Gene Expression Regulation

Question 0

Promoter

Answer 0

The CIS regulatory element that sits immediately 5’ proximal to the transcription starts site of RNA
Sequence bound by transcriptional apparatus

Directional

Question 1

Regulatory elements

Answer 1

Promoters
Enhancers
Repressors/silencers
Insulators

Question 2

Enhancer

Answer 2

Distal regulatory elements 
Distance independent 
Orientation independent 
Can be in introns 
Long range acting enhancer is within 1 mega base , vast majority are 100 kb

Question 3

Repressors/Silencers

Answer 3

The flimsier of enhancers

Functioning to repress rather than activate

Question 4

Insulator

Answer 4

Enhancer blocking

Question 5

Discovery of CIS regulatory elements

Answer 5

Sequence based methods
DNAse I hypersensitivity mapping
ChIP seq
Cap analysis of gene expression

Question 6

Predicting promoters

Answer 6

Readily identifiable 
In the presence if gene annotation 
(5' end of gene)
Some sequence elements like TATA box
More constrained

Question 7

Predicting enhancer

Answer 7

They can be anywhere so hard to find from sequence
More constrained
Motif based finding is uninformative in large genomes (short and degenerative)
Gotta use combination

Question 8

DNAse I hypersensitivity mapping

Answer 8

Regions of the genome that are bound by DNA binding proteins are relatively open and therefore accessible to nucleases
Identifies any region of open chromatin

Question 9

ATAC-seq

Answer 9

DHS newer method

Question 11

ChIP-Seq

Fix

Answer 11

Chromatin immunoprecipitation
Can be used to characterize the genomic distribution of DNA interacting protein
not restricted to sequence specific transcription factors

Question 12

HOT regions

Answer 12

highly open chromatin regions

Question 13

What fraction of ChIP binding sites are functional?

Answer 13

~89% OF ChIP TFBS at promoters were not functional

what does this mean?

Question 14

CAGE

Answer 14

Cap Analysis of Gene Expression
developed at RIKEN
technique for mapping transcription start sites(TSS)
Takes advantage of the fact that many RNAs have a 5’ cap(7-methylguanylate)

Question 15

RAMPAGE

Answer 15

1) Reverse transcription
2) Oxidation & Biotinylation
3) RNAse I digestion
4) Pulldown and cDNA release
5) PCR and size selection(SPRI beads)

Question 16

Application of CAGE

Answer 16

Annotation of TSSs by FANTOM5 consortium
CAGE in 975 human and 399 mouse samples
Identified thousands of new TSSs, characterized their cell type specificity
Identification of enhancers
–>often produce lowly expressed bidirectional transcription(eRNAs)

Question 17

Methods to quantify the function of CREs

Answer 17

reporter assays
enhancer traps
STARR-seq

Question 18

CRE reporter assays

Answer 18

luciferase assays
Principle: Clone sequence of interest into reporter vector that will drive expression of a quantifiable protein such luciferase or GFP lacZ , HRP
transfect cells of interest, measure protein expression

Question 19

Pros of reporter assays

Answer 19

demonstrate suffuciency
used for easy site directed mutagenesis
can be performed in cell culture, bacteria, plants, animals and yeast
typically lack chromatin context (get around this by using large clone reporters and stable transgenic reporters)

Question 20

Fine scale functional characterization via reporters

Answer 20

each coexpressed enhancer has a different arrangement of TFBS
this arrangement and activity is conserved over large evolutionary distance

Question 21

enhancer traps

Answer 21

principle: ‘randomly’ clone genomic fragments into individual reporter vectors, test for activity
often carried out in developing embryos, looking for spatially restricted expression patterns

Question 22

STARR-seq

Answer 22

principle: modify the traditional enhancer trap to leverage NGS as a quantitative read out
1) Fragment the genome
2) Clone in 3’ UTR of reporter
3) RT-PCR and Sequence enhancer

Seq counts are proportional to expression

Question 23

Chromosome Conformation Capture and its derivatives

Answer 23

[345]C
Hi-C
Chia-PET

Principle: Use proximity ligation to capture 3D DNA intereactions

1) Cross link DNA
2) Restriction digest
3) Intramolecular Ligation
4) Reverse Cross-link
5) Method Specific processing
6) Product quantification

Question 24

3C

Answer 24

uses PCR to amplify and quantify target pairs of interest. Assumes both targets are known.
1 vs 1

Question 25

4C

Answer 25

uses a 2nd restriction digest and inverse PCR to build an NGS library. Assumes 1 target is known.
1 vs all

Question 26

5C

Answer 26

Similar to 3C bult multiplexes target pairs, couples with NGS
many vs many

Question 27

Hi-C

Answer 27

All vs ALL

Question 28

Chia-PET

Answer 28

Principle: Identifies 3d dna interactions by proximity libration and immunoprecipitation of a protein of interest
Method: Crosslink, sonicate, IP, ligate, create NGS library, sequence paired tags

Question 29

Identify 3d interactions from correlation matrices

Answer 29

Principle: The activity of interacting enhancers and promoers is correlated across cell types or conditions

1) Start with map of DHS site across a large number of diverse cell types
2) Classify sites as proximal and distal
3) Quantify correlation between cites across cell types, identify significantly correlated site

Question 30

DHS correlations vs 3C

Answer 30

Interacting DHS IDENTIFied by correlation well replicated by targeted 3c approach
identifies several known long range interactions

Question 31

Future directions in gene regulation

Answer 31

How often do regulator elements “skip” genes?
Combinatorics of gene regulation: Do multiple promoters interact with the same promoter?
How do architectural proteins function to facilitate looping, chromatin domains etc?
How does seq variation within cis regulatory elements affect gene expression and complex traits?