IT1 - DNA Packaging

Question 1

What is the chromatosome?

Answer 1

Nucleosome core particle + H1

Question 2

What enzymes catalyse H3K4me1, and what is its role?

Answer 2

MLL3 and MLL4; mark active or primed enhancers, help prime enhancers and stabilise chromatin remodeller binding.

Question 3

How did we confirm whether chromosomes would either form ‘spaghetti’ or ‘dumplings’ in cells?

Answer 3

UV irradiation experiments showed that chromosomes form distinct territories, as predicted by Boveri.

FISH then showed that chromosome territory positioning is non-random; gene-rich chromosomes are positioned towards the center, and gene-poor towards the periphery.

Question 4

Describe the structure of centromeric chromatin. How is CENP-A localized?

Answer 4

CENP-A histone H3 variant
Other CENP proteins forms a network of interactions with the DNA and CENP-A nucleosomes.

CENP-A contains a targeting domain that binds a CENP-A specific histone chaperone (HJURP) which deposits it on the centromeric DNA. FACT is involved in its incorporation.

Question 5

What is the HUSH complex?

Answer 5

The HUSH complex consists of three core components: TASOR , MPP8*, and MORC2. These proteins are thought to work together to recruit additional factors that promote transcriptional repression of retroviral elements.

MORC2 is an ATPase that is thought to compact chromatin.

*MPP8 forms an alternative complex with SETDB1 to spread methylation on transposable elements.

Question 6

What is the structure of B-form DNA?

How does under-winding or over-winding impact the structure?

Answer 6

~10.5 bp per turn
Right-handed double helix

Under-winding: negative supercoils (right-handed)

Over-winding: positive supercoils (left-handed)

Question 7

How can H3K4me3 affect promoter function?

Give examples of 3 proteins that can bind this modification and what their functions are.

Answer 7

1. Affects nucleosome-DNA interactions.
2. Recruits proteins that bind acetylated histones:

SAGA acetyltransferase complex binds H3K4me3 to open up the chromatin more.

CHD1 (chromatin remodeling enzyme) helps RNAPII pass the nucleosomes by partially unwinding the upstream DNA and pumping the DNA towards the polymerase.

TAF3 is part of the TFIID complex which must bind to form the PIC containing RNAPII.

Question 8

What are early- and mid-late replicating domains?

Answer 8

Early replicating domains (ER) correspond to regions of the genome that are replicated during the first half of S phase. These domains are characterized by open chromatin structure, high gene density, and are enriched for active chromatin marks such as H3K4me3 and H3K27ac. i.e., R-bands.

Mid-late replicating domains (MLR) correspond to regions of the genome that are replicated during the second half of S phase. These domains are characterized by condensed chromatin structure, low gene density, and are enriched for repressive chromatin marks i.e., G-bands.

Question 9

How does gene body chromatin differ from gene promoter chromatin? What machinery is involved?

Answer 9

Gene promoter: H3K4me3
Gene body: H3K36me3

During transcription initiation, Ser5 on the CTD of RNAPII is phosphorylated, recruiting SET1. SET1 deposits H3K4me3 on gene promoters.

As RNAPII transitions into elongation, the CDT is phosphorylated at Ser2. SET2 can bind this and deposit H3K36me3 in the gene bodies.

Question 10

How do modifications to chromatin influence its function?

Give an example for each.

Answer 10

1. Act as a binding site for reader proteins that then effect function.
   E.g., Su(var)3-9 methylation promotes HP1 binding that dimerizes to hold the nucleosomes in a more compact configuration.
2. Directly influence nucleosome structure and interaction.
   E.g., acetylation neutralizes the positive charge on histone lysines, reducing the thermostability of the nucleosome (if acetylated on the tail) and its affinity for DNA (if core is acetylated).

Question 11

Describe the histone-fold structure and how this is used to form histone dimers.

Answer 11

The histone-fold structure consists of three alpha helices separated by two loops, and it is highly conserved across different histone proteins and species.

The skewed u-shaped folds of the histone monomers allows for them to slot together via a ‘handshake’ to form dimers.

Question 12

How has the positioning of histones on DNA been studied? What did this reveal about histone occupancy across the genome.

