The winding road toward transcriptional repression.

Enhancers are known for their role in mediating transcriptional activation. In this issue, Vermunt et al.1 report the unexpected finding that genes can undergo a sequential transition between distinct enhancers to mediate progressive downregulation of expression.

Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.

Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.

UTX inhibition as selective epigenetic therapy against TAL1-driven T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation.

The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.

Alteration of CTCF-associated chromatin neighborhood inhibits TAL1-driven oncogenic transcription program and leukemogenesis.

Aberrant activation of the TAL1 is associated with up to 60% of T-ALL cases and is involved in CTCF-mediated genome organization within the TAL1 locus, suggesting that CTCF boundary plays a pathogenic role in T-ALL. Here, we show that -31-Kb CTCF binding site (-31CBS) serves as chromatin boundary that defines topologically associating domain (TAD) and enhancer/promoter interaction required for TAL1 activation. Deleted or inverted -31CBS impairs TAL1 expression in a context-dependent manner. Deletion of -31CBS reduces chromatin accessibility and blocks long-range interaction between the +51 erythroid enhancer and TAL1 promoter-1 leading to inhibition of TAL1 expression in erythroid cells, but not T-ALL cells. However, in TAL1-expressing T-ALL cells, the leukemia-prone TAL1 promoter-IV specifically interacts with the +19 stem cell enhancer located 19 Kb downstream of TAL1 and this interaction is disrupted by the -31CBS inversion in T-ALL cells. Inversion of -31CBS in Jurkat cells alters chromatin accessibility, histone modifications and CTCF-mediated TAD leading to inhibition of TAL1 expression and TAL1-driven leukemogenesis. Thus, our data reveal that -31CBS acts as critical regulator to define +19-enhancer and the leukemic prone promoter IV interaction for TAL1 activation in T-ALL. Manipulation of CTCF boundary can alter TAL1 TAD and oncogenic transcription networks in leukemogenesis.

Proteomic/transcriptomic analysis of erythropoiesis.

PURPOSE OF REVIEW: Erythropoiesis is a hierarchical process by which hematopoietic stem cells give rise to red blood cells through gradual cell fate restriction and maturation. Deciphering this process requires the establishment of dynamic gene regulatory networks (GRNs) that predict the response of hematopoietic cells to signals from the environment. Although GRNs have historically been derived from transcriptomic data, recent proteomic studies have revealed a major role for posttranscriptional mechanisms in regulating gene expression during erythropoiesis. These new findings highlight the need to integrate proteomic data into GRNs for a refined understanding of erythropoiesis. RECENT FINDINGS: Here, we review recent proteomic studies that have furthered our understanding of erythropoiesis with a focus on quantitative mass spectrometry approaches to measure the abundance of transcription factors and cofactors during differentiation. Furthermore, we highlight challenges that remain in integrating transcriptomic, proteomic, and other omics data into a predictive model of erythropoiesis, and discuss the future prospect of single-cell proteomics. SUMMARY: Recent proteomic studies have considerably expanded our knowledge of erythropoiesis beyond the traditional transcriptomic-centric perspective. These findings have both opened up new avenues of research to increase our understanding of erythroid differentiation, while at the same time presenting new challenges in integrating multiple layers of information into a comprehensive gene regulatory model.

A non-canonical role for the proneural gene Neurog1 as a negative regulator of neocortical neurogenesis.

Neural progenitors undergo temporal identity transitions to sequentially generate the neuronal and glial cells that make up the mature brain. Proneural genes have well-characterised roles in promoting neural cell differentiation and subtype specification, but they also regulate the timing of identity transitions through poorly understood mechanisms. Here, we investigated how the highly related proneural genes Neurog1 and Neurog2 interact to control the timing of neocortical neurogenesis. We found that Neurog1 acts in an atypical fashion as it is required to suppress rather than promote neuronal differentiation in early corticogenesis. In Neurog1-/- neocortices, early born neurons differentiate in excess, whereas, in vitro, Neurog1-/- progenitors have a decreased propensity to proliferate and form neurospheres. Instead, Neurog1-/- progenitors preferentially generate neurons, a phenotype restricted to the Neurog2+ progenitor pool. Mechanistically, Neurog1 and Neurog2 heterodimerise, and while Neurog1 and Neurog2 individually promote neurogenesis, misexpression together blocks this effect. Finally, Neurog1 is also required to induce the expression of neurogenic factors (Dll1 and Hes5) and to repress the expression of neuronal differentiation genes (Fezf2 and Neurod6). Neurog1 thus employs different mechanisms to temper the pace of early neocortical neurogenesis.

