Genetic dissection of the α-globin super-enhancer in vivo

Hay, Deborah; Hughes, Jim R.; Babbs, Christian; Davies, James O. J.; Graham, Bryony; Hanssen, Lars; Kassouf, Mira; Oudelaar, A. Marieke; Sharpe, Jacqueline A.; Suciu, Maria; Telenius, Jelena; Williams, Ruth M.; Rode, Christina; Li, Pik-Shan; Pennacchio, Len A.; Sloane-Stanley, Jackie; Ayyub, Helena; Butler, Sue; Sauka‐Spengler, Tatjana; Gibbons, Richard J.; Smith, Andrew J.H.; Wood, W. G.; Higgs, Douglas R.

doi:10.1038/ng.3605

Cited by 335 publications

(435 citation statements)

References 43 publications

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“…However, it is very likely that many such clusters will not act as a single unit (Hay et al 2016;Shin et al 2016), and we find many of them carrying both "with" and "without" peaks (Supplemental Fig. S6).…”

Section: Wwwgenomeorgmentioning

confidence: 88%

Binding of nuclear factor κB to noncanonical consensus sites reveals its multimodal role during the early inflammatory response

et al. 2016

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Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extracellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. Here, we monitor the early phase of this cascade at high spatiotemporal resolution in TNF-stimulated human endothelial cells. NF-κB, the transcription factor complex driving the response, interferes with the regulatory machinery by binding active enhancers already in interaction with gene promoters. Notably, >50% of these enhancers do not encode canonical NF-κB binding motifs. Using a combination of genomics tools, we find that binding site selection plays a key role in NF-κΒ-mediated transcriptional activation and repression. We demonstrate the latter by describing the synergy between NF-κΒ and the corepressor JDP2. Finally, detailed analysis of a 2.8-Mbp locus using sub-kbp-resolution targeted chromatin conformation capture and genome editing uncovers how NF-κΒ that has just entered the nucleus exploits pre-existing chromatin looping to exert its multimodal role. This work highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression.[Supplemental material is available for this article.]Mammalian cells, embedded in a multicellular environment, require intricate mechanisms in order to interpret, integrate, and ultimately respond to extracellular stimuli. Inflammatory signaling constitutes a well-studied example (Bhatt and Ghosh 2014; Smale and Natoli 2014); tumor necrosis factor (TNF) rapidly remodels gene expression programs through its main effector, nuclear factor κB (NF-κB) (Hayden and Ghosh 2008;Bhatt et al. 2012). TNF activates the same genes across cell types (Moynagh 2005), but the cascade needs to unfold against the ongoing transcriptional program of the stimulated cell. Along these lines, recent work in adipocytes showed that NF-κB is involved in gene repression via redistribution of prebound factors . This is tightly linked to the choice of NF-κB binding in vivo, but the specific principles that guide this choice in three-dimensional (3D) nuclear space and over time are still not well understood, despite the wealth of data on the different phases of the inflammatory response. For example, we now know that signaling directs binding of its downstream effectors to already-active cis-regulatory ele-

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Section: Wwwgenomeorgmentioning

confidence: 88%

Binding of nuclear factor κB to noncanonical consensus sites reveals its multimodal role during the early inflammatory response

et al. 2016

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“…Similarly, individual deletion of three constituent enhancers of the Wap super-enhancer revealed their partially redundant role in regulating transcription of Wap (Shin et al 2016). Furthermore, Hay et al (2016) showed by deletions within the Hba super-enhancer that each constituent enhancer acts independently in an additive manner to regulate expression of Hba genes in vivo. In ES cells, deletion of Dppa5aEC-Up or Dppa5aEC-Dn alone caused only a 36% and 16% reduction, respectively, in the levels of Dppa5a; deletion of both regions caused a 99% reduction.…”

Section: Enhancer and Super-enhancer Equivalencementioning

confidence: 99%

“…The SCR overlaps a predicted super-enhancer and was found to be required for >85% of the transcript levels of Sox2 in ES cells. Similarly, other studies have revealed the role of select super-enhancers in gene regulation (Mansour et al 2014;Hnisz et al 2015;Hay et al 2016;Huang et al 2016;Shin et al 2016). At the Sox2 locus, an additional super-enhancer was predicted surrounding the Sox2 gene; however, deletion of the enhancers within that region revealed that they are not required for Sox2 transcription in ES cells, thus raising questions about the super-enhancer prediction model (Zhou et al 2014).…”

mentioning

confidence: 95%

“…In fact, the two classic robust distal regulatory elements, the LCRs of the Hbb and hemoglobin subunit alpha (Hba) genes, are strikingly similar to super-enhancers; they contain multiple DHSs or are enhancer clusters (ECs), with each enhancer bound by multiple transcription factors (Grosveld et al 1987;Tuan et al 1989;Higgs et al 1990;Chen et al 1997;Mitchell et al 2012). Furthermore, both the Hbb and Hba LCRs were recently determined to fit the bioinformatic criteria for super-enhancers (Hay et al 2016).…”

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confidence: 99%

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Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes

et al. 2016

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Transcriptional enhancers are critical for maintaining cell-type-specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.

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“…35 While the concept of super enhancers is relatively new and requires substantial validation, their emerging role may be important in cell identity both in health and disease. 36 …”

Section: Transcriptional Codementioning

confidence: 99%

Cellular identity at the single-cell level

2016

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A single cell creates surprising heterogeneity in a multicellular organism. While every organismal cell shares almost an identical genome, molecular interactions in cells alter the use of DNA sequences to modulate the gene of interest for specialization of cellular functions. Each cell gains a unique identity through molecular coding across the DNA, RNA, and protein conversions. On the other hand, loss of cellular identity leads to critical diseases such as cancer. Most cell identity dissection studies are based on bulk molecular assays that mask differences in individual cells. To probe cell-to-cell variability in a population, we discuss single cell approaches to decode the genetic, epigenetic, transcriptional, and translational mechanisms for cell identity formation. In combination with molecular instructions, the physical principles behind cell identity determination are examined. Deciphering and reprogramming cellular types impact biology and medicine.

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Genetic dissection of the α-globin super-enhancer in vivo

Cited by 335 publications

References 43 publications

Binding of nuclear factor κB to noncanonical consensus sites reveals its multimodal role during the early inflammatory response

Binding of nuclear factor κB to noncanonical consensus sites reveals its multimodal role during the early inflammatory response

Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes

Cellular identity at the single-cell level

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