gene expression r-loops chromatin structure · margot ladislas isabella concina. rna polymerase...
TRANSCRIPT
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R-loops GENE EXPRESSION
CHROMATIN STRUCTUREPRESENTED BY:Violina POTLOG
Margot LADISLASIsabella CONCINA
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RNA Polymerase
single-stranded DNA
R-loop structure
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Rare and accidental byproducts of transcription ?
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Rare and accidental byproducts of transcription ?NO
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WITHOUT / WITH R-loops ?
Nascent Connections: R-Loops and Chromatin Patterning Frédéric Chédi
Basic Determinants of Co-Transcriptional R-Loop Formation
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Methods for R-loops Mapping2● By using Bisulfite treatment
● By using immunoprecipitation
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Methods for R-loops Mapping2● By using Bisulfite treatment
C Bisulfite U= deamination
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Methods for R-loops Mapping2● By using Bisulfite treatment
C TBisulfite
(+ sequencing)
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Methods for R-loops Mapping2● By using Bisulfite treatment
C Bisulfite T= True only with
single strand DNA
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Methods for R-loops Mapping2● By using Bisulfite treatment
ATACGACCGTATGCTGGC
ATACGACCGTATGCTGGC
Bisulfite
No Denaturation
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Methods for R-loops Mapping2● By using Bisulfite treatment
NO R-loops SAME sequence
Bisulfite
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Methods for R-loops Mapping2● By using Bisulfite treatment
ATACGACCGTATG CTGGC G A C C G
ATACGATTGTATGCTGGC
Bisulfite
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Methods for R-loops Mapping2● By using Bisulfite treatment
Presence of R-loops
Long strand-specific
C to T conversion
Bisulfite
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Methods for R-loops Mapping2● By using Bisulfite treatment
= low-throughput single-molecule approach
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Methods for R-loops Mapping2- By using immunoprecipitation
CHIP-seq DRIP-seq= DNA-RNA immunoprecipitation
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Methods for R-loops Mapping2- By using immunoprecipitation
RNA Polymerase
- precipitation- sequencingS9.6 Antibody
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Methods for R-loops Mapping2- By using immunoprecipitation
Precipitation + DNAse
= DRIP-RNA-seq higher resolution + strand specific
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Methods for R-loops Mapping2● By using immunoprecipitation
= high-throughput population average
technique
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Function ?
R-loops are more prevalent than we thought:
10.000 loci = 150 Mb= 5% of the genome
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Where’s R-l ps?
- in promoter regions- in terminator regions- in gene bodies
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Hp: R-loops interfere with nucleosome redesposition behind the advancing RNAP
H3.3 = histone variant that dynamically replaces H3 lost owing to nucleosome disruption
R-loops locally open the chromatin structure by regulating nucleosome:
● occupancy,● positioning ● turn-over
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Identification of a set of histone modifications associated with R-loop chromatin under normal conditions
R-loops = additional layer of epigenetic information!
Histone modification signatures of R-loop chromatin
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Promoter regions markers
Active transcription:
● H3K4me1● H3K4me3● H3 acetylation
Transcription elongation:
● H3K36me3
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● H3K4me1 common mark of R-loop regions
● p300 acetyltransferase● CTCF-cohesin complex
R-loop regions = enhancer-like chromatin state
Terminal R-loops markers
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R- loops favor the recruitment of chromatin - modifying complexes
What is the mechanism of R-loop mediated patterning ?
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H3K36me3Observations:
● SETD2 histone methyltransferase interact with CTD of RNAP
● levels of H3K36me3 + RNAP density at R-loop(+) promoters
R-loops facilitate recruitment of SETD2 to RNAP complexes by transiently stalling the transcription machinery
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H3K4me1/3
CFP1 recruitment
SET1/COMPASS
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Histone acetylation
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Observation in mESCs:
Tip60-p400 chromatin remodelling and acetylase complex in mESCs expressing RNaseH1
Tip60-p400 targets form R-loops
R-loops contribute to recruiting the Tip60-p400 complex to chromatin.