Answer 12

EM images and digestion assays show homogeneous and uniform coverage of DNA…

Nucleosome mapping (e.g., DNase seq) showed nucleosome occupancy is high, with regular arrays that lack defined phasing. But some regions have more regular phasing with low or no occupancy at TSS.

The same can be said for other non-gene elements, such as enhancers, insulators and origins of replication.

Question 13

What is a plectoneme?

Answer 13

A structural feature of super-coiled DNA, forming a twisted structure that resembles a series of loops.

[Think of when you over twist some rope and it forms individual loops coming off the central strand.]

Question 14

Describe the tandem repeat structure of centromeric alphoid DNA.

Answer 14

Comprised of a monomer repeat unit of approximately 171 base pairs (bp).

Monomers are organized in tandem arrays, repeated head-to-tail multiple times.

Monomers are grouped into higher-order repeat units (HORs), typically ranging from 2 to over 30 monomers per unit.

These HORs are themselves repeated in tandem, forming arrays that can span hundreds of kilobases to several megabases.

Question 15

What are the roles of H3K36me3 in gene body chromatin architecture and function?

Answer 15

1. Transcription - recruits demethylaters and HDACs.
2. DNA methylation - recruits HMTs to reinforce DNA methylation in gene bodies.
3. Splicing - recruits RNA splicing factors to the gene body, facilitating proper splicing of nascent transcripts via co-transcriptional splicing.
4. DNA repair - recruits proteins that are involved in mismatch repair and homologous recombination.

Question 16

What is a Barr body?

Answer 16

the heterochromatic structure formed by the inactive X-chromosome in mammals.

Question 17

What are TADs?

Answer 17

Topologically-associated domains are self-interacting genomic regions that are thought to regulate gene expression by limiting the enhancer-promoter interactions to each TAD. These are formed by CTCF and cohesin loop extrusions.

Question 18

Which nucleoid-associated proteins function to bend DNA?

Describe the structure and function of each.

Answer 18

Factor for inversion stimulation (FIS):
- Dimer
- Binds major groove of DNA
- Bends DNA between 50-90 degrees

Integration host factor (IHF):
- Heterodimer of alpha and beta subunits
- Prefers to bind AT-rich DNA
- Beta-ribbon arms protrude into the minor groove to kink the DNA 160 degrees.

Question 19

How do prokaryotes and eukaryotes utilize DNA supercoiling to compact their DNA?

Answer 19

Prokaryotes use DNA gyrases to negatively supercoil their DNA, forming the nucleoid with plectonemes.

Eukaryotes use supercoiling to wrap their DNA around histone octamers and compact the DNA 6-7 fold.

Question 20

Compare type I and II alpha satellite DNA.

Answer 20

Type I:
- Rich in CENP-B binding sites and CENP-A nucleosomes
- Assembly site for kinetochore
- ‘open’ chromatin

Type II:
- fewer CENP-B binding sites
- heterochromatic
- enriched in condensin and cohesin

Question 21

What is the role of centromeric alphoid DNA in centromere formation?

Answer 21

Centromeric alphoid DNA (alpha-satellite) is a type of tandem repeat DNA sequence that is found in the centromeric regions of human chromosomes.

It’s AT-rich, and contains CENP-B and CENP-A binding domains which are needed for de novo centromere formation.

Question 22

What is a t-loop, found within telomeres? How do we know this exists?

Answer 22

Its consists of a single-stranded DNA overhang at the 3’ end of the telomere that invades the double-stranded telomeric DNA and forms a displacement loop (D-loop) structure. This D-loop structure is stabilized by the shelterin complex, which includes proteins such as TRF1, TRF2, and POT1. The t-loop structure is important for protecting the chromosome ends from being recognized as double-stranded breaks, which could lead to DNA damage responses and chromosomal instability.

It has been visualized using both EM and super-resolution imaging.

Question 23

How are centromeres and kinetochores distinguished? What are their basic functions?

Answer 23

Centromere: chromatin structure under the kinetochore that specifies where the kinetochore will form.
- Segregates chromosomes
- Holds sister chromatids together
- Coordinates the above processes
Composed of DNA sequence and proteins.

