Control Cell Identity Research Articles

From Genotype to Phenotype: How Enhancers Control Gene Expression and Cell Identity in Hematopoiesis.

Blood comprises a wide array of specialized cells, all of which share the same genetic information and ultimately derive from the same precursor, the hematopoietic stem cell (HSC). This diversity of phenotypes is underpinned by unique transcriptional programs gradually acquired in the process known as hematopoiesis. Spatiotemporal regulation of gene expression depends on many factors, but critical among them are enhancers-sequences of DNA that bind transcription factors and increase transcription of genes under their control. Thus, hematopoiesis involves the activation of specific enhancer repertoires in HSCs and their progeny, driving the expression of sets of genes that collectively determine morphology and function. Disruption of this tightly regulated process can have catastrophic consequences: in hematopoietic malignancies, dysregulation of transcriptional control by enhancers leads to misexpression of oncogenes that ultimately drive transformation. This review attempts to provide a basic understanding of enhancers and their role in transcriptional regulation, with a focus on normal and malignant hematopoiesis. We present examples of enhancers controlling master regulators of hematopoiesis and discuss the main mechanisms leading to enhancer dysregulation in leukemia and lymphoma.

CTCF coordinates cell fate specification via orchestrating regulatory hubs with pioneer transcription factors

CCCTC-binding factor (CTCF), a ubiquitously expressed architectural protein, has emerged as a key regulator of cell identity gene transcription. However, the precise molecular mechanism underlying specialized functions of CTCF remains elusive. Here, we investigate the mechanism through integrative analyses of primary hepatocytes, myocytes, and B cells from mouse and human. We demonstrate that CTCF cooperates with lineage-specific pioneer transcription factors (TFs), including MyoD, FOXA, and PU.1, to control cell identity at 1D and 3D levels. At the 1D level, pioneer TFs facilitate lineage-specific CTCF occupancy via opening chromatin. At the 3D level, CTCF and pioneer TFs form regulatory hubs to govern the expression of cellidentity genes. This mechanism is validated using MyoD-null mice, CTCF knockout mice, and CRISPR editing during myogenic differentiation. Collectively, these findings uncover a general mechanism whereby CTCF acts as a cell identity cofactor to control cell identity genes via orchestrating regulatory hubs with pioneer TFs.

Mitotic bookmarking by SWI/SNF subunits.

For cells to initiate and sustain a differentiated state, it is necessary that a 'memory' of this state is transmitted through mitosis to the daughter cells1-3. Mammalian switch/sucrose non-fermentable (SWI/SNF) complexes (also known as Brg1/Brg-associated factors, or BAF) control cell identity by modulating chromatin architecture to regulate gene expression4-7, but whether they participate in cell fate memory is unclear. Here we provide evidence that subunits of SWI/SNF act as mitotic bookmarks to safeguard cell identity during cell division. The SWI/SNF core subunits SMARCE1 and SMARCB1 are displaced from enhancers but are bound to promoters during mitosis, and we show that this binding is required for appropriate reactivation of bound genes after mitotic exit. Ablation of SMARCE1 during a single mitosis in mouse embryonic stem cells is sufficient to disrupt gene expression, impair the occupancy of several established bookmarks at a subset of their targets and cause aberrant neural differentiation. Thus, SWI/SNF subunit SMARCE1 has a mitotic bookmarking role and is essential for heritable epigenetic fidelity during transcriptional reprogramming.

Pattern formation on dynamic membranes.

Pattern formation defines cell identities and plays a crucial role in arguably all membrane functionality such are cell division, motility, communication, adhesion, etc. Cell often evolves by changing their shape, which translates to membrane dynamics, hence leading to pattern formation on dynamic surfaces. While pattern formation on static shapes has been intensively studied both theoretically and experimentally, the understanding of patterning on moving shapes remains largely elusive. We study force distribution on dynamic but initially spherical or cylindrical shapes by our surface dynamics equations. For simplicity, we consider small deformation of incompressible sphere and cylinder and show that shape dynamics induce nonuniform force distributions, therefore clarifying spontaneous polarisation, symmetry breaking, and rich static and moving pattern formations. Based on surface dynamics equations we derive a generic Young-Laplace equation which indicates that practically any static and dynamic patterns are possible in incompressible systems and if so, the question is what is the underlying mechanism that controls cell identity? The answer to the problem is again a surface dynamics equation that guarantees mass conservation. In other words, dynamic surfaces induce dynamic surface mass density that is governed by a generic mass balance equation and the last one controls the identity. While mass balance itself is regulated by an active or passive influx of material particles on the surface. The last statement for cell membrane means that membrane mass balance is in full correlation with cell-governed reaction, synthesis, and diffusion. The same statement can be reached for the charge balance equation which is also part of surface dynamics equations.

