Membrane to nuclear signalling - Lectures 25-35 (Christopher Pin) Flashcards

Question

What does inhibition of TGFB promote?

Answer 1

It appears that inhibition of TGFB promotes cancer. overexpression of DN-TBRII in the pancreas: - increased proliferation, but impaired maintenance of the differentiated state - inhibition of TGFB after determination results in acinar cells reverting to a more progenitor-like state - pre-malignant precursors, what you see in precursors of pancreatic cancer *TGFB maintains differentiation, so with inhibition, get de-differentiation

Answer 2

- affects only a subset of TGF-B regulated target genes and functions - SMAD4 was first identified as DPC4 = deleted in pancreatic cancer 4 - some events downstream of TGFB are SMAD4 independent (possibly due to non-canonical pathways)

Answer 3

Appears that loss of some components of TGFB signalling may unlock other functions - decreased TGFB signalling, promotes proliferation and prevents differentiation - gain of TGFB function and decreased TGFB signalling are both linked to cancer

Answer 4

1- Evading growth suppressors 2- Avoiding immune destruction (several of these hallmarks are targeting cells that are not part of the tumour, may be part of signalling pathway) 3- Enabling replicative immortality 4- Tumor-promoting inflammation (tumour also consists of supportive cells such as cancer associated fibroblasts, tumour-associated macrophages, and endothelial cells 5- Activating invasion and metastasis 6- Inducing or accessing vasculature 7- Genome instability and mutation 8- Resisting cell death 9- Deregulating cellular metabolism 10- Sustaining proliferative signalling * There are also 4 emerging ones

Answer 5

Epithelial-mesenchymal transition

Answer 6

Areas of gene silencing (dark areas, other than nucleolus)

Answer 7

Areas of gene expression (light areas)

Answer 8

- region for ribosome synthesis - involved in cell response to stress - assembly of signal recognition particles

Answer 9

- made up of two phospholipid bilayers (outer and inner) - the outer is continuous with the endoplasmic reticulum - structural support on inner surface by the nuclear lamina (proteins)

Answer 10

- protein meshwork (inside nuclear envelope) - mostly made up of lamins and lamin associated proteins (spiderweb of interaction - coats entire inner layer - keeps structure) lamins and lamin associated proteins (LAP): - connect to the inner nuclear membrane - connect to chromatin

Answer 11

- type of intermediate filament found in the nucleus (found almost exclusively at the periphery of the nucleus) - A-type- all types encoded from alternate splicing of the LMNA gene - B-type - encoded by two genes, LMNB1 and LMNB2

Answer 12

A- type lamins: higher expression in mechanical type tissues, skeletal and cardiac muscle (cells with very rigid nuclei, constantly contracting - need to be able to survive this stress) B-type lamins: function may be disposable but more highly expressed in undifferentiated cells (embryonic stem cells) (cells can move around, migrate, more flexible, often in development) - structural support and shape to the nucleus - associate with chromosomes - mechanotransduction - regulate gene expression`

Answer 13

- linker of nucleoskeleton and cytoskeleton - affects signalling pathways and gene regulation - another mechanism for rapid signalling from the plasma membrane - reacts to change that are more mechanical based -e.g. integrin (embedded in plasma membrane and interacts with stroma around cells)

Answer 14

Diseases linked to mutations within lamin genes 1. Emery-Dreifuss muscular dystrophy - mutations in LINC genes inc. LMNA - muscle degeneration, weakness and atrophy - cardiac defect 2. Dunnigan-type familial partial lipodystrophy - various missense mutations in LMNA - adipose tissue redistribution, insulin resistant diabetes mellitus (increased potential) 3. Charcot-Marie-Tooth disease type 2B1 -recessive mutations in LMNA - mutation in connexin gene too - motor and sensory neuropathy - slight or absent reduction of nerve-conduction velocities 4. Hutchinson-Gilford progeria syndrome - LMNA deletion - early onset aging (look much older than they are), alopecia (hair loss), fragile skin, atherosclerosis - nucleus falls apart, no structure to it - most famous

Answer 15

- Lamin A or lamin C expression is down-regulated in leukemias, lymphomas, breast cancer, colon cancer, gastric carcinoma and ovarian carcinoma. (not straightforward, diff cancers can have increase/decrease) - reduced levels of A-type lamins are predicted to result in more malleable nuclei, which could facilitate extravasation and invasion of malignant cells through narrow constrictions (this is similar to how white blood cells work - squeeze through walls to migrate) - expression of A-type lamins is upregulated in skin and ovarian cancers (need upregulated structure + integrity for primary tumours here - supports mechanical stress that the skin undergoes for example, protects them) - higher lamin levels could support the increased mechanical stress within solid tumours - changes in lamin expression could modulate cell proliferation, differentiation, epithelial-to mesenchymal transition and migration