Kinetochore: protein complex that microtubules attach to during chromosome segregation.

Question 24

What is the constitutive centromere-associated network (CCAN)?

Answer 24

The constitutive centromere-associated network (CCAN) is a protein complex that’s responsible for various functions at the centromere, including kinetochore assembly.

It’s formed of many types of CENP proteins, hence the ‘alphabet soup’ name!

Question 25

What are neocentromeres, and how are they formed?

Answer 25

Neocentromeres are a type of centromere that forms in non-centromeric regions of chromosomes i.e., centromeric DNA is not only insufficient, but isn't actually needed to make a centromere. Formed via: 1. Deletion or inversion of the original centromere 2. Epigenetic reprogramming to acquire centromeric features 3. Translocation of centromeric DNA to other regions

Question 26

Define the following terms: - Euchromatin - Heterochromatin - Constitutive heterochromatin - Facultative heterochromatin

Answer 26

Euchromatin: 'open' chromatin Heterochromatin: 'closed' chromatin Constitutive heterochromatin: constantly 'closed' chromatin Facultative heterochromatin: silenced chromatin that can be activated when needed

Question 27

Where can DNA methylation be found in the genome, and what does it do?

Answer 27

Primarily found on CpG dinucleotides within vertebrate genomes. Short stretches of CpG islands escape DNA methylation (CpG islands) and these are normally associated with vertebrate gene promoters. Active enhancers tend to be hypomethylated. It protects cells from retrotransposon elements jumping into coding elements.

Question 28

Describe the structure of budding yeast centromeres.

Answer 28

The centromere core contains 3 elements: CDEI, II and III. CBF1 is a sequence-specific DNA binding protein that binds CDEI. CBF3 is a protein complex that binds a motif in CDEIII. CDEII is the least conserved, but is highly AT-rich and is bound by Cse4 nucleosome.

Question 29

How is chromatin organized and modified at gene promoters?

Answer 29

1. Contain histone variants H2AZ and H3.3, making the nucleosomes intrinsically less stable and are dynamic. 2. H3K4me3

Question 30

What is the structure and role of histone H1 in the histone octamer?

Answer 30

Structure: - Flexible N-terminus - Intrinsically-disordered C-terminus that's highly basic - Lacks histone fold - Globular domain sits on the dyad and interacts with linker DNA where it exits the nucleosome. Function: - Holds DNA in a more rigid and compact conformation - Can be rapidly exchanging with other H1 molecules to alter chromatin structure of function, aiding in chromatin dynamics

Question 31

How is DNA bent around the histone octamer? How is this stabilized?

Answer 31

DNA is bent around the histone at AT rich sequences through narrowing of the minor groove. - It's sequence-independent. - Uses hydrogen bonds. This is stabilized by the high-degree of positive charge in the histone octamer, coming from the >20% lysine and arginine residues.

Question 32

How are transposable elements suppressed in vertebrate genomes?

Answer 32

1. General methylation (prevents TF binding and recruits HDACs to maintain compaction) 2. SETDB1-H3K9me3 pathway (potentially recruits HP1 for compaction, or recruits the HUSH complex)

Question 33

What enzyme catalyses H3K36me3, and what is its function?

Answer 33

SETD2; marks active gene bodies, recruits splicing factors, DNA methyltransferases, and mismatch repair proteins.

Question 34

What experiment has suggested that the 30nm fiber doesn't actually exist?

Answer 34

SAXS measurements on mitotic chromosomes from various cell types showed that it was ribosome contamination primarily causing the 30nm SAXS peaks. When ribosomes were washed away, most cells showed no 30nm peak...

Question 35

Which nucleoid-associated proteins function to bridge and loop DNA? Describe the structure and function of each.

Answer 35

Histone-like nucleoid structuring proteins: - short protein - binds AT-rich DNA via C-terminus - N-terminus contains self-associating domains to form 'daisy chain' filaments which stiffen the DNA SMC: - 2 SMC monomers and a Kleisin subunit form a ring - The ring captures DNA to enable looping - Head domains have ATPase activity to pull DNA through the ring

Question 36

How are new nucleosomes synthesized and deposited on DNA?

