Nolan Shelley
48682108
Due: Sept 21, 2015
Questions for Sept 21 Reading
1. The article summarizes a whole suite of experimental results of investigations into the
3D structure of the genome. What kind of approach (out of the approaches we have
seen in class) did most of these studies take? What is the value of such investigations?
The majority of the studies utilized the “remove something” approach to
determine the dependence of target gene expression on various transcription binding
factors and their interactions with enhancer sequences. For example, RNAi studies, in
which small RNA sequences are used to interfere with target mRNA sequences, have
been used to determine the specific enhancer binding proteins required for enhancergene looping in the -globin locus control region (LCR). The “remove something”
approach has also been used to determine whether topologically associated domains
(TADs) are functionally important for long-range enhancer activation in genes. For
example, the deletion of a TAD border in the Xist locus resulted in long-range
transcription misregulation, suggesting that such TADs are indeed functionally
relevant. Yet another example of the “remove something” approach has been the use
of small interfering RNAs (siRNAs) to deplete enhancer RNAs (eRNAs), resulting in
the reduction of target gene expression, suggesting that such eRNAs are actually
important in regulating gene expression.
Several of the studies also used something similar to the “look” approach to
determine a wide variety of enhancer-gene interactions. These studies used highthroughput sequencing methods in tandem with chromatin conformation capture to
determine enhancer sequences that were found to be in close proximity to various
target sequences. For example, the Hi-C technique revealed that long-range
interactions between genomic loci in embryonic stem cells occur mainly within
topologically associating domains (TADs).
I was also able to find an example of a study using the “add something” approach
in the review article. It was found that upon treatment with lipopolysaccharide
endotoxin (LPS), “inactive” enhancers of macrophages marked by H3K4me1 and
tissue-specific transcription factors were able to activate inflammatory response target
genes.
The value of such investigations that look into the role that enhancers play in gene
regulation is enormous. They enable us to determine the numerous ways in which
misregulation of genes (and the misregulation of enhancers more specifically) lead to
various diseases. Determining the mechanisms at play can provide possible methods
of treatment for such diseases, such as the forced demethylation of enhancers to enable
the activation of previously inactive genes. These investigations also provide valuable
insight into how genes are regulated throughout development and the various ways in
which genes can be activated or deactivated in response to cellular differentiation.
2. A study in mouse mentioned in the article identified ~90,000 enhancers that had
particular characteristics. How is this possible, when the mouse genome has
approximately 23,000 genes?
Nolan Shelley
48682108
Due: Sept 21, 2015
If there are roughly four times as many enhancers as there are genes in the mouse
genome, this implies that some genes require multiple enhancers for regulation
(indeed, there are also enhancers that regulate multiple genes). One example of the use
of multiple enhancers for a single gene occurs in the HoxD cluster, near a TAD border,
where certain genes have been observed to switch enhancer contacts from one TAD to
another as gene expression changes during limb development. Furthermore, enhancer
usage by commonly expressed genes such as Myc and Pim1 varies among cell types,
suggesting that such genes use different tissue-specific enhancers and transcriptions
factors throughout development.
3. What do you think a ‘latent’ enhancer is? Is there any evidence that it is possible to
activate/reactivate latent enhancers?
“Latent” enhancers are defined as genomic regions that are not sensitive to
nucleases, are unbound by transcription factors and lack the histone marks
characteristic of active enhancers, but do acquire these features in response to
stimulation. Such regions may be remnants of ancestral enhancer sequences that have
undergone various epigenetic changes over many generations. The fact that they can
become enhancers in response to stimulation suggests that they do not differ from
normal enhancer sequences genetically, but rather, epigenetically, especially since
enhancers require transcription factors that bind to very specific DNA sequences.
There is certainly evidence that it is possible to activate/reactivate latent enhancers, as
supported by experiments in which exposure to lipopolysaccharide endotoxin (LPS) in
macrophages lead to latent enhancers acquiring attributes of active enhancers and
initiating gene transcription. Furthermore, once unveiled, many such enhancers did not
return to the latent state, but instead produced a faster and stronger response upon
restimulation.