Answer 16

- composed of a membrane associated scaffold, central transport channel, cytoplasmic ring, nuclear ring, and 8 filaments attached to each ring - for the most part where things enter and leave the nucleus - areas of heterochromatin depletion - not symmetrical or equal distribution of these pores - key for facilitated transport - multi-protein structure, multiple copies of over 30 different proteins (30 are not fixed - variability exists) - not ubiquitous composition of proteins between cell types, allowing for cell type specific transport

Answer 17

- nucleotides for DNA synthesis and RNA synthesis - transcription factors - proteins required for nuclear architecture - ions that may help in protein function e.g. calcium (second messenger)

Answer 18

- passive diffusion: molecules less than 40 kDa and ions - facilitated active transport: molecules more than 40 kDa

Answer 19

Nuclear pore proteins - why so many? - different proteins between cell types, allow for cell type specific transport (change selectivity) - some components of the pores change in response to signals

Answer 20

- importins bind transcription factors at the NLS - interaction between importin (dimerization) and transcription factors leads to nuclear localization (import) - in the nucleus, the transcription factor is released when Ran-GTP binds to importin - this leads to translocation of importins back to the cytoplasm - in the cytoplasm Ran-GTP is converted to Ran-GDP by GTPase Activating Proteins (GAPs) leading to release of the importin - Ran-GDP translocates back into the nucleus where it is converted back to Ran-GTP by guanine exchange factors (GEFs) *NF-AT is a transcription factor that interacts with importin *CRM1 is an exporter

Answer 21

- soluble carrier proteins that facilitate active transport - exportins (e.g. CRM1) and importins (all the proteins that import and export) - requires phosphorylation

Answer 22

- macromolecules that do not bind to nucleoporins do not diffuse across the nuclear pore complex - macromolecules that bind to nucleoporins increase their residence time at the entrance of the central tube - diffusion across the nuclear pore complex is greatly facilitated - cytoplasmic filaments have role (select molecules)

Answer 23

1. Genes - introns, exons 2. Promoters 3. Enhancers (silencers) 4. Intergenic regions - regulatory regions, transcribed areas

Answer 24

- position is fixed - upstream (5') of the transcriptional start site (TSS) - lose functionality if flipped for example - generally 150-500 bp in length - contains the consensus DNA sequences TATA/CAAT (generally, 70%), - tells where to transcribe - proximal control elements such as TFIIB recognition element (BRE) and initiator sequences (INR) - binds and starts transcription - binds general (basal) transcription factors - may bind tissue specific transcription factors (TSTF) - tissue specific itself transcription factor ( can have enhancer, promoter, embedded in gene)

Answer 25

- TFIID (transcription factor 2 D) binds the core promoter region and may bind additional sequences near the TSS, such as the INR (initiator sequence) - TFIIA and TFIIB (recruited) help to stabilize TFIID binding - TFIIB helps recruit RNA polymerase II - TFIIF binds RNA polII and TFIIB which aids in recruitment of RNA polymerase II (promoter can remain in this state) - TFIIE and TFIIH are recruited. These factors help to melt and unwind the promoter region, and activate the polymerase to begin RNA synthesis - TFIIH phosphorylates RNA polymerase II, the polymerase releases from the general transcription factors leading to transcriptional elongation - tissue specific transcription factors often aid in long interactions between different chromosomal regions

Answer 26

- can be upstream or downstream - can be on different chromosomes (trans-regulation) - "position" independent (within reason) - can move along genome and it still function, can flip as well - affect DNA folding and interactions (helps with unfolding of chromatin)

Answer 27

- have identified that chromatin is non-randomly distributed within the nucleus in interphase (specific organization - put genes don't want into a "corner") - distribution is cell type specific - identified gene sequences (LADs) that bind to lamin associated proteins - can repress gene expression (inner nuclear membrane) - DNA looping can bring enhancer and promoter regions to regulate gene expression (bring close to eachother) - binding of co-activators or co-repressors to the same region will regulate expression from a distance (affect interactions - proximity changes) - DNA looping and long range interactions is a mechanism for coordinated gene expression (topologically associated domains, TAD, regions brought close together)

Answer 28

- co-linear expression of globins - two chromosomes with genes that must be transcribed in parallel - both genes coordinated by same transcriptional factors - the two haemoglobin genes are localized to the same area within the nucleus - enhancer can regulate multiple genes (coordinate together)

Answer 29

Promoter analysis

Answer 30

- easily detectable - not normally expressed in the cells or tissues to be analyzed - not quickly degraded - not subject to post transcriptional or post translational regulation - examples include: green fluorescent protein (GFP) or other fluorescent proteins (YFP, mCherry, etc), luciferase, and B-galactosidase (turns blue)

Answer 31

- reporter gene replaces the gene of interest, but get same expression (still has enhancer/ repressor, and promoter) - emission of light - stable - does not affect the cells - quantifiable (can tell how strong = how much protein) - allows to identify important regions (e.g. activator, enhancer, repressor, start site, etc.) through deletion of areas and measuring outcome

Answer 32

- very stable - easily detectable - in vivo analysis - does this promoter region recapitulate complete expression in the pancreas? (can analyze this)

