Tonon: Lecture V

Question 1

What was the main focus of the ENCODE Project Consortium (ENCyclopedia Of DNA Elements)?

Answer 1

to develop and apply high-throughput approaches to detect all sequence elements that have a biological function

focused on 30 megabases (∽1%)

Question 2

What tools did ENCODE use to have a full perspective of the “junk” DNA?

Answer 2

DNAse digestion
microarray hybridization
DNA replication sites
Chip-chip
cis-regulatory elements (promoters, transcription factors binding sites)
long-range regulatory elements (enhancer, repressors/silencers, insulators)

Question 3

Name the steps scientists took to identify “junk” DNA and what they found:

Answer 3

1. chose 44 genomic regions
2. chose 30 megabases (15 known, 15 unknown)

they found “functional elements”, which is a genome segment that actually encoded for a defined product

DNA is no longer thought to be composed of mainly “junk”

Question 4

How are the important elements of a genome identified?

Answer 4

by comparing the homology of organisms

signatures are important, whether alone or in combination, in order to identify sequences with important functions

Question 5

Why did it take scientists so long to detect “junk” DNA? List the 4 reasons:

Answer 5

1. genes are not as conserved
2. functional elements were made of small or fragmented sequences that can be interrupted with other unspecific sequences
3. can lie in repetitive regions of the genome, making their recognition difficult
4. evolve very rapidly and can be nearly neutral to evolutionary processes

Question 6

What were the experimental targets of ENCODE in relation to the 2012 experiment?

Answer 6

assign functions to as many DNA regions as possible

Question 7

DNA methylation

Answer 7

regions layered with chemical methyl groups, which regulate gene expression

Question 8

open chromatin

Answer 8

areas in which the DNA and proteins that make up chromatin are accessible to regulatory proteins

Question 9

RNA binding

Answer 9

positions where regulatory proteins attach to RNA

Question 10

RNA sequences

Answer 10

regions that transcribed into RNA

Question 11

CHIP-SEQ experiment

Answer 11

technique that reveals where proteins bind to DNA

Question 12

Modified Histone

Answer 12

histone proteins, which package DNA into chromosomes, modified by chemical marks

Question 13

Transcription Factors

Answer 13

proteins that bind to DNA and regulate transcription

Question 14

What was a limitation to the ENCODE approach?

Answer 14

some cells could be open and others closed, so the cells would be replicating at different points resulting in the analyzation of the average

also the preparation across different countries was not uniform even if the protocol was in place due to the slight variations that could occur in materials

Question 15

What is an observation based on the image in regards to H3K27ac?

Answer 15

H3K27ac is a marker used for enhancer expression

what can be observed is that the enhancers undergo silencing, so they are not a stabilizing feature of the genome as they can undergo epigenetic changes

Question 16

What are 2 limitations to this study?

Answer 16

1. transcription factors were not taken into consideration
2. they overlooked the complexity of the organism and simply focused on definite cells and definite cell lines

Question 17

What did ENCODE find?

Answer 17

80.4% of the human genome participates in at least 1 biochemical RNA and/or chromatin-associated event in at least one cell type

Question 18

Explain the Manhattan plot and why it is used:

Answer 18

it represents the position of the gene in the genome in relation to its p-value

the red line is the threshold and everything above the red line is significant

used to see the association between a certiain region of the genome with a certain disease

Question 19

Explain the Manhattan plot and why it is used:

Answer 19

it represents the position of the gene in the genome in relation to its p-value

the red line is the threshold and everything above the red line is significant

used to see the association between a certain region of the genome with a certain disease

used to show Genome Wide Association Studies (GWAS)

Question 20

What is GWAS?

Answer 20

https://www.youtube.com/watch?v=b7VTJv77f24

Question 21

What should be used as an enhancer for a manhattan plot studying GWAS?

Answer 21

microarray to visualize intergenic regions (Regions outside of the genes)

Question 22

What does GWAS identify?

Answer 22

association between disease trait and surrogate marker (tag SNP)

Question 23

What is interesting about the findings related to GWAS?

Answer 23

only 3% of the genomic variants are located within genes

most are found in intronic or intergenic genes

Question 24

How do we analyze Crohn’s patients?