4. How do you think chromatin loops form?
As evidenced by the review article, it appears that chromatin loop formation
between enhancer and promoter sequences occurs through a complex set of
interactions between lineage-specific transcription factors (TFs), the transcriptional
coactivator Mediator, RNA polymerase II (Pol II) and ubiquitous architectural proteins
such as CTCF and cohesin. Topologically associated domains (TADs) and enhancer
RNAs (eRNAs) may play additional roles in chromatin loop formation.
Firstly, it has been shown that specific enhancer binding proteins are required for
enhancer-gene looping, such as LDB1 in the -globin locus control region (LCR),
whose dimerization is capable of driving loop formation in immature erythroid cells
where this does not normally occur. Other studies of the MYB loci have shown that the
architectural proteins CTCF/cohesin cooperate with such enhancer binding proteins to
facilitate loop formation. Furthermore, the basal transcriptional machinery such as Pol
II and Mediator has been implicated in enhancer-promoter interactions. Lastly, it has
been shown that chromatin looping occurs preferentially within (but not between)
TADs and that eRNAs interact with cohesin, with both looping to target genes and
cohesin occupancy reduced after eRNA inhibition. All this evidence indicates just how
Nolan Shelley
48682108
Due: Sept 21, 2015
complex the formation of chromatin loops is, and the exact interaction between the
various parts that give rise to such loops remains a mystery. However, it seems likely
that, first, lineage-specific transcription factors bind to demethylated enhancer
sequences, which then in turn bind to cohesin. Cohesin and eRNAs together possibly
act as some sort of bridge between the specific TFs and the basal transcription
machinery, facilitating the loop formation.
5. What was the most confusing part of this article that you would really like to
understand?
I think the most challenging part of the article for me was the section on
topologically associated domains (TADs). I am still confused as to what exactly a
TAD is and how one identifies TAD borders. The description of these very large
sequence domains was somewhat vague and yet the results were quite specific, such as
the conclusion that looping between promoters and enhancers happens preferentially
within and not between enhancer-promoter units (EPUs) that overlap with defined
TADs. I would really like to learn about the difference between TADs and sub-TADs
as this would contribute to my knowledge of the overall structure of chromatin in the
nucleus and how these structures contribute to local chromatin looping events such as
those involved in facilitating transcription.
Nolan Shelley
48682108
Due: Sept 21, 2015
Notes on Enhancer Function: Mechanistic and Genome-Wide Insights Come Together
(Plank and Dean, 2014)
- Enhancers are cis-regulatory elements that establish spatial or temporal patterns
of gene expression that are critical for development
- High-throughput sequencing studies place enhancer-gene interactions within the
3D context of chromosome folding, inviting a new look at enhancer function and
stimulating provocative new questions
- They integrate these whole-genome studies with recent mechanistic studies to
illuminate how enhancers physically interact with target genes, how enhancer
activity is regulated during development, and the role of noncoding RNAs
transcribed from enhancers in their function
Introduction
- Enhancers are cis-acting DNA regulatory elements that increase the
transcriptional output of target genes to influence the destiny of cells during
development and differentiation
- They may reside far from their in vivo targets interacting through proposed
mechanisms such as enhancer looping with linking by large protein complexes
- Chromosome conformation capture (3C) technology allowed physical interaction
frequencies between specific enhancers and target genes to be determined,
establishing that enhancers need proximity to the genes they activate
- Enhancer DNA sequences have plenty of binding sites for transcription factors
whose occupancy confers upon them tissue specificity
o Thus, long been proposed that factors binding to enhancers and genes
could stabilize chromatin loops through homotypic or heterotypic
interactions
o Looping interactions of chromosome-organizing proteins, such as the
insulator binding protein CTCF and its partner cohesin can facilitate
enhancer-gene chromatin contacts
o Lineage-specific activators and CTCF/cohesin engage in interactions
with components of RNA polymerase II and with the Mediator