Answer 33

- may determine the factors that promote expression - may aid in defining genetic mutations that have a distinctive phenotype but are not within a gene - can be used as a tool to drive the expression of other genes in a cell, tissue and time specific fashion - e.g. viral introduction for gene therapy

Answer 34

- initiates gene expression - TFII proteins - bind all genes - RNA polymerase II is NOT considered a transcription factor

Answer 35

- increases transcription rate and efficiency for a specific gene - binds to a subset of genes in a cell and time specific fashion - is required for complete expression of a gene

Answer 36

- short region in gene - short domain of the protein required for nuclear localization (get into nucleus) - generally basic or positive charge (because nucleus is negative?) - all have basic amino acids - lysine (K), Arginine (R)

Answer 37

Transactivation domain - required for expression of protein - can work by itself or targeted to a different protein (there are two different TADs)

Answer 38

- radiolabel a known sequence of DNA (20-30 bp) - radiolabelling ensures specific binding - mix with purified protein - interaction leads to a "shift" - shift is lost when adding excess unlabeled sequence - supershift when you include and antibody against the protein - proteins don't migrate as far with DNA binding domain

Answer 39

- show binding of co-activator or other TFs - suggest that two proteins interact - purify one proteins from an extract with an antibody specific to that protein - maintain interacting proteins - assess what other proteins are pulled out by the first protein - assess by western blot (targeted) or mass spectrometry (see many at once) e.g. ask if protein x binding protein y? - use protein A agarose beads to pull protein of interest away from others (like a magnet) - brings protein it is interacting with as well - do a western blot + test antibodies for y to make sure it is present

Answer 40

- often classified based on their protein binding motifs - 5 classes (ordered by binding, smaller to larger regions of DNA, larger regions = more general functions) Super classes: 1. Basic 2. Zinc-coordinating DNA-binding domains 3. Helix-turn-helix 4. Beta-scaffold factors with minor groove contacts 5. Orphan transcription factors

Answer 41

Named after what they look like: - Leucine zipper (ZIP) - Helix-loop-helix (HLH) - HLH-ZIP bHLH proteins bind the consensus DNA sequence CANNTG (short region, not too specific) distinct roles (specific) e.g. Basic helix-loop-helix (bHLH): MyoD and MYF5 are bHLH proteins expressed in myogenic precursors. loss of both results in no muscle

Answer 42

Zinc molecule at base - zinc fingers or zinc clusters - provide specificity for DNA sequences (pre-cursor to CRISPR-CAS9) - LIM proteins composed of two contiguous zinc finger domains - name based on motif found in Lin-1, Isl-1, and Mec-3 e.g. Lim1: transcription factor expressed during brain development

Answer 43

- Homeodomain - Paired box - Fork head - specificity of DNA recognition lies within the homeodomain e.g. regulates insulin gene: MODY - maturity onset diabetes of the young - 5-10% of those patients with type II diabetes - autosomal dominant - mutations of these proteins causes incomplete expression of insulin gene = diabetes

Answer 44

- include the TFII transcription factors - wrap around - STAT: signal transducer and activator of transcription. involved in inflammation and cancer. trigger by jun kinase (JNK) - NFAT: nuclear factor of activated T-cells. involved in immune response. triggered by increased calcium. -STAT and NFAT are generally outside the nucleus and are brought in - stabilizing binding factors. - P53: represses the cell cycle. mutated in many cancers. involved in DNA damage repair. generally localized to nucleus, affects transcription directly + DNA repair.

Answer 45

- HMGB - bends DNA to facilitate binding of other transcription factors (such as p53) - affects how chromatin bends

Answer 46

- synthesis - transcription/translation (mRNA can be targeted for degradation) - phosphorylation - can occur at multiple levels (post-translational modifications) - prevent nuclear localization (cellular localization - cytoplasm to nucleus) - protein degradation - access to DNA (chromatin structure - heterochromatin vs euchromatin) - ligand binding (of receptors at the cel surface trigger signalling cascade) - availability of co-activators and co-repressors

Answer 47

- nuclear receptors translate external signals that directly bind TFs that go (C) "cytoplasm" (binds receptor in the cytoplasm, cause to dissociate with inhibitor complex and go into nucleus) or are at (D) "direct" the target genes (go straight into nucleus and binds receptor in nucleus) - nuclear receptor superfamily is comprised of 48 genes (plus splice variants) in humans - receptors shuttle to/ or already present in the nucleus upon ligand interaction - regulate transcription by binding to specific DNA sequences - can bind as monomers, homodimers or heterodimers - divided into four classes based on key characteristics such as dimerization, DNA binding motifs and specificity, and ligand binding - classifications based on sequence alignments and DNA binding behaviour

Answer 48

- contains the activation function 1 (AF-1) whose action is independent of the presence of ligand. - first function of protein - highly variable in sequence between various nuclear receptors-

Answer 49

- highly conserved domain - contains two zinc fingers that bind to specific sequences of DNA called hormone response element (HRE)

Answer 50

- connects the DBD with the LBD (ligand binding domain) - influences intracellular trafficking and sub cellular distribution - affects how protein is folded and whether it is stable or not

