Brian J. Abraham

Super-Enhancers in the Control of Cell Identity and Disease

Super-enhancers are large clusters of transcriptional enhancers that drive expression of genes that define cell identity. Improved understanding of the roles that super-enhancers play in biology would be afforded by knowing the constellation of factors that constitute these domains and by identifying super-enhancers across the spectrum of human cell types. We describe here the population of transcription factors, cofactors, chromatin regulators, and transcription apparatus occupying super-enhancers in embryonic stem cells and evidence that super-enhancers are highly transcribed. We produce a catalog of super-enhancers in a broad range of human cell types and find that super-enhancers associate with genes that control and define the biology of these cells. Interestingly, disease-associated variation is especially enriched in the super-enhancers of disease-relevant cell types. Furthermore, we find that cancer cells generate super-enhancers at oncogenes and other genes important in tumor pathogenesis. Thus, super-enhancers play key roles in human cell identity in health and in disease.

Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers

Super-enhancers and stretch enhancers (SEs) drive expression of genes that play prominent roles in normal and disease cells, but the functional importance of these clustered enhancer elements is poorly understood, so it is not clear why genes key to cell identity have evolved regulation by such elements. Here, we show that SEs consist of functional constituent units that concentrate multiple developmental signaling pathways at key pluripotency genes in embryonic stem cells and confer enhanced responsiveness to signaling of their associated genes. Cancer cells frequently acquire SEs at genes that promote tumorigenesis, and we show that these genes are especially sensitive to perturbation of oncogenic signaling pathways. Super-enhancers thus provide a platform for signaling pathways to regulate genes that control cell identity during development and tumorigenesis.

An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element

In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, that recruit much of the cell’s transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits its H3K27 acetylase–binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, which suggests a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells. Leukemia-associated mutations drive cell growth by creating a powerful transcriptional enhancer upstream of an oncogene. [Also see Perspective by Vähärautio and Taipale] A super-enhancer in leukemia development Human cancer genome projects have provided a wealth of information about mutations that reside within the coding regions of genes and drive tumor growth by functionally altering protein products. However, this mutational portrait of cancer is incomplete: A growing number of mutations are being found within gene regulatory regions. Mansour et al. present an intriguing example of this in a study of a childhood cancer, T-cell acute lymphoblastic leukemia (see the Perspective by Vähärautio and Taipale). An oncogene known to drive the growth of this cancer is expressed at high levels in the leukemic cells because the cells harbor mutations that create a powerful superenhancer (a DNA sequence that activates transcription) upstream of the oncogene. Science, this issue p. 1373; see also p. 1291

Transcription factor trapping by RNA in gene regulatory elements.

Noncoding RNA helps protein binding Besides reading the coding regions of genes, RNA polymerase generates RNA at promoter-proximal and -distal DNA elements, but the function of these molecules is largely unknown. Sigova et al. show that these RNAs facilitate interactions between gene regulators and the regulatory elements they occupy. Nascent RNA associates with the transcription factor YY1 and increases its ability to bind DNA. Thus, transcription at active regulatory elements may provide a positive feedback loop that reinforces regulatory elements contributing to the stability of gene expression programs. Science, this issue p. 978 Nascent RNAs facilitate interactions between gene regulators and regulatory elements. Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs.

The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

Multiple Structural Maintenance of Chromosome Complexes at Transcriptional Regulatory Elements

Summary Transcription factors control cell-specific gene expression programs by binding regulatory elements and recruiting cofactors and the transcription apparatus to the initiation sites of active genes. One of these cofactors is cohesin, a structural maintenance of chromosomes (SMC) complex that is necessary for proper gene expression. We report that a second SMC complex, condensin II, is also present at transcriptional regulatory elements of active genes during interphase and is necessary for normal gene activity. Both cohesin and condensin II are associated with genes in euchromatin and not heterochromatin. The two SMC complexes and the SMC loading factor NIPBL are particularly enriched at super-enhancers, and the genes associated with these regulatory elements are especially sensitive to reduced levels of these complexes. Thus, in addition to their well-established functions in chromosome maintenance during mitosis, both cohesin and condensin II make important contributions to the functions of the key transcriptional regulatory elements during interphase.

YY1 Is a Structural Regulator of Enhancer-Promoter Loops

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

Models of human core transcriptional regulatory circuitries

A small set of core transcription factors (TFs) dominates control of the gene expression program in embryonic stem cells and other well-studied cellular models. These core TFs collectively regulate their own gene expression, thus forming an interconnected auto-regulatory loop that can be considered the core transcriptional regulatory circuitry (CRC) for that cell type. There is limited knowledge of core TFs, and thus models of core regulatory circuitry, for most cell types. We recently discovered that genes encoding known core TFs forming CRCs are driven by super-enhancers, which provides an opportunity to systematically predict CRCs in poorly studied cell types through super-enhancer mapping. Here, we use super-enhancer maps to generate CRC models for 75 human cell and tissue types. These core circuitry models should prove valuable for further investigating cell-type-specific transcriptional regulation in healthy and diseased cells.

Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions.

Significance More than a thousand proteins are thought to contribute to mammalian chromatin and its regulation, but our understanding of the genomic occupancy and function of most of these proteins is limited. We have used a chromatin proteomic profiling approach to produce a catalogue of proteins associated with genomic regions whose chromatin is marked by specific modified histones. A substantial number of the newly identified proteins are associated with human disease. Future chromatin proteomic profiling studies should prove valuable for identifying additional chromatin-associated proteins in a broad spectrum of cell types. More than a thousand proteins are thought to contribute to mammalian chromatin and its regulation, but our understanding of the genomic occupancy and function of most of these proteins is limited. Here we describe an approach, which we call “chromatin proteomic profiling,” to identify proteins associated with genomic regions marked by specifically modified histones. We used ChIP-MS to identify proteins associated with genomic regions marked by histones modified at specific lysine residues, including H3K27ac, H3K4me3, H3K79me2, H3K36me3, H3K9me3, and H4K20me3, in ES cells. We identified 332 known and 114 novel proteins associated with these histone-marked genomic segments. Many of the novel candidates have been implicated in various diseases, and their chromatin association may provide clues to disease mechanisms. More than 100 histone modifications have been described, so similar chromatin proteomic profiling studies should prove to be valuable for identifying many additional chromatin-associated proteins in a broad spectrum of cell types.