Transcription analysis and extragenic transcription Flashcards
Genome-wide transcription analysis and ncRNA regulation (51 cards)
1
Q
What information might we want to find out about transcription?
A
- Which genes are transcribed in which cells/conditions?
- Where does transcription start and terminate for known genes?
- Are there additional transcription units not previously identified?
2
Q
What methods can be used for genome-wide transcription analysis?
A
- RNA-seq
- RNA polII ChIP-seq
- GRO-seq
- NET-seq
3
Q
RNA-seq method:
A
- Isolate RNA
- Convert it to a cDNA library
- Fragment cDNA
(or fragment RNA and convert to cDNA after) - Ligate adapter sequences onto cDNA fragments complementary to primers
- Amplify fragments using PCR
- Sequence fragments
- Map sequences onto the genome
4
Q
What would we first do to cellular RNA when studying gene expression using RNA-seq?
A
Do a polyA selection so just mRNA is enriched to be isolated.
5
Q
What results does RNA-seq give?
A
- Which genome sequences are transcribed (present in the RNA pool of a cell)
- How frequent this transcription is (abundance of RNA).
6
Q
What do gaps in RNA-seq data read represent?
A
Introns - not present in mRNA.
7
Q
RNA-seq advantages:
A
- Technically straightforward (can be done using a commerical kit or sequencing service)
- Gives information on stable / mature RNAs; allows us to see where splicing occurs and the splicing efficiency
8
Q
RNA-seq disadvantages:
A
- Doesn’t retain strand information (don’t know which strand of cDNA was original mRNA)
- Unstable RNAs are not reported
9
Q
What is stranded RNA-seq?
A
RNA-seq but retaining strand information (which strand of cDNA was mRNA).
10
Q
How can we do stranded RNA-seq?
A
- Ligate the RNA adaptors prior to conversion to cDNA (5’ and 3’ adaptor always in the same orientation)
- Include dUTP in the second strand cDNA synthesis; allows us to then degrade second strand or avoid amplifying it.
11
Q
What RNA-seq data implies a gene is transcribed?
A
Peaks on the genomic map. Level of transcription indicated by peak height.
12
Q
What does an RNA-seq peak not corresponding to a gene mean?
A
It is a stable uncharacterised transcript (SUT).
13
Q
RNA polII ChIP-seq method:
A
- Cross-link proteins to DNA
- Isolate chromatin
- Sonicate to fragment chromatin
- Incubate with RNA polII antibody
- Isolate the Ab-chromatin complexes
- Isolate the DNA from the complexes
- Ligate on adapters
- Amplify fragments using PCR
- Sequence fragments
- Map sequences onto the genome
14
Q
RNA polII ChIP-seq advantages:
A
- Gives information on unstable RNAs
15
Q
RNA polII ChIP-seq disadvantages:
A
- Prescence of of RNAPII does not necessarily prove functional transcription (polymerase may have stalled)
- No information on directionality (don’t know which way polymerase is transcribing)
16
Q
What results does RNA polII ChIP-seq give?
A
- Which genome sequences are transcribed (regions of the genome that RNA polII is associated with)
- How frequent this transcription is (how much polymerase is associated with that region)
17
Q
Why would we use different antibodies for RNA polII ChIP-seq?
A
The RNApolII tail can be dynamically modified.
18
Q
What modification does RNA polII have during transcription initiation (promoter)?
A
Serine 5 phosphorylation
19
Q
What modification does RNA polII have during transcription elongation (gene body)?
A
Serine 2 phosphorylation
20
Q
GRO-seq method:
A
- Chill cells to arrest polymerases
- Lyse cells and isolate nuclei
- Incubate with bromoUTP labelled nucleotide and sarkosyl drug
- Increase temperature, allowing transcription to ‘run on’ using bromoUTP
- Newly transcribed RNA contains bromoUTP analog
- Use antibody to bromoUTP to pull down newly synthesised RNA
- Hydrolyse into small fragments
- Ligate on adapters
- Convert to cDNA
- Amplify fragments using PCR
- Sequence fragments
- Map sequences onto the genome
21
Q
GRO-seq advantages:
A
- Reports active transcription with directionality (shows transcription levels in both DNA strands)
- Reveals unstable transcripts
22
Q
GRO-seq disadvantages:
A
- Complex method
23
Q
What does GRO-seq stand for?
A
Global run on sequencing.
24
Q
What results does GRO-seq give?
A
Rates of transcription - tells us about newly synthesised RNA.
25
What does Sarkosyl do?
Block new polymerases from binding to DNA so we only se transcription that is already happening.
26
What does NET-seq stand for?
Native elongating transcript sequencing.
