day-old plants as well as embryos - Wt Columbia, FRI and FRI lec1 - Similar results seen in older plants as embryos

Answer 353

LEC1 activity is required to re-activate FLC expression during embryogenesis

Answer 354

Gene silencing

Answer 355

It does this by blocking the binding of transcription factors or by binding MECP2 (methyl CpG binding protein 2) which recruits histone modifying complexes which promote silent chromatin formation

Answer 356

Detection of hemi methylated DNA by DNMT1

Answer 357

The one carbon cycle - The product of this is DNMT (DNA methyltransferase) which is the source of methylation (SAM is an important methyl donor)

Answer 358

The one carbon cycle - Many components are supplied by the diet including Folate, vitamins B6 and B12 and methionine - Diets high in this methyl donating nutrients can rapidly alter gene expression at the whole genome level. Isn’t gene expression - This has the largest impact during early development when the epigenome is first being established

Answer 359

Dominguez-Salas et al, 2013 - In some countries, diet changes throughout the year depending on the seasonal food availability - Can see in Gambia, there are monthly and seasonal changes in protein intake which are correlated with folate, B2 and DMG (components of one carbon metabolism pathway)

Answer 360

- Agouti gene encodes paracrine-signalling molecule ASIP (Agouti signalling peptide) - When its promoter is un-methylated, the gene is on, ASIP protein is abundant, and mice show the typical yellow coat and tendency to be obese. - ASIP inhibits hypothalamic signalling making mice hyperphagic (eat a lot) - Maternal diet therefore dictates the activity of this gene and the offspring phenotype

Answer 361

Morgan et al, 1999 - Mice were supplemented with vitamin B12, folic acid, choline and betaine during pregnancy results in thin, brown pups. - Without supplementation, the pups were yellow and hyperphagic

Answer 362

Transmission of epigenetic information through the germ line that affects phenotypic traits in more than one generation without changes in DNA sequence

Answer 363

Intrauterine exposure does not require the passage of an environmental memory through the germ line

Answer 364

- To rule out the possibility of genetic change (difficult in large genomes such as humans) - Rule out direct exposure (the developing foetus wasn’t exposed to the environment)

Answer 365

- The foetus (F1) is exposed to the environment that the mother (F0) is - The foetus will also have developed their oocytes (F3) and therefore exposed to the environment too by the grandmother. This is intrauterine exposure - For TEI to be correct, would have to look at the effect of the third generation (F3)

Answer 366

- The males (F0) experience cells and his reproductive cells (F1) also experience the stress - The grandson however will not have experienced the stress so TEI could be shown at the F2 generation

Answer 367

The MTRR protein is responsible for the activation of methionine synthase through the reductive methylation of its vitamin B12 cofactor and is essential for folate metabolism

Answer 368

Made a knockdown transgenic line of MTRR (mtrrgt). This resulted in increased plasma levels of homocysteine. The knockdown was successful and resulted in fewer methyl groups and a build-up of one of the cycle components

Answer 369

The grand progeny of these mice showed a variety of developmental defects including neural tube defects, developmental delay and heart defects

Answer 370

The genotype of the grandmother effects the phenotype of the grand progeny - When the grandmother was heterozygous for knockdown gene resulted in developmental issues - Healthy grandmother resulted in healthy mice grand progeny. - Normal genotypes of offspring still had developmental defects if the grandmother was heterozygote

Answer 371

These congenital malformations independent of maternal environment persisted for five generations – TEI

Answer 372

- The agouti gene encodes a signalling molecule (hormone) that blocks melanin production in hair follicles. - The tip of the fur is dark due to the presence of melanin and the base of the hair is yellow due to agouti blocking melanin. - These mice appear light brown and give the agouti colour. This is wild type

Answer 373

- A – melanin is always blocked so mouse is yellow - a – hormone is not produced so black - A vy () – Brown to yellow transient expression

Answer 374

- There is a IAP retro transposon upstream to the transcription start site of the agouti gene. - o When this transposon is heavily methylated, the agouti gene is silenced and they are pseudo agouti but when demethylated, the gene is active and they look yellow. - This methylation is not just in the coat but in many tissues: the brain testis and lung ect

Answer 375

- Gave vitamin B12 supplementation to mother and looked at the colour of the offspring - Supplemented diet gives the pseudo-agouti phenotype (agouti is off) due to IAP being more methylated

Answer 376

- Counted the number of sites methylated - In unsupplemented, the methylation is usually less than 30% - Supplemented mice had increased methylation - They then mapped the percentage methylation to the phenotype seen e.g. 80% methylation – pseudo-agouti - Dietary supplements can therefore change the methylation level of the transposable element in mice

Answer 377

Synthetic hormone | - BPA is widely used in plastic production and leaches out of plastics over time

Answer 378

- BPA exposure to rodents results in increased body weight, breast/prostate cancer and meiotic defects - BPA increases the frequency of yellow phenotype in Agouti vy mice

Answer 379

- BPA was added to the diet of virgin wild type female mouse and they were mated with A vy/a males - The A vy/a offspring were scored for coat colour and methylation status CpG sites downstream of the transposon insertion

Answer 380

BPA supplement reduced the DNA methylation frequency increasing the expression of the A vy gene and the mice became fatter and yellower.

