Sudbery

Question 1

Why do we care about transcriptomics?

Answer 1

• 98.5% of protein coding seq is the same human to mouse
• 1-2% of the genome is coding (hence why we are so diff from mice)
• metazoan genomes are not selected for size → much is repetitive seq for decaying pseudogenes
• every cell has the same DNA, but cells are diff –> dep on what genes are active/exp levels

Question 2

What did ENCODE find about ‘junk’ DNA?

Answer 2

• most of what was thought to be junk has a function in controlling something

Question 3

How much of the genome did ENCODE claim was functional?

Answer 3

• 80% (CRMs)

Question 4

What are cis-regulatory modules?

Answer 4

• inc promoters, enhancers, silencers and insulators

- regions of DNA that bind DNA BPs (eg. TFs) and reg gene exp

Question 5

What seq motifs do DNA BPs bind, and what is the result of this?

Answer 5

• bind degenerate sequence motifs
• binding sites vary, but certain seqs are more likely
• but just because seq is present doesn’t mean it will bind
• eg. 8 mil GATA1 binding sites in the genome, only 0.2% bound by GATA1 (ChIP-seq)

Question 6

Does all DNA exist as heterochromatin or euchromatin?

Answer 6

• no, sliding scale

Question 7

What is hetero and euchromatin?

Answer 7

• heterochromatin = tightly packed

- euchromatin = loosely packed

Question 8

What regions tend to be nucleosome free, or have v few nucleosomes?

Answer 8

• CRMs

Question 9

How tightly packed is chromatin in transcribing genes?

Answer 9

• intermediate

Question 10

How can nucleosome free regions of genome be mapped?

Answer 10

• map w/ DNase-seq
• DNase only cuts where there are no nucleosomes, can use to build up genome wide pic of where nucleosome free regions are

Question 11

What did ENCODE measure and how?

Answer 11

• RNA expression –> RNA-seq, CAGE-seq and RNA-PET
• DNA/protein interactions –> ChIP-seq
• chromatin accessibility –> DNase-seq and FAIRE-seq
• 3D structure –> ChIA-PET and 5C
• methylation –> RRBS

Question 12

How does ChIP-seq work?

Answer 12

• prots bind to DNA, and use crosslinking to see where binds to DNA, chop up DNA and use Ab to select for DNA which is crosslinked to a prot, so can separate this DNA, seq it and work out where in genome prot binds

Question 13

What assays were carried out on what cells in ENCODE?

Answer 13

• tier 1 = all assays
• tier 2 = a selected subset of assays
• tier 3 = everything else, eg. a specific assay or combination

Question 14

What did ENCODE prod?

Answer 14

• lots of data sets and continues to gen new data

Question 15

What did ENCODE claim?

Answer 15

• vast majority (80.4%) of human genome participates in at least 1 biochemical RNA and/or chromatin-assoc event in at least 1 cell type
• 19.4% covered by at least 1 DHS or TF ChIP-seq peak across all cell lines

Question 16

What was wrong w/ ENCODEs claim that 80% of the genome is functional?

Answer 16

• 100% of genome participates in replication
• about 60% of this 80% is transcription and about half of this is introns (which are NOT coding, and these are much bigger than exons so account for a signif proportion of the genome), and we would not necessarily say introns have a function

Question 17

Was ENCODEs claim that 19.4% of the genome covered by at least 1 DHS or TF ChIP-seq peak more sensible, why?

Answer 17

• yes
• assuming half the elements from TF and cell-type diversity sampled, could estimate a min of 20% of genome participates in these specific functions, w/ the likely figure signif higher

Question 18

What is the question at the debate over ENCODEs finding?

Answer 18

• what do we mean by functional?

Question 19

What are the definitions of function?

Answer 19

• causal role = a seq has a function if seq causes the function
• selected effect = a seq has a function is that seq exists because of this function
• also the genetic role = a seq has a function if it is req for that function (doesn’t have to be visible to natural selection)

Question 20

How is biological function created?