R-loops recruit Tip60-p400
Histone acetylation
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R-loops influence chromatin modification states by:
Summarising...
chromatin regulators
affecting nucleosome
density
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CIS or TRANS R-loops
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CIS or TRANS R-loops
1) In 90% of cases R-loops are formed co-directionally with transcription;2) R-loops levels correlate with expression levels and respond to transcriptional
changes;3) DNA based and RNA based R-loops maps agree well with each other;4) Genes showing allele-specific expression show allele specific R-loops.
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CIS or TRANS R-loops
1) RNA strand transcribed from a locus can hybridize onto a distant locus through regions of RNA-DNA complementary;
2) This mechanism likely requires proteins to catalyse strand invasion and assist in homology searching;
3) Evidence obtained in different models and provides an attractive mechanism for directing lncRNA to potential targets.
CIS or TRANS R-loops
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CIS or TRANS R-loops
1) R-loops might provide a ready-made ssDNA landing pad on the non-template strand for lncRNA to interact with in trans;
2) RNA-DNA hybrid formation might be easier to achieve mechanistically and only requires the activity of a protein with strand annealing capacity (Rad52).
CIS or TRANS R-loops
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Aberrant R-loops and chromatin condensation
I.Defects in the THO mRNA export complex in yeast trigger marked genomic instability and increased R-loops formation.
II.Increase of R-loops is accompanied by higher levels of H3S10P which is a marker of condensed chromosomes during mitosis.
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Aberrant R-loops and chromatin condensation
I. Defects in the THO mRNA export complex in yeast triggers marked genomic instability and increased R-loops formation.
https://jbiol.biomedcentral.com/track/pdf/10.1186/jbiol217
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Aberrant R-loops and chromatin condensation
II. Increase of R-loops is accompanied by higher levels of H3S10P which is a marker of condensed
chromosomes during mitosis.
Castellano-Pozo, M. et al. (2013) R loops are linked to histone H3S10 phosphorylation and chromatin
condensation. Mol. Cell 52, 583–590
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Defining aberrant R-loops formation under pathological conditions
R-loops association with genomic instability is clear in the context of defects in a variety of factors:
1) involved in co-transcriptional processes (splicing mRNA, export, 3’ RNA processing, transcription elongation);
2) which are thought to prevent R-loops formation ( topoisomerase);
3) which are thought to mediate R-loops resolution ( ribonucleaseH, RNA/DNA elicase).
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Defining aberrant R-loops formation under pathological conditions
Assumptions about increased genic R-loops LEVELS in pathological situation may NOT be correct
Alterations of R-loops TURNOVER RATES may lead to persistent R-loops and threaten genome stability.
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R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator GenesKonstantina Skourti-Stathaki, Elena Torlai Triglia, Marie Warburton, Philipp Voigt, Adrian Bird, and Ana Pombo
Polycomb group proteins (PcG)= Epigenetic regulators= Transcription repressor
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PRC1RING1B
PRC2EZH2
H3H2A
K119ub1 K27me2/3
Polycomb repressive complex:
monoubiquitinylation di-trimethylation
IntroductionIntroduction
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H3H2AH3H2AH3H2A
= Repressed chromatin
silence CpG-rich developmental regulator genes in mESCs
maintain patterns of gene expression established during cell commitment
IntroductionIntroduction
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IntroductionIn mESC cells :PcG prevent the inappropriate expression of specific differentiated cell genes
H3K27me2/3H3K9ac/H3K4me2/3
Introduction
BUT Those genes also have active marks on the chromatin
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IntroductionIn mESC cells :PcG prevent the inappropriate expression of specific differentiated cell genes
BUT Those genes also have active marks on the chromatin
Though Poised polymerase = NO mRNA
RNA Polymerase
Ser5PNO Ser2P and Ser7P = NO productive elongation
Introduction
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PcG recruitment:Gene silencing = recruitment of PCR2 to CGI promoters
PCR1 occur after trimethylation of H3K27 by PCR2
PRC2EZH2
PRC1RING1B
monoubiquitinylationTrimethylation
Introduction
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R-loops x PcG ?→ Correlations
Introduction
human ESCs
mouse fibroblast
How does R-loops regulate PcG repression?