Complexity of enhancer networks predicts cell identity and disease genes revealed by single-cell multi-omics analysis.

Many enhancers exist as clusters in the genome and control cell identity and disease genes; however, the underlying mechanism remains largely unknown. Here, we introduce an algorithm, eNet, to build enhancer networks by integrating single-cell chromatin accessibility and gene expression profiles. The complexity of enhancer networks is assessed by two metrics: the number of enhancers and the frequency of predicted enhancer interactions (PEIs) based on chromatin co-accessibility. We apply eNetalgorithm to a human blood dataset and find cell identity and disease genes tend to be regulated by complex enhancer networks. The network hub enhancers (enhancers with frequent PEIs) are the most functionally important. Compared with super-enhancers, enhancer networks show better performance in predicting cell identity and disease genes. eNet is robust and widely applicable in various human or mouse tissues datasets. Thus, we propose a model of enhancer networks containing three modes: Simple, Multiple and Complex, which are distinguished by their complexity in regulating gene expression. Taken together, our work provides an unsupervised approach to simultaneously identify key cell identity and disease genes and explore the underlying regulatory relationships among enhancers in single cells.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Super-enhancers conserved within placental mammals maintain stem cell pluripotency

Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share an evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that displayed remarkable conservation in placental mammals and were sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR-Cas9 approach severely impaired stem cell pluripotency. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.

Dissecting super-enhancer heterogeneity: time to re-examine cancer subtypes?

The heterogeneity of transcriptional regulations by super-enhancers (SEs) is poorly understood in human cancers. Herein, we summarize a bioinformatics workflow for genome-wide SE profiling and identification of subtype-specific SEs and regulatory networks. Dissecting SE heterogeneity provides new insights into cancer biology and alternative therapeutic strategies for cancer precision medicine.

DIRECT-NET: An efficient method to discover cis-regulatory elements and construct regulatory networks from single-cell multiomics data.

The emergence of single-cell multiomics data provides unprecedented opportunities to scrutinize the transcriptional regulatory mechanisms controlling cell identity. However, how to use those datasets to dissect the cis-regulatory element (CRE)–to–gene relationships at a single-cell level remains a major challenge. Here, we present DIRECT-NET, a machine-learning method based on gradient boosting, to identify genome-wide CREs and their relationship to target genes, either from parallel single-cell gene expression and chromatin accessibility data or from single-cell chromatin accessibility data alone. By extensively evaluating and characterizing DIRECT-NET’s predicted CREs using independent functional genomics data, we find that DIRECT-NET substantially improves the accuracy of inferring CRE-to-gene relationships in comparison to existing methods. DIRECT-NET is also capable of revealing cell subpopulation–specific and dynamic regulatory linkages. Overall, DIRECT-NET provides an efficient tool for predicting transcriptional regulation codes from single-cell multiomics data.

Inference of age-associated transcription factor regulatory activity changes in single cells.

Transcription factors (TFs) control cell identity and function. How their activity is altered during healthy aging is critical for an improved understanding of aging and disease risk, yet relatively little is known about such changes at cell-type resolution. Here we present and validate a TF activity estimation method for single cells from the hematopoietic system that is based on TF regulons, and apply it to a mouse single-cell RNA-sequencing atlas, to infer age-associated differentiation activity changes in the immune cells of different organs. This revealed an age-associated signature of macrophage dedifferentiation, which is shared across tissue types, and aggravated in tumor-associated macrophages. By extending the analysis to all major cell types, we reveal cell-type and tissue-type-independent age-associated alterations to regulatory factors controlling antigen processing, inflammation, collagen processing and circadian rhythm, that are implicated in age-related diseases. Finally, our study highlights the limitations of using TF expression to infer age-associated changes, underscoring the need to use regulatory activity inference methods.

The molecular understanding of super-enhancer dysregulation in cancer.