transcriptional coactivator complex to tie enhancer loops directly to
the transcription apparatus
- Studies across developmental stages detail the dynamic regulation of enhancers
and enhancer looping during development
- Enhancers can be transcribed into enhancer RNA (eRNA) as part of the
mechanism of gene activation and possible looping
- Studies raise questions about how chromosome folding occurs, what drives the
changes in enhancers during development and how enhancer looping and
transcription activation are integrated physically and spatially in the nucleus to
achieve a unique expression pattern
- They discuss the basis for enhancer-gene loops in individual loci and across
chromosomes. Then they discuss how enhancer activity is regulated during
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development to modulate gene expression and conclude with understanding of
eRNA
Enhancers engage in long-range interactions with target genes through lineagespecific factors and ubiquitous architectural proteins
- At select loci, 3C and RNAi studies have shown that specific enhancer binding
proteins are required for enhancer-gene looping
o For example, a complex including GATA1 and cofactor FOG1 along
with TAL1, LMO2, and LDB1 is required for -globin locus control
regions (LCR) looping to globin genes and for transcriptional
activation in mature erythroid cells
o The dimerization domain of LDB1 underlies the enhancer-gene
proximity and this domain alone, when linked to the -globin
promoter is capable of driving loop formation and partially activating
transcription in immature erythroid cells where this does not
normally occur, arguing for the causality of the loop in the activation
- Architectural proteins CTCF/cohesin can participate directly in enhancer-gene
looping
o For example, enhancers that bind the lineage-specific factor T-BET in
TH1 cells are interspersed with CTCF/cohesin binding elements in the
IFN locus
o As naïve T cells differentiate, CTCF promotes a TH1 cell-specific IFN
locus looped conformation joining both kinds of sites and activating IFN
transcription
o CTCF occupancy and looping are dependent on enhancer binding by TBET, but whether they interact is unknown
o Not only do lineage-specific transcription activators mediate enhancergene looping, but so do ubiquitous looping factors (CTCF) to drive a celltype-specific regional architecture conducive to transcription
- Enhancer-gene interactions are dismantled during cell-division
o However, cohesin and some lineage-specific factors remain associated
with select sites on mitotic chromosomes, some of which have enhancer
markings, suggesting that enhancers may be appropriate repositories for
mitotic bookmarks to re-establish these long-range interactions
Enhancer loops are tied to the RNA Polymerase II transcription complex and
transcription activation
- The enhancer’s main job to increase transcriptional output could manifest at different
stages, including initiation, elongation or termination
- Enhancer-promoter interactions can involve components of the basal transcription
machinery
- Moreover, Mediator occupies enhancers of many ES cell genes together with
pluripotency factors OCT4, SOX2, and NANOG and bridges the enhancers to Pol II at
target promoters by direct interaction with cohesin
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o Mediator is a multiprotein complex that functions as a transcriptional
coactivator in all eukaryotes. It has been shown to associate with general
transcription factors as well Pol II (RNA polymerase II) and is essential
for activator-independent transcription, implying that it may provide
fundamental control of the formation of the initiation complex
o These studies clearly link enhancer loops to transcription initiation
o New work now shows that certain enhancers specifically function to
release Pol II pausing and allow elongation
o Anti-pause enhancers loop to target promoters and permit activation of the
P-TEFb complex, which is required for release of Pol II into elongation
Further insight emerges from a ChIA-PET study that determined interactions centered
on Pol II across the genome in human cell lines
o Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET)
incorporates chromatin immunoprecipitation (ChIP)-based enrichment,
chromatin proximity ligation, paired-end tags and high throughput
sequencing to determine de novo long-range chromatin interactions
genome-wide
o Networks of interacting promoters and promoter/enhancers were revealed
and these interactions were significantly enriched for cell-type-specific
genes
o Gene families regulated by a common transcription factor were