I) Impact of R-loops on PcG recruitment
II) Impact of R-loops on PcG-repressed genes
III) Impact of PcG on R-loops and PcG-target genes
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Introduction
PcG target = repressed
active, with R-loops
active, no R-Loops
● Msx1, Math1, Nkx2.2, Nkx2.9, Gata4
● B-actin
● CyclinB1
● Myf5 inactive, no PcG
Sidenote : The genes mentioned in the article
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ResultsResultsR-loops DRIP : R-loops form over PcG-repressed genes
With AntibodyWithout Antibody
PcG target = repressed active, with R-loops no R-loops, no PcG
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ResultsResults
There are R-loops at PcG repressed genes
But do they have a role in the repression?
Do we have derepression when we remove R-loops?→ quantify RNA after RNAse H1 treatment
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ResultsResultsRT-PCR of mature RNA :
PcG target = repressed active, with R-loops no R-loops, no PcG
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ResultsResults
There are R-loops at PcG repressed genes
They have a role in the repression
This role is linked to PcG?
Does R-Loops Co-occupy Chromatin with PcG Enzymes ?
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ResultsResults
PRC2EZH
2
Sequential Native Chip : R-loops → EZH2
First hypothesis : R-loops and PcG co-occupy the chromatin
Second hypothesis : R-loops and PcG don’t co-occupy the chromatin
PRC2EZH
2
PRC2EZH
2
PRC2EZH
2
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ResultsResults
First hypothesis : R-loops and PcG co-occupy the chromatin
![Page 54: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/54.jpg)
ResultsResults
First hypothesis : R-loops and PcG co-occupy the chromatin
![Page 55: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/55.jpg)
ResultsResults
There are R-loops at PcG repressed genes
They have a role in the repression
This role is linked to PcG?
R-Loops Co-occupy Chromatin with PcG Enzymes
![Page 56: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/56.jpg)
ResultsResults
There are R-loops at PcG repressed genes
They have a role in the repression
This role is linked to PcG?
Does R-loops help recruitment of PcG?
![Page 57: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/57.jpg)
ResultsResultsRNAse H1 treatment + CHIP: EZH2 (PRC2)→ If we remove R-loops, will PcG still be recruited ?
![Page 58: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/58.jpg)
ResultsResultsRNAse H1 treatment + CHIP: RING1B (PRC1)→ If we remove R-loops, will PcG still be recruited ?
![Page 59: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/59.jpg)
ResultsResults
There are R-loops at PcG repressed genes
They have a role in the repression
R-loops facilitate binding of PcG
![Page 60: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/60.jpg)
The effect of R-loop (loss) on RNA Pol II activation
(Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7)52
![Page 61: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/61.jpg)
Basic concepts
PcG repressed genes Active genes
The effect of R-loop (loss) on RNA Pol II activation
![Page 62: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/62.jpg)
Determine whether the transcriptional activation of PcG target genes observed on R-loop removal is linked to changes in CTD modification.
ChIP with Ser7P Ab
Remember: Ser7P = active genes!
Goal
The effect of R-loop (loss) on RNA Pol II activation
![Page 63: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/63.jpg)
Results
PcG target genes exhibit an increase in Ser7P Pol II levels after R-loops resolution
The effect of R-loop (loss) on RNA Pol II activation
PcG target = repressed active, with
R-loops no R-loops,no PcG
Removal of R-loops at PcG target genes leads to a specific change in Pol II CTD modification
Loss of R-loops increase of Ser7P
Pol II levels at PcG target genes
![Page 64: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/64.jpg)
R-loop depletion has NO detectable effect on Ser5P occupancy levels over PcG target genes
Results
The effect of R-loop (loss) on RNA Pol II activation
PcG target = repressed active, with R-loops
Ser5P is not affected by decreased occupancy of PcG enzymes upon R-loop depletion
Ser5P precedes R-loop formation
![Page 65: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/65.jpg)
The effect of R-loop (loss) on RNA Pol II activation
Ser5P is NOT affected by the R-loops resolution
R-loops removal leads to an increase of Ser7P
R-loops contribute to the transcriptional repression of PcG target genes via changes that affect not only PcG stability on chromatin but also Pol II
activation
![Page 66: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/66.jpg)
What is the role of PcG on R-loop formation and gene repression?
