Epigenomics Intro

Question 1

Epigenetics Outline

Answer 1

Chemical changes that allow for changes in gene expression in response to environment. No changes to overall structure (reversible). Allows for cell differentiation

Question 2

2 Epigenetic Mechanisms of Gene Expression Control

Answer 2

Acetylation & Methylation

Question 3

Acetylation MOA

Answer 3

Histone Acetyltransferase takes acetyl group from Co A. Acetyl group binds to histone making it less negative, loosening histone, increasing transcription.

Question 4

Methylation MOA

Answer 4

DNA Methyltransferase (DNMT) takes methyl group from SAM. Attaches methyl group to CpG regions to prevent RNA polymerase II attaching, reducing transcription

Question 5

Hypermethylation Outline

Answer 5

Methylation in CpG sites, prevents transcription factor binding, decreases transcription

Question 6

Hypomethylation Outline

Answer 6

Reduced methylation in CpG binding sites, enables better transcription factor binding, increases transcription

Question 7

Cancer Epigentics

Answer 7

Hypomethylation at oncogene CpG sites, overpromotes transcription. Hypermethylation at silencer gene CpG sites, suppresses transcription. Results in hyperproliferatiom

Question 8

Methylation Assessment Methods

Answer 8

Tissue biopsy are treated with demethylating agents. See if gene expression changes as if so then methylation is main cause (if not secondary complication)

Question 9

Basic Principle of DNA Methylation Assesment

Answer 9

If Cytosine is Methylated then bisulfate treatment can’t convert Cs to Ts and Cs will be present in PCR. If not then all Ts are converted to T and no Cs will be present in PCR output

Question 10

How is DNA Methylisation Evaluated

Answer 10

MBD2 and 5-methylcytosine antibodies are used into microarray (methylated DNA chips). Limitation: hypomethylation is lost

Question 11

Use of Methylation Assays

Answer 11

Patient stratification, personalised medicine

Question 12

Methyl Capture Based Sequence

Answer 12

Target regions, smaple library, bisulfate conversion, PCR, SeqCap, Probe Solution capture, PCR and Sequence

Question 13

Library Prep Outline

Answer 13

DNA Sonification (breaking up DNA), End repair 9fixing DNA strands), primers added and DNA applied to sequencing platform

Question 14

Benefits of Whole Genome Bisulfate Sequencing

Answer 14

Assay all mappable CpG sites. Genome sequence automatically produced at same time

Question 15

Disadvantages of Whole Genome Bisulfate Sequencing

Answer 15

Effected by depth of sequencing (requires a lot of coverage for comparability small quantitation ability), expensive, low throughput and not straightforward

Question 16

Increasingly Thorough Methodology

Answer 16

HM450, RRBS, MBDCap-Seq and WGBS

Question 17

All Active (Transcription) Histone Modifications

Answer 17

Acetylation, Arginine & Lysine Methylation, Phosphorylation, Lysine Ubiquilation (H2BK120)

Question 18

All Repressed (transcription) Histone Modifications

Answer 18

Cytosine Methylation, Lysine Methylation, Lysine Sumoylation and Lysine Ubiquilation

Question 19

CHIP Outline

Answer 19

Chromatin Immuno Precipitaion, fragments of DNA binds to chip and is sequenced. Metod of measuring histone modifications

Question 20

How are specififc histone marks found in genome

Answer 20

ATAC-Seq: Tn5 binds transpossases on open chromatin, tagging and DNA Fragmentation PCR and next gen sequencing

Question 21

DNA Library Prep

Answer 21

Adaptor Ligation, Adaptor Blocking, Circulisation, Dimer Removal, reverse Transcription and PCR aplification and size selection

Question 22

Analytical Pipeline small RNA discovery

Answer 22

miRNA isolation, Sample Prep, Library Prep, Sequencing, Sequence filtering, QA, Sequence mapping, mapping sequancing, quantitative calculation, normalisation, Catalogue miRNA, miRNA dysfunctional ID, differential expression, correlation testing, Biomarker ID