L3 Chromatin Structure

Describe regulation of transcription by activators

• promote binding of an additional activator

• stimulate complex assembly

• release stalled RNA pol II

  • however, DNA is not naked in eukaryotes- packaged in CHROMATIN

how long is the human genome

2 metres in length- has t be packaged into a nucleus a few microns in diameter

whats the basic function of chromatin

to compact DNA

what is chromatin composed primarily of

histones

• small basic proteins

Describe N-terminal tail

Highly basic: rich in Lys and Arg

describe globular domain

alpha helices and loops

what are the repeating units that core histones form

nucleosomes

describe the structure of a nucleosome

~147 bp of DNA wrapped twice around an octamer of histone proteins

describe the structure of an octamer

central H3-H4 tetramer + 2 flanking H2A-H2B dimers

describe how nucleosomes are organised

• DNA passes directly from one nucleosome to the next (10nm fibres)

• linker histones bind to the DNA between nucleosomes

give an example of a linker histone

histone H1

what do in vitro histones result in

the formation of a thicker 30nm fibre (in vivo structure?)

describe the in vitro experiments that’s evidence suggests chromatin inhibits transcription

• RNA pol II + transcription factors + naked DNA template → transcription

• RNA pol II + transcription factors + chromatin template   → no transcription

describe how the in vivo nucleosome positioning experiments provide evidence that chromatin inhibits transcription

in vivo nucleosome positioning experiments

• nucleosomes are disrupted or lost during transcriptional activation

Describe how genetic studies in Saccharomyces cerevisiae have shown evidence that chromatin inhibits transcription

• singly engineered yeast strain

• chromosomal copies of H4 genes were deleted and a plasmid expressing H4 under the control of a ‘regulatable’ promoter (GAL4)

• when glucose was added to the medium, the expression of H4 was rapidly shut off

• resulting in nucleosome depletion and the expression of many inducible genes

when is the GAL4 promoter off

when there is glucose in the medium

when is GAL4 promoter on

when there is galactose in the medium

what are the conflicting roles in the nucleosome

• compaction of DNA forms a template for DNA transcription

• chromatin structure is dynamic and this lecture is a load of waffle

what are the 3 major mechanisms for modulating the structure of chromatin

• histone variants

• post-translational modification of histones

• ATP dependent chromatin remodelling

how are histone variants expressed

very low levels

which histones have variants

all conventional histones except H4

give examples of the variant histones for H2A

• H2AZ

• H2AX

• macroH2A

give an example of the variant histone for H2B

hTSH2B

Give examples of the variant histones for H3

• CENP-A

• H3.3

what do histone variants confer

novel structural and functional properties of the nucleosome which affect the chromatin dynamics

give examples of post translational modifications histones are subjected to

• acetylation

• methylation

• ubiquitylation

• phosphorylation

what has histone modification state been proposed to constitute

code that sets transcriptional state

what are the possibilities for histone modification to constitute a code that sets its transcriptional state

• directly alter chromatin folding/structure

• control the recruitment of non histone proteins to chromatin- influences the recruitment/function of the transcriptional machinery

describe histone lysine acetylation

• mediated by histone acetyl transferases (HATs)

• readily reversible by histone deacetylases (HDACs)

highly dynamic

what mediates acetylation

histone acetyl transferases (HATs)

what reverses acetylation

histone deacylases (HDACs)

what happened in the 1960s for histone acetyl transferases

correlation between high levels of acetylation and transcription

what happened in the 1990s for histone acetyl transferases

the first nuclear HAT was shown to be homologous to yeast GCN5

• GCN5 functions as a transcriptional activator

  • acetylation is a key component of transcriptional activation

what is the function of nuclear HATs now

function in large multisubunit complexes (GNAT and MYST)

what are examples of the GNAT family complex

• yeast SAGA

• human STAGA

• human PCAF

what is the catalytic subunit of yeast SAGA

yGCN5

what is the catalytic subunit of human STAGA

hGCN5

what is the catalytic subunit of human PCNF

PCAF

what is the substrate of yeast SAGA

H3/H2B

what is the substrate of human STAGA

H3/H2B

what is the substrate of human PCAF

H3/H4

give examples of the MYST family complex

• yeast NuA4

• humanTip60

what is the catalytic subunit of yeast NuA4

Esa1

what is the catalytic subunit of humanTip60

Tip60

what is the substrate of yeast NuA4

H4/H2A

what is the substrate of human Tip 60

H4/H2A

what do activators recruit HATs to

specific promoters

many HAT complexes contain a specific subunit that interacts with…

activators

give an example of a HAT complex containing a specific subunit that interacts with activators

Tra1 (yeast) TRRAP (mammals)

what are some HATs part of

general transcription machinery

how does acetylation mediate transcriptional activation

• directly influence on chromatin structure

• directs recruitment of BROMODOMAIN proteins

what are specific acetylated lysine residues recognised by

proteins with bromodomains

what do bromodomains often promote

transcription

how does Bdf1 work as a bromodomain protein promoting transcription

binds acetylated H4 and recruits TFIID

how does TAFII250 work as a bromodomain protein promoting transcription

TFIID subunit, binds acetylated H4

where does histone modification occur

Lysine

• (arginine residues- less well understood)

what are histone lysine methyl transferases (HKMTs)

SET domain proteins

• lysine demethylases have opposite effect

methylation is not readily reversible but

demethylases exist

methylation does not affect charge so

only minor influence on chromatin structure

how do HKMTs manipulate lysines

mono, di or tri methylated (by HKMTs containing a SET domain)

give examples of methyl-lysine binding modules

• chromo-domains

• PHD fingers

• ankyrin repeats

• tudor domains

• PWWP domains

• WD repeats

• MBT domains

what can methyl-lysine residues either function as

activating or repressing marks

give examples of methyl-lysine residue repressing marks

• H3 Lys9

• H3 Lys27

give examples of methyl-lysine residue activating marks

• H3 Lys4

• H3 Lys36