L4 Chromatin remodelling and transcriptional repression

What are the major mechanisms in the post-translational modification of histones

• acetylation

• methylation

• ubiquitylation

• phosphorylation

give an example of enzymes that write the histone code

• histone acetyl transferases

• histone methyl transferases

• kinases

give an example of enzymes that erase the histone code

• histone deacetylases

• demethylases

• phosphatases

give an example of enzymes that read the histone code

• bromodomain proteins

• chromodomain proteins

• PWWP proteins

• PhD

What do all multiple remodeling complexes have

Snf2-related ATPase

• helicase and NTP driven nucleic acid translocase superfamily 2 (SF2)

describe ATP-dependent chromatin remodelling

• sliding

• unwrapping

• eviction

• spacing

• histone variant exchange

what was the first complex to be isolated

SWI/SNF from yeast

what is the catalytic subunit of SWI/SNF called

Snf2 (or Swi2)

what is Snf2 related to

DNA helicases

describe the function of Snf2

a molecular motor that uses energy from ATP hydrolysis to track along DNA and induce torsion

• results in disruption of histone-DNA interactions and movement of the nucleosome

what do SWI/SNF and the GCN5 HAT regulate the same genes in

yeast

what are HATs and ATP-dependent remodellers commonly recruited to

the same promoters

what do bromodomains in Snf2 help tether HATs and ATP-dependent complexes to

acetylated nucleosomes

how do HATs and ATP-dependent remodellers function

co-operatively

what mSWI/SNF complexes do the products of the 29 SWI/SNF genes assemble into

• canonical BAF

• polybromo-associated BAF

• noncanonical BAF

  • compromise common and complex-specific subunits

what catalytic subunits do the mSWI/SNF complexes contain

SMARCA4 or SMARCA2 (referred to as BRG1 or BRM)

what are the human mSWI/SNF complexes essential for

regulating gene expression, cell differentiation, DNA repair

what are the mutations that genes encoding SWI/SNF associated with

• Neurodevelopmental disorders

• Cancers

give examples of neurodevelopmental disorders with mutation genes encoding SWI/SNF

• coffin-siris syndrome

• nicolaides-baraitser syndrome

give examples of cancers with mutation genes encoding SWI/SNF

• Rhabdoid tumors

• gynecologic cancers

how many genes encoding subunits of SWI/SNF complexes are recurrently mutated in cancer 

9

how many cancers are SWI/SNF mutations present in

almost 25%

what is the tumour-suppressor activity of the SWI/SNF complexes most likely due to

roles in facilitating transcription factor function

what is the identification of potential therapeutic vulnerabilities that arise from SWI/SNF gene mutations is leading to

new areas of clinical investigation

what do cells commonly exploit to bring about transcriptional repression

chromatin structure

what is repression of transcription mediated by

the recruitment of chromatin modifying factors

give examples of chromatin modifying factors

• histone deacetylases (HDACs)

• ATP-dependent remodellers

• histone methylases (heterochromatin)

describe active regions of the genome

hyperacetylated

describe repressed regions of the genome

hypoacetylated

what is deacetylation mediated by

histone deacetylases (HDACs)

what do HATs function as

transcriptional co-activators

what do HDACs function as

co-repressors

what are the major groups of classical HDACs (zinc dependent)

• Class I

• class II

• class IV

what is the major group of Sir2 family (SIRTURNS)- require NAD as a co-factor

Class III

what do HDACs commonly function in the context of

large multi-subunit complexes

give an example of a HDAC in a large multi-subunit complex

SIN3 CO-REPRESSOR complex

• conserved from yeast to mammals

how are HDAC large multi-subunit complexes recruited to promoters

interaction with site-specific DNA binding proteins

what do some ATP-dependent remodellers commonly mediate

transcriptional repression

give an example of an ATP-dependent remodeller mediating transcriptional repression

the NuRD complex belongs to the Mi2/CHD family

what is the NuRD complex highly conserved in

plants and animals

what is the NuRD complex broadly expressed in

most tissues

• plays roles in normal differentiation and tumourogenesis

what have several oncogenic transcription factors been shown to recruit

the NuRD complex to suppress the transcription of tumour suppressor genes

describe euchromatin

• gene rich

• potential to be transcribed

describe heterochromatin

• gene poor

• repetitive regions 

• transcriptional silencing

give an example of heterochromatin

• centromeres and telomeres

• mating type genes in yeast

• homeotic genes in drosophila

• X-chromosome inactivation in humans

what are the biochemical features of heterochromatin

• hypoacetylation

• specific histone H3 methylation (e.g. Lys9 and Lys27)

• association of specific silencing factors

what protein structure is heterochromatin protein 1

chromodomain

describe chromodomains

recognise and bind to methylated lysine residues

what is the chromodomain of HP1 specific for

H3 Lys9me2/3

what is the binding of HP1 thought to compact

nucleosomal arrays

what does binding of HP1 act as

a platform for the recruitment of further activities that prevent recruitment/activity of RNA pol II

how does euchromatin convert to heterochromatin

• H3K9 methylation

• HP1 binding

describe reporter silencing assay analysis of heterochromatin of the ‘normal’ wild type (fission yeast)

• ade6 gene expressed

• white colonies

describe reporter silencing assay analysis of heterochromatin of the silencing reporter strain (fission yeast)

• ade6 silenced

• red colonies*

describe reporter silencing assay analysis of heterochromatin of the mutation in gene encoding a component of heterochromatin (fission yeast)

• ade6 silencing alleviated

• white/pink colonies

how do red colonies result

from a build up of a red pigment which is an adenosine biosynthetic intermediate

what chromosomes do females have

two X chromosomes (one of which is inactivated)

what does the inactivation of one of the female X chromosomes do

equalises the number of X-linked genes expressed in males and females

what is the inactivated X-chromosome seen in the nucleus as

a condensed structure (Barr Body) that is assembled into a specific form of heterochromatin

what is the formation of a Barr body controlled by

non-coding RNAs Xist and Tsix

describe the process of X-chromosome inactivation

• Xist and Tsix are opposing (early development in embryonic cells)

• Xist is upregulated (later in development)

• Xist coats the chromosome

• Xist recruits H3K27 methylase

• recruitment of silencing factors

• Inactive X (Xi)

what happens if Xist isnt upregulated

Active X chromosome (Xa)