Answer 36

1. HSP70/Hsc70/NASP promote folding and dimerization of H3/H4, whilst ASF1 binds H3 to prevent tetramerization. 2. MCM2 shields DNA binding interface.. 3. Delivery to CAF-1 in the nucleus allows binding to the PCNA and supports tetramerization and deposition on the DNA. 4. NAP1/FACT deposits H2A/H2B dimers on the tetrasome. 5. Chromatin-remodelling enzymes wrap the rest of the DNA around the nucleosome. H1 deposition is less well understood.

Question 37

What is the role of acidic patches in 30nm fiber formation?

Answer 37

The acidic patches on the face of one tetranucleosome unit interacts with the basic tail of H4 on the adjacent tetranucleosome.

Question 38

What's the difference between point centromeres and regional centromeres?

Answer 38

Point: highly defined sequence recruiting sequence-specific DNA binding protein e.g., yeast Regional: complex tandem arrays of sequences lacking specific binding factors.

Question 39

What is the Polycomb complex? What does it do?

Answer 39

A repressive complex composed of two chromatin modifying enzymes: PRC1 and PRC2. - PRC1 is a H2A ubiquitin ligase - PRC2 is a H3 methyltransferase This complex binds non-methylated DNA in CpG islands that aren't actively transcribed, initiating a feedback system that leads to Polycomb chromatin domain formation and spreading. This is achieved through PRC1 and PRC2 modifications causing the recruitment of more PRC1 and PRC2 machinery.

Question 40

How does SETDB1 know to methylate parasitic DNA elements? How does this methylation then spread?

Answer 40

It relies on a family of KRAB domain zinc finger DNA binding proteins that recognize the sequences in transposable elements first. These proteins interact with an adaptor protein which recruits SETDB1 to nucleate H3K9me3. KRAB DBPs are rapidly evolving, likely in a 'red-queen' fashion to keep up with the hundreds of new types of invasions. SETDB1 forms an alternative complex with MMP8 that binds H3K9me3 to cause spreading of methylation.

Question 41

What is STORM, and how has it aided in determining chromatin organization in cells?

Answer 41

STORM is a super-resolution microscopy technique that uses fluorescent probes that can be turned on and off to obtain highly precise spatial information about the positions of individual fluorescent molecules in a sample. STORM has been used to visualize the higher-order organization of chromatin, suggesting there's no evidence for a regular 30nm fiber.

Question 42

What enzymes catalyse H3K27ac, and what is its function?

Answer 42

CBP and p300; mark active enhancers, promote transcriptional elongation by recruiting BRD4

Question 43

How is centromeric and pericentric chromatin organized in the nucleus?

Answer 43

Centromeric and pericentric chromatin are organized in a distinct manner in the nucleus. In interphase, centromeric chromatin is often located at the nuclear periphery or near the nucleolus. Pericentric chromatin is generally found clustered in distinct regions of the nucleus known as heterochromatic domains. Human centromeric DNA is extremely long, yet there are only a few nucleosomes involved. It's thought that the DNA folds in 3D space to form a platform with centromeric proteins concentrated on one face for kinetochore assembly.

Question 44

What is the Shelterin complex and how does it function in t-loop formation?

Answer 44

The Shelterin complex is a protein complex that binds to telomeres and regulates their structure and function. It is composed of six proteins including TRF1, TRF2 and POT1. TRF1 and TRF2 form homodimers that bind double-stranded DNA with nanomolar affinity. POT1 binds to the single-stranded overhang of the telomere, preventing it from being recognized as damaged DNA and protecting it from degradation. Together, these proteins form a protective structure called the t-loop, where the single-stranded overhang is folded back and base-paired with the double-stranded telomeric DNA. It's able to do this because the telomeric DNA is made up of the same repeats!

Question 45

Which nucleoid-associated proteins have functions beyond packaging DNA? What are these functions?

Answer 45

IHF can bend the DNA upstream of promoters to allow DNA elements bound by TFs get closer to the gene promoter - GENE REGULATION. SMC complexes are loaded onto newly replicating daughter chromosomes to support chromosomes individualization and segregation into daughter cells - CHROMOSOME SEGREGATION.

