chromatin, epigenetics, and the histone code

state of DNA in cells

• not naked

• associated with histone proteins (H2, H3, H4) that bind it strongly and wind it up into nucleosomes

nucleosomes

higher-order structures of DNA bound to histone proteins that are necessary to package the genomic DNA into the nucleus in the form of chromosomes

heterochromatin

• condensed form of chromatin that localizes at the nuclear envelope often near nuclear pores

• considered transcriptionally inactive

facultative heterochromatin

can turn into euchromatin

constitutive heterochromatin

always heterochromatin

euchromatin

• delicate and thread-like

• abundant in actively transcribing cells

• may represent DNA that is unwound to provide a transcriptional template

control of mating type in yeast

• HMLα and HMRa loci must be silenced otherwise the cells will be diploid a/α and cannot mate

• transcriptional repression depends on silencer sequences

• either α or a will be recombined at MAT locus and that trait will be expressed

RAP1

binds to DNA in silencer region and binds repetitive sequence in telomeres

SIR1 (silent information regulator)

cooperates with RAP1 and is important for binding the silencer region in the silent mating type loci

SIR2, SIR3, and SIR4

• SIR3 and SIR4 bind to hypoacetylated histone tails (H3 and H4) and recruits SIR2

• forms large complexes with telomeric DNA

function of SIR2

histone deacetylase that exposes lysines, which become attracted to DNA causing DNA to compact

histone deacetylation complexes (HDACs)

remove acetyl groups from histone tails, resulting in a positive charge that interacts electrostatically with DNA phosphate groups

Rpd3p

histone deacetylase whose specific targeting requires Ume6p (which binds URS) and Sin3p

co-repressor

needs to be directed by DNA binding proteins

histone acetyl transferases (HATs)

• acetylation of histone tails neutralizes the electrostatic interaction and permits complex formation

• some transcriptional activators can overcome the repressed chromatin state by inducing acetylation of histone tails through associated proteins (Gcn4p and Gcn5p, CBP, p300)

LacI-VP16

• very strong activation domain

• histone acetylase and chromatin-remodeling complexes

• can trigger decondensation of chromatin

SWI/SNF

chromatin remodellers and histone acetyltransferases

pioneer transcription factors

• DNA binding transcriptional activators that function by interaction with DNA via sequences exposed on the outside of the DNA-wound histone octamer

• recruit enzymes that alter the configuration of the neighbouring histone tails

histone code

specific modifications on tails of H3 and H4 induce changes in chromatin structure typical of euchromatin and heterochromatin, but do not always have the same result

chromatin immunoprecipitation (ChIP) with antibodies against modified histone tails

1. isolate and shear chromatin

2. add antibody specific for modified N-terminal histone tail

3. immunoprecipitate

4. release immunoprecipitated DNA and assay by PCR or subject it to NGS

next generation sequencing following ChIP

entire genome can be surveyed to provide information about the genes (loci) affected by the marks

ChIPs performed with antibodies against transcription factors

help to delineate enhancer positions and other key regulatory elements

epigenetic traits

transmitted independently of the DNA sequence itself

types of epigenetic traits

• inactive X (Xist, histone methylation and heterochromatin spreading)

• developmental restrictions

• imprints (DNA methylation)

histone marks

often heritable following cell divisions

epigenetic writers

histone methyltransferases that repress gene activity across an entire genetic region nucleated by histone marks

epigenetic readers

proteins that recognize epigenetic marks and act upon them following division

H3K9me3

recognized by a specific histone methyltransferase to methylate neighbouring naive histones, this HMT therefore acts as an epigenetic reader and writer