Journal ClubJournal Club

December 3rd, 2013

LCBGDr. Hunter Lab

Farhoud Faraji Kent Hunter, PhD

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.

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

The spatial arrangement of chromatin regulates of gene expression

Barton & Emerson. Genes Dev. 1994Spilianakis. Nature. 2005

Cavali & Misteli. Nat Struct Mol Biol. 2013

The “chromosome territories” in which a gene resides determine:• Whether it is expressed – on/off• The level of expression – tuning

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”

Sandhu et al. Cell Rep. 2012Schoenfelder et al. Nat Genet. 2010

Spector & Lamond. Cold Spring Harb Perspect Biol. 2011

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

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 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

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 disruption of a gene loop abrogates transcription of interacting members

3. Restoration of gene loop rescues cotranscription of multigene complex

TNFα

TNFα non-responsive genes

Model

“TranscriptionFactory”

-single focus of multiple RNA polymerases

TNFα responsive genes

SAMD4A, TNFAIP2, and SLC6A5 genes associate in an NF-κB-dependent multigene complex in a subset of cells

3C data: Chromosomal contacts occur ~1.5kb downstream of TSS

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.

In a fraction of HUVEC population, SAMD4A, TNFAIP2, and SLC6A5 may be constrained within a TAD prior to TNFα induction

Are these gene loci in proximity prior to TNFα activation?

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?

Transcriptional response to TNFα is asymmetric: 1) Not all cells respond to TNFα in the same way

84% 16% Never ObservedPhenotypicFrequency:

small n – insufficient for this highly stochastic system

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 transcribed when SAMD4A transcribed

• SLC6A5 transcribed when:1. SAMD4A transcribed

-or-2. SAMD4A and TNFAIP2

transcribed

Transcriptional asymmetry and colocalization data suggest hierarchical regulation:

1)SAMD4A2)TNFAIP23)SLC6A5

Suggests that chromosomal contact favors cotranscriptional activation.

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

TALENs directed to loci of chromosomal contact

Single cell assay:1)Disrupt individual gene loops at sites of chromosomal contact2)Visualize transcriptional activity of other genes in multigene complex via RNA-FISH

Hypothesis:Should loop-mediated contact be required for cotranscription, DSB would rupture chromosome contact and, thus, disrupt cotranscription.

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 still expressed

2)Production of protein product

Does it impact expression of cotranscribed genes in the multigene complex?

Disruption of TNFα non-responsive gene and cis-locus 5’ to SAMD4A has no effect on SAMD4A, TNFAIP2, or SLC6A5 expressionExcludes possibility that reductions in expression are due to cell cycle arrest

Disruption of SLC6A5 has no effect on SAMD4A or TNFAIP2 expression or colocalization

Disruption of TNFAIP2 abrogates SLC6A5 expression and TNFAIP2/ SLC6A5 colocalization

Disruption of SAMD4A abrogates TNFAIP2 and SLC6A5 expression and TNFAIP2/ SLC6A5 colocalization

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

SAMD4A TALEN does not impact transcriptional status of TNFα non-responsive gene RCOR1

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 SLC6A52) TNFAIP2 – promotes expression of SLC6A53) 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?

Recap

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

Lee. Genes Dev. 2009Orom et al. Cell . 2010

Brown et al. Nature. 1994Guttman & Rinn. Nature. 2012

Alternative Model

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

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

Conclusions1. 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

Thanks!

Questions?