ABSTRACTAbnormalities in the regulation of gene expression are associated with various pathological conditions. Among the distal regulatory elements in the genome, the activation of target genes by enhancers plays a central role in the formation of cell type–specific gene expression patterns. Super-enhancers are a subclass of enhancers that frequently contain multiple enhancer-like elements and are characterized by dense binding of master transcription factors and Mediator complexes and high signals of active histone marks. Super-enhancers have been studied in detail as important regulatory regions that control cell identity and contribute to the pathogenesis of diverse diseases. In cancer, super-enhancers have multifaceted roles by activating various oncogenes and other cancer-related genes and shaping characteristic gene expression patterns in cancer cells. Alterations in super-enhancer activities in cancer involve multiple mechanisms, including the dysregulation of transcription factors and the super-enhancer–associated genomic abnormalities. The study of super-enhancers could contribute to the identification of effective biomarkers and the development of cancer therapeutics targeting transcriptional addiction. In this review, we summarize the roles of super-enhancers in cancer biology, with a particular focus on hematopoietic malignancies, in which multiple super-enhancer alteration mechanisms have been reported.

Improving prediction of core transcription factors for cell reprogramming and transdifferentiation

Identification of transcription factors (TFs) that could induce and direct cell conversion remains a challenge. Though several hundreds of TFs are usually transcribed in each cell type, the identity of a cell is controlled and can be achieved through the ectopic overexpression of only a small subset of so-called core TFs. Currently, the experimental identification of the core TFs for a broad spectrum of cell types remains challenging. Computational solutions to this problem would provide a better understanding of the mechanisms controlling cell identity during natural embryonic or malignant development, as well as give a foundation for cell-based therapy. Herein, we propose a computational approach based on over-enrichment of transcription factors binding sites (TFBS) in differentially accessible chromatin regions that could identify the potential core TFs for a variety of primary human cells involved in hematopoiesis. Our approach enables the integration of both transcriptomic (single-cell RNA sequencing, scRNA-seq) and epigenenomic (single-cell assay for transposable-accessible chromatin, scATAC-seq) data at the single-cell resolution to search for core TFs, and can be scalable to predict subsets of core TFs and their role in a given conversion between cells.

Super-Enhancers Dysregulations in Hematological Malignancies.

Hematological malignancies affecting either the lymphoid or the myeloid lineages involve epigenetic mutations or dysregulation in the majority of cases. These epigenetic abnormalities can affect regulatory elements in the genome and, particularly, enhancers. Recently, large regulatory elements known as super-enhancers, initially identified for their critical roles in cell-type specific expression regulation of genes controlling cell identity, have been shown to also be involved in tumorigenesis in many cancer types and hematological malignancies via the regulation of numerous oncogenes, including MYC. In this review, we highlight the existing links between super-enhancers and hematological malignancies, with a particular focus on acute myeloid leukemia, a clonal hematopoietic neoplasm with dismal outcomes, resulting in an uncontrolled proliferation of myeloblasts, abnormally blocked during differentiation and accumulating within the patient’s bone marrow. We report recent works, performed during the last few years, treating this subject and consider the possibility of targeting oncogenic regulatory elements, as well as the effectiveness and limitations reported so far for such strategies.

Insights on life cycle and cell identity regulatory circuits for unlocking genetic improvement in Zygosaccharomyces and Kluyveromyces yeasts.

ABSTRACTEvolution has provided a vast diversity of yeasts that play fundamental roles in nature and society. This diversity is not limited to genotypically homogeneous species with natural interspecies hybrids and allodiploids that blur species boundaries frequently isolated. Thus, life cycle and the nature of breeding systems have profound effects on genome variation, shaping heterozygosity, genotype diversity and ploidy level. The apparent enrichment of hybrids in industry-related environments suggests that hybridization provides an adaptive route against stressors and creates interest in developing new hybrids for biotechnological uses. For example, in the Saccharomyces genus where regulatory circuits controlling cell identity, mating competence and meiosis commitment have been extensively studied, this body of knowledge is being used to combine interesting traits into synthetic F1 hybrids, to bypass F1 hybrid sterility and to dissect complex phenotypes by bulk segregant analysis. Although these aspects are less known in other industrially promising yeasts, advances in whole-genome sequencing and analysis are changing this and new insights are being gained, especially in the food-associated genera Zygosaccharomyces and Kluyveromyces. We discuss this new knowledge and highlight how deciphering cell identity circuits in these lineages will contribute significantly to identify the genetic determinants underpinning complex phenotypes and open new avenues for breeding programmes.

