Regulation of Expression 2 Flashcards
Name 4 methods for genome-wide analysis of transcription
1) RNAseq
2) RNAPII ChIP-seq
3) GRO-seq
4) NET-seq
Name steps of RNA-seq
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
Name two ways in which RNAseq can be made strand-specific
1) Ligate RNA adapters prior to conversion to cDNA
2) Include dUTP during synthesis of the second strand of cDNA, then degrade it
Name two pros of RNA seq
1) Technically straightforward
2) Gives information on stable/mature RNA, provides information on upstream RNA maturation events e.g. splicing
Name a con of RNA seq
Does not report on unstable RNAs
Name two common PTMs of RNA pol II CTD and transcription stages they are associated with
Ser5 phosphorylation: initiation
Ser2 phosphorylation: elongation
Name a pro of RNAPII ChIP seq
Can reveal transcription of unstable RNAs
Name two cons of RNAPII ChIP seq
1) presence of RNAPII does not necessarily prove transcription is occurring
2) no strand information
What is the main difference between nuclear run-on assay and GRO-seq?
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
Name steps of GRO-seq
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
Name two pros of GRO-seq
1) Reports all nascent transcription - both stable and unstable
2) Has orientation information
Name a con of GRO-seq
Technically complex
What is the main difference between RNAPII ChIP-seq and NET-seq?
RNAPII ChIP-seq pulls down the DNA associated with RNApolII, while NET-seq pulls down the RNA associated with RNApolII
Name steps of NET-seq
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
Name pros of NET-seq
Same as GRO-seq (all nascent transcription with strand information), plus:
1) Fewer manipulations of material
2) Gives single nucleotide resolution
3) Relatively simple
Which features need to be considered when choosing a transcriptome analysis method?
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?
Define eRNA
Enhancer RNA - short, unstable, non-coding RNA expressed at both directions from an enhancer
Define CUT/PAR/PROMPT sequences
Antisense transcripts produced upstream from a gene promoter. Short, unstable and non-coding
Name 4 similarities between eRNAs and PARs/PROMPTs
1) Very short - typically <1kb
2) Expressed at low levels
3) Highly unstable due to exosomal degradation
4) Postulated to contribute to transcriptional activation
Name two types of methods in which PROMPTs and eRNAs can be studied
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)
Why is transcription at promoters bidirectional?
Nucleosome positioning and recruitment of TFs allows RNApolII to bind to either strand
Name three mechanisms that enforce transcription directionality
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
How do introns promote directionality of transcription?
Formation of spliceosome complexes prevents nearby termination complexes from forming
How do chromatin modifications promote transcription directionality?
Gene body markers such as H3K79me promote elongation
How does gene looping promote transcription directionality?
Association between promoter and terminator promotes RNApolII progression in the direction of the loop
Name two similarities between enhancers and promoters in the context of transcription initiation
Both contain NDRs
Both recruit TFs
Name three proposed functionalities of transcription at enhancer sites
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
Name three proposed functions of eRNA
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