Answer 51

- highly conserved in structure between the nuclear receptors (can't be completely conserved, wouldn't get specificity for ligand) - binds ligand leading to dimerization and coactivators/ corepressors - contains the activation function 2 (AF-2) - cause second function of protein

Answer 52

- highly variable in sequence - might be responsible for binding other things as well

Answer 53

1. Class I: steroid receptors (homodimer) 2. Class II: RXR heterodimers (typically) 3. Class III: Dimeric orphan receptors (DOR) - homodimer 4. Class IV: Monomeric orphan receptors (MOR)

Answer 54

- localized to the cytoplasm - bound to an inhibitor such as Heat shock protein (HSP) - chaperone - typically activated by hormones such as thyroid hormone, estrogen, androgens glucocorticoids and mineralocorticoids - bind as homodimers to activate gene expression (inverted + direct repeats, mirror eachother) - steroid hormones - type C (bind in the cytoplasm and transported into nucleus - too large to go into alone) - roles in: maintenance of cellular homeostasis, gene expression, regulation in embryogenesis (large role, sex hormones involved), and tissue development. - bind hormone response elements (HREs) consisting of two half-sites separated by a variable length of DNA - second half-site has a sequence inverted from the first (inverted repeat)

Answer 55

Deletion of ERa or ErB (knockout of estrogen receptors): - mice are viable but... - leads to infertility - CL, corpus luteum: evidence of ovulation and produces progesterone for early maturation of oocyte - HC, hemorrhagic cyst - large cysts occur, influx of blood, no corpus luteum

Answer 56

- inducible systems for deleting genes in a tissue and spatial-restricted pattern - Cre-recombinase targets specific DNA sequences and causes recombination (used to delete specific genes) - How do you make this cell or tissue specific? - use promoter - express a cre recombinase from a promoter that generates the specificity nedded - modify cre driver with estrogen receptor on end - add tamoxifen, makes it have fidelity - removes stop codon - express the modified creERT (estrogen receptive, tamoxifen-inducible) in a tissue specific fashion - deliver tamoxifen at the time required - DCLK1 is only expressed in cells at the base of villi - LacZ+ cells don't need to express DCLK1 AFTER recombination (change is permanent)

Answer 57

- localized at the gene (in the nucleus) - bound to gene and maintains the gene in an off state - typically activated by ligands and metabolites that can passively enter the cell and nucleus - bind as heterodimers to activate gene expression, most commonly with Retinoid X receptor (RXR) - most common binding - roles in: gene expression, regulation in embryogenesis, tissue development, and processing vitamins, medicine, and foreign substances - larger family than class I - bind response elements consisting of two half-sites separated by a variable length of DNA - sites can be RARE (retinoic acid response elements), PPRE (PPAR responsive elements), etc. - second half-site can be a direct repeat (DR), an inverted repeat (IR) or everted repeat (ER) -have flexibility, allows a lot of diff partners - involve switching genes from off to on states - changes the affinity of the existing heterodimer from a co-repressor complex to a co-activator complex - many of these complexes affect the packaging of DNA i.e. epigenetic regulation

Answer 58

e.g. retinoic acid - metabolite of vitamin A that mediates the vitamin A functions required for proper development - loss of retinoic acid affects multiple developmental processes

Answer 59

- androgen (AR) - estrogen (ER) - progesterone (PR) - glucocorticoids (GR) - mineralocorticoids (MR)

Answer 60

- fatty acids and derivatives (PPAR) - cholesterol and derivatives (LXR, FXR) - retinoic acid (RAR) - vitamin D3 (VDR) - steroids and xenobiotics (PXR, CAR) - thyroid hormones (THR)

Answer 61

- start of November 1944 to the late spring of 1945 - people lived on 30% of their normal calorie intake (pregnant women underwent famine) - cohort A: pregnant mothers in the third trimester had babies that were smaller and remained smaller their entire life - cohort B: pregnant mothers in the first trimester had babies with a higher rate of obesity - children in cohort B showed increased risk for metabolic disease including diabetes, cardiovascular disease and mental health (during pregnancy if there is deficit in methyls, miss label genes, so those that may usually be turned off, now are not) - children of cohort B also showed increased risk for these diseases (epigenetic reprogramming - catch up growth, learned to grow in starvation, now with propre nutrients, store in body, extra fat) - methylation was affected by the dutch famine because: limited substrates for obtaining methyl groups in the diet; decreased intake leads to decreased availability. Folic acid is now provided as a supplement to pregnant women

Answer 62

Heritable changes in gene expression that do not involve any change in DNA sequence 1. Modification of histone core proteins (could be phosphorylation, ubiquination, methylation, sumylation and acetylation, repression or activation, includes chromatin remodelling proteins) 2. DNA methylation (generally at cytosine residues, usually associated with gene repression) 3. non-coding RNAs, ncRNA (affect transcription, silence genomic regions or alter RNA processing all leading to changes in RNA accumulation and expression - non-coding RNA

