HC 4.1 - Omics and Gene expression: Functional Genomics Flashcards
hoorcollege 4
What are important factors for the binding of the Pol II preinitiation complex (PIC) to the promotor/DNA?
-DNA methylation status
-Chromatin remodelling before PIC assembly
Gene activation can occur at a distance with the help of regulators. What do these do?
direct the assembly of the mediator, chromatin remodelling complexes, histone modifying enzymes, TFs and Pol II
Describe the factors that influence the transcription process: pre-initiation
-Methylation status of DNA
-Promotor accessibility by chromatin remodeling
-Pol II preinitiation complex assembly
Describe the factors that influence the transcription process: Initiation
-Transcription regulators and factors are assembled
-Looping of DNA enables contact between activator and transcription initiation complex bound to promotor
Describe the factors that influence the transcription process: elongation/termination
RNA synthesis
Capping
Polyadenylation
Splicing
Describe the factors that influence the transcription process: abundance
Balance between transcription rate and degradation rate
Describe the factors that influence the transcription process: translation
the process of translating mRNA sequence to AA-sequence during protein synthesis
Measuring transcription processes genome wide: pre-initiation
-DNA methylation
-Open chromatin
-histone modification
Measuring initiation
transcription factors and enhancers
Measuring elongation and termination
detect nascent transcription from Pol II (GRO-seq) or nascent long read RNA sequences (FLEP-seq_
Measuring RNA abundance
RNA seq
Measuring translation
Ribo-seq
Measuring DNA methylation
Bisulfite seq
Measuring open chromatin
ATAC seq
Measuring histone modifications
Chip-seq
Measuring transcription factors
ChIP-seq
Measuring promotors-enhancers
Hi-C
What kind of chromatin is measured with ATAC seq?
Euchromatin
The chromatin remodelling is a … process
dynamic process
How is ATAC seq performed?
With the use of Tn5 transposase which is connected to transposons in the cell.
-Tagmentation method: integration in genome and coupling of adapters and cutting in a single reactions by Tn5 transposases
-Transposases cut in open chromatin and adds adapers for limited PCR in open genomic area.
-Fragmentation and tagging by Tn5 transposase in one step
-Results in tagmented DNA fragments > DNA fragment purification and PCR amplification, library prep and seq
Interpreatation of sequencing peaks with ATAC seq
Peaks correspond to open chromatin after alignment
> info about open chromatin DNA
> shows alterations in open chromatin between 2 groups
For measuring which factors is ChIP-seq used?
Histone modifications and transcription factors
What does ChIP seq isolate?
DNA fragments bound to proteins
Workflow ChIP seq
-All proteins bound to DNA are crosslinked to DNA with formaldehyde
-Cut the DNA into small fragments with restriction enzymes
-Capture protein of interest using an antibody (attached to magnetic bead)
-Isolate proteins bound by antibody
-Separate DNA from proteins by reverse crosslinking (heating)
-Isolate DNA by washing away proteins and histones
-Prepare library and PCR and seq
-Alignment and location indentification
Control sample for ChIP seq (for the procedure)
Same procedure without enrichment with antibody > isolate all the DNA with all the proteins
Alignment ChIP seq for TFs
Both the TF sample and control sample are mapped against reference genome
ChIP seq for histones workflow differences with for TFs
-Histones are also crosslinked during the crosslinking step (also with other protocol)
-use antibodies against (not) modified histones
-library prep, sequencing, mapping
-readouts for histone modifications at the peaks of alignment > for certain modification if isolation for it > location shown
Purpose Hi-C
Identify long range chromatin interactions (enhancer-promotor identification)
Workflow Hi-C
-Fixing cells with formaldehyde
-Crosslinking DNA when loci are close to each other
-Cells are lysed and fragmentation DNA with restriction enzyme
-Biotinylated residue is incorporated, as the 5’-overhangs are filled in (biotin at 5’ overhangs)
-Blunt-end ligation is performed, that favors ligation event between crosslinked DNA fragment > connects DNA that was earlier distanced DNA
-library is sheared, junctions are pulled down with streptavidin magnetic beads
-purified junctions are analysed by sequencer after PCR
-Alignment of reads
What does one Hi-C read contain?
Sequence of associated loci A and B with adapters at both ends which were added during library prep
Interpretation Hi-C heatmap
A dark box means a lot of mapping at this site > follow diagonal lines to x-axis for locations of associated fragments
The results of the Hi-C are …
chromosome wide
> chromosome confirmation capture
Hi-C contact map
Diagram with association and the similar chromosomes at each axis
> total contact map combines maps per chromosome
Gene expression is the process by which the information encoded by a gene is turned into a function. Gene expression is not mRNA abundance but mRNA abundance is … (regarding gene expression)
mRNA abundance is a step towards gene expression
