L4 ENCODE and Negative Selection

Question 1

ENCODE Project

Answer 1

• Encyclopedia of DNA elements

- aims to identify all the functional elements of the genome

Question 2

Encode over-interpretation

Answer 2

• Dan Graur said that ENCODE did not consider the evolutionary aspect of genetics
• over-interpret “biochemical function”
  car hood analogy

Question 3

What is a function?

Answer 3

• if you were to lost it, there would be a fitness cost
• does the sequence conservation tell us anything about function; if it is conserved, it is likely to have a function
• sequence conservation reflects function

Question 4

Selective constraint

Answer 4

purifying selection/negative selection

Constraint is due to purifying selection - there is a function associated to it

Question 5

How can we tell which proteins have a function related to it?

Answer 5

See OneNote

Compare species that are closely related

Question 6

Phylogenetic footprinting

Answer 6

See OneNote

Phylogenetic footprinting is a technique used to identify transcription factor binding sites (TFBS) within a non-coding region of DNA of interest by comparing it to the orthologous sequence in different species.

Question 7

DNase1 Footprinting

Answer 7

protein modification protection assay

Question 8

Transgenic experiment

Answer 8

See OneNote

• use reporter gene
• Does our reporter gene show an expression pattern that is consistent with the gene that we are interested in?

Question 9

ApoA

Answer 9

See OneNote

Question 10

Extreme levels of conservation

Answer 10

• conserved non-genic sequences (CNG)
• conserved but not transcribed
• doesn’t show substitution patterns of coding or ncRNA sequences

Question 11

CNG characteristics

Answer 11

• Has to be at least in 10 different genomes
• CNG are mutating slowly than protein coding sites
• many of them but each is unique
• often clustered
• often in gene deserts
• many are near genes that are involved in regulation of transcription or development
• some overlap with exonic and these are highly enriched for genes involved in RNA binding and splicing regulation

Question 12

Function of non-coding conserved sequences?

Answer 12

See OneNote

Regulator modules?
- reporter constructs; Pax6 - eye development

CNG associated with complex genes e.g. genes with alternate splicing

Question 13

Functional conservation without sequence conservation

Answer 13

See OneNote

• even-skipped gene stripe 2 enhancer; regulatory module composed of multiple TF binding sites
• Limited conservation of stripe 2 enhancer sequence BUT if you place the different stripe 2 enhancer sequence in front of a stripe 2 reporter gene, it still drives the expression of stripe 2
• expresses at the correct place and rescues to the same extent
  BUT
• chimeric enhancers don’t recapitulate pattern

The sequences can change over time as long as you keep the same number of bicoid and hunchback sites

Question 14

Some lessons from eve

Answer 14

• enhancer function can be conserved without sequence conservation
• TF binding sites change relative position over evolutionary time

Question 15

ENCODE debate

Answer 15

See OneNote