Recapitulation of erythropoiesis in congenital dyserythropoietic anaemia type I (CDA-I) identifies defects in differentiation and nucleolar abnormalities.

The investigation of inherited disorders of erythropoiesis has elucidated many of the principles underlying the production of normal red blood cells and how this is perturbed in human disease. Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is a rare form of anaemia caused by mutations in two genes of unknown function: CDAN1 and CDIN1 (previously called C15orf41), whilst in some cases, the underlying genetic abnormality is completely unknown. Consequently, the pathways affected in CDA-I remain to be discovered. To enable detailed analysis of this rare disorder we have validated a culture system which recapitulates all of the cardinal haematological features of CDA-I, including the formation of the pathognomonic 'spongy' heterochromatin seen by electron microscopy. Using a variety of cell and molecular biological approaches we discovered that erythroid cells in this condition show a delay during terminal erythroid differentiation, associated with increased proliferation and widespread changes in chromatin accessibility. We also show that the proteins encoded by CDAN1 and CDIN1 are enriched in nucleoli which are structurally and functionally abnormal in CDA-I. Together these findings provide important pointers to the pathways affected in CDA-I which for the first time can now be pursued in the tractable culture system utilised here.

KMT1 family methyltransferases regulate heterochromatin-nuclear periphery tethering via histone and non-histone protein methylation.

Euchromatic histone methyltransferases (EHMTs), members of the KMT1 family, methylate histone and non-histone proteins. Here, we uncover a novel role for EHMTs in regulating heterochromatin anchorage to the nuclear periphery (NP) via non-histone methylation. We show that EHMTs methylate and stabilize LaminB1 (LMNB1), which associates with the H3K9me2-marked peripheral heterochromatin. Loss of LMNB1 methylation or EHMTs abrogates heterochromatin anchorage at the NP We further demonstrate that the loss of EHMTs induces many hallmarks of aging including global reduction of H3K27methyl marks and altered nuclear morphology. Consistent with this, we observe a gradual depletion of EHMTs, which correlates with loss of methylated LMNB1 and peripheral heterochromatin in aging human fibroblasts. Restoration of EHMT expression reverts peripheral heterochromatin defects in aged cells. Collectively, our work elucidates a new mechanism by which EHMTs regulate heterochromatin domain organization and reveals their impact on fundamental changes associated with the intrinsic aging process.

Tissue-specific splicing of a ubiquitously expressed transcription factor is essential for muscle differentiation.

Alternate splicing contributes extensively to cellular complexity by generating protein isoforms with divergent functions. However, the role of alternate isoforms in development remains poorly understood. Mef2 transcription factors are essential transducers of cell signaling that modulate differentiation of many cell types. Among Mef2 family members, Mef2D is unique, as it undergoes tissue-specific splicing to generate a muscle-specific isoform. Since the ubiquitously expressed (Mef2Dα1) and muscle-specific (Mef2Dα2) isoforms of Mef2D are both expressed in muscle, we examined the relative contribution of each Mef2D isoform to differentiation. Using both in vitro and in vivo models, we demonstrate that Mef2D isoforms act antagonistically to modulate differentiation. While chromatin immunoprecipitation (ChIP) sequencing analysis shows that the Mef2D isoforms bind an overlapping set of genes, only Mef2Dα2 activates late muscle transcription. Mechanistically, the differential ability of Mef2D isoforms to activate transcription depends on their susceptibility to phosphorylation by protein kinase A (PKA). Phosphorylation of Mef2Dα1 by PKA provokes its association with corepressors. Conversely, exon switching allows Mef2Dα2 to escape this inhibitory phosphorylation, permitting recruitment of Ash2L for transactivation of muscle genes. Thus, our results reveal a novel mechanism in which a tissue-specific alternate splicing event has evolved that permits a ubiquitously expressed transcription factor to escape inhibitory signaling for temporal regulation of gene expression.

Single-cell fate decisions of bipotential hematopoietic progenitors.

PURPOSE OF REVIEW: In hematopoiesis, rapid cell fate decisions are necessary for timely responses to environmental stimuli resulting in the production of diverse types of blood cells. Early studies have led to a hierarchical, tree-like view of hematopoiesis with hematopoietic stem cells residing at the apex and serially branching out to give rise to bipotential progenitors with increasingly restricted lineage potential. Recent single-cell studies have challenged some aspects of the classical model of hematopoiesis. Here, we review the latest articles on cell fate decision in hematopoietic progenitors, highlighting single-cell studies that have questioned previously established concepts and those that have reaffirmed them. RECENT FINDINGS: The hierarchical organization of hematopoiesis and the importance of transcription factors have been largely validated at the single-cell level. In contrast, single-cell studies have shown that lineage commitment is progressive rather than switch-like as originally proposed. Furthermore, the reconstruction of cell fate paths suggested the existence of a gradient of hematopoietic progenitors that are in a continuum of changing fate probabilities rather than in a static bipotential state, leading us to reconsider the notion of bipotential progenitors. SUMMARY: Single-cell transcriptomic and proteomic studies have transformed our view of lineage commitment and offer a drastically different perspective on hematopoiesis.

