Chromatin and Transcription

How does chromatin affect transcription?

Chromatin inhibits DNA transcription in vitro. Its structure affects all stages of transcription (initiation, elongation, termination).

Experiment: What happens if you add nucleases to chromatin?

Chromatin restricts the accessibility of DNA to nucleases.

Why is Chromatin compacted?

Compacted through higher order structures to fit into the nucleus e.g. higher order chromatin. The more compacted it is, the less accessible.

How do histone modifications distribute around transcribed genes?

Complex patterns of distribution!

Features of heterochromatin?

Condensed Gene poor Less expressive

What factors are involved when chromatin inhibits transcription elongation?

FACT (facilitates chromatin transcription). Histone chaperone that removes H2A/H2B from chromatin.

14-3-3 proteins...

Promote transcription elongation in Drosophila.

How might chromatin lead to epigenetic changes in bees?

Queen bees: Larger than worker bees, Lives 20x longer than workers, Can lay her own bodyweight in eggs every day.

Give an example of how a histone modification affects chromatin structure indirectly.

Serine phosphorylation introduces a negative charge (serine is catalysed by kinase to form phosphoserine).

What are some features of histones?

Small, basic proteins (10-15kDa) Highly conserved Simple helical structure They associate with each other

Why do cells need to be 'dynamic'?

So that cells and whole biological systems can respond quickly to an unpredictable environment.

How can you know what a histone modification does to chromatin?

1. Reconstitute modified chromatin and test its properties in vitro. 2. Pulldown modified chromatin and analyse (high-throughput sequencing) the DNA sequences it is associated with. 3. Pulldown modified chromatin and analyse (mass spec) the proteins it associates with.

Chromatin is strongly implicated in epigenetics. Why is this important?

Affects ALL nuclear processes including DNA transcription. Cancer - not just a disease, but an epigenetic disease. Development.

Lysine methylation by HMTs can create...

3 forms of lysine methylation: moni-, di-, and tri-methyllysine. (NB: does not neutralise the charge on the lysine in any form)

What is Epigenetics?

A stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence. I.e. DNA sequence alone is not enough, CONTEXT is important.

How can chromatin be active or silent (carry info)?

Amino acid sequence of histone H3: ARTK. Histones can be post-transcriptionally modified. Some lysines (K) converted to acetyllysine. New AA, new properties, new info!!

Why would you want to chromatin to signal at all? Compare bacteria and humans...

Bacteria manage to control their genes with just TFs. Humans would need to many TFs because they are multicellular and have many more genes, therefore require additional regulatory mechanisms.

What are HMG proteins?

Bend DNA upon binding. Open up chromatin structure.

What is Histone H1?

Binds nucleosome DNA entry exit site. Promotes chromatin 30nm fibre formation.

Acetylated lysines are recognized and read by...

Bromodomains

Methyllysines are recognized by...

CHROMODOMAINS

Why is it useful for histone modification 'writers' to contain domains which bind to the modification thy add e.g. bromodomains on HATs?

Can modify the other histone copy within the same nucleosome. Can spread along between nucleosomes to modify a larger domain.

Histone modifications change chromatin structure by...

Changing the biophysical properties of chromatin. Acting as signalling molecules to recruit downstream effectors.

What is regarded as the definitive epigenetic mark and why?

DNA methylation (uses DNMT = DNA methyltransferase) Can be stably maintained across replication! And altered DNA methylation can lead to epigenetic changes!

Histone modification 'writers' often contain...

Domains that bind to the modification they catalyze.

How is altered promotor structure sufficient to affect transcriptional activation?

During initiation, the IFN-ß promotor nucleosome is repositioned to expose TATA box. This induces Interferon (IFN) activation.

What are the two types of chromatin that exist?

Heterochromatin Euchromatin

What other DNA binding proteins are important components of chromatin?

Histone H1 HMG proteins

How might histone modifications change chromatin structure by acting as signalling molecules to recruit downstream effectors?

Histone modifications act as binding sites for other proteins.

What forms the protein component of nucleosomes?

Histones

How does chromatin provide context to genetic information?

In the same DNA sequence, repressed chromatin with silence the DNA, whilst active chromatin will transcribe it.

What epigenetic affect does Royal Jelly have on bees?

Larva + pollen = worker bee Larva + Royal jelly = queen bee Royal jelly = 5% (w/w) 10HDA = histone deacetylase inhibitor Histone deacetylases change chromatin structure.

Features of euchromatin?

Less condensed Gene rich More expressed

What is the ultimate higher order structure?

The metaphase chromosome

Histones contain...

Long flexible tail domains (<1/3 the size of a histone). They are unstructured and highly charged --> bind to DNA. Extensively modified by post-translational modifications.

Lots of histone marks =

Lots of binding proteins! Affinity of modified histone binding proteins tend to be weak (high µM to low mM Kd)

Give three examples of histone marks?

Lysine acetylation Lysine methylation Serine phosphorylation

Post-translational histone modifications: how is this a good way to respond quickly?

Lysine converted to acetyllysine by HAT (histone acetyltransferase). Acetyllysine converted to lysine by HDAC (hisone deacetylase). --> Doesn't change your DNA, just the way you use it :-)

Histone tail domains are subject to extensive modifications, for example...

Methylation, acetylation, phosphorylation, citrullination...

Where would a histone modification take place that would affect accessibility?

Near the DNA entry-exit site.

What would extensive lysine acetylation to cause?

Neutralises the positive charge on lysine by converting to acetyllysine using HAT. Since binding of histone tails to DNA is electrostatic, this binding decreases, making the chromatin structure more open.

What do histone tails bind?

Nucleosomal liner DNA (therefore nucleosomes lacking histone tales are unstable).

What is DNA methylation?

Occurs within CpGs -> (methyl CpG), Distributed non-randomly (CpG islands), Bound by methyl-CpG binding domains (MBDs), DNA methylation associated with transcriptional repression.

What happens when histones compete with transcription factors for binding to DNA?

Steric hindrance

How does chromatin affect transcription termination?

Strains lacking chromatin remodelling enzymes fail to terminate transcription efficiently.

What is the nucleosome?

The basic repeating unit of chromatin. Nucleosomes compact DNA filaments.

What is Chromatin?

The complex of DNA and associated binding proteins found in the nucleus.

Summary of messages so far...

The genetic info of euks is contained within its chromatin NOT just its DNA. Epigenetics influences development. Histones are major contributors to chromatin structure. Chromatin structure affects transcription both positively and negatively.

Why is DNA within a nucleosome core particle less accessible?

The inner surface of DNA is not solvent exposed. Nucleosomal DNA is bent and twisted.

Promotor chromatin structure changes during...

Transcriptional activation. By nucleosome repositioning or nucleosome eviction.

How to nucleosomes prevent TFs binding to cryptic binding sites?

Upon cellular senescence histone levels are reduced. TFs bind new sites. Can speed up senescence.

What are the higher order chromatin structures?

With increase compactness and decreasing accessibility: Nucleosomes coiled into a chromatin fibre. Further condensation of chromatin. Duplicated chromosome.

Does chromatin ultrastructure change?

Yes, during development. E.g. from Proerythroblast to Late Erythroblast there are noticeable changes.

Can one genotype really give rise to many phenotypes?

Yes, for example: Embryo can either be a neuron or a muscle? Or a queen as opposed to a worker?