significantly overrepresented
o Spatial chromatin connectivity based on Pol II linking of select
coregulated genes showed the several pluripotency genes in ES cells are
connected within one major hub, suggestive of a transcription factory
These studies strongly advocate for enhancer looping as key and likely required for,
transcription activation
Supported by data showing formation of new enhancer loops precedes transcription in
the -globin locus
Reasonable to propose that long-range enhancer-gene interactions collectively drive
nuclear relocalization and clustering, underlying dynamic focal accumulation of Pol II
and the appearance of transcription factories
o Transcription factories describe the discrete sites where transcription
occurs in the nucleus – they contain RNA polymerase and the necessary
transcription factors for transcription
Enhancer loops within the global 3D organization of the nucleus
- Thousands of enhancer-gene interactions associated with the unique transcriptional
profile of cells have been observed in 5C studies
- Hi-C revealed that long-range interactions between genomic loci in ES cells and
certain differentiated cell types occur predominantly within circumscribed topological
domains or topologically associating domains (TADs)
o These TADs are largely conserved across a range of cell types and during
development
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o Long-range interactions within the same TAD are much more frequent
than between TADs and intradomain loops are more variable among cell
types, suggesting that intra-TAD contacts are involved in cell-specific
transcription activation
o TADs transcriptional activity can change during development or
differentiation when gene activity changes
TAD-type organizational structure has been widely observed across multiple mouse
tissue and cell types
o Clusters of coregulated enhancers and promoters were revealed that were
defined as enhancer-promoter units (EPUs)
o Hi-C confirmed looping between the identified promoters and enhancers
preferentially within and not between EPUs that overlap with defined
TADs
TADs also encompass “super enhancers” in ES cells
o These large domains of up to 50 kb contain clusters of individual enhancer
elements that are highly occupied by Mediator and pluripotency factors –
enhancers within these regions are consistently associated with genes that
encode important regulators of cell identity and interact with these targets
with high frequency
Sub-TADs also vary in a tissue specific manner with CTCF/cohesin and Mediator
playing roles at different levels of long-range interactions that contribute to TAD or
sub-TAD organization, although a complete description of these borders differ from
one another is still missing
5C experiments in HoxA genes suggest enhancers and genes interact with each other
through contacts between sub-TADs possible mediated by CTCF and cohesin
Deletion of a TAD border in the Xist locus resulted in new ectopic contacts and longrange transcription misregulation, suggesting that TADs are functionally relevant for
proper long-range enhancer activation of genes
o Moreover, CTCF (but not cohesin) reduction in HEK293T cells resulted in
a gain of inter-TAD interactions suggesting that CTCF helps to maintain
TAD borders
o Cohesin reduction in postmitotic thymocytes had no effect on TAD
organization, but in astrocytes, relaxation of TAD structure and gene
misregulation was observed upon cohesin reduction
An ectopic -globin LCR reestablished contact with the -globin gene and activated
transcription outside its normal TAD context and inter-TAD interactions may be
significant for enhancer function
o In the HoxD cluster, situated near a TAD border, certain genes are
observed to switch enhancer contacts from one TAD to another as gene
expression changes across the locus during limb development, indicating a
certain fluidity of the border
Within TADs, enhancers cluster together with relevant gene targets. EPUs, super
enhancers, and sub-TADs may all be manifestations of the same enhancer-dependent
clustering phenomenon
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48682108
Due: Sept 21, 2015
- Although descriptive, this insight is mechanistically important, because it means
enhancers and promoters need only scan a limited nuclear neighborhood to
successfully and efficiently encounter each other
- Enhancer-gene clustering within TADs and association of TADs of similar
transcriptional character nucleate transcription factories argues the possibility that
transcription precipitates clustering
Temporal and spatial regulation of enhancer activity
