Regulation of Expression 2

Question 1

Name 4 methods for genome-wide analysis of transcription

Answer 1

1) RNAseq
2) RNAPII ChIP-seq
3) GRO-seq
4) NET-seq

Question 2

Name steps of RNA-seq

Answer 2

1) Extract RNA (total cell or poly-A enriched)
2) Convert to short cDNA fragments by reverse transcription and sonication
3) Ligate adapters to either end
4) Amplify the library by PCR using adaptor sequences
5) Perform NGS
6) Map the reads against a reference genome

Question 3

Name two ways in which RNAseq can be made strand-specific

Answer 3

1) Ligate RNA adapters prior to conversion to cDNA

2) Include dUTP during synthesis of the second strand of cDNA, then degrade it

Question 4

Name two pros of RNA seq

Answer 4

1) Technically straightforward

2) Gives information on stable/mature RNA, provides information on upstream RNA maturation events e.g. splicing

Question 5

Name a con of RNA seq

Answer 5

Does not report on unstable RNAs

Question 6

Name two common PTMs of RNA pol II CTD and transcription stages they are associated with

Answer 6

Ser5 phosphorylation: initiation

Ser2 phosphorylation: elongation

Question 7

Name a pro of RNAPII ChIP seq

Answer 7

Can reveal transcription of unstable RNAs

Question 8

Name two cons of RNAPII ChIP seq

Answer 8

1) presence of RNAPII does not necessarily prove transcription is occurring
2) no strand information

Question 9

What is the main difference between nuclear run-on assay and GRO-seq?

Answer 9

Nuclear run-on assay relies on radioactive labelling and hybridization on gel to reveal nascent RNA; GRO-seq is more high-throughput as it uses a 5-bromouridine label that can be used to pull down only newly synthesised RNA

Question 10

Name steps of GRO-seq

Answer 10

1) Chill cells to arrest transcription
2) Add 5-bromouridine and sarkosyl to inhibit engagement of new RNA polymerases
3) Raise the temperature and allow transcription to run on
4) Isolate RNA and hydrolyse into small fragments
5) Extract only Br-UTP containing RNA by affinity purification using an anti-Br-UTP Ab
6) Prepare a sequencing library and perform NGS

Question 11

Name two pros of GRO-seq

Answer 11

1) Reports all nascent transcription - both stable and unstable
2) Has orientation information

Question 12

Name a con of GRO-seq

Answer 12

Technically complex

Question 13

What is the main difference between RNAPII ChIP-seq and NET-seq?

Answer 13

RNAPII ChIP-seq pulls down the DNA associated with RNApolII, while NET-seq pulls down the RNA associated with RNApolII

Question 14

Name steps of NET-seq

Answer 14

1) Flash freeze and lyse cells to arrest polymerase and release contents
2) Digest chromatin
3) Pull down RNApolII using an anti-RNApolII Ab
4) Isolate associated RNA
5) Generate a library for NGS

Question 15

Name pros of NET-seq

Answer 15

Same as GRO-seq (all nascent transcription with strand information), plus:

1) Fewer manipulations of material
2) Gives single nucleotide resolution
3) Relatively simple

Question 16

Which features need to be considered when choosing a transcriptome analysis method?

Answer 16

1) Do we want to analyse all RNA in cell or only newly produced RNA?
2) Do we need orientation information?
3) Are we analysing stable, mature mRNA or unstable RNA like eRNA?
4) Do we need single nucleotide resolution?

Question 17

Define eRNA

Answer 17

Enhancer RNA - short, unstable, non-coding RNA expressed at both directions from an enhancer

Question 18

Define CUT/PAR/PROMPT sequences

Answer 18

Antisense transcripts produced upstream from a gene promoter. Short, unstable and non-coding

Question 19

Name 4 similarities between eRNAs and PARs/PROMPTs

Answer 19

1) Very short - typically <1kb
2) Expressed at low levels
3) Highly unstable due to exosomal degradation
4) Postulated to contribute to transcriptional activation

Question 20

Name two types of methods in which PROMPTs and eRNAs can be studied

Answer 20

1) Methods that study nascent transcription: GRO-seq, NET-seq, RNAPII ChIP-seq
2) RNAseq when exosomes are suppressed (e.g. via KD or RNAi)

Question 21

Why is transcription at promoters bidirectional?

Answer 21

Nucleosome positioning and recruitment of TFs allows RNApolII to bind to either strand

Question 22

Name three mechanisms that enforce transcription directionality

Answer 22

1) Transcription machinery helps orient RNApolII to some extent
2) Upstream termination sites suppress non-coding transcription
3) Downstream factors: introns, gene body chromatin markers and gene looping

Question 23

How do introns promote directionality of transcription?

Answer 23

Formation of spliceosome complexes prevents nearby termination complexes from forming

Question 24

How do chromatin modifications promote transcription directionality?

Answer 24

Gene body markers such as H3K79me promote elongation

Question 25

How does gene looping promote transcription directionality?

Answer 25

Association between promoter and terminator promotes RNApolII progression in the direction of the loop

Question 26

Name two similarities between enhancers and promoters in the context of transcription initiation

Answer 26

Both contain NDRs

Both recruit TFs

Question 27

Name three proposed functionalities of transcription at enhancer sites

Answer 27

1) No function - eRNA is made due to chance RNApolII binding and is rapidly degraded
2) Binding of RNApolII causes changes to nucleosome patterning which allows additional TFs to bind: transcription, but not eRNA is functional
3) eRNA has its own function, e.g. as a cofactor

Question 28

Name three proposed functions of eRNA

Answer 28

1) Facilitates enhancer-promoter looping by bringing together enhancer and mediator
2) Aids recruitment of TFs through weak eRNA-TF interactions at the enhancer site
3) Regulates activity of other factors, e.g. by releasing autoinhibition of CBP histone acetyltransferase