Results
![Page 67: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/67.jpg)
What is the role of PcG presence on R-loop formation and gene repression at PcG-repressed genes?
Is R-loops formation affected upon PRC2 and H3K27me3 loss?
Results
![Page 68: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/68.jpg)
What is the role of PcG presence on R-loop formation and gene repression at PcG-repressed genes?
Is R-loops formation affected upon PRC2 and H3K27me3 loss?
Results
![Page 69: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/69.jpg)
What is the role of PcG presence on R-loop formation and gene repression at PcG-repressed genes?
Does EZH2 KO cause transcriptional derepression of R-loop (+) PcG-repressed genes?
Results
![Page 70: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/70.jpg)
What is the role of PcG presence on R-loop formation and gene repression at PcG-repressed genes?
Results
Does EZH2 KO cause transcriptional derepression of R-loop (+) PcG-repressed genes?
![Page 71: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/71.jpg)
What is the role of PcG presence on R-loop formation and gene repression at PcG-repressed genes?
Is PRC1 recruitment affected upon PRC2 KO?
Results
![Page 72: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/72.jpg)
What is the role of PcG presence on R-loop formation and gene repression at PcG-repressed genes?
Is PRC1 recruitment affected upon PRC2 KO?
Results
![Page 73: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/73.jpg)
● EZH2 KO does NOT affect R-loop formation
● EZH2 KO does NOT affect derepression of R-loop(+) PcG genes
● EZH2 KO does NOT affect RING1B recruitment on R-loop(+) PcG genes
Results
What is the role of PcG on R-loop formation and gene repression?
Presence of R-loops and RING1B explains the lack of derepression on R loop(+) PcG genes.
![Page 74: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/74.jpg)
What if we chemically interfere with both EZH1 and EZH2 methyltransferase
activity?
Results
![Page 75: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/75.jpg)
What if we chemically interact with both EZH1 and EZH2 methyltransferase activity?
Levels of H3K27me3 upon EZH1-2 activity removal
Results
![Page 76: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/76.jpg)
What if we chemically interact with both EZH1 and EZH2 methyltransferase activity?
Levels of EZH2 binding upon EZH1-2 activity removal
Results
![Page 77: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/77.jpg)
What if we chemically interact with both EZH1 and EZH2 methyltransferase activity?
Are R-loop levels altered upon EZH1-2 activity removal?
Results
![Page 78: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/78.jpg)
What if we chemically interact with both EZH1 and EZH2 methyltransferase activity?
Are R-loop levels altered upon EZH1-2 activity removal?
Results
![Page 79: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/79.jpg)
What if we chemically interact with both EZH1 and EZH2 methyltransferase activity?
Are mRNA levels altered upon EZH1-2 activity removal?
Results
![Page 80: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/80.jpg)
What if we chemically interact with both EZH1 and EZH2 methyltransferase activity?
Are mRNA levels altered upon EZH1-2 activity removal?
Results
![Page 81: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/81.jpg)
What if we chemically interact with both EZH1 and EZH2 methyltransferase activity?
Is RING1B altered upon EZH1-2 activity removal?
Results
![Page 82: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/82.jpg)
What if we chemically interact with both EZH1 and EZH2 methyltransferase activity?
Is RING1B altered upon EZH1-2 activity removal?
Results
![Page 83: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/83.jpg)
What if we chemically interfere with both EZH1 and EZH2 methyltransferase
activity?
● NO loss of R-loops: R-loops form before EZH1-2 activity or presence
● R-loop(+) genes derepression
● Reduced recruitment of PRC1 on R-loop(+) genes
Results
![Page 84: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/84.jpg)
Do R-loops and EZH2 activity act through parallel pathways or do they
have synergistic effects?
Results
![Page 85: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/85.jpg)
Do R-loops and EZH2 activity act through parallel pathways or do they have synergistic effects?
Combination of R-loop removal and EZH2 inhibition
Results
![Page 86: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/86.jpg)
Do R-loops and EZH2 activity act through parallel pathways or do they have synergistic effects?
