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5bby0205 > L14+15 HISTONE N CHROMATIN > Flashcards

L14+15 HISTONE N CHROMATIN Flashcards

1
Q

epigenetics

A
  • Inheritance of pattern of gene expression and gene function
  • not changing primary sequence of DNA
  • inherited by cells after cell division
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2
Q

how epigenetics is regulated

A

done by histone modifications to change chromatin - can be inherited between daughter cells of mitosis
- also can be done by dna methylation to affect chromatin structure

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3
Q

chromatin role

A

Package and condense DNA into a small volume to fit into cell nucleus

tightly coiled to produce the chromatid of a chromosome

Regulate access of TFs and transcription machinery to DNA sequence during transcription
and replication

Lock-in patterns of gene expression levels - the transcription of mRNA and its translation into protein

contribute to Tissue-specific variation in gene expression - which parts of the genome are transcriptionally active

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4
Q

chromatin, chromatid and chromosome

A

In mitosis metaphase, chromatin condenses to form chromosomes which split into two identical strands called chromatids. Each chromatid then becomes a chromosome in each new daughter cell that is formed

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5
Q

smallest unit of chromatin

A

nucleosome -form when DNA wraps around histone proteins.
1.8 turns of DNA wrap around nucleosome core

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6
Q

length of chromatin fibre

A

30nm. Further condensation during mitosis

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7
Q

chromatins then become…

A

Chromatin fibre - nucleosome coil into 30 nm chromatin fibre

after this, Chromosome scaffold proteins provide higher order organisation

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8
Q

nucleosome structure

A

Five histone subunits at nucleosome core:
H1, H2A,H2B, H3 and H4
- an octameric structure

Nucleosome coreis assembled sequentially from 2x H2A-H2B dimers plus H3-H4 tetramer –

Histone H1 binds to the linker region

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9
Q

Matrix-attachment regions (MARs)

A

also known as Scaffold-attachment regions (SAR)

are anchored to the nuclear matrix and induce the radial loop of chromatin - influence arrangement of gene expression

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10
Q

2 forms of chromatin in interphase nuclei

A

highly condensed heterochromatin : INACTIVE chromatin(TF can’t access)

less condensed euchromatin : ACTIVE chromatin (open to TF influence and transcription machinery)

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11
Q

meaning of active chromatin

A

the chromatin of active genes/ genes being expressed

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12
Q

reason for 2 forms

A

to facilitate the desired or required changes in gene transcription - for CHROMATIN REMODELLING: changing structure and position of the nucleosomes

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13
Q

how chromatin remodelling is done

A

by Chromatin remodelling complexes e.g. SWI/SNF

Hydrolysis of ATP provides energy

change how tight the DNA is bound and location of nucleosome
Promotes the exchange of histones in nucleosomes

these movings in histone placement allow different areas of DNA to open and be accessible

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14
Q

histone modification

A

N-terminal tails of core nucleosome proteins (H2A, H2B, H3 and H4) modified

Acetylation of lysines:
- Histone acetyltransferase (HAT)
- Histone deacetylases (HDAC)

Methylation of lysines and arginines:
- Mono-, di- and tri-methyl derivatives are possible
- Specific histone methytransferases (HMT) and demethylases

Phosphorylation of serines and threonines
Ubiquitination of lysines

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15
Q

histone modification

A

Each nucleosome contains histones that are
potentially modified in a large number of different
ways.

modification can turn gene on or off.

Pattern of modification is duplicated during DNA
replication.

modification is inherited between cells following mitosis

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16
Q

HAT properties and effect on histone

A

histone acetyl transferase is
a CBP (CREB-binding protein) complex - transcriptional activator - the response of cAMP activation

hyperacetylation of histones leads to active /open chromatin - active genes

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17
Q

hyperacetylation of histone effects:

A

induces nucleosome to bind to the DNA less tightly.

Lysine is positively charged. Acetylation changes charge of lysine. DNA is negatively charged and bound to lysine. Acetylation induces DNA to be less tightly bound = gene turned on.

Removal of acetyl group allows the same lysine to be methylated instead
leading to condensation and inactivation of chromatin = repression - transcription off

H3K9ac – activation

H3K9me - repression

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18
Q

drug target example

A

changing gene expression is a significant drug target.

SAHA - HDAC inhibitor
Binds to the active site of HDAC and chelates zinc ions involved in the enzyme reaction
Inhibits the removal of acetyl groups and chromatin remains in open state – genes stay active

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19
Q

histone methylation

A

unlike acetylation, methylation does not affect the charge of the amino group

methylation can induce active or inactive chromatin - dependent on lysine being methylated

Trithorax group (trxG) proteins methylates H3 lysine 4 (H3K4) = active chromatin

Polycomb group (PcG) proteins methylates H3 lysine 9 (H3K9) = inactive chromatin

20
Q

DNA methylation - how

A

methylate (5’-methyl cytosine), ~5% bases mostly found in CpG dinucleotides

Hyper-methylation of CpG islands located at promoters linked with inactive chromatin

done by DNA methyltransferase (DNMT1, DNMT2, DNMT3).
enzymes transfer methyl group.

affects chromatin structure

21
Q

DNA methylation - why,how and disorder

A

one of the epigentic contributions in controlling
tissue gene expression.

methylation of cpgs is inherited - maintained during DNA replication

Methylated CpG binds to MeCP2 (methyl CpG binding Protein 2)
MeCP2 recruits HDAC which leads to inactive chromatin

Mutation in MeCP2 gene causes neurodevelopmental disorder Rett syndrome

22
Q

drug affecting gene expression example

A

5-azacytidine is a cytidine
analogue – gets
incorporated into DNA but
cannot be methylated.
for prevention of tumours becoming resistant to chemotherapy , due to a gene expression epigenetic change

23
Q

X-chromosome inactivation

A

At fertilization female cells 2 X chromosomes are active

50% cells active maternal and 50% cells active paternal X
This is due to Xist - non coding RNA
Xist binds to Inactive X chromosome to trigger whole chromosome inactivation and to turn chromosome into heterochromia.
Xist recruits polycomb group methylases
inactive X chromosome is fully condensed as chromatin. it can be identified as so-called the Barr body - a very condensed X chromosome that doesn’t change during the cell life cycle.

