Gene Expression Regulation Flashcards
Promoter
The CIS regulatory element that sits immediately 5’ proximal to the transcription starts site of RNA
Sequence bound by transcriptional apparatus
Directional
Regulatory elements
Promoters
Enhancers
Repressors/silencers
Insulators
Enhancer
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
Repressors/Silencers
The flimsier of enhancers
Functioning to repress rather than activate
Insulator
Enhancer blocking
Discovery of CIS regulatory elements
Sequence based methods
DNAse I hypersensitivity mapping
ChIP seq
Cap analysis of gene expression
Predicting promoters
Readily identifiable In the presence if gene annotation (5' end of gene) Some sequence elements like TATA box More constrained
Predicting enhancer
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
DNAse I hypersensitivity mapping
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
ATAC-seq
DHS newer method
ChIP-Seq
Fix
Chromatin immunoprecipitation
Can be used to characterize the genomic distribution of DNA interacting protein
not restricted to sequence specific transcription factors
HOT regions
highly open chromatin regions
What fraction of ChIP binding sites are functional?
~89% OF ChIP TFBS at promoters were not functional
what does this mean?
CAGE
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)
RAMPAGE
1) Reverse transcription
2) Oxidation & Biotinylation
3) RNAse I digestion
4) Pulldown and cDNA release
5) PCR and size selection(SPRI beads)
Application of CAGE
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)
Methods to quantify the function of CREs
reporter assays
enhancer traps
STARR-seq
CRE reporter assays
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
Pros of reporter assays
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)
Fine scale functional characterization via reporters
each coexpressed enhancer has a different arrangement of TFBS
this arrangement and activity is conserved over large evolutionary distance
enhancer traps
principle: ‘randomly’ clone genomic fragments into individual reporter vectors, test for activity
often carried out in developing embryos, looking for spatially restricted expression patterns
STARR-seq
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
Chromosome Conformation Capture and its derivatives
[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
3C
uses PCR to amplify and quantify target pairs of interest. Assumes both targets are known.
1 vs 1
4C
uses a 2nd restriction digest and inverse PCR to build an NGS library. Assumes 1 target is known.
1 vs all
5C
Similar to 3C bult multiplexes target pairs, couples with NGS
many vs many
Hi-C
All vs ALL
Chia-PET
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
Identify 3d interactions from correlation matrices
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
DHS correlations vs 3C
Interacting DHS IDENTIFied by correlation well replicated by targeted 3c approach
identifies several known long range interactions
Future directions in gene regulation
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?