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MBB267 > chromatin > Flashcards

chromatin Flashcards

1
Q

What are the key components of chromatin?

A

Chromatin packages DNA with core histones, non histone proteins and RNA (roughly 50% protein by mass)

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2
Q

How does chromatin appear in both low salt conditions no Mg2+ and physiological conditions?

A

IN low salt conditions, chromatin is visualized as ‘beads on a string’ - nucleosomes linked by linker dna - nucleosomes are roughly 10nm
in physiological salt conditions, nucleosomes closer to 30nm, and no consensus on 3d structure - two proposed structures are zig zag rope structure and solenoid structure.

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3
Q

What are chromosome territories?

A

The space that individual chromosomes occupy in the nucleus.

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4
Q

Describe transcription decondensation of chromosome territories

A

Regions to be actively transcribed are found looped out rom the rest of the DNA

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5
Q

What is the structure of a nucleosome?

A

nucleosomes have an octomeric (8) core of histone proteins - H2A,H2B H3 and H4.
147bp DNA makes 1.7 left handed turns around core

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6
Q

What is the function of H1 histone

A

H1 binds DNA as it enters and leaves the nucleosome, nucleosomes are not permanently fixed.

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7
Q

What are some core histone variants and their functions?

A

H2AX - found in DNA repair
CENPA - attachment of centromeres to mitotic spindle
H3.3 - found in highly actively transcribed genes (close to nuclear pores)

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8
Q

What are four post transcriptional modifications of histone tails?

A
  • Acetylation of Lys residues
  • Methylation of Lys/Arg residues (mono,di,tri methylated)
  • Phosphorylation of Ser/Thr residues
  • Ubiquitylation - Lys residues
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9
Q

Which enzymes assist the transcriptional activation/repression of chromatin structure and how?

A

Histone acetylation complexes (HATS) promote acetylation of lysine residues. This promotes chromatin decondensation, promoting transcription. H3K4 methyl transferase has same function
Histone deacetylase complexes (HDACS) deacetylate lysine residues, and Histone Methyl Transferases Methylate residues, block acetylation and promote chromatin condensation (prevents transcription)

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10
Q

What is the difference between facultative and constitutive heterochromatin?

A

Constitutive heterochromatin includes repetitive DNA sequences, such as telomeres, centromeres and satellite DNA. Stably inherited in cell division.
Facultative heterochromatin is densely packaged but can readily decondense.

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11
Q

What are the key epigenetic markers or heterochromatin and euchromatin?

A 
Euchromatin = H3K9Ac and H3K4Me
Heterochromatin = H3K9Me and H3K27Me
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12
Q

What is the difference between trans and cis histone modifications?

A

Trans affects histone bound by factors

cis affects nucleosome interactions

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13
Q

What can cHIP indicate about gene landscapes?

A

cHIP can show the histone modification status of genes, allows the analysis of protein and DNA interactions in vivo.

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14
Q

How does cHIP work?

A

Protein DNA interaction are stabilized by crosslinking - eg Formaldehyde
DNA is fragmented and protein/DNA complexes purified by immunoprecipitation
DNA analysed by PCR or microarrays

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15
Q

What is a chromodomain?

A

Chromodomain is a region in proteins that can bind methylated histone tails at specific lysine residues (ie heterochromatin)

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16
Q

What is an example of a protein containing a chromodomain?

A

HP1 - allows nucleosome code to be read

17
Q

What is another region which a protein containing a chromodomain might have?

A

A chromoshadow domain. This allows interactions with other chromoshadow domains - mediates nucleosome interactions (eg can bring 2 nucleosomes closer together - even more densely packaged)

18
Q

What domains does the SUV3-9 protein contain and what do these domains allow it to do?

A 
Contains both a chromodomain and a SET domain. 
SET domain is catalytic domain of methyl transferase.
allows binding (CD) to methylated regions, and brings SET domain to adjacent lysine residues to be methylated = heterochromatin formation. methylated neighbours will be linked by HP1.
19
Q

What are boundary elements and insulators?

A

Boundary elements define where heterochromatin should end - recognized by DNA binding elements and prevent heterochromatin spreading.
Insulators protect further from heterchromatin spreading particularly to actively transcribed genes.

20
Q

How do matrix attachment regions affect chromatin?

A

MARS tether chromatin to the nuclear matric and mark transcriptional units
Recruitment of facultative MARS is associated with transcriptional activation

21
Q

Discuss where you might find nuclease sensitive areas in chromatin

A

Nuclease hypersensitive areas are found upstream of actively transcribed genes, where nucleosome disruption has occurred.

22
Q

How can you carry out a nuclease sensitivity assay?

A

Digest chromatin with micrococcal nuclease or DNase1
Resolves through an agarose gel.
Gene specific probes can show whether a gene is nuclease resistant or nuclease sensitive

23
Q

What are the key features and function of the SAGA complex in yeast?

A

SAGA complex acts as a transcriptional coactivator
Forms histone acetyl transferase complex
-SAGA is recruited to upstream activation sequence by interactions with coactivators Gcn4/Gal4 bound to UAS
-acetylation of histones = chromatin decondensation, provides recognition sites for Bromodomain proteins.
-GCN5 is catalytic component of SAGA

24
Q

What type of proteins contain a bromodomain?

A

Chromatin remodeling machines, general transcription factor TF2D (preinitiation complex)

25
Q

What is a homologue to Gcn5 in higher eukaryotes?

A

CBP - creb binding protein. Means it acts as acetyl transferase.

26
Q

Discuss a chromatin remodeling complex that acts as transcriptional activator

A

SW1/SNF complex can slide nucleosomes along DNA via ATP dependent helicase activity. Has a bromodomain and can interact with hyperacetylated histones.

27
Q

Discuss a chromatin remodeling complex which acts as a represor

A

SIN3/Rpd3 acts as a corepressor in eukaryotes. Has histone deacetylase activity.
Rpd3 is the catalytic subunit - allows interaction with Ume6 which is a transcriptional repressor bound to the upstream repressor sequence.

28
Q

Discuss the methylation of cytosine residues in higher eukaryotes

A

DNA methylation of C5 in CpG islands correlates to transcriptional repression.
M5C sites recognized by m5CG biding proteins (MECP2). recruites HDACS and HMTS, heterochromatin formation.

29
Q

What is position effect variegation?

A

This is when genes that are normally actively transcribed are repositioned close to heterochromatic regions. Can cause potential transcriptional repression.

30
Q

How can mosaic allele patterns arise?

A

Some chromatin boundaries are softer than others - leads to some genes being heterochromatic in some cells but not others eg mottle white eye locus in drosophila

MBB267 (13 decks)

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