Answer 63

- small proteins, 100-300 amino acids mostly positively charged - subunits have N-terminal tails that sticks out of the octomer core, allow covalently modifications that change DNA wrapping - nucleosomes are separated by a short piece of linker DNA - length of linker DNA can be modified - histones are translationally modified in many ways: acetylation, methylation, sumoylation (associated with gene silencing), phosphorylation, and ubiquitination

Answer 64

- erasers (marks removed): HDACs, KDMs - writers (marks added): HATs, HMTs or PRMTs - readers (recognize marks): bromodomains (binds acetylated lysines- associated with gene expression), chromodomains (binds methylated lysines - active/repressive expression), Tudor domains (domains within protein), SANT domains (bind unmodified histones - potentially open it up) *BRG1/Baf60c has a SANT domain required for "opening" up DNA

Answer 65

- catalyzed histone acetyl transferases (HATs) transfers an acetyl from acetyl-CoA to histone tails - HAT A enzymes: activate only in the nucleus - HAT B enzymes: active in the cytoplasm and modifies new histones prior to incorporation into nucleosomes - generally acetylation causes DNA to be unwound to allow for transcription (euchromatin)

Answer 66

- removes acetyl group from histones - class I HDACs (1-3, 8): are located only in the nucleus - class II HDACs (4-7, 9, 10): are active in the nucleus and cytoplasm and shuttle between compartments - unlike transcription factors, HATs and HDACs don't directly bind DNA but are recruited by transcription factors - have large role in cancers - in many hallmarks of cancer including differentiation, proliferation, metastasis, angiogenesis, apoptosis, cell cycle and inflammation * clinical trials are testing HDAC specific inhibitors but many roadblocks (poor availability, ineffective for solid tumours, off target toxicity, affects healthy proliferating cells

Answer 67

- histone methyltransferases (HMTs) methylate arginine or lysine by transferring a methyl group from the donor S-adenosylmethionine (SAM); carried out by the SET domain regions within the HMT - unlike acetylation, methylation can be both an active or repressive mark depending on the residue marked - trimethylation of H3K4 is considered an active mark - mixed lineage leukemia proteins (MLLs) catalyze methylation of H3K4 (also known as lysine methyltransferases - KMTs) - lysine demethylase of K4 = repressive (KDM) - trimethylation of H3K27 is considered a repressive mark - Polycomb repressor complex 2 catalyzes the methylation of H3K27 - lysine demethylation (KDM) of K27 is considered an active mark - most methylation eventually end as trimethylation, but mono and di (me2) methylation also occur - H3K4me1: associated with transcriptional sliencing in myoblasts, macrophages and human embryonic stem cells. Role in establishing boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters - H3K4me2: marks enhancers where transcription factors bind

Answer 68

- part of the polycomb repressor complex 2 (PRC2) - establishes histone trimethylation of H3K27 - H3K27Me3 is associated with genomic regions that are weakly transcribed or silent - upregulation or inactivation of Ezh2 have deleterious consequences: upregulated in breast, pancreatic, and prostate cancer. somatic LOF mutations in various leukemias.

Answer 69

-H3H3me3: considered an active mark. mixed lineage leukemia proteins (MLLs) catalyze methylation of H3K4 (also known as lysine methyltransferases - KMTs) H3K4me1: associated with transcriptional silencing in myoblasts, macrophages, and human embryonic stem cells. establishes boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters. - H3K4me2: marks enhancers where transcription factors bind (recruits complexes, associated more with activation of genes) - H3K27me3: associated with genomic regions that are weakly transcribed or silent. polycomb repressor complex 2 (PRC2) promotes H3K27me3. enhancer of zeste homologue 2 (EZH2) carries SET domain. upregulated in breast, pancreatic and prostate cancer. somatic LOF mutations in various leukemias. - H3K36me3: associated with gene transcription - active mark - H3K9me2/3: associated with heterochromatin; define LADs (lamin associated domains)

Answer 70

- exists on developmentally important genes or immediate response genes - maintains the possibility for transcription to occur - genes are "primed" or "poised" - this allows specific type of gene expression to be fixed in - prevent impropre activation of genes (limits the effects of activators and repressors) - can keep genes in a "ready" state (inactive but primed, can be activated rapidly)

Answer 71

- DNA can be modified by methylation of cytosines (generally) - functional relevance of non cytosine methylation is still unclear - primary human fibroblast cell line demonstrated that 4.25% of total cytosines in genomic DNA are methylated - However, 99.98% of DNA methylation occurs in CpG dinucleotides and ~75% of these DpGs are methylated - related to gene regulation, found in areas (promoters, enhancers) that regulate expression *Roles for DNA methylation: - recruitment of factors that allow for inheritance of histone modifications - include interacting with histone modifying enzymes or preventing binding of transcription factors - inactivation of the X chromosome in females - imprinting - mono-allelic gene expression of maternal or paternal gene (having both alleles expressed is not good, have to shut one off, only express one) - repression of DNA translocation - repression of gene expression

