HC 4.1 - Omics and Gene expression: Functional Genomics

Question 1

What are important factors for the binding of the Pol II preinitiation complex (PIC) to the promotor/DNA?

Answer 1

-DNA methylation status
-Chromatin remodelling before PIC assembly

Question 2

Gene activation can occur at a distance with the help of regulators. What do these do?

Answer 2

direct the assembly of the mediator, chromatin remodelling complexes, histone modifying enzymes, TFs and Pol II

Question 3

Describe the factors that influence the transcription process: pre-initiation

Answer 3

-Methylation status of DNA
-Promotor accessibility by chromatin remodeling
-Pol II preinitiation complex assembly

Question 4

Describe the factors that influence the transcription process: Initiation

Answer 4

-Transcription regulators and factors are assembled
-Looping of DNA enables contact between activator and transcription initiation complex bound to promotor

Question 5

Describe the factors that influence the transcription process: elongation/termination

Answer 5

RNA synthesis
Capping
Polyadenylation
Splicing

Question 6

Describe the factors that influence the transcription process: abundance

Answer 6

Balance between transcription rate and degradation rate

Question 7

Describe the factors that influence the transcription process: translation

Answer 7

the process of translating mRNA sequence to AA-sequence during protein synthesis

Question 8

Measuring transcription processes genome wide: pre-initiation

Answer 8

-DNA methylation
-Open chromatin
-histone modification

Question 9

Measuring initiation

Answer 9

transcription factors and enhancers

Question 10

Measuring elongation and termination

Answer 10

detect nascent transcription from Pol II (GRO-seq) or nascent long read RNA sequences (FLEP-seq_

Question 11

Measuring RNA abundance

Answer 11

RNA seq

Question 12

Measuring translation

Answer 12

Ribo-seq

Question 13

Measuring DNA methylation

Answer 13

Bisulfite seq

Question 14

Measuring open chromatin

Answer 14

ATAC seq

Question 15

Measuring histone modifications

Answer 15

Chip-seq

Question 16

Measuring transcription factors

Answer 16

ChIP-seq

Question 17

Measuring promotors-enhancers

Answer 17

Hi-C

Question 18

What kind of chromatin is measured with ATAC seq?

Answer 18

Euchromatin

Question 19

The chromatin remodelling is a … process

Answer 19

dynamic process

Question 20

How is ATAC seq performed?

Answer 20

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

Question 21

Interpreatation of sequencing peaks with ATAC seq

Answer 21

Peaks correspond to open chromatin after alignment
> info about open chromatin DNA
> shows alterations in open chromatin between 2 groups

Question 22

For measuring which factors is ChIP-seq used?

Answer 22

Histone modifications and transcription factors

Question 23

What does ChIP seq isolate?

Answer 23

DNA fragments bound to proteins

Question 24

Workflow ChIP seq

Answer 24

-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

Question 25

Control sample for ChIP seq (for the procedure)

Answer 25

Same procedure without enrichment with antibody > isolate all the DNA with all the proteins

Question 26

Alignment ChIP seq for TFs

Answer 26

Both the TF sample and control sample are mapped against reference genome

Question 27

ChIP seq for histones workflow differences with for TFs

Answer 27

-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

Question 28

Purpose Hi-C

Answer 28

Identify long range chromatin interactions (enhancer-promotor identification)

Question 29

Workflow Hi-C

Answer 29

-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

Question 30

What does one Hi-C read contain?

Answer 30

Sequence of associated loci A and B with adapters at both ends which were added during library prep

Question 31

Interpretation Hi-C heatmap

Answer 31

A dark box means a lot of mapping at this site > follow diagonal lines to x-axis for locations of associated fragments

Question 32

The results of the Hi-C are …

Answer 32

chromosome wide
> chromosome confirmation capture

Question 33

Hi-C contact map

Answer 33

Diagram with association and the similar chromosomes at each axis
> total contact map combines maps per chromosome

Question 34

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)

Answer 34

mRNA abundance is a step towards gene expression