ChIRP and related methodology
for revealing the role of IncRNAs
Presented by Duo Li
23 September 2014
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RNA world
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Genomic organization of coding and noncoding transcripts
Long ncRNAs: longer than 200 nucleotides.
Mercer, T.R., et al, Nature reviews, 2009
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Modular principles of long ncRNA (lncRNA)
genes
Guttman, M., et al, Nature, 2012
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Classification of lncRNA function
Guttman, M., et al, Nature, 2012 5
Functions lncRNAs
Mercer, T.R., et al, Nature reviews, 2009
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Chromatin Isolation by RNA Purification (ChIRP)
HOTAIR, human lncRNA,
affect PRC2 occupany,
which mediate
H3K27me3 on hundreds
of genes genome wide.
20-mer (probes)
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ChIRP Enriches for TERC RNA and Detects TERCassociated Telomere DNA and TCAB1 Protein
DNA
Protein
TCAB1: subunit of the telomerase holoenzyme,
facilitates telomerase trafficking
lnc TERC function as the template and scaffold for the telomerase complex.
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ChIRP-Seq Reveals roX2 Binding Sites on X
Chromosome
Binding site for
roX-MSL complex
308 binding site
Drosophila, male cells upregulate teh expression of genes on their single X chromosome by 2
fold, this requires a ribonucleoprotein complex containing MSL proteins, and two lncRNA roX1
and roX2.
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ChIRP-Seq Reveals roX2 Binding Sites on X
Chromosome
ChIRP
ChIP
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TERC ChIRP-Seq
TERT can bind and
coactivate Wnt target
genes
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HOTAIR ChIRP-Seq Suggests Mechanisms of
HOTAIR Recruitment of PRC2
PRC2 subunits:EZH2,
SUZ12, H3K27Me3
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HOTAIR Binds Chromatin in a PRC2independent Manner
HOTAIR actively recruit PRC2 or
serve as a scaffolding molecule?
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Summary
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Split pools of tiling oligonucleotide probes and glutaraldehyde crosslinking.
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First genome-wide views of ncRNA occupancy on the human genome.
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IncRNA binding sites are focal, specific and numerous.
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IncRNA access the genome through specific DNA sequences.
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Comparison of IncRNA occupancy map with chromatin state maps can reveal the
order and logic of the regulatory cascade.
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Capture hybridization analysis of RNA targets
(CHART)
• C-oligo target roX2 RNA available
for hybridization
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3 25-mer DNA oligonucleotides
• C-oligo chemistries ( locked nucleic
acid or O2‘-methylated ribonucleotides
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CHART allows specific enrichment of roX2
along with its associated targets
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NEAT1 CHART, but not MALAT1 CHART,
specifically enriches NEAT1 RNA along with its
protein and DNA targets
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roX2 CHART-seq reveals robust enrichment of
roX2 on chrX and precise localization to sites
of MSL binding
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Summary
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Allows determinaton of RNA targets. CHART was successfully applied to
lncRNAs of different lengths from two different organisms.
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CHART appears similar to ChIP in enrichment and resolution.
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The limitation of CHART also overlap with those of ChIP. Neither provides
information regarding the stoichiometry of binding at eatch genomic locusonly enrichment values. No guarantee that different target loci will be
enriched with equal efficiency.
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dChIRP uses antisense oligonucleotides to
purify specific RNA domains and associated
RNAs, proteins and chromatin
Domain-specific oligonucleotide
pools, 20-mer oligonucleotides
Aim: to dissect the functional
domains of an RNA interest
within its native cellular context
1%glutaraldehyde/formalehyde
+3%formaldehyde
DNA to 500bp for sequencing
RNA 200-500nt
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dChIRP RNA co-recovery reveals roX1’s
topological architecture
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U: tertiary RNA structure or
interacting proteins
• D: long, linear stem-loop
Validate the data with the even/odd pools, as well as in HOTAIR system.
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roX1 D domains interact with the MSL complex
and chromatin on the X chromosome
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MSL ribonucleoprotein
complex: roX1, roX2
lncRNAs and 5 proteins
(MSL1-3, MLE, MOF)
Chromatin entry sites
(CESs) are co-occupied by
the MSL proteins.
CLAMP, a zinc finger
protein, bind to MSL
recognition site (MRE,
GAGA rich) within CES.
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dChIRP boosts genomic occupancy signal
relative to traditional ChIRP-seq
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dChIRP boosts genomic occupancy signal
relative to traditional ChIRP-seq
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CESs cluster together in a dosagecompensation territory of the nucleus
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roX1’s D domains are independent, functional
RNA subunits
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Summary
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dChIRP traps endogenous RNA-chromatin interactions in living cells and
then breaks the RNA apart to explore the fuction of each domain.
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No mutant constructs are required. The number of configurations tested
depends on the number of oligonucleotide pools.
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The findings allows them to identify D3 as the smallest RNA unit sufficient
for chromosome-wide dosage compensation.
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CLIP method
Analysis of 340 Nova CLIP tags
Nova-dependent regulation of JNK2
C
PAR-CLIP methodology
B
RNA recognition by PUM2 protein
RNA recoginition by QKI protein
RNA Recognition by the IGF2BP Protein Family
AGO Protein Family and TNRC6
Family PAR-CLIP
AGO PAR-CLIP Identifies miRNA SeedComplementary Sequences in HEK293 Cells
Relationship between Various Features of
miRNA/Target RNA Interactions and mRNA Stability