Answer 72

- compose 1% of the genome; ~25,000 in the genome - have ~ 10-fold higher frequency of the CpG dinucleotide than the rest of the genome - often (but not always) is associated with the promoter regions of genes; >50% of all mammalian genes are associated with CpG islands - generally thought to be actively protected from DNA methylation to allow for appropriate regulation of transcription

Answer 73

1. DNA methyltransferase 1 (DMNT1): - maintenance methyltransferases - maintains previously methylated DNA - primary role is to copy DNA methylation patterns during DNA synthesis as well as repair of DNA methylation patterns - during cell replication, DNMT1 replicates the methylation patterns to the new strand - 5-azacytidine inhibits DMNT activity 2. DNA methyltransferase 3 (DMNT3A) and DMNT3B - De novo methyltransferases (lay down initial marks) - patterns of methylation are established during development - capable of methylating native DNA, regardless of whether the DNA is in a replicative state or not *DNMT3L regulates DNMT3A and B function

Answer 74

1. Passive: based on cell division and inhibition of DNMT1 (no requirement of energy) 2. Active: this can occur through the removal or conversion of methylcytosine and usually involved either base excision repair or nucleotide excision (involves damage to DNA - cutting out/ breaking DNA backbone)

Answer 75

- the x chromosome that makes more Xist becomes the Xi chromosome; this chromosome is silenced - different methylation modifications of histone tail at the Xist promoter contributes to this X-inactivation - H3K4 results in upregulated Xist and stable expression of Xist which causes inactive X

Answer 76

1. Change histone profile to recessive (remove H3K4me3, add H3K27me3) 2. Promote histone deacetylation (HDACs) 3. DNA methylation 4. Add H3K9me3 *last three steps can work in either direction

Answer 77

- chemotherapeutic targets are being silenced by epigenetics - now testing inhibitors for therapy in order to prevent silencing of pathway, so it can potentially respond to another therapy e.g. pro-inflammatory pathway - activate immune system - alter pathway to see tumour as foreign so immune system fights against

Answer 78

Induced pluripotent stem cells - reprogramming somatic cells to become embryonic stem cells - factors required for reprogramming include Oct4, cMyc, Klf4 and Sox2 - these factors are called pioneer factors since they can promote loosening of the heterochromatin and allow access for other factors - factors come to open up DNA, and restore stem-like quality (can be used for stem cell therapies) - activate more genes than normal

Answer 79

- increase in Nanog protein - decrease in lamin A (lamin A associated with large areas of heterochromatin, less heterochromatin in stem cells) - increase in acetylation marks associated with gene activation (H3ac, H4ac, H3K9ac, H3K27ac, H4K5ac) - increase in methylation marks associated with gene activation (H3K4me3, H3K36me2) - increase in y-H2AX (gamma) - associated with DNA repair (open DNA is more susceptible to damage, thus increased expression of repair factor makes sense) - decrease in methylation marks associated with foci number (H3K9me3, HP1a) - more foci in heterochromatin - increase in methylation associated with nucleoplasmic intensity (H3K9me3, HP1a) - becoming more diffuse - H3K27me3 doesn't change, this is a priming of genes. - this means that more genes are given the possibility to be activated but are not actually all being activated.

Answer 80

- known protein coding gene exons compose less than 3% of the human genome - based on the encode* database approximately 76% of the human genome is transcribed (more than 3/4 transcribed, but only 3% becomes proteins) - most of these transcripts do not code for protein, called non-coding RNA - separated into two classes: short ncRNAs and long ncRNAS - long ncRNA are arbitrarily defined as being longer than 200 nucleotides

Answer 81

- transfer RNA (tRNA) - ribosomal RNA (rRNA) - small nucleolar RNA (snoRNA); guide modifications of other RNAs - small nuclear RNA (snRNA); aid in processing of pre-mRNA - piwi-interacting RNA (piRNA); gene silencing of retrotransposons - extracellular RNA (exRNA); possibly cell communication - small interfering RNA (siRNA); promotes mRNA degradation - microRNA (miRNA); RNA silencing and post-transcriptional regulation - long non-coding RNA (lncRNA) - circular RNA (circRNA); secreted

Answer 82

Small nucleolar RNA - type of small non coding RNA - responsible for posttranscriptional modification and maturation of rRNAs, snRNAs and other cellular RNAs - most snoRNAs are encoded in the intros of protein-coding or non-coding genes (within the body of genes, can also be in 3' untranslated region) - typically 60-170 nt long - can regulate mRNA splicing (alternative splicing) - primarily accumulate in the nucleoli (site where lots of ribosomal RNA is produced, helpful with this)

Answer 83

Small nuclear RNA - average size of 150 nt - exist with proteins in small ribonucleoprotein particles - catalyze the splicing of pre-mRNA - allow precise alignment and correct excision of introns - found in Cajal bodies within the nucleus (RNA + protein association = cajal bodies)

Answer 84

Piwi-interacting RNA - approximately 24-32 nucleotides - bind to members of the piwi protein family - largest class of small non-coding RNA molecules expressed in animal cells - influence transposon silencing, spermiogenesis, genome rearrangement, epigenetic regulation, protein regulation, and germ stem-cell maintenance

