Dosage Compensation

Question 1

What is epigenetics?

Answer 1

The study of mitotically heritable changes in gene function that cannot be explained by changes in DNA sequence. (NOT novel mutations in genome)

Question 2

What are the 2 major mechanisms of epigenetic regulation?

Answer 2

o Methylation of DNA
o Modification of histone proteins

o Both of these modifications modulate the transcriptional potential of genes (i.e. they regulate gene expression)

Question 3

What is DNA Methylation?

Answer 3

• The formation of 5-methylcytosine
  o When DNA methyl transferase catalyzes addition of methyl group onto cytosine
• Occurs in CpG islands which are typically found in the upstream regulatory regions of eukaryotic genes (promoters)
• It is associated with REPRESSION of transcription

o i.e. genes where the promoter has been methylated are typically inactive.
- Responsible for genomic imprinting

Question 4

How does DNA Methylation block transcription?

Answer 4

-Doesn’t interfere with H bonding between bases-

o Represses gene expression through 2 mechanisms:

   - Prevents binding of transcription factors to promoter by adding methyl group into major groove of DNA so transcription factor doesn’t recognize promoter
   - Recruits MBD proteins which recruit chromatin remodeling proteins leading to formation of “repressive” chromatin

Question 5

What is genomic imprinting?

Answer 5

DNA methylation of either the maternal or paternal allele of an autosomal gene results in the origin of the allele determining its phenotype.

Question 6

What are 2 examples of genomic imprinting?

Answer 6

Prader-Willi syndrome

Angelman Syndrome

Question 7

Prader-Willi Syndrome

Answer 7

o Characterized by uncontrolled appetite
o Results from deletions in the q11-q13 region of chromosome 15 on the PATERNAL chromosome

o HOW?

   - Deletion in q11-q13 region of chromosome 15 leads to loss of 2 genes
   - In EVERY HUMAN maternal copy of gene SNRPN is methylated
   - In EVERY HUMAN paternal copy of gene UBE3A is methylated
   - This means that in all humans those 2 genes are hemizygous, because they only active on one chromosome
   - Loss of SNRPN function = Prader-Willi syndrome
   - So if paternal deleted, SNRPN only on maternal chromosome where it is methylated.. so its function is lost.

Question 8

Angelman Syndrome

Answer 8

o Characterized by behavioral issues
o Results from deletions in the q11-q13 region of MATERNAL chromosome 15…

o HOW?

   - Deletion in q11-q13 region of chromosome 15 leads to loss of 2 genes
   - In EVERY HUMAN maternal copy of gene SNRPN is methylated
   - In EVERY HUMAN paternal copy of gene UBE3A is methylated
   - This means that in all humans those 2 genes are hemizygous, because they only active on one chromosome
   - Loss of UBE3A function = Angelman syndrome
   - So if maternal deleted, UBE3A function lost

Question 9

Chromatin Structure

Answer 9

• DNA is complexed by histone proteins (H2A, H2B, H3 & H4) into nucleosomes
• Nucleosomes condense together to form chromatin
• Chromatin structure is dynamic
• Degree to which nucleosomes are packed can regulate transcription

Question 10

What is repressive chromatin?

Answer 10

Condensed chromatin when nucleosomes are tightly packed. Represses transcription

Question 11

What is Open Chromatin?

Answer 11

Permissive, loose nucleosome packing. Increases transcription.

Question 12

2 types of post-translational histone modification

Answer 12

o Methylation = Adds methyl group - condenses chromatin

o Acetylation = Adds acetyl group - opens chromatin

Question 13

SWI/SNF Chromatin Remodeler

Answer 13

o Methylation of lysine 9 on Histone 3 (H3K9Me) by Hp1 compacts chromatin into heterochromatin.

o SWI/SNF chromatin remodeler recognizes acetylation of lysine 9 on Histone 3 (H3K9Ac) opens up chromatin structure and makes it easy for basal factors to access promoter region of genes in that area

Question 14

2 strategies for dosage compensation

Answer 14

1. Increased transcription of X chromosome (Drosophila)

2. Inactivation of 1 X chromosome (Mammals)

Question 15

What are the 5 Male Sex Lethal proteins which form the MSL complex?

Answer 15

1. MLE (maleless) – RNA helicase
2. MSL1 (male sex lethal 1) – Binds chromatin
3. MSL2 – Binds chromatin
4. MSL3 – Binds methylated histones
5. MOF (males absent on first) – Histone acetyltransferase

• MSL proteins localize to the X chromosome in male flies
• Loss of any one MSL proteins greatly reduces binding of the others to X
  o i.e. all 5 need to be there for the complex to form

Question 16

What are the key players in Drosophila Compensation?

Answer 16

1. MSL proteins in the MSL Complex

2. Non-protein coding RNAs (rox1 & rox2)

Question 17

Evidence for an RNA component of the MSL complex

Answer 17

1. MLE protein contains an RNA binding domain (therefore it must have an RNA mol to bind to)
2. Treatment with RNAse abolishes binding of MLE protein to X chromosome

Question 18

rox RNA

Answer 18

• rox RNAs don’t encode proteins and co-localize with MSL proteins on the X chromosome in males
• rox1 and rox2 are redundant (i.e. you don’t need both)

Question 19

What is the process of the MSL complex formation?

Answer 19

o First transcription of rox RNA
o Binds to X chromosome from which its transcribed
o Leads to recruitment of MLE protein (has RNA binding domain)
o Once MLE is bound, the other 4 proteins in the complex bind

Question 20

Mechanism of dosage compensation in Drosophila

Answer 20

• Binding of MSL complex leads to hyperacetylation of H4K16 in males
  o This is catalyzed by MOF which is a histone acetyl-transferase.
• Because of this Males have high levels of a specific histone marker (H4K16Ac) on the nucleosomes along the entire length of the X chromosome
• This marker is recognized by chromatin remodelers which open up the chromatin structure
  o Because this histone marker is present on all nucleosomes along the entire chromosomes, the entire chromosome is opened.
• This allows basal transcription machinery to more easily access gene promoters
• So transcription rate of genes on male X chromosome is higher (2x level of single x chromosome in females).

Question 21

What prevents dosage compensation from occurring in Drosophila females?

Answer 21

• Sxl binds to the 3’ UTR of the MSL2 mRNA blocking translation = no MSL2 protein produced -> no dosage compensation occurs.

Question 22

What are the parallels between dosage compensation in Drosophila and in mammals?

Answer 22

1. Involves determining number of X chromosomes
2. Involves X-linked non-protein coding RNAs (in mammals = Xist & Tsix)
3. Xist work in cis (meaning they bind to the X chromosome from which they are transcribed)
4. Leads to large scale modification of Lys residues in histone tails along the entire length of X chromosome in mammals

Question 23

What is the mechanism of X chromosome inactivation in mammals?

Answer 23

• Large non-coding RNA’S are overlapping
• No free Xist RNA in the nucleus because transcription of Tsix leads to dsRNA (because it overlaps with Xist) so it is degraded
• In Xa (active X chromosome) Xist expression is turned off and Tsix is left on
• In Xi (inactivated X chromosome) Xist expression is left on and Tsix is turned off.
  o Means that Xist is transcribed and not ‘mopped up’ to form dsRNA by Tsix.
• Xist remains attached to X chromosome it was transcribed from and serves as scaffold to recruit PRC2 complex which deposits a specific histone marker (Tri-methylation of H3K27)
• PRC1 complex (chromatin remodeler) recognizes histone marker and it compacts chromatin down to form barr body (highly dense and transcriptionally silent)