Lecture 30: Chromatin Remodeling and DNA Methylation

How is transcription primarily regulated in bacteria and eukaryotes?

by transcription factor proteins that bind to DNA sequences associated with any given gene

In eukaryotes, how else is transcription regulated?

by the structure of the chromatin surrounding the gene

What two levels is chromatin structure controlled at?

• Modification of the histones associated with the DNA

• Methylation of the DNA itself

At any given time, what is most of the DNA in a eukaryotic cell wound around?

nucleosomes

What happens if a core promoter or other essential regulatory sequences are pressed against histones?

the core promoter or essential regulatory sequences can be blocked

What is chromatin remodeling?

a process in which nucleosomes are either moved or disassembled to expose the blocked sequences

What activates chromatin remodeling?

pioneer TFs

How do yeast regulate expression of metabolic enzymes?

regulation is based on the nutrients available in the environment

When galactose is present in yeast, what happens?

the transcription factor Gal4 activates transcription of a set of genes required for galactose metabolism

What does the Gal4 homodimer bind to?

the UAS

What is the UAS?

Gal4’s upstream activating sequence

What does the cis-regulatory region of the Gal1 gene contain?

4 UASg enhancers

What must happen for Gal4 to activate Gal1 transcription?

Gal4 must bind one or more of the UAS sequences

True or False: Eukaryotic enhancers often have multiple binding sites for the same transcription factor; the more molecules of Gal4 that are bound, the higher the rate of Gal1 transcription.

true

How does Gal4 gain access to the UAS binding sites?

The UASg enhancer contains binding sites for the RSC chromatin remodeling complex

What does the RSC insure?

that the enhancer DNA is associated with partially unwound nucleosome which leaves the Gal4 binding sites accessible

What happens when Gal4 is bound to the UASg enhancer?

Gal4 uses its activation domain to recruit a second chromatin remodeling complex called SWI/SNF

What does the SWI/SNF complex do?

displaces a nucleosome that covers the core promoter and +1 start site of target gene Gal1

What does Gal4 do with its activation domain?

Gal4 interacts directly with the general transcription factor TFIID and the mediator complex—attracting them to the Gal1 promoter

What does the recruitment of TFIID and the mediator complex to the promoter lead to?

the recruitment of RNA Pol II and the activation of Gal1 transcription

How else can chromatin be remodeled?

by chemical modifications of the histones that form the nucleosome core

How can chromatin structure be modified?

by the acetylation, phosphorylation, and/or methylation of amino acid R groups on the N-terminal tail domains of the core histones

What did Dr. Marie Maynard Daly identify?

the amino acid sequence of histones and discovered that regions were lysine rich

What do chemical modification of histones influence?

the rate of gene transcription

What does the acetylation and phosphorylation of histone tails promote?

the transcription of the neighboring genes

What does methylation of histone tails do?

repress the transcription of the neighboring genes but the effect of methylation can vary depending on which amino acid is methylated

What is the most common form of histone modification?

histone acetylation

What enzyme covalently bonds an acetyl group to a lysine R group?

histone acetyltransferase (HATs)

What removes acetyl groups from R groups?

histone deactylases (HDACs)

What do acetylation and deacetylation change?

the net electrical charge of the histone protein subunit

True or False: Heterochromatin has little or no transcription, and euchromatin has a high level of transcription.

true

What are the N-terminal histone tails required for?

formation of the 30 nm fiber

True or False: The positively charged H4 proteins are said to be important for the formation of the 30 nm fiber too.

true

What does acetylation of H4 by HAT do?

HAT negates H4’s positive charge—causing disassembly of the 30 nm fiber and locally increasing the rate of transcription

True or False: Deacetylation by an HDAC can reverse the negation of H4’s positive charge.

true

Once histone tails are acetylated, what can they do?

recruit other proteins that contain the bromodomain motif

What does the bromodomain motif include?

nucleosome remodeling proteins and additional histone acetyltransferases

What recruits chromodomain motifs?

methylated histone tails

What do chromodomain proteins help to?

silence transcription of the surrounding DNA

What are the different functions of DNA methylation in different organisms?

• E. coli uses DAM methylase to distinguish new and parent DNA strands during base mismatch repair

• In addition, methylation protects bacterial DNA from the cell’s own restriction enzymes

• DNA methylation does not play either of those roles in eukaryotes; the primary role of DNA methylation in animals is to regulate chromatin structure and silence gene transcription

What does DNA methyltransferase do?

add a methyl group to carbon 5 of the cytosine ring

Is there an alteration in the base pair sequence when cytosine is methylated?

no, methyl-C still base pairs with G

What is the main target for DNA methylation?

CpG

True or False: To mark the gene for silencing, a methyltransferase methylates both strands of the CpG.

true

How many CpGs are methylated in an adult?

70%

Where are the majority of CpG sites clustered?

in the cis-regulatory regions of protein-coding genes called CpG islands

True or False: While methylation of a single CpG has little or no effect on the gene, methylation of multiple sites within a CpG island can result in gene silencing.

true

What does removing CpG methylation do?

activates the transcription of silenced genes

True or False: In the KM20 tissue culture cell line, the neurotensin gene is transcriptionally silent.

true

What do KM20 tissues show a lot of?

CpG methylation of the NT promoter

How can CpG methylation be blocked?

with the drug 5-azacytidine

What does 5-azacytidine do?

eliminates DNA methylation and reactivates transcription of the neurotensin gene

True or False: DNA methylation and chromatin remodeling reinforce one another in regulating chromatin structure.

true

What does epigenetic mean?

information is being passed along without any modification in the DNA sequence of the gene(s)

True or False: When a cell divides, its pattern of DNA methylation is reliably passed along to both daughter cells.

true

What does DNA replication of methylated CpG produce?

two hemi-methylated CpGs

What does maintenance methyltransferase do?

follows the replication fork, restoring the fully methylated CpGs in the same gene(s) of both daughter cells

True or False: If men and women expressed X chromosome genes at the same level, women would synthesize twice as much of all 2,000 proteins, and this genetic imbalance would be lethal.

true

True or False: XX females silence the transcription of 1 of the 2 X chromosomes in each cell, but the inactivated X actually retains about 5% of the transcriptional activity of an active X.

true

What does inactivation of a female X chromosome involve?

hypermethylation of X chromosome DNA and hypoacetylation of its histones

What will the inactivated chromosome condense into?

a heterochromatin structure called a Barr body

When does inactivation occur?

when the embryo is composed of 32-64 cells

True or False: Inactivation is random; the maternal X or the paternal X can be inactivated in any given cell.

true

True or False: Once an embryonic cell has inactivated one X chromosome, that chromosome remains inactive in subsequent cell divisions, an example of epigenetic gene regulation.

true

What is anhidrotic ectodermal dysplasia?

A human disease condition caused by a loss-of-function allele of an x-linked gene essential for sweat gland and hair development

Why is X chromosome inactivation not inherited from mother to child?

If it were, one half of the XY offspring would inherit the mother’s inactive X chromosome and that would be lethal

What minimizes the inheritance of gene silencing from parent to child?

global demethylation of genomic DNA within the germ cells

True or False: DNA methylation is not erased in somatic cells, so the inactivated X chromosome gets reactivated before it is passed along to children.

true