Answer 20

• evolution and selection for that function

Question 21

What definition of function did ENCODE use, and what is the problem w/ this?

Answer 21

• causal definition
• but surely if a seq is important it will be selected for and if it has no effect on function then it is not important as far as evolution is concerned
• but ENCODE estimates of functionality were way above the amount of DNA known to be selected (at time best estimate was around 5% under -ve selection)

Question 22

How can the amount of the genome under selection be investigated?

Answer 22

• compare seqs from 2 species and find regions w/ fewer diffs than expected

Question 23

What is the problem w/ comparing seqs of 2 species to see how much of the genome is under selection?

Answer 23

• what is expected?
• if distant species used, only find function conserved across long time
• if close species used, not enough mutations to find conserved regions

Question 24

How can problems in investigating how much of the genome is under selection by comparing seqs be overcome?

Answer 24

• use indels –> compare 2 species, look at the gaps in alignment, are there regions w/ fewer gaps than expected?
• had to work out how much seq conserved now in humans, can extrapolate this to plot human mouse, human horse, human chimp etc., then find fit line and extrapolate to 0

Question 25

If just look at -ve selection to see how much of the genome is under selection, then what’s missing?

Answer 25

• +ve selection
• non coding seqs
• compensatory evolution (applies to non-coding seq)

Question 26

How does +ve selection affect genome selection?

Answer 26

• seq changes because of new function
• coding seq: dN/dS (comp synonymous to nonsynonymous changes and if lots of nonsynonymous then prob selecting for new function)

Question 27

How can selection of noncoding seqs in genome be investigated?

Answer 27

• intra-species diversity comp to inter-species diversity

- using 1000 genomes, approx 4%

Question 28

What is the effect of compensatory evolution on genome selection?

Answer 28

• TF sites control exp of gene, if lose a binding site then fitness reduced from 1 to eg. 0.8 (80% exp)
• isn’t fatal so allows time to get another mutation to gain binding site and fitness again increased to 1
• over years of evolution get many diff seqs that perform same function and give same fitness, but look quite diff

Question 29

What evidence if there for compensatory evolution?

Answer 29

• mainly anecdotal, eg. from fly embryos
• systematic evidence –> took 4 species of yeast, counted amount of TF binding in regulatory regions of genes, and looked at how much seq changed between 2 species, T is like evolutionary time, get much more changes in seq than binding energy, so conservation of function w/o conservation of sequence

Question 30

Are ENCODE elements conserved?

Answer 30

• some signal of selection, but quite weak
• melanocyte DHSs are depleted in somatic mutations in whole cancer genomes –> didn’t find somatic mutations in ENCODE elements
• cancer function = unselected function?
• so cancer needs seq but body doesn’t, does this make it functional?

Question 31

What are some eg.s of function w/o conservation?

Answer 31

• eye colour genes –> not under selection, but is genetic
• disease causing mutations such as AD –> no evolutionary advantage to stopping these mutations, as affects after reproductive age

Question 32

What did an experiment looking at enhancer and promoter evolution do and find?

Answer 32

• experiment took livers from 20 mammals and mapped where enhancers and promoters are
• promoters generally in conserved location
• enhancers move between species, not v conserved between species
• are they under seq constraint?
  promoters and enhancers have some conservation but much less than exons

Question 33

What evidence is there for function of promoters and enhancers?

Answer 33

• 98% of DHSs are linked to a promoter in ChIA-pet experiments
• genes closer to predicted enhancers tend to have higher expression levels in correct cell type
• ENCODE tested a no. of elements in enhancer reporter assays “over half of the elements showing activity, often in the corresponding tissue type”
• 65% of predicted human heart enhancers drove heart expression in mice

Question 34

What is the significance of evidence for function os enhancers and promoters being TF binding sites?

Answer 34

• does not imply TF binding
• does not imply enhancer state
• does not imply contact w/ promoter
• does not imply regulation of a promoter
• does not imply phenotypic consequence for the cell
• does not imply phenotypic consequence for the organism