Answer 24

find associations with SNPs that are overlapping GATA2-binding site

Question 25

What was found out about alternative splicing with ENCODE?

Answer 25

most genes undergo alternative splicing, during transcription with a local effect when the RNA is still bound to DNA

Question 26

What is known about Poly(A)?

Answer 26

there are many poly(A) for certain genes

Question 27

How many human genes have a Poly(A)?

Answer 27

at least 50%

Question 28

In the example with CD47, what happens if the poly(A) tail is on a short 3'UTR?

Answer 28

CD47 protein doesn’t translocate to the membrane because the tail has nothing to bind to RNA is driven to the axon

Question 29

In the example with CD47, what happens if the poly(A) tail is on a long 3'UTR?

Answer 29

binds the RNA to a few components and brings the whole set to the surface RNA remains in the soma

Question 30

What do 3'UTRs act as?

Answer 30

a scaffold to regulate membrane protein localization

Question 31

Highlight 3'UTR to cancer:

Answer 31

short 3'UTR → increased proliferation → alternative cleavage and polyadenylation → oncogenes activated in cancer cells

Question 32

Why is the shorter 3'UTR associated with proliferation?

Answer 32

ncRNAs will have the ability to bind to it and prevent translation or induce elimination

Question 33

What are 2 important factors in the discovery of transcription factors with ENCODE?

Answer 33

transcription factors work together found in both promoter regions and in intergenic regions, where apparently there are no genes (probably bound to regulatory regions, exerting some activities)

Question 34

Describe the hierarchy of chromatin modifications in epigenetics:

Answer 34

DNA sequence & DNA methylation → nucleosomes → histone modification (compactin of chromatin) → nuclear organization and transcription factors

Question 35

What is the nomenclature of histones?

Answer 35

first letter: histone (with number) second letter: residue number: number of histone type of modification number of residues

Question 36

What are some exceptions for the nomenclature of histones?

Answer 36

acetylation does not have a number for the number of residues because there can only be 1

Question 37

What is H3K27ac a typical modification of?

Answer 37

enhancers

Question 38

What are the 3 types of chromatin modifiers and what do they do?

Answer 38

writers: put residue on lysine 4 erasers: readers

Question 39

What is the most important histone?

Answer 39

H3

Question 40

What are the most important residues?

Answer 40

K4, K9, K27, and K36

Question 41

Are writers and erasers specific in their activity?

Answer 41

no, they add many different residues to different amino acids or remove many different histone modifications

Question 42

What composes histone modifying genes?

Answer 42

writers erasers readers

Question 43

What is the most frequent histone modification of promoters?

Answer 43

it is the 3 methylation of lysine residue number 4 in histone 3 (H3K4me3)

Question 44

Which lysine residue number is almost always associated with active transcription of genes?

Answer 44

K4

Question 45

What is on the promoters of active genes?

Answer 45

H3K4me3 (trimethylation)

Question 46

What is typical of active enhancers?

Answer 46

H3K4me1 (monomethylation)

Question 47

What is a classical heterochromatin marker that represses chromatin?

Answer 47

H3K9me3

Question 48

What is associated with repressed genes?

Answer 48

H3K27me3

Question 49

Review the chart of active and repressed transcription in a normal cell:

Answer 49

Question 50

active gene promoter →

Answer 50

H3K4me3

Question 51

gene promoter not expressed →

Answer 51

H3K27me3

Question 52

What is the function of chromatin in regards to histones?

Answer 52

it is an accessibility barrier, so if there are no histones near the promoter, there is no H3K4me3, so it's marker for active promoters are "on their side"

Question 53

What is a technique that is used to find all chromatin that is opened?

Answer 53

ATAC-seq, which is a DNA-seq

Question 54

Why is ATAC-seq important?

Answer 54

if chromatin is open, it almost always implies there is active transcription in that region

Question 55

What happens when you look at CHIP-seq for H3K4me3?

Answer 55

since there are no histones, signals are not present

Question 56

What does it mean if important genes have bivalent promoters?

Answer 56

they have both H3K27me3 and H3K4me3 on the same region in order to go both directions (about 20% in ES cells)