lf206 lecture 4 - eukaryotic genomes, histones

approx. 40% of our genome is derived from ______________.

what are they?

retrotransposons

can copy and paste themselves using RNA intermediate

why do we have multiple haemoglobin genes?

allows for gradual change from foetal to adult haemoglobins - requires temporal transcriptional regulation

protein-coding genes have relatives of which they share common _______.

some genes exist in families and _____-families

some are pseudogenes - what are these?

within a genome, families can be dispersed or c_______. maintenance of clusters implies _________

ancestry

super-families

non-functional copies of a gene - they look like the gene but are not functional.

functional co-ordination/regulation

expressed genes are found in what formation?

euchromatin

genes not expressed are usually found in what conformation?

facultative heterochromatin

difference between facultative and constitutive heterochromatin

facultative can switch into euchromatin - so is not always packed

constitutive is permanently not expressed - these genes are silent.

three specific structures of constitutive heterochromatin

origins of replication

telomeres

centromeres

what is this?

euchromatin

what is a nucleosome?

147bp dsDNA wrapped around a octameric histone core (of 8 histone proteins)

how many and what histone subunits per nucleosome

8 subunits

2x H2A

2x H2B

2x H3

2x H4

how to dissect nucleosome core particle to release histones

digest linker with DNAse

high salt dissociation to remove octameric histone core and 147bp dsDNA

rebuilding euchromatin/nucleosome

1. express each histone separetly in E.coli

2. purify each histone

3. combine in different combination to explore interactions

   1. H3 and H4 interact to form a dimer, which binds to a tetramer, which can bind DNA

4. palindromic dsDNA sequence of 146bp on a recombinant plasmid maintained in E.coli

5. H3-H4 tetramer:DNA complex binds 2 separate H2A:H2B dimers

6. reconstituting nucleosomes in vitro which pack evenly during crystallisation = high-resolution structure

nucleosomes are not static. they are ___________ to make DNA more/less accessible to ______ proteins, general transcription ______, mediator ______ and RNA _________ II

thus histone modification ______ transcription

remodelled (removed/re-positioned/replaced/covalently modified)

activator

factors

proteins

polymerase

regulate

histone tails can be modified.

specific ______ (K) residues can be modified by _________ (Ac).

this is a code to ‘_____ the chromatin structure’

lysine

acetylation

relax

how can a gene be silenced?

some _____ (K) and arginine (___) residues can be modified by _________ (Me) on the histone ____/tail

this can relax or compact chromatin depending on

• which residue is methylated

• the ________ of methylation (mono-, di-, tri-)

lysine

R

methylation

code

degree

phosphorylation (P) of some _____ (S) and _______ (T) residues promotes ________

____________ (Ub) of some _______ (K) on H2A are __________ sites for DNA ______

serine

threonine

transcription

ubiquitylation

arginine

recruitment

repair

define epigenetics

heritable changes in gene expression that are not mediated at the DNA sequence levels

(e.g. DNA modifications, histone modifications)

how can histone modifications be maintained during transcription

epigenetic marks are preserved

RNA Pol II transcribes naked DNA quickly but transcribes ‘beads on a string’ slowly with pauses

1. RNA Pol II stalls at nucleosome A

2. RNA Pol II unwinds about half of the DNA from nucleosome A

3. transcribed DNA is looped and starts to wind around nucleosome A whilst unwinding continues

identifying origins of replication in yeast

ARS region?

autonomously replicating sequences

DNA sequences that can replicate independently and are functional origins of replication

origin of replication?

site on DNA where replication begins

cell cycle phases

INTERPHASE

G1

S

G2

MITOTIC PHASE

mitosis

cytokinesis

G₁

• an origin of replication has a pre-bound complex of ___ proteins (_______). they remain ______ throughout the cycle

• Cdc6 and Cdt1 associate with ___

• ___ helicases are recruited to form the _______ ______ (pre-RC)

• in G₁ pre-RC is formed but not _______

ORC (ORC 1-6)

associated

ORC

Mcm

prereplicative complexes

activated

S phase

• Cdk2 (_____ ________ ____ 2) phosphorylates ____

• proteasomal __________ of phosphorylated Cdc6

• phosphorylation of ___ = RC is activated but new ones cannot form (no Cdc6)

• Mcm _____ open the DNA allowing for recruitment of primase clamp _____ and DNA _______.

cyclin dependent kinase 2

Cdc6

degradation

helicases

loader

polymerases

what happens to histones during replication

replication machinery displaces H2A-H2B dimers.

unclear whether H3-H4 tetramers are displaced or reused?

but both new strands inherit them

large increase in expression of histones in S phase

new subunits acetylated to promote open structure

NAP-1 (nucleosome assembly protein 1) chaperones H2A-H2B dimers

CAF-1 (chromatin assembly factor) chaperones H3-H4 tetramers

assembles nucleosomes in an open chromatin structure

fresh acetylations are removed, ‘reader-writer complex’ copies epigenetic information from neighbouring nucleosomes.

thus daughter cells maintaining parental cell histone modifications

cytosines in DNA can be methylated.

methylated DNA means __ ___ _____

methylated histones can recruit DNA _____ that methylate _____ residues

methylated DNA can recruit histone _______ = ______ by condensing chromatin

histone code and DNA methylation work together

do not express

methylases

cytosine

methylases

silencing

what is SINE

short interspersed nuclear elements

80-500bp long

a type of retrotransposon

how can SINEs regulate gene expression (non-epigenetic and epigenetic)

non-epigenetic: act as enhancers or alternative promoters to recruit transcription factors & promote expression

epigenetic: SINEs are GC-rich so hotspot for DNA methylation, silence nearby genes by stimulating chromatin condensation