27
NET-seq method:
- Chill cells to arrest polymerases
- Lyse cells and isolate nuclei
- Sonicate to fragment RNA
- Incubate with RNA polII antibody
- Isolate the Ab-RNA complexes
- Isolate the RNA from the complexes
- Ligate on adapters
- Convert to cDNA
- Amplify fragments using PCR
- Sequence fragments
- Map sequences onto the genome
28
NET-seq advantages:
- Reports active transcription with directionality (shows transcription levels in both DNA strands)
- Allows sequencing of introns (pulls down premRNA)
- Reveals unstable transcripts
- Fewer manipulations than GRO-seq; introduces fewer artifacts.
- Gives single nucleotide resolution as we know the nucleotides at the 3' end.
29
What are examples of unstable transcripts?
- Antisense RNA upstream of a promoter.
- Cryptic Unstable Transcripts (CUTs) in yeast
30
What is pervasive transcription?
The idea that much more of the eukaryotic genome is transcribed (75%) than expected when looking at how much of the genome contains genes (30%).
31
What does ENCODE stand for?
Encyclopedia of DNA elements.
32
What does the ENCODE project tell us?
How much of the genome actually contains genes.
33
What does the idea of pervasive transcription tell us?
There are lots of non-coding RNAs in the genome.
34
What are PARs and PROMPTs?
Promoter Associated RNAs and PROMoter uPstream Transcripts - ncRNAs with bidirectional transcription.
35
What are eRNAs?
Enhancer RNAs - ncRNAs with bidirectional transcription.
36
Which ncRNA makes up the majority of extragenic transcripts?
eRNAs.
37
How many genes are PROMPTs found at?
>50%
38
Properties of PARs / PROMPTs / eRNAs?
- <1kb
- Typically expressed at low levels
- Highly unstable (degraded by exosome)
- Thought to contribute to transcriptional activation
39
How do we know eRNAs and PROMPTs exist?
- Nascent transcription detection
- Suppression of degradation
40
Why do we need PROMPTs?
To enforce transcription in just 1 direction; promoters are inherently bidirectional; polymerase can bind either strand.
41
What suggests promoter directionality evolved over time?
Bidirectional transcription is most prominent at newly evolved promoters.
42
How is transcription directionality at promoters enforced?
- Assembly of core promoter elements help loading of polymerase in the right direction.
- Upstream termination sites mean wrong direction transcripts are stopped early and degraded.
- Binding of U1 SnRP to 5’ splice site can suppress nearby transcription termination sites (introns act as a go signal for transcription in that direction).
- H3K79 methylation is associated with elongation and enriched in the gene body. More methylations are added every round so transcription continues. Positive feedback.
- Gene looping brings together 5’ and 3’ ends of a gene to help with polymerase recycling and loading of it in the correct orientation.
43
Why might some bidirectionality be maintained?
- Difficult to avoid; takes lots of energy to completely shut down.
- Increases local pool of TFs
- Negative supercoiling behind transcribing RNAPII helps unwind promoter DNA to increase efficiency of initiation
- Facilitates evolution of functional transcripts
44
How are enhancers and promoters similar?
- Nucleosome depleted regions
- Have binding sites for transcriptional activators
- Transcription machinery binds and initiates transcription
45
Properties of eRNA transcription:
- Bidirectional
- Terminated quickly in both directions
46
What are proposed mechanisms of eRNA function?
- Noise
- Transcription-dependent effects
- RNA-dependent effects
47
Are eRNAs likely to just be noise?
No.
- Enhancer transcription frequently correlates with, and precedes, adjacent gene transcription, and then drops off when mRNA starts being produced.
- Enhancer transcription is also broadly correlated with H3K4 methylation.
- This appears to be more regulated than just noise
48
How might enhancer transcription increase their activity?
Chromatin remodelling for transcription improves availability of enhancer sequences. Transcribing polymerases recruit Set1 Compass proteins (H3K4 methyl transferases) so there is a positive feedback loop of enhancer transcription and activation.
49
What might be the function of eRNA products themselves?
- Activation of target genes; a study showed eRNA KD suppressed gene activation, and artifical eRNA recruitment without transcription (tethering experiment (Lecture 4, slide 25)) restored gene activation.
- Recruitment of other TFs (with (e)RNA binding domains) e.g. YY1. Again eRNA KD and artificial recruitment (Lecture 4, slide 26) effects YY1 activity.
50
How is eRNA thought to activate gene transcription?
- Plays a role in promoter-enhancer looping; brings them together.
- Binds TFs bringing them into close proximity with their DNA binding site therefore increasing binding.
51
How is CBP (a H3K27 histone acetyl transferase) regulated by eRNAs?
Usually when bound to enhancers the active site is blocked by an activation loop. eRNA binding to CBP displaces activation loop and allows H3K27 acetylation.