Combination of R-loop removal and EZH2 inhibition
Results
![Page 87: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/87.jpg)
Do R-loops and EZH2 activity act through parallel pathways or do they
have synergistic effects?● Both R-loop and EZH2 catalytic activity contribute to PcG
repression
Results
![Page 88: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/88.jpg)
Do R-loops and EZH2 activity act through parallel pathways or do they have synergistic effects?
What is the role of the reduced recruitment of RING1B?
Results
![Page 89: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/89.jpg)
Do R-loops and EZH2 activity act through parallel pathways or do they have synergistic effects?
What is the role of the reduced recruitment of RING1B?
Results
![Page 90: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/90.jpg)
Do R-loops and EZH2 activity act through parallel pathways or do they
have synergistic effects?● Both R-loop and EZH2 catalytic activity contribute to PcG
repression
● Both R-loop and RING1B recruitment on chromatin are important to repress R-loops(+) PcG targets genes
● RING1B recruitment can be regulated by R-loop formation in absence of PRC2 activity
Results
![Page 91: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/91.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop
forming PcG target genes?
Results
![Page 92: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/92.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop forming PcG target genes?
Can R-loops removal induce activation in EZH2 absence?
Results
![Page 93: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/93.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop forming PcG target genes?
Can R-loops removal induce activation in EZH2 absence?
Results
![Page 94: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/94.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop forming PcG target genes?
Can R-loops removal induce activation in EZH2 absence?
Results
![Page 95: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/95.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop
forming PcG target genes?● R-loops can act as transcriptional repressors in PcG system
independently of EZH2 occupancy on chromatin
Results
![Page 96: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/96.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop forming PcG target genes?
Is transcriptional change in EZH2 KO R-loops - due to RING changes?
Results
![Page 97: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/97.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop forming PcG target genes?
Is transcriptional change in EZH2 KO R-loops - due to RING changes?
Results
![Page 98: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/98.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop forming PcG target genes?
Is transcriptional change in EZH2 KO R-loops - due to RING changes?
Results
![Page 99: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/99.jpg)
Is the EZH2 occupancy on chromatin important for gene repression in R-loop
forming PcG target genes?● R-loops can act as transcriptional repressors in PcG
system independently of EZH2
● R-loops are important for RING1B recruitment specifically at the subset of PcG-repressed genes that form R-loops
Results
![Page 100: GENE EXPRESSION R-loops CHROMATIN STRUCTURE · Margot LADISLAS Isabella CONCINA. RNA Polymerase single-stranded DNA R-loop structure. Rare and accidental byproducts of transcription](https://reader033.vdocument.in/reader033/viewer/2022042916/5f56ca8969381740df4e1402/html5/thumbnails/100.jpg)
Let’s sum it up
➔ R-loops are important for both active and repressed genes
➔ In active genes: R-loops help the recruitment of chromatin modifying enzymes
REVIEW
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Let’s sum it up
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Let’s sum it up
➔ In mESC, R-loops correlate with PcG repressed genes
➔ R-loops collaborate with RING1B for the repression of the PcG repressed genes
➔ R-loops form independently of EZH1-2, but together they contribute to PcG repression
➔ R-loops can act as transcriptional repressors in PcG target genes independently of EZH2 occupancy on chr
ARTICLE
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Let’s sum it up
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https://ifunny.co/picture/me-and-my-friend-after-our-powerpoint-presentation-IlhStuB17
THANK YOU FOR YOUR
ATTENTION!
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REFERENCESImages without references on the slides are either made by the presenter, or are from the review and article studied.
REVIEW:
Chédin, Frédéric. 2016. « Nascent Connections: R-Loops and Chromatin Patterning ». Trends in Genetics 32 (12): 828‑38. https://doi.org/10.1016/j.tig.2016.10.002.
ARTICLE:
Skourti-Stathaki, Konstantina, Elena Torlai Triglia, Marie Warburton, Philipp Voigt, Adrian Bird, et Ana Pombo. 2019. « R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator Genes ». Molecular Cell 73 (5): 930-945.e4. https://doi.org/10.1016/j.molcel.2018.12.016.