Question 46

What leads to nucleoid formation in prokaryotes?

Answer 46

1. Formation of negative supercoils by topoisomerases, such as DNA gyrase. 2. Histone and nucleoid-associated proteins that organize the DNA into a compact structure that's more resistant to damage.

Question 47

How do remodeling enzymes move nucleosomes?

Answer 47

They use ATP to translocate or slide DNA over the surface of the nucleosome: They anchor themselves to histones where the ATPase can then grab onto the DNA and translocate it towards the nucleosome dyad. This process disrupts local histone-DNA interactions and as the DNA is pushed towards the dyad, the distortion is translated around the nucleosome to cause DNA translocation.

Question 48

What is the role of telomerase RNA?

Answer 48

Template for the synthesis of telomeric DNA by reverse transcriptase activity of telomerase. It also has structural roles.

Question 49

What is the function of CENP-A and how is it inherited?

Answer 49

- Epigenetic mark to define centromere position. During cell division, CENP-A is inherited in a semi-conservative manner, meaning that it is passed down from the mother and father to daughter cells. New CENP-A proteins are added through a positive feedback group, where CENP-A nucleosomes recruit CENP-C and HJURP to deposit new CENP-A nucleosomes.

Question 50

What is the end replication problem and how is it solved? What was the evidence for this?

Answer 50

The end replication problem refers to the inability of DNA polymerase to fully replicate the ends of linear chromosomes. This is made worse by the 3' overhang found in telomeric DNA. To overcome this, cells use telomerase. EVIDENCE: 1. Tetrahymena were used to show telomere length is variable and telomeric sequences can be seeded de novo. 2. Tetrahymena telomeric DNA was stably and heritably maintained in yeast cells via the addition of yeast telomeres. Hence, telomeres are made de novo. 3. Biochemical isolation of the telomere lengthening activity. Addition of protease OR RNase abolished activity...the enzyme requires RNA. 4. The killer experiment: changing the RNA sequence alters the telomere DNA sequence i.e., the RNA is the template.

Question 51

What is the function of centromeric DNA, and how is it organized?

Answer 51

Centromeric DNA is highly organized and consists of a conserved DNA sequence called the centromere core, which is typically several kilobases in length. The centromere core is flanked by variable amounts of heterochromatic DNA that often contain transposable elements, satellite DNA, and other repetitive sequences. The centromere core is composed of CENP-A histone H3 variants. Centromeric DNA is made of alpha-satellite DNA that forms higher-order repeat units of 171 bp monomers.

Question 52

What enzymes catalyse H3K4me3, and what is its function?

Answer 52

SET1 and MLL1; mark active promoters, promote transcription initiation, and recruit acetyltransferases and transcription machinery.

Question 53

How is telomere homeostasis maintained?

Answer 53

The longer the telomere becomes, the less active telomerase is. When telomeres are short, then telomerase is more active. This is mediated by Shelterin, as the more Shelterin there is, the less telomerase activity there is.

Question 54

What is the end protection problem and how is this overcome? What experiment showed this?

Answer 54

The end protection problem refers to the vulnerability of chromosome ends to be recognized as double-stranded breaks and activate DNA damage response pathways, which could lead to genomic instability and cell death. - Shelterin complex - Formation of protective T-loops Protein fractionation was used to isolate proteins from nuclear extracts that specifically bound telomeric DNA sequences. The protein was purified and mass spec was used to identify it as TRF1.

Question 55

Which two histone modifications are associated with repressed chromatin?

Answer 55

H3K9me3 and H3K27me3

Question 56

Give 2 examples of chromosomes that are 'exceptional'.

Answer 56

1. The giant polytene chromosome in flies These are multiple copies of chromosomes that are then held together in parallel bundles. 2. Giant lampbrush chromosomes Highly elongated chromosomes that are arranged in loops, visible as thin fibres extending from a central axis.

Question 57

What are chromatin remodeling enzymes? Describe their structure.

Answer 57

Chromatin remodeling enzymes are a class of proteins that use the energy from ATP hydrolysis to alter the structure and position of nucleosomes on DNA. There are several families of chromatin remodeling enzymes, each with their own unique structure and function. - 2 conserved lobes for movement along the DNA - ATPase domain + auxilliary domains - ATPase domain powers movement (like 2 hands walking along the DNA)

Question 58

What is the role of the centromere in cell division?