Differential genomic targeting of the transcription factor TAL1 in alternate haematopoietic lineages.

TAL1/SCL is a master regulator of haematopoiesis whose expression promotes opposite outcomes depending on the cell type: differentiation in the erythroid lineage or oncogenesis in the T-cell lineage. Here, we used a combination of ChIP sequencing and gene expression profiling to compare the function of TAL1 in normal erythroid and leukaemic T cells. Analysis of the genome-wide binding properties of TAL1 in these two haematopoietic lineages revealed new insight into the mechanism by which transcription factors select their binding sites in alternate lineages. Our study shows limited overlap in the TAL1-binding profile between the two cell types with an unexpected preference for ETS and RUNX motifs adjacent to E-boxes in the T-cell lineage. Furthermore, we show that TAL1 interacts with RUNX1 and ETS1, and that these transcription factors are critically required for TAL1 binding to genes that modulate T-cell differentiation. Thus, our findings highlight a critical role of the cellular environment in modulating transcription factor binding, and provide insight into the mechanism by which TAL1 inhibits differentiation leading to oncogenesis in the T-cell lineage.

Maintenance of gene silencing by the coordinate action of the H3K9 methyltransferase G9a/KMT1C and the H3K4 demethylase Jarid1a/KDM5A.

Chromatin remodeling is essential for controlling the expression of genes during development. The histone-modifying enzyme G9a/KMT1C can act both as a coactivator and a corepressor of transcription. Here, we show that the dual function of G9a as a coactivator vs. a corepressor entails its association within two distinct protein complexes, one containing the coactivator Mediator and one containing the corepressor Jarid1a/KDM5A. Functionally, G9a is important in stabilizing the Mediator complex for gene activation, whereas its repressive function entails a coordinate action with the histone H3 lysine 4 (H3K4) demethylase Jarid1a for the maintenance of gene repression. The essential nature of cross-talk between the histone methyltransferase G9a and the demethylase Jarid1a is demonstrated on the embryonic E(y)-globin gene, where the concurrent introduction of repressive histone marks (dimethylated H3K9 and dimethylated H3K27) and removal of activating histone mark (trimethylated H3K4) is required for maintenance of gene silencing. Taken together with our previous demonstration of cross-talk between UTX and MLL2 to mediate activation of the adult ß(maj)-globin gene, these data suggest a model where "active" and "repressive" cross-talk between histone-modifying enzymes coexist on the same multigene locus and play a crucial role in the precise control of developmentally regulated gene expression.

UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis.

Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically to establish tissue-specific patterns of gene expression. The PcG protein Ezh2 facilitates repression by catalysing histone H3-Lys27 trimethylation (H3K27me3). For expression, H3K27me3 marks are removed and replaced by TrxG protein catalysed histone H3-Lys4 trimethylation (H3K4me3). Although H3K27 demethylases have been identified, the mechanism by which these enzymes are targeted to specific genomic regions to remove H3K27me3 marks has not been established. Here, we demonstrate a two-step mechanism for UTX-mediated demethylation at muscle-specific genes during myogenesis. Although the transactivator Six4 initially recruits UTX to the regulatory region of muscle genes, the resulting loss of H3K27me3 marks is limited to the region upstream of the transcriptional start site. Removal of the repressive H3K27me3 mark within the coding region then requires RNA Polymerase II (Pol II) elongation. Interestingly, blocking Pol II elongation on transcribed genes leads to increased H3K27me3 within the coding region, and formation of bivalent (H3K27me3/H3K4me3) chromatin domains. Thus, removal of repressive H3K27me3 marks by UTX occurs through targeted recruitment followed by spreading across the gene.

Cell fate decision in erythropoiesis: Insights from multiomics studies.

Every second, the body produces 2 million red blood cells through a process called erythropoiesis. Erythropoiesis is hierarchical in that it results from a series of cell fate decisions whereby hematopoietic stem cells progress toward the erythroid lineage. Single-cell transcriptomic and proteomic approaches have revolutionized the way we understand erythropoiesis, revealing it to be a gradual process that underlies a progressive restriction of fate potential driven by quantitative changes in lineage-specifying transcription factors. Despite these major advances, we still know very little about what cell fate decision entails at the molecular level. Novel approaches that simultaneously measure additional properties in single cells, including chromatin accessibility, transcription factor binding, and/or cell surface proteins are being developed at a fast pace, providing the means to exciting new advances in the near future. In this review, we briefly summarize the main findings obtained from single-cell studies of erythropoiesis, highlight outstanding questions, and suggest recent technological advances to address them.

