Chromatin and gene regulation 1 & 2

Question 1

A: What’s the fraction of coding genes in the genome?
B: What’s the fraction of gene regulatory sequences in the genome?

Answer 1

A: 2%
B: 33%

Question 2

During what phase in the cell cycle is chromatin condensed and typically karyotyped?

Answer 2

M-phase.

Question 3

What’s the histone structure like?

Answer 3

Histones are octamers consisting of two tetrameric sub-assemblies in which 2x (H3 +H4) dimerize and 2x (H2A + H2B) dimerize. The two tetrameric dimers are brought together to form the histone core.

Each subunit hosts three alpha-folds and long N-terminal tails which are rich in arginine. The tails can be modified and the nucleosome density can change.

Question 4

What’s the structure of chromosomes, small –> large?

Answer 4

1. NUCLEOSOMES: 147 bp of DNA is wrapped around histones consisting of 8 subunits (H2A-H2B, H3-H4 octamers). This corresponds to 1.65 laps.
2. 10 NM FIBERS. The zigzag beads on a string illustration. Histones carry 147 bases of DNA each.
3. 30 NM FIBERS.
4. CHROMOSOMES.

Question 5

Why is arginine important in histones?

Answer 5

Arginine is often methylated, thus, it’s important for histone regulation.

Question 6

What’s the function of histone H2A.X and H1?

Answer 6

H2A.X gets phosphorylated when DNA damage occurs, it proceeds to recruit the DNA repair machinery.

H1 is a linker histone that wraps around the free bases linking the nucleosomes together in order to stabilize the structure. H1 is enriched in heterochromatin.

Question 7

What are topologically associated domains (TADs)?

Answer 7

TADs are regions of DNA that are tightly bound together via zin finger proteins like CTCF. These domains are thought to be co-regulated. Hi-C is used to find TADs.

Question 8

How do histones slide on the DNA and what effects does it have?

Answer 8

The sliding is mediated by the nucleosome-remodeling complexes which shift the histones using ATP.

Since the linker DNA is transcribable, shifting the histones regulate which DNA is available.

Question 9

What do the following acronyms mean?
HDAC
HAT
HMT
HDM

Answer 9

HDAC = Histone deacetylase
HAT = Histone acetylase
HMT = Histone methyl transferase
HDM = Histone demethylase

Question 10

What domain do proteins need to host in order to have the ability to read histone modifications?

Answer 10

chromo- or Bromo domains.

Question 11

Does the histone profile get inherited during meiosis?

Answer 11

50% of the histones in each parental gamete will be conserved in the progeny. (50% maternal and 50% paternal histones are inherited).

DNA methylations are wiped clean twice during embryogenesis.

Question 12

What’s the most common base to be modified in the DNA?

Answer 12

Cytosine. It’s sometimes found in CpG islands. If a CpG island gets DNA modified it recruits methyl-CpG-binding proteins that in turn recruit histone deacetylases and induce heterochromatin in the surrounding histones.

Question 13

How can you study DNA methylations

Answer 13

Bisulfite sequencing

(look into this if you have time).

Question 14

What are DNA activators and repressors?

Answer 14

1. They bind enhancer regions in DNA.
2. Hosts a DNA-binding domain and an activating domain. The activating domain often recruits other proteins to mediate its function. The activating domain is “sticky” meaning it can form complexes with lots of different proteins. The results are fuzzy compelxes which in the best case recruits parts of RNApol2 or the mediator.
3. Often dimers.
4. Often recognise helix inserts, allows for major groove interaction.
5. You’ll find activators / repressors downstream of multiple pathways like JAK-STAT.

Question 15

Wht are the four most common DNA binding domains found on activators?

Answer 15

1. HMG - Minor groove interaction.
2. Zinc finger domains - DNA binding stabilized by zinc.
3. Helix-loop-helix
4. Homeodomain - Regulates hox genes. The recognition sequence is identified via three helices.

Question 16

Describe the six models of gene regulation.

Answer 16

1. Activator makes it easier for the mediator to dock at the promoter sterically or chemically.
2. Histone tail modifications. Acetylation –> unwinding, methylations –> winding.
3. Activators can help form PIC/PEC, and repressors can inhibit them.
4. Activators can ensure 147 bases aren’t available for histones to wrap around, making the bases free.
5. Insulators - DNA sequences that bind CTCF proteins. Insulators inhibit upstream sequences from interacting with downstream sequences.
6. Locus control regions (LCRs). Complex regulatory landscapes which generate complex gene clusters, for example: Globin- and hox-clusters.

Question 17

What’s MNase seq?

Answer 17

Very similar to ribosome profiling. The method assays chromsome accessibility. All linker DNA is cleaved, histone-DNA interactions remain and are subsequently sequenced.