Answer 58

During cell division, the centromere serves as the attachment point for spindle fibers, which are microtubules that extend from opposite poles of the cell and attach to the kinetochores located at the centromere of each chromosome. The spindle fibers then pull the chromosomes apart, so that each daughter cell receives an identical set of chromosomes.

Question 59

What is the 30nm fiber, and how was it discovered?

Answer 59

The 30nm fiber is a compacted and more condensed form of chromatin that is formed when nucleosomes are further organized and packed together. This was discovered using EM to visualize chromatin at higher resolution, demonstrating that increased cation charge partially shielded the DNA negative repulsion to create these higher order fibers.

Question 60

What are nuclear bodies?

Answer 60

Specialized membrane-less structures within eukaryotic nuclei that serve diverse functions, but are mainly involved in RNA processing and ribonucleoprotein formation. They form stochastically from 'seeds' and then sequester protein factors. [Also known as bimolecular condensates]

Question 61

What is the role of pericentric domains in the maintenance of chromosome integrity and sister chromatic cohesion?

Answer 61

The pericentric regions are the site of kinetochore assembly, which is necessary for proper chromosome segregation during cell division. In addition, the pericentric regions are rich in heterochromatin, which helps to stabilize chromosome structure and prevent the formation of DNA breaks or aberrations. The cohesion between sister chromatids is also maintained by proteins that are associated with the pericentric regions, including cohesin complexes and condesin.

Question 62

What histone modification characterizes enhancer elements? What protein deposits these?

Answer 62

H3K4me1. This modification is catalyzed by the histone methyltransferase complex MLL3/4 and is specifically enriched at enhancer elements rather than promoters. In addition to H3K4me1, enhancer elements may also be marked by other histone modifications, such as H3K27ac. H3K27ac is caused by CBP/P300 which can help to support PIC formation and enable transition of RNAPII into elongation mode.

Question 63

How can we study mesoscale chromatin organization?

Answer 63

HiC and super-resolution microscopy.

Question 64

Which two histone modifications are associated with active enhancers?

Answer 64

H3K4me1 and H3K27ac

Question 65

Q: Which histone modification is increased around the promoter of an induced gene?

Answer 65

H3K4me3

Question 66

How do H2A and H2B molecules interact in the 30nm fiber?

Answer 66

The interaction between H2A-H2B dimers in the 30nm fiber is thought to occur primarily through electrostatic interactions and hydrogen bonding between amino acid residues on the surface of the nucleosome. This mediates stacking in the tetranucleosome unit.

Question 67

How do insulators work to regulate genes?

Answer 67

They can form cohesin rings that uses ATP to loop the DNA. The extrusion is then halted by CTCF interacting with a defined component of the cohesin ring. Within regions of extrusion, communication can occur between enhancers and promoters, but CTCF blocks this communication between adjacent loops e.g., TAD domains.

Question 68

How does t-loop formation and the Shelterin complex prevent: - NHEJ - HDR

Answer 68

TRF2 blocks ATM kinase and NHEJ. POT1 binds ssDNA to block HDR from occurring at the 3' overhang. [NB: DNA damage signaling kinases are actually needed to recruit and activate telomerase...]

Question 69

What is TSA, and how has it been used to study the effects of acetylation on chromatin structure in vivo?

Answer 69

TSA inhibits HDAC activity and leads to histone hyperacetylation. Super-resolution imaging shows a more uniform distribution of chromatin after TSA treatment.

Question 70

What is a nucleoid?

Answer 70

Meaning nucleus-like, it contains the genetic material of prokaryotic cells.

Question 71

What is chromEM tomography, and how has it aided in determining chromatin organization in cells?

Answer 71

Combines cryo-EM with computational methods to generate 3D models of chromatin. It showed that chromatin is far more heterogeneous than originally anticipated, with no evidence for a 30nm fiber.

Question 72

Which biophysical forces are at play to shape chromatin?

