Journal Club

December 3rd, 2013

Farhoud Faraji

Kent Hunter, PhD

LCBG
Dr. Hunter Lab
Gene Expression "Programs"
• Def: A gene network whose coordinated expression
impacts the overall phenotype of a cell.
– P...
The genome is topologically organized in
3-dimensional space
• Chromosomes occupy defined locations
(“territories”) within...
The spatial arrangement of chromatin
regulates of gene expression

The “chromosome territories” in which a gene resides de...
Chromosomes form functional long-range
cis- and trans- contacts via “looping”

Cavali & Misteli. Nat Struct Mol Biol. 2013
Chromatin loops also localize co-regulated genes to
specialized “transcription factories”

Example of transcription factor...
Chromatin Conformation Capture
(3C, 4C, 5C, Hi-C, ChIA-PET)
However, nuclear organization is
inherently stochastic
• Large heterogeneity observed in 3C-based
data for global chromati...
Question

Are chromosomal contacts required for
co-transcription in multigene complexes?
The model system

Papantonis et al. EMBO J. 2012
Agenda
1. Transcriptional response to TNFα of
coregulated genes in a multigene complex is
asymmetric
2. TALEN-mediated dis...
Model

TNFα nonresponsive genes

TNFα responsive
genes

TNFα

“Transcription
Factory”
-single focus o
multiple RNA
polymer...
SAMD4A, TNFAIP2, and SLC6A5 genes associate in an NFκB-dependent multigene complex in a subset of cells
3C data:
Chromosom...
Overlapping SAMD4A, TNFAIP2, and SLC6A5
RNA FISH foci colocalize with active RNA PolII

Collectively, data suggest that co...
Are these gene loci in proximity prior to TNFα
activation?

In a fraction of HUVEC
population, SAMD4A,
TNFAIP2, and SLC6A5...
Recap
First clue to a heterogeneous response to TNFα:
•Observed overlapping cotranscription of TNFα
responsive genes in 5%...
Transcriptional response to TNFα is asymmetric:
1) Not all cells respond to TNFα in the same way
small n – insufficient fo...
Transcriptional response to TNFα is asymmetric:
2) TNFα-responsive genes are not always coexpressed
Transcriptional response to TNFα is asymmetric:
3) Certain TNFα-responsive genes expressed more pervasively
• TNFAIP2 tran...
Agenda
1. Transcriptional response to TNFα of
coregulated genes in a multigene complex is
asymmetric
2. TALEN-mediated dis...
TALENs directed to loci of chromosomal contact
Hypothesis:
Should loop-mediated contact be required for cotranscription, D...
TALEN-induced DSBs are highly specific
SAMD4A targeted TALENs induce DSBs
at the SAMD4A transcription unit

Does DSB induction disrupt transcription of the gene?
TALEN-induced DSB does not disrupt
SAMD4A transcription up to the DSB
Biallelic DSB abrogates
SAMD4A protein expression

Transcript from non-disrupted
allele is still produced
Recap
TALEN-induced DSBs abrogate:
1)Production of a full length transcript of the
disrupted allele
– Non-disrupted allele...
Disruption of TNFα nonresponsive gene and cislocus 5’ to SAMD4A has no
effect on SAMD4A,
TNFAIP2, or SLC6A5
expression
Exc...
TALEN-induced
bi-allelic DSBs at:
SAMD4A abrogate protein
expression of TNFAIP2 and SLC6A5

TNFAIP2 abrogate protein expre...
SAMD4A TALEN does not impact transcriptional
status of TNFα non-responsive gene RCOR1
Recap
TALEN-mediated disruption of a single gene loop
and associated chromosomal contacts in a
multigene complex alters th...
Major Flaw (in my naïve opinion)
Authors have not excluded possibility that
transcriptional activation may not be directly...
Agenda
1. Transcriptional response to TNFα of
coregulated genes in a multigene complex is
asymmetric
2. TALEN-mediated dis...
Repair Strategy:
Splice site flanked IRES-eGFP
TNFα-induced activation of SAMD4A induces
eGFP expression
Rationale for using RNA FISH probes instead of
fluorescence to detect eGFP positive cells
Restored SAMD4A gene expresses SAMD4A 5’terminus and full-length eGFP transcripts
Repairing disrupted SAMD4A gene loop restores
transcription of genes in the multigene complex
Hypothetical model for hierarchical transcription
between coregulated genes in a multigene complex
Conclusions
1. First direct demonstration for the requirement of cis- and trans“chromosomal contacts” for coexpression of ...
Thanks!