Xist is 17kb transcript expressed from inactive X gene

24
Q

Genomic imprinting

A

Normally, Diploid cells two alleles are both active for genes on autosomes (non-sex chromosomes)

Some genes are imprinted - one allele is repressed/ methylated, while the active allele is unmethylated.
Parental origin of a region of chromosome/ gene determines which gene is active

E.g. Insulin-like growth factor 2 (IGF2) only expressed from
paternal allele. only this locus of chromosome is imprinted

Epigenetic regulation is fixed due to parental origin.

Mutations can affect the epigenetic modification of imprinted genes e.g. Angelman syndrome and Prader-Willi syndrome

25
Q

charge of DNA and histone of chromatin

A

DNA negative
Histone positive

26
Q

Behaviour of Chromosomes during interphase of mitosis

A

chromosomes are not randomly distributed or positioned in the nucleus - occupy discrete chromosome territories

27
Q

what are discrete chromosome territories

A
  • 1-2 microns in diameter
  • interphase chromosomes occupy discrete territories in the nucleosome with limited overlap (but some intermingling at the borders)
28
Q

discrete chromosome territories’ 2 regions

A
  • dense interior of territory = transcriptionally INACTIVE
  • periphery of territory = transcriptionally ACTIVE - more condensed, the more nucleosomes open up and are localised - easier access
29
Q

dysfunction of territories

A

if there are neighbouring chromosomes occupying adjacent regions =
potential chromosomal translocations - fusion into each other, can lead to cancers

30
Q

territories are dependent on

A

the gene content of each chromosome and the transcriptional activity

31
Q

high and low densities of chromosomes mean…

A

chromosomes that have a high density of genes tend to localise towards the interior of the nucleus, where most of the factors that are required for transcription are located.

chromosome of lower density of genes = less active - less transcription.

32
Q

what is the nucleus organised by

A

chromatin compartmentalisation:
chromosome territories’ different compartments (A and B):
- Euchromatin compartment/ A compartment: less tightly packed area towards the interior of chromosome = transcriptionally active
- heterochromatin compartment/ B compartment = more tightly packed chromatin towards the periphery of chromosome = transcriptionally inactive

A and B compartments tend to alternate on the chromosome

33
Q

How we visualise genes in interphase

A

FISH - fluorescence in situ hybridisation

34
Q

what binds to promoter sequence

A

pre initiation complex, RNA polymerase and elongation factors will bind

35
Q

gene regulatory sequences role and position

A
  • can be upstream or downstream of promoter
  • include TFs, chromatin remodellers or histone modification enzymes to change DNA structure to loop out intervening DNA to make contact with the promoter
36
Q

3 types of distal regulatory elements

A
  • enhancers - dna sequences that increase transcription rate
  • silencers - sequences decreasing transcription rate
  • insulators - sequences block effects of enhancers and silencers from the promoter. Can separate defined chromatin regions - organisation of chromatin domains of gene activity.

distal regulatory elements make contact w promoter

37
Q

sequence of insulators

A

very GC rich sequence motif.
these motifs bind a TF called CTCF.
CTCF has 11 zinc fingers enabling homodimerisation and binding to DNA often with a cohesion complex (forms a ring-like structure)

38
Q

how do we know that both silencers and enhancers are different to promoters?

A

both function in either orientation
. e.g enhancer pointing towards or away from the promoter, it will activate transcription still.

both function either upstream or downstream gene and will still activate transcription

39
Q

enhancers contain…

A

clusters of TF binding sites

40
Q

enhancers are located…

A

upstream or downstream gene

can be from few to hundred of kBases away from gene

can be intronic

41
Q

enhancers act under restricted circumstances such as…

A

in a specific cell type, specific developmental stage, extracellular signalling cues

42
Q

DNAse I Hypersensitive Sites (HS)

A

Found in regions of DNA where a gene is being actively transcribed. These regions correlate with histone tail acetylation. Is a characteristic of open active chromatin.

43
Q

example of a gene that is regulated by several regulatory elements

A

SHH gene - sonic hedgehog gene.
regulated by distal enhancers located very upstream. Shows that different regulatory elements drive transcription in different scenarios e.g.
- cell specificity
- stage of development
- cell cycle control
- stimuli response

44
Q

methods of measuring chromatin interactions, silencers and enhancers

A

ChIP-seq
For protein-DNA interactions.
Coupled with NGS (next gen sequencing) allows genome-wide identification of TF binding sites.
Limitation: only provides linear info of protein binding sites on chromosomes
POI (point of interest) can be histone modification or TF

3C:
analyses long range chromatin interactions in nucleus
for understanding structural features of chromosomes, cataloguing of chromatin loops and increased understanding of transcriptional regulation mechanisms.
Limited detection scope

45
Q

what do Chromosome conformation capture techniques do

A

analyze the spatial organization of chromatin in a cell.

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