Answer 85

Extracellular RNA - possibly cell communication - a class of RNA - non coding - these can include miRNAs that lead to gene silencing in target cells (produce RNA, packaged, go to another cell, can silence genes in other cell)

Answer 86

- hairpin derived RNAs ~ 20-24 nucleotides long - typically binds the 3' UTR of mRNA leading to repression - computational analysis suggests that 60% of protein-coding RNAs can be targeted by miRNAs (some can be targeted by multiple, and some miRNA can target multiple coding RNA) - typically produces subtle (<2-fold) reduction in protein levels (small changes, minimal role that tweaks expression) - have been linked to regulation of developmental processes, fine tuning gene expression, disease progression and chemotherapeutic resistance - miRNAs can exist, and be transcribed as a cluster. Therefore, multiple miRNAs produced (conserves on energy for multiple pathways for example)

Answer 87

Small interfering RNAs - originates from dsRNA and is usually 21-22 nucleotides long - primary role in repression is to cleave mRNA (recognize + promote degradation) - often the response to viral infection and provide a perfect match to its target (specific - typically only has a single target, different from miRNA) - often used as a research tool to knock down expression by as much as 85-90% (knock-out = removal of gene, knock-down = target mRNA with siRNA to reduce expression) - unsure if siRNA is produced in mammalian cells

Answer 88

- cleaves double-stranded RNA molecules into short double stranded fragments of around 20 nucleotides

Answer 89

RNA-induced silencing complex - integration of one strand into a RISC complex - RISC then targets the complementary sequences in mRNA - Argonaute (AGO) is then activated and depending on the isoform, can cause mRNA cleavage or translation inhibition AGO 1-4 = translational repression or de-adenylation (from miRNA) AGO2 = endonucleolytic cleavage (from siRNA)

Answer 90

- over 200 nucleotides in length; can reach up to several 100 thousand nucleotides in length - originally thought of as transcriptional noise of RNA polymerases with low fidelity (actually has a function though) - almost 15,000 described transcripts in the human genome in 2012 - some lcRNAs have begun to be classified for functions: regulating expression of neighbouring protein-coding genes, chromatin modification, transcription and post-transcriptional processing

Answer 91

- first described by Dr. Murray Barr (western prof) - represents the inactive X chromosome - the entire X chromosome is not inactivated

Answer 92

- Xist is a lncRNA that is transcribed from a region of one of the X-chromosomes - the X-chromosome that makes more Xist becomes the Xi chromosome; this chromosome is silenced - Xic contains the X-chromosome controlling element (Xce) - Xce contributes to the X chromosome that will be inactivated - represents a form of imprinting in which only one chromosome or allele is expressed even though two exist (don't want two copies) - other regions of the genome are also imprinted through paternal or maternal inheritance and lncRNA also play a role in that type of regulation e.g. HOTAIR

Answer 93

- should be random which is deleted - X inactivation "skewing" means that the same X chromosome is inactivated >70% of the time (happens in 35% of women) - extreme skewing is >90% of the time (happens in 7% of women) - could be the underlying cause for variability in X-linked disease. Example: if Rett syndrome (RTT) which is due to mutations in MeCP2 - RTT patients show significant variability of symptoms likely due to differences in X-inactivation (incomplete)

Answer 94

1. different variants of the Xce gene (4 variants of Xce protein that have different potentials for recruiting Xist) 2. mutations in the Xist promoter 3. mutations in an X-linked gene that affects cell survival

Answer 95

- normal transcription, proliferation, etc. (suggested that up to one million DNA lesions can occur every day - majority are fixed) - environmental agents (UV light, chemical exposure, chemotherapy) - internal stress (inflammation, reactive oxygen species -ROS) *Can lead to genomic instability, apoptosis, or senescence Genomic instability can predispose individuals to cancer, neurological disorders and cardiovascular diseases

Answer 96

- modified nucleotide - mismatched nucleotide - DNA adducts (bubbles in chromosome) - missing nucleotide - double strand breaks

Answer 97

- direct reversal - MMR = mismatched repair - NER = nucleotide excision repair - BER = base excision repair - HR = homologous repair - NHEJ = non-homologous end joining

Answer 98

1. identify the mistake - type, extent, etc. 2. remove the mistake - mismatch, unmatched base pair, non-cohesive ends 3. trim back the DNA backbone 4. repair the sequence 5. ligate the backbone of DNA

Answer 99

- single step process with no excision of nucleotides - does not break the phosphate backbone of DNA - error-free and preserves genetic information (not changing base pair, just editing modification e.g. methylation) - most energy efficient type of repair - multiple pathways that are specific to the modified nucleotide - mammalian O6-methylguanine-DNA methyltransferase (MGMT) removes DNA adducts by transferring alkyl group to a cysteine - AlkB can repair other modified nucleotides

