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. 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. 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. Chromosomes form functional long-range
cis- and trans- contacts via “looping”
Cavali & Misteli. Nat Struct Mol Biol. 2013
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
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
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
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. 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. 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. 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. 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
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. 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. 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
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. 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. 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. SAMD4A TALEN does not impact transcriptional
status of TNFα non-responsive gene RCOR1
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. 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. 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
38. Hypothetical model for hierarchical transcription
between coregulated genes in a multigene complex
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
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.