DeepSE: Detecting super-enhancers among typical enhancers using only sequence feature embeddings

Super-enhancer (SE) is a cluster of active typical enhancers (TE) with high levels of the Mediator complex, master transcriptional factors, and chromatin regulators. SEs play a key role in the control of cell identity and disease. Traditionally, scientists used a variety of high-throughput data of different transcriptional factors or chromatin marks to distinguish SEs from TEs. This kind of experimental methods are usually costly and time-consuming. In this paper, we proposed a model DeepSE, which is based on a deep convolutional neural network model, to distinguish the SEs from TEs. DeepSE represent the DNA sequences using the dna2vec feature embeddings. With only the DNA sequence information, DeepSE outperformed all state-of-the-art methods. In addition, DeepSE can be generalized well across different cell lines, which implied that cell-type specific SEs may share hidden sequence patterns across different cell lines. The source code and data are stored in GitHub (https://github.com/QiaoyingJi/DeepSE).

Super Enhancer Profiles Identify Key Cell Identity Genes During Differentiation From Embryonic Stem Cells to Trophoblast Stem Cells Super Enhencers in Trophoblast Differentiation.

Trophoblast stem cells (TSCs) are derived from blastocysts and the extra-embryonic ectoderm (ExE) of post-implantation embryos and play a significant role in fetal development, but the roles that TSCs play in the earlier status of fetal diseases need further exploration. Super enhancers (SEs) are dense clusters of stitched enhancers that control cell identity determination and disease development and may participate in TSC differentiation. We identified key cell identity genes regulated by TSC-SEs via integrated analysis of H3K27ac and H3K4me1 chromatin immunoprecipitation sequencing (ChIP-seq), RNA-sequencing (RNA-seq) and ATAC-sequencing (ATAC-seq) data. The identified key TSC identity genes regulated by SEs, such as epidermal growth factor receptor (EGFR), integrin β5 (ITGB5) and Paxillin (Pxn), were significantly upregulated during TSC differentiation, and the transcription network mediated by TSC-SEs enriched in terms like focal adhesion and actin cytoskeleton regulation related to differentiation of TSCs. Additionally, the increased chromatin accessibility of the key cell identity genes verified by ATAC-seq further demonstrated the regulatory effect of TSC-SEs on TSC lineage commitment. Our results illustrated the significant roles of the TSC-SE-regulated network in TSC differentiation, and identified key TSC identity genes EGFR, ITGB5 and Pxn, providing novel insight into TSC differentiation and lays the foundation for future studies on embryo implantation and related diseases.

H3K4 di-methylation governs smooth muscle lineage identity and promotes vascular homeostasis by restraining plasticity.

Epigenetic mechanisms contribute to the regulation of cell differentiation and function. Vascular smooth muscle cells (SMCs) are specialized contractile cells that retain phenotypic plasticity even after differentiation. Here, by performing selective demethylation of histone H3 lysine 4 di-methylation (H3K4me2) at SMC-specific genes, we uncovered that H3K4me2 governs SMC lineage identity. Removal of H3K4me2 via selective editing in cultured vascular SMCs and in murine arterial vasculature led to loss of differentiation and reduced contractility due to impaired recruitment of the DNA methylcytosine dioxygenase TET2. H3K4me2 editing altered SMC adaptative capacities during vascular remodeling due to loss of miR-145 expression. Finally, H3K4me2 editing induced a profound alteration of SMC lineage identity by redistributing H3K4me2 toward genes associated with stemness and developmental programs, thus exacerbating plasticity. Our studies identify the H3K4me2-TET2-miR145 axis as a central epigenetic memory mechanism controlling cell identity and function, whose alteration could contribute to various pathophysiological processes.

Single-nuclei chromatin profiling of ventral midbrain reveals cell identity transcription factors and cell-type-specific gene regulatory variation

BackgroundCell types in ventral midbrain are involved in diseases with variable genetic susceptibility, such as Parkinson’s disease and schizophrenia. Many genetic variants affect regulatory regions and alter gene expression in a cell-type-specific manner depending on the chromatin structure and accessibility.ResultsWe report 20,658 single-nuclei chromatin accessibility profiles of ventral midbrain from two genetically and phenotypically distinct mouse strains. We distinguish ten cell types based on chromatin profiles and analysis of accessible regions controlling cell identity genes highlights cell-type-specific key transcription factors. Regulatory variation segregating the mouse strains manifests more on transcriptome than chromatin level. However, cell-type-level data reveals changes not captured at tissue level. To discover the scope and cell-type specificity of cis-acting variation in midbrain gene expression, we identify putative regulatory variants and show them to be enriched at differentially expressed loci. Finally, we find TCF7L2 to mediate trans-acting variation selectively in midbrain neurons.ConclusionsOur data set provides an extensive resource to study gene regulation in mesencephalon and provides insights into control of cell identity in the midbrain and identifies cell-type-specific regulatory variation possibly underlying phenotypic and behavioural differences between mouse strains.