Yin Yang 1 regulates cohesin complex protein SMC3 in mouse hematopoietic stem cells.

ABSTRACT: Yin Yang 1 (YY1) and structural maintenance of chromosomes 3 (SMC3) are 2 critical chromatin structural factors that mediate long-distance enhancer-promoter interactions and promote developmentally regulated changes in chromatin architecture in hematopoietic stem/progenitor cells (HSPCs). Although YY1 has critical functions in promoting hematopoietic stem cell (HSC) self-renewal and maintaining HSC quiescence, SMC3 is required for proper myeloid lineage differentiation. However, many questions remain unanswered regarding how YY1 and SMC3 interact with each other and affect hematopoiesis. We found that YY1 physically interacts with SMC3 and cooccupies with SMC3 at a large cohort of promoters genome wide, and YY1 deficiency deregulates the genetic network governing cell metabolism. YY1 occupies the Smc3 promoter and represses SMC3 expression in HSPCs. Although deletion of 1 Smc3 allele partially restores HSC numbers and quiescence in YY1 knockout mice, Yy1-/-Smc3+/- HSCs fail to reconstitute blood after bone marrow transplant. YY1 regulates HSC metabolic pathways and maintains proper intracellular reactive oxygen species levels in HSCs, and this regulation is independent of the YY1-SMC3 axis. Our results establish a distinct YY1-SMC3 axis and its impact on HSC quiescence and metabolism.

Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

GSK3 temporally regulates neurogenin 2 proneural activity in the neocortex.

The neocortex is comprised of six neuronal layers that are generated in a defined temporal sequence. While extrinsic and intrinsic cues are known to regulate the sequential production of neocortical neurons, how these factors interact and function in a coordinated manner is poorly understood. The proneural gene Neurog2 is expressed in progenitors throughout corticogenesis, but is only required to specify early-born, deep-layer neuronal identities. Here, we examined how neuronal differentiation in general and Neurog2 function in particular are temporally controlled during murine neocortical development. We found that Neurog2 proneural activity declines in late corticogenesis, correlating with its phosphorylation by GSK3 kinase. Accordingly, GSK3 activity, which is negatively regulated by canonical Wnt signaling, increases over developmental time, while Wnt signaling correspondingly decreases. When ectopically activated, GSK3 inhibits Neurog2-mediated transcription in cultured cells and Neurog2 proneural activities in vivo. Conversely, a reduction in GSK3 activity promotes the precocious differentiation of later stage cortical progenitors without influencing laminar fate specification. Mechanistically, we show that GSK3 suppresses Neurog2 activity by influencing its choice of dimerization partner, promoting heterodimeric interactions with E47 (Tcfe2a), as opposed to Neurog2-Neurog2 homodimer formation, which occurs when GSK3 activity levels are low. At the functional level, Neurog2-E47 heterodimers have a reduced ability to transactivate neuronal differentiation genes compared with Neurog2-Neurog2 homodimers, both in vitro and in vivo. We thus conclude that the temporal regulation of Neurog2-E47 heterodimerization by GSK3 is a central component of the neuronal differentiation "clock" that coordinates the timing and tempo of neocortical neurogenesis in mouse.

A method for cell type marker discovery by high-throughput gene expression analysis of mixed cell populations.

BACKGROUND: Gene transcripts specifically expressed in a particular cell type (cell-type specific gene markers) are useful for its detection and isolation from a tissue or other cell mixtures. However, finding informative marker genes can be problematic when working with a poorly characterized cell type, as markers can only be unequivocally determined once the cell type has been isolated. We propose a method that could identify marker genes of an uncharacterized cell type within a mixed cell population, provided that the proportion of the cell type of interest in the mixture can be estimated by some indirect method, such as a functional assay. RESULTS: We show that cell-type specific gene markers can be identified from the global gene expression of several cell mixtures that contain the cell type of interest in a known proportion by their high correlation to the concentration of the corresponding cell type across the mixtures. CONCLUSIONS: Genes detected using this high-throughput strategy would be candidate markers that may be useful in detecting or purifying a cell type from a particular biological context. We present an experimental proof-of-concept of this method using cell mixtures of various well-characterized hematopoietic cell types, and we evaluate the performance of the method in a benchmark that explores the requirements and range of validity of the approach.