Questions?
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12.03.13 - Journal Club

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  • Hypothesis. NFκB (green) is usually cytoplasmic, and genes 1, 3, and 5 are transcribed in a factory (blue sphere) while TNFα-responsive genes 2, 4 and 6 are unattached and inactive. Only 3 of the ∼16 sequences attached to a factory are shown (Cook, 2010). TNFα induces phosphorylation of the p65 subunit of NFκB (now purple), import into the nucleus, binding to responsive promoters and/or the factory, and—once relevant promoters diffuse through the nucleoplasm and collide with the factory—transcription of responsive genes in what has become a ‘specialized' factory (green sphere). As a result, gene 2 now lies near other responsive NFκB-binding genes. Gene 1 is still attached and transcribed, but may later be replaced by responsive gene 6. If this model applies, then TNFα stimulation should bring gene 2 close to other responsive genes.
  • 12.03.13 - Journal Club

    1. 1. Journal Club December 3rd, 2013 Farhoud Faraji Kent Hunter, PhD LCBG Dr. Hunter Lab
    2. 2. Gene Expression "Programs" • Def: A gene network whose coordinated expression impacts the overall phenotype of a cell. – Proliferation, differentiation, immunogenic response – Co-expressed in some cases • Regulated by a concerted interplay between transcription factors, epigenetic modulation, and signaling mediators.
    3. 3. The genome is topologically organized in 3-dimensional space • Chromosomes occupy defined locations (“territories”) within the nucleus. • What are the functions of the spatial structure of chromatin? Lieberman-Aiden. Science. 2009
    4. 4. The spatial arrangement of chromatin regulates of gene expression The “chromosome territories” in which a gene resides determine: • Whether it is expressed – on/off Barton & Emerson. Genes Dev. 1994 • The level of expression – tuning Spilianakis. Nature. 2005 Cavali & Misteli. Nat Struct Mol Biol. 2013
    5. 5. Chromosomes form functional long-range cis- and trans- contacts via “looping” Cavali & Misteli. Nat Struct Mol Biol. 2013
    6. 6. Chromatin loops also localize co-regulated genes to specialized “transcription factories” Example of transcription factories: the nuclear speckle (interchromatin granule) • A transcriptionally active subnuclear compartment Multigene complexes are the main modality of transcription in metazoan cells. • >95% of transcription occurs in multigene complexes Sandhu et al. Cell Rep. 2012 Schoenfelder et al. Nat Genet. 2010 Spector & Lamond. Cold Spring Harb Perspect Biol. 2011
    7. 7. Chromatin Conformation Capture (3C, 4C, 5C, Hi-C, ChIA-PET)
    8. 8. However, nuclear organization is inherently stochastic • Large heterogeneity observed in 3C-based data for global chromatin interactions – Suggests a subset of cells within population are enriched for specific chromosomal interactions • Necessitates use of single-cell analysis in global interactome and gene loop studies
    9. 9. Question Are chromosomal contacts required for co-transcription in multigene complexes?
    10. 10. The model system Papantonis et al. EMBO J. 2012
    11. 11. Agenda 1. Transcriptional response to TNFα of coregulated genes in a multigene complex is asymmetric 2. TALEN-mediated disruption of a gene loop abrogates transcription of interacting members 3. Restoration of gene loop rescues cotranscription of multigene complex
    12. 12. Model TNFα nonresponsive genes TNFα responsive genes TNFα “Transcription Factory” -single focus o multiple RNA polymerases
    13. 13. SAMD4A, TNFAIP2, and SLC6A5 genes associate in an NFκB-dependent multigene complex in a subset of cells 3C data: Chromosomal contacts occur ~1.5kb downstream of TSS
    14. 14. Overlapping SAMD4A, TNFAIP2, and SLC6A5 RNA FISH foci colocalize with active RNA PolII Collectively, data suggest that cotranscription of these genes at PolII foci may occur in only a small fraction of HUVEC population.
    15. 15. Are these gene loci in proximity prior to TNFα activation? In a fraction of HUVEC population, SAMD4A, TNFAIP2, and SLC6A5 may be constrained within a TAD prior to TNFα induction
    16. 16. Recap First clue to a heterogeneous response to TNFα: •Observed overlapping cotranscription of TNFα responsive genes in 5% of cell population •Unspecified fraction of TNFα responsive genes colocalize prior to TNFα induction – Are these “TAD restricted genes” the genes that will be transcribed upon TNFα induction? • DNA-FISH: RNA-FISH ratio? • Concurrent DNA-RNA FISH on uninduced and induced cells?
    17. 17. Transcriptional response to TNFα is asymmetric: 1) Not all cells respond to TNFα in the same way small n – insufficient for this highly stochastic system Phenotypic Frequency: 84% 16% Never Observed
    18. 18. Transcriptional response to TNFα is asymmetric: 2) TNFα-responsive genes are not always coexpressed
    19. 19. Transcriptional response to TNFα is asymmetric: 3) Certain TNFα-responsive genes expressed more pervasively • TNFAIP2 transcribed when SAMD4A transcribed • SLC6A5 transcribed when: 1. SAMD4A transcribed -or2. SAMD4A and TNFAIP2 transcribed Transcriptional asymmetry and colocalization data suggest hierarchical regulation: 1)SAMD4A 2)TNFAIP2 3)SLC6A5 Suggests that chromosomal contact favors cotranscriptional activation.