- High-throughput studies across development show that enhancers are progressively
modified to activate specific transcriptional programs, particularly by acquisition of
the H3K27ac mark
o For example, “poised” developmental enhancers in ES cells are
characterized by p300 and BG1 occupancy, H3K4me1 and low
nucleosome density together with Polycomb Repressive Complex 2
(PRC2) and the associated H3K27me3 mark
o At later developmental stages, these enhancers lose PRC2 and H3K27me3
and acquire H3K27ac and the ability to activate gene expression
o Additional “inactive” state of ES cell enhancers have high nucleosome
density but occupancy by the Pol II elongation factor ELL3. These
inactive enhancers must harbor ELL3 or they will be unable to activate
target genes upon differentiation
 Thus, ELL3 may mark enhancers for later activation of genes by
Pol II promoter recruitment, which requires cohesin, consistent
with looping
- As ES cells differentiate, enhancers of pluripotency genes are inactivated to silence
their target genes
o One study invoked activity of the LSD1 histone H3k4/9 demethylase to
remove the H3K4me1 enhancer mark
o In ES cells lacking LSD1, enhancers of pluripotency genes fail to undergo
histone demethylation associated with differentiation and their target
genes are not repressed
o Another study demonstrated that PRC2 deposits the H3K27me2 mark at
enhancers in ES cells, thereby precluding the active H3K27ac mark and
inappropriate enhancer activation
- Differentiating cells also require the function of previously inactive enhancers
o During transition from naïve to primed stem cells, the transcription factor
OTX2 pioneers new enhancer sites allowing OCT4 to relocalize and
activate a new set of target genes that permit cells to exit the naïve state
- Epigenetic profiling of the H3K27ac active enhancer mark in different developing
mouse tissues identified approximately 90,000 enhancers that exhibited precise tissueand stage-specific windows of predicted activity
- Enhancer usage varied widely among cell lineages in an additional study and was
linked to recruitment of lineage-determining transcription factors
Nolan Shelley
48682108
Due: Sept 21, 2015
- Enhancer usage by commonly expressed genes such as Myc and Pim1 also varies
among cell types, suggesting that such genes make use of different tissue enhancers
and transcription factors as cells progress through development
- Enhancers can respond to external stimuli to active appropriate genes
o For example, TGF signaling in pro-B cells or myotubes results in
SMAD2/3 recruitment to silent enhancers occupied by PU.1 or MYOD,
respectively and target genes are activated
o In macrophages, upon treatment with lipopolysaccharide endotoxin (LPS)
“inactive” enhancers marked by H3K4me1 and tissue-specific
transcription factors, including PU.1, are rapidly occupied by p300 and
activate inflammatory response target genes
o A set of “latent” enhancers in macrophages are not sensitive to nucleases
and lack both H3K4me1 and PU.1
 However, upon exposure to LPS, latent enhancers acquire
attributes of active enhancers and initiate gene transcription
 Although transcription factors can mark inactive enhancers in
unstimulated differentiated cells that will be required for later
activation of target genes, it is not clear how latent enhancers can
be recognized in order to be “unveiled”
- Both active and poised enhancers in IMR90 cells are equally likely to be engaged in
looping before gene activation by signaling to TNF--responsive enhancers,
suggesting that it is not just the establishment of loops to target promoters that activate
genes via enhancers
- Thus, it will be important in the future to focus on the precise temporal relationship of
enhancer recognition, poising and activation with enhancer looping and transcriptional
activation
DNA methylation as a modulator of enhancer activity
- Genome-wide studies imply that enhancers are negatively impacted by DNA
methylation consistent with the fact that methylation interferes with transcription
factor binding
- A study of the murine methylome in ES cells established a significant association of
DNA hypomethylation at regions distal to promoter CpG islands with transcription
factor binding, enhancer histone marks and the activity of enhancers
- DNA hypomethylation at enhancers is associated with open chromatin state, p300
occupancy and target gene expression
- Large study showed lineage-specific gene silencing through asymmetric loss of
enhancers by methylation
o For example, in human ES cells, enhancers destined to regulate genes
required for neural specification are poised, unmethylated, marked by