Answer 72

1. Nucleosomal charge 2. Phase separation 3. Loop extrusion 4. Transcriptional activity

Question 73

What do bisulfite-seq and ChIP-seq reveal about chromatin structure?

Answer 73

Bisulfite seq: Bisulfite treatment converts C to U, but leaves methylated C alone. NGS can then show the methylation status and identify CpG islands. ChIP-seq: Proteins (e.g., nucleosomes) are fixed on the DNA and immunoprecipitated to allow for identification of the regions of DNA to which they bind.

Question 74

How does acetylation influence inter-nucleosome interactions?

Answer 74

The acidic patch on the face of one tetranucleosome unit interacts with the basic tail of H4 on the adjacent tetranucleosome. Acetylation of that H4 tail prevents the interaction from occurring, resulting in a de-compaction that is similar to removing the H4 tail entirely.

Question 75

What dictates nucleosome phasing and organization? How was this shown?

Answer 75

1. AT-rich sequence of DNA for nucleosome binding and bending. Shown via modeling predictions and testing these patterns. [Other gene promoter sequences can also influence phasing.] 2. Chromatin-remodeling enzymes. The models weren't enough, but adding ATP to the patterns made them much more like in vivo patterns, suggesting ATP dependent processes...aka chromatin remodeling enzymes. Deletions of the genes encoding these enzymes in yeast resulted in far less-defined nucleosome phasing. 3. DNA binding factors. High occupancy of DNA binding factors competes with nucleosome occupancy, mostly at promoters, enhancers, insulators and origins. e.g., CTCF in mammals which binds insulators and causes high levels of phasing.

Question 76

How was centromeric DNA and proteins discovered in budding yeast? How were centromeric proteins then discovered in humans?

Answer 76

A genetic assay for chromosome loss made it possible to isolate centromeric DNA from yeast. The yeast genome was digested and fragments put into plasmids and transfected into cells. Only plasmids that contains centromeric DNA could replicate stably, and so these cells had their plasmids sequenced. The centromeric DNA was then used to isolate centromeric proteins, using an EMSA gel that showed heavier DNA fragments i.e., DNA bound by protein. Human centromere proteins were found when patients with scleroderma were shown to have antibodies that recognized the centromere proteins (CENP). Subsequent studies identified the individual proteins within the CENP complex: CENP-A, CENP-B and CENP-C.

Question 77

What enzyme catalyses H3K9me3, and what is its role?

Answer 77

SETDB1; promotes repressive heterochromatin by recruiting HP1, compacting chromatin and repressing transposable elements with the HUSH complex.

Question 78

What are the individual roles of the 3 human centromeric proteins?

Answer 78

CENP-A: histone H3-like protein CENP-B: sequence-specific DNA binding protein CENP-C: large DNA binding protein

Question 79

Give an example of a protein encoded by a Su(var) gene. What is its function?

Answer 79

Su(var)3-9 encodes a histone methyltransferase that uses SAM to methylate K9 on H3 tails.

Question 80

How is telomerase expression regulated, and how does this link to cancer?

Answer 80

In normal somatic cells, telomerase expression is tightly regulated and telomerase activity is low or absent, leading to progressive telomere shortening with each cell division. In contrast, most cancer cells have high telomerase activity, which allows them to maintain their telomeres and continue to divide indefinitely. Upregulation of telomerase activity is therefore a hallmark of cancer cells, making telomerase an oncogene.

Question 81

What is the role of CTCF in mammals?

Answer 81

A DNA binding factor that functions to insulate the genome, blocking the capacity of enhancers to activate promoters from different regions of the genome.

Question 82

Which histone modification is increased in the transcribed region of an induced gene?

Answer 82

H3K36me3

Question 83

How was telomeric DNA discovered? What is its sequence and structure?

Answer 83

Functional cloning - yeast genome was digested and ligated to gene sequences. Only fragments that encoded telomeric DNA would make the gene sequences stable. These stable sequences could then be isolated and sequenced. SEQUENCE: Telomeric DNA consists of sequence repeats. In humans, this is TTAGGG, but it varies across species. STRUCTURE: Telomeric DNA is double-stranded, with a G-rich single-stranded 3' overhang that forms a T-loop to protect the end of the chromosome.