    20. 20. Agenda 1. Transcriptional response to TNFα of coregulated genes in a multigene complex is asymmetric 2. TALEN-mediated disruption of a gene loop abrogates transcription of interacting members 3. Restoration of SAMD4A gene loop rescues cotranscription of multigene complex
    21. 21. TALENs directed to loci of chromosomal contact Hypothesis: Should loop-mediated contact be required for cotranscription, DSB would rupture chromosome contact and, thus, disrupt cotranscription. Single cell assay: 1)Disrupt individual gene loops at sites of chromosomal contact 2)Visualize transcriptional activity of other genes in multigene complex via RNA-FISH
    22. 22. TALEN-induced DSBs are highly specific
    23. 23. SAMD4A targeted TALENs induce DSBs at the SAMD4A transcription unit Does DSB induction disrupt transcription of the gene?
    24. 24. TALEN-induced DSB does not disrupt SAMD4A transcription up to the DSB
    25. 25. Biallelic DSB abrogates SAMD4A protein expression Transcript from non-disrupted allele is still produced
    26. 26. Recap TALEN-induced DSBs abrogate: 1)Production of a full length transcript of the disrupted allele – Non-disrupted allele still expressed 1)Production of protein product Does it impact expression of cotranscribed genes in the multigene complex?
    27. 27. Disruption of TNFα nonresponsive gene and cislocus 5’ to SAMD4A has no effect on SAMD4A, TNFAIP2, or SLC6A5 expression Excludes possibility that reductions in expression are due to cell cycle arrest Disruption of SAMD4A abrogates TNFAIP2 and SLC6A5 expression and TNFAIP2/ SLC6A5 colocalization Disruption of TNFAIP2 abrogates SLC6A5 expression and TNFAIP2/ SLC6A5 colocalization Disruption of SLC6A5 has no effect on SAMD4A or TNFAIP2 expression or colocalization
    28. 28. TALEN-induced bi-allelic DSBs at: SAMD4A abrogate protein expression of TNFAIP2 and SLC6A5 TNFAIP2 abrogate protein expression of SLC6A5 but not SAMD4A SLC6A5 does not impact protein expression of SAMD4A or TNFAIP2
    29. 29. SAMD4A TALEN does not impact transcriptional status of TNFα non-responsive gene RCOR1
    30. 30. Recap TALEN-mediated disruption of a single gene loop and associated chromosomal contacts in a multigene complex alters the transcriptional status of other genes occupying the complex in a hierarchical manner. 1) SAMD4A – promotes expression of TNFAIP2 and SLC6A5 2) TNFAIP2 – promotes expression of SLC6A5 3) SLC6A5 – does not impact expression of SAMD4A or TNFAIP2 - And 4) Chromosomal contact favors cotranscriptional activation – – Do not formally show that chromosomal contact is disrupted upon DSB TALEN induced DSB -> DNA-FISH and/or 3-C -> Reduced frequency of chromatin contact?
    31. 31. Major Flaw (in my naïve opinion) Authors have not excluded possibility that transcriptional activation may not be directly mediated by chromatin contact. •SAMD4A transcript could recruit transcriptional machinery of TNFAIP2/SLC6A5 in cis via a tethering mechanism. – A proposed and validated mechanism of transcriptional regulation by lncRNAs such as XIST and eRNAs Alternative Model Lee. Genes Dev. 2009 Orom et al. Cell . 2010 Brown et al. Nature. 1994 Guttman & Rinn. Nature. 2012
    32. 32. Agenda 1. Transcriptional response to TNFα of coregulated genes in a multigene complex is asymmetric 2. TALEN-mediated disruption of a gene loop abrogates transcription of interacting members 3. Restoration of a gene loop rescues cotranscription of multigene complex
    33. 33. Repair Strategy: Splice site flanked IRES-eGFP
    34. 34. TNFα-induced activation of SAMD4A induces eGFP expression
    35. 35. Rationale for using RNA FISH probes instead of fluorescence to detect eGFP positive cells
    36. 36. Restored SAMD4A gene expresses SAMD4A 5’terminus and full-length eGFP transcripts
    37. 37. Repairing disrupted SAMD4A gene loop restores transcription of genes in the multigene complex
    38. 38. Hypothetical model for hierarchical transcription between coregulated genes in a multigene complex
    39. 39. Conclusions 1. First direct demonstration for the requirement of cis- and trans“chromosomal contacts” for coexpression of multigene complexes – Unanticipated transcriptional regulatory hierarchy of these interactions suggests a mechanism for upstream regulation of multigene complex expression – “Chromosomal contacts” = non-DSB disrupted chromatin loops – Even rescue experiment does not exclude possibility that SAMD4A and TNFAIP2 transcript tethers transcriptional activators to multigene complex • Reconstitution of SAMD4A also rescues transcription of the full length SAMD4A message Cellular Systems are Inherently Stochastic 2. Employed an informative method to interrogate intrinsically heterogeneous cellular phenotypes – Single cell expression assessment over a representative population Elowitz et al. Science. 2002
    40. 40. Thanks! Questions?

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