Answer 100

- repairs mismatched bp that may arise from mistakes in DNA replication or recombination - failure results in microsatellite instability (high concentration of DNA mismatches more likely to have damage occur) - DNA mismatches are recognized by MUTS and MUTL - MUTL causes an incision (nick) in the DNA - exonuclease (EXO) removes the mismatched nucleotide and up to 100 bps more - DNA polymerase restores the correct nucleotide and the DNA ligase joins/ restores the DNA backbone

Answer 101

- repair of modified base pairs or nucleotides which is the most common DNA insult - typically repairs a single nucleotide (short patch) but can replace up to 13 nucleotides (long patch) - removal of the damaged nucleotide by glycosylases - strand incision by endonuclease APE1 - nucleotide(s) inserted by DNA polymerase - ligation of DNA - FEN1 helps repair the 5' flap that develops in this process - DNA polymerases work only in 5' to 3' direction

Answer 102

- repairs DNA adducts that distort the double helix, including those induced by UV light, chemotherapy Two types: 1. transcription-coupled (TC)-NER 2. global genome (GG)-NER - four successive steps: lesion detection (different for the two processes) local unwinding and damage verification excision of the DNA surrounding the lesion DNA synthesis and ligation - XP = xeroderma pigmentosum; rare genetic disorder linked to mutations in genes involved in NER

Answer 103

- uses the matching chromosomes as a template - DSB are recognized by a MRE11-RAD50-NBS1 (MRN) complex - MRN complex is essential for maintaining genomic integrity, cell viability and checkpoint activation - recruits ataxia telangiectsia mutated (ATM) protein which phosphorylates factors involved in repair (including BRCA1 and p53) - BRCA1 is required for the recruitment of proteins such as Rad51, BRCA2 and BARD1 to sites of DSBs (double strand breaks)

Answer 104

1. Crossing over - swapping gene regions between chromosomes (happens in normal development) - results in non-mendelian inheritance 2. loss of heterozygosity of a specific gene - result of HR leads to identical genomic regions - possible duplication of adverse genetic events

Answer 105

Core protein components include: -Ku subunits (Ku70 and Ku80) interact with DNA- PKcs and potentially form a docking site for other proteins - Artemis is involved in end trimming of the DSB - may lead to microdeletions - LigIV/ XRCC4 complex serves to perform the ligation and final step of NHEJ

Answer 106

- HR accounts for the repair of 10% of DSBs, while NHEJ accounts for the repair of 90% of DSBs - HR is active in S and G2 phases of the cell cycle, NHEJ is active in G1 - HR provides a complete repair, NHEJ is error prone as small deletions and insertions occur - HR relies on the pairing of one of the broken strands with a complimentary region on the sister chromatid while NHEJ does not

Answer 107

- mice lacking Exo1 displayed reduced survival and increased susceptibility to the development of lymphomas - sterile because of meiotic defect - hereditary nonpolyposis colorectal cancer = HNPCC; is an autosomal dominant disorder (Exo2 lacking in humans) - germline mutations in mismatch repair genes have been found in the majority of families with HNPCC - germline mutations in EXO1 appear in atypical HNPCC (with deficiency inEXO1, end up with more breaks and less repair)

Answer 108

- XRCC1 = X-ray repair cross complimenting 1 - deletion in mice results in lethality before gastrulation - biallelic mutations in humans leads to ocular motor apraxia, axonal neuropathy and progressive cerebellar ataxia - point mutations found to increase risk for head and neck cancers - FEN1= flap endonuclease 1 - removes the "flaps" during DNA repair - deletion leads to early embryonic lethality (mice) - human mutations results in autoimmunity, chronic inflammation and cancer - leads to a lot/ frequent of spontaneous mutations/ damage - can't get rid of flaps

Answer 109

- human mutations in CSA and CSB (help unwind DNA) genes results in Cockayne syndrome; recessive genetic disorder - Cockayne syndrome results in abnormally small head, reduce growth and delayed development - complete loss of ERCC1 in mice is embryonic lethal - mutations in ERCC1 result in cerebro facial skeletal defect (facial deformities)

Answer 110

- Ku70/80 sense double stranded DNA breaks - mice deficient in Ku70/80 both are viable and fertile (survive- other mechanisms to repair break- just not as effective) - both show growth retardation and severe combined immunodeficiency (Scid) - both mouse mutants exhibit premature aging - only Ku70 mouse mutants show susceptibility for cancer - inhibiting Ku function has been suggested as a therapy for some cancers

Answer 111

- compensation by other pathways - may not be as efficient or correct in repair process

Answer 112

- PARP = poly(ADP-ribose) polymerases (inhibitors have been effective in BRCA genes - main form of chemo) - effective in cancers that have mutations in BRCA 1/2 - this includes breast and ovarian cancers * if BRCA1/2 is defective, PARP will target repair to a different pathway (MMEJ repair) and away from NEHJ, which is more error prone and likely to lead to further genome instability and cell death

Answer 113

- decreased blood cell production - multiple congenital somatic abnormalities, bone marrow failure and cancer susceptibility (presence of blasts - immature blood cells should not be found here - bone marrow failure)