H3K4me1 and occupied by pluripotency factors
o Upon differentiation to neuroectoderm, these enhancers become activated,
lose pluripotency factors and acquire neural lineage transcription factors
o However, upon differentiation into mesoderm or endoderm, pluripotency
factors are lost and the enhancers become highly methylated
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Due: Sept 21, 2015
o Loss of DNA methylation happens concomitantly with the transition to
H3K4me1 and either H3K27me3 or H3K27ac to poise or activate
enhancers, respectively
- Vestigial enhancers are early enhancers that retain a hypomethylated landscape in
adult tissues, possibly as a memory mark
o They exist in closed chromatin and are heavily marked by repressive
H3K27me3, which may be inhibitory to methylation of local DNA
residues
o This demonstrates that DNA hypomethylation is a common hallmark of
active enhancers but not sufficient for activity of enhancers
o Loss of LSH, a modulator of DNA methylation, results in decreased in
enhancer methylation and increased target gene expression
- It is unknown whether DNA methylation is the cause of inactivation or a step
following other silencing mechanisms
Regulated production of transcripts from enhancers
- It has long been known that transcription occurs at enhancer elements, but it was
widely assumed to be ancillary to transcription activation of the target genes
- Transcription of eRNA upstream of the Arc gene requires the intact promoter and gene
hinting that eRNA transcription and enhancer-promoter communication are linked
- Androgen receptor, p53, MYOD, and MYOG positively regulate eRNAs by directly
binding to enhancers
- REV-ERB signaling can negatively regulate target eRNAs, as evidenced by reduced
eRNA transcription following Rev-Erb overexpression
- Demethylation of mature B cell enhancers accompanies their transcription into eRNAs
and their activation
- In each example, the regulation of eRNA transcription was associated with the
appropriate positive or negative effect on mRNA transcription of target genes,
supporting a mechanistic link
Mechanism by which eRNA may function to regulate target gene expression
- High-throughput studies established a significant association between eRNAs,
enhancer-promoter interactions and target gene expression
- Small interfering RNAs (siRNAs) were used to reduce eRNAs transcribed from
estrogen receptor-alpha-sensitive enhancers resulting in target gene expression
reduction
o The eRNAs were associated with cohesin, and both looping to the genes
and cohesin occupancy were reduced after eRNA inhibition, suggesting
that eRNAs and cohesin stabilize the contacts
o eRNAs also interact with the Mediator complex with the knockdown of
MED1 and Med12 compromising eRNA activation of target genes,
decreasing loop formation between genes and enhancers
o These studies suggest a direct role of eRNAs in formation or stabilization
of loops between enhancers and target genes
Nolan Shelley
48682108
Due: Sept 21, 2015
- Other studies have questioned the role of eRNAs in looping
o One study found that the transcribed enhancers physically interacted with
multiple target genes whose expression was reduced after eRNA depletion
at p53-dependent enhancers
o However, the loops were not diminished after p53 or eRNA reduction
o Similarly, chemical inhibition of transcription elongation of eRNAs
activated by ER binding did not reduce looping
o In these studies, enhancer-gene loops are maintained after reduction
eRNAs, but they do not rule out a requirement for eRNAs to initially form
loops or for another function relating to target gene activation
- eRNAs have also been implicated in chromatin remodeling at target genes with
reduction of eRNAs regulating Myod1 expression resulting in decreased promoter Pol
II occupancy and DNaseI hypersensitivity
Conclusions
- Computational improvements in Hi-C and greater sequencing depth in ChIA-PET
bring us closer to goal of identifying enhancer-gene pairs with greater resolution
- However, candidate enhancers must be functionally tested
o Genome editing with TALE nucleases has been deployed to delete or
inactivate enhancers, as have the use of CRISPR/Cas9 methodologies
o However, these mechanisms are low-throughput and vastly complicated
by the fact that multiple enhancers can be used for one gene and vice versa
- Sequence variation or other disruption of distant enhancers is a critical aspect of
misregulation of gene expression in disease with both sequence and methylation
polymorphisms in enhancers likely to be important with the better correlation with
altered target gene expression of the latter compared to the former