Epigenetics

Question 1

stable, long-term alterations in the transcriptional potential of a cell

Answer 1

epigenetics: heritable changes in gene expression without change in DNA sequence

epigenome varies from one cell type to another and can be reprogrammed

Question 2

Describe the effect of DNA methylation? What residues are methylated?

Answer 2

C residues in CG dinucleotides are methylated (both strands)
most promoter sequences contain CpG islands

DNA methylation [typically] turns OFF gene expression

Question 3

what are the two ways in which methyl-CpG binding domain proteins (MBD) can repress gene expression?

Answer 3

1. prevent binding of transcription factors
2. recruit depressors and histone modifying enzymes to promoter

Question 4

what makes it possible for DNA methylation patters to be transmitted to daughter cells?

Answer 4

DNA replication is semi-conservative

DNMT1 (DNA methyltransferase enzyme) recognizes hemi-methylated DNA (of daughter strand) and makes it match the parent strand

Question 5

describe the process of de novo DNA methylation during embryogenesis

Answer 5

1. wave of global demethylation
2. embryonic methylation pattern established via de novo methyltransferase enzymes (DNMT3a/b)

** de novo DNA methyltranferases can methylate both strands of DNA

Question 6

To study changes in the epigenome, you can use either methylation-sensitive restriction enzymes or bisulfite-mediated conversion of DNA. Contrast how these work

Answer 6

methylation-sensitive restriction enzymes - do NOT cut methylated DNA … compare with control

Bisulfite-mediated conversion of DNA - converts NONmethylated C to U, which appears as a T (U binds A, A bind T) after PCR amplification … any C’s from control that were converted to T’s after bisulfite treatment must have been epigenetically methylated

Question 7

what are the characteristic features and cause of Rett Syndrome

Answer 7

X-linked dominant neurodegenerative disorder in females with spontaneous monogenic mutations in MECP2 gene (binds methylated DNA and facilites gene silencing)

normal development until 6-18 months, then severe seizures, lung infections, autism, failure to walk and speak

Question 8

Fast forward and you are a pediatric neurologist. During rounds a resident presents this patient to you:

Pt is a 16mo F presenting with severe seizures and a minor lung infection. Pt underwent cognitive and behavioral exam which indicated autism. Pt shows delayed developmental progress of walking and speaking. Genetic analysis indicates a mutation in MECP2.

What is your diagnosis?

Answer 8

Rett Syndrome: X-linked dominant (females affected) neurodegenerative disorder

spontaneous mutation in MECP2 gene which binds methylated DNA and facilitates gene repression

causes inappropriate over-expression of genes

Question 9

what is the function of MECP2 protein in epigenetics

Answer 9

MECP2 = methyl-CpG binding protein 2

binds methylated DNA via MBD domain and represses transcription from methylated gene promoters

LOF —> inappropriate over-expression, esp in brain where MECP2 is most abundantly expressed

Question 10

what epigenetic mechanism causes imprinting?

Answer 10

DNA methylation of one parental allele

whether a specific allele is imprinted depends on whether it was inherited from male or female parent

parental imprint pattern is erased during gametogenesis to allow inheritance of imprinted genes

imprint pattern is reset to correspond to sex of developing fetus

Question 11

what is a potential clinical consequence of gene imprinting

Answer 11

causes mono-allelic expression

if expressed allele is nonfunctional or deleted, leads to complete LOF

ex: Prader-Willi disease and Angelman disease

Question 12

the causes of Prader-Willi and Angelman syndromes have this in common

Answer 12

caused by mono-allelic expression due to gene imprinting

Question 13

what are 4 ways by which histones can be modified, and what is the general impact of each of these

Answer 13

1. acetylation - turn ON
2. phosphorylation - turns ON
3. methylation - turn OFF
4. ubiquitination - turns OFF

Question 14

describe the effect and mechanism of histone acetylation (and name the enzymes involved!)

Answer 14

histone acetyltransferases (HATs) neutralize (+) charge of lysine residues in histones

acetylated histones have lower affinity for (-) charged DNA

nucleosome is looser, DNA more accessible —> gene expression turned ON

HDAC (histone deacetylases) reverse the process

Question 15

describe the effect and enzymes involved in histone methylation

Answer 15

histone methyltransferase (HMT) promotes heterochromatin formation because methyl groups are very hydrophobic —> gene turned OFF

histone demethylase (HDMT) reverses the processes

Question 16

During rounds, a resident presents two patients, one with AML (acute myeloid leukemia) and another with myelodyplastic syndrome (MDS). The resident wants to start them both on either Decitabine or Azacytidine, both inhibitors of DNMT.

Will these drugs be effective and why?

Answer 16

yes (hopefully) - common epigenetic features of cancer cells include loss of histone acetylation and CpG island hypermethylation

inhibiting DNA methyltransferase may inhibit CpG island hypermethylation

Question 17

Why might an oncologist recommend an HDAC inhibitor for a patient with T-cell lymphoma (Vorinostat) or multiple myeloma (Panobinostat)?

Answer 17

common epigenetic features of cancer cells include CpG island hypermethylation and loss of histone acetylation

HDACi will inhibit function of histone deacetylases —> genes turned back ON

Question 18

this type of genetic material is not translated into protein, but plays an important role in X chromosome inactivation. What is?

Answer 18

non-coding RNA (ncRNA)

long non-coding RNA (lncRNA) is important in formation of Barr body (inactivated X)

Xic (X inactivation center) controls process - includes Xist (binds X to inactivate) and Tsix (antisense transcript of Xist to repress it in active X)

Question 19

what is the biological purpose of x-inactivation in females

Answer 19

dosage compensation (males only have 1 X)

entire chromosome is silenced

female is mosaic of X chromosomes

Question 20

what is the role of MECP2 in gene silencing?

Answer 20

MECP2 binds to methylated DNA and represses transcription from methylated gene promoters

MECP2 LOF —> Rett syndrome (neurological development disorder) with inappropriate overexpression of genes