Answer 381

- Fed pregnant mice with methyl donors and tried to increase the silencing of the agouti gene - The control diet was similar to the methyl donor supplemented BPA diet showing that that methyl donor supplementation can rescue BPA exposure

Answer 382

Genome wide study (Kim et al, 2014) in the liver - Progeny of pregnant mice that had even been fed control, small amounts of bisphenol A or large amounts of bisphenol A - Some genes are unaffected, some show modest differences to the control. Others show heavy unmethylation and some acquire methylation - Can see that the function of many of the genes that have affected methylation are involved in metabolism

Answer 383

That prenatal environments can affect adult metabolic health and that of subsequent generations using male mice as an example

Answer 384

F1 generation - Control group: F0 were fed during pregnancy week 1, 2 and 3 - UN group which had a reduction in food in week 3. These pups are born with low weight (F1) F2 - Take control females and mate with one of the undernourished males (F1) and feed normally - The F2 mice are either CC (control, control) or (control undernourished) - The control undernourished had many developmental disorders despite them not being undernourished them selves

Answer 385

- Immunoprecipitation experiments in F1 male sperm showed difference in DNA methylation profiles at the genome level between CC (control) and UC - Some genes in the sperm were hypomethylated and some were hypermethylated compared to the control

Answer 386

- The F2 had dysregulation of metabolism in the over and by 8 months they had glucose intolerance and high white adipose tissue. - However, the differential methylation pattern seen in sperm had been lost but the dysregulation of genes neighbouring the DMRs is observed in F2 offspring - The mechanism of this is unknown

Answer 387

Chen at al, 2016 - Offspring were affected by sperm small tRNAs paternal dietary conditions in mammals and influence the metabolic phenotypes of offspring - They injected the 5’ fraction of sperm tRNA from males who had not been fed a high-fat diet into normal fertilised oocytes (not the parents). - The progeny has various metabolic disorders – not inherited as parents weren’t fed differently – must be because of the tRNA - Altered gene expression in metabolic pathway and obesity

Answer 388

Also investigated how offspring were affected by sperm small RNAs - Observed a reduction in abundant tRNA-derived small RNAs in sperm from mice on a low protein diet

Answer 389

Weaver et al, 2004

Answer 390

- High nurturing mothers raise nurturing offspring and low nurturing mothers raise low nurturing offspring - Whether the pup grows up to the anxious or relaxed depends on the mother that raises it not maternal mother

Answer 391

- Pup licking and grooming | - Arched back nursing

Answer 392

Glucocorticoid receptor - Encoded by GR gene - Glucocorticoids are primary stress hormones that regulate physiological processes - Ligand occupied GR induces or represses the transcription of thousands of genes

Answer 393

- The 5’ of the GR promotor has a big difference in methylation (high grooming has less methylation) - Low grooming mothers were associated with H3K9 acetylation and NGF1-A binding to the promotor of the GR gene in the hippocampus - Long term stress was quantified by looking at HPA responses and behaviour in grown up pups

Answer 394

- They tested the rats with a TSA (HDAC inhibitor) and observed changes in GR expression and stress responses. However, this isn’t specific to the GR gene, it will target all genes acetylated by HDACs - GR expression did change in response to this

Answer 395

- Difficult to study due to variation in genetic, ecological and cultural inheritance - Difficult to prove just through observation studies

Answer 396

- Parental nutrition and smoking behaviour are linked to cardiovascular, metabolic diseases, schizophrenia and antisocial personality disorders in the offspring

Answer 397

- Investigated a link between plasma folate level (methyl donor) and asthma at age 3 - 1062 participants and chose 18 Caucasians in each category

Answer 398

- Global analysis if CpG sites and found three DMRs • AHRR • CYP1A1 • GFI1 - AHRR and CYPA1 play a key role in the aryl hydrocarbon receptor signalling pathway which mediated the detoxification of tobacco smoke - Epigenetic may play a role in the observed poor health outcomes of children born to smoking woman – only an association

Answer 399

Show that genotype and environment can also change behaviour - Used two strains of mice which differ in colour and behaviour: B6 and BALB

Answer 400

Then tested their behaviour using open field test. Rodents naturally have an aversion to open areas due to predators but also like to explore new areas. This conflict allows us to explore their anxiety levels. The amount of time spent on the outer edge or in the middle can determine their anxiety levels.

Answer 401

- The white (BALB) mice are generally more anxious then the black (B6) mice which are more exploratory. If BALB mice were prenatally transferred into B6 mice or if B6 mice were prenatally transferred into BALB mice but then spent with B6 mice then they have B6 behaviour - Despite genotype, both pre and post-natal periods affected their behaviour. The B6 mice who spent time with white mice were much more anxious then would naturally be

Answer 402

Investigated associations between prenatal exposure to maternal smoking and offspring DNA methylation

Answer 403

- Methylation at 15 CpG sites in seven gene regions including AHRR and CYP1A1 was associated with maternal smoking - Shown to be dose-dependent in relation to smoking duration and intensity - Compared paternal and maternal smoking and found stronger maternal associations

Answer 404

- More likely an example of direct exposure rather than TEI | - Maternal environment is still important

Answer 405

Predictive Adaptive Response

Answer 406

- Fetal malnutrition (e.g. low protein condition) sets the metabolism to be prepared for an environment where food is scarce and use every calorie and downregulate essential organs to increase chance of reproduction - Until recently, such predictions were mostly true enabling the individual to survive to reproductive age while staying small and thin due to nutrition in later life matching the nutrition of the mothers

Answer 407

With increasingly available food supplies within a single generation, the prediction often become false, and the individual is more likely to be obese and diabetic and to have high risk of heart disease due to their lower set metabolism

Answer 408

Changes in gene expression | - DNA methylation is an epigenetic modification which enables such changes

MBB6346 Genetic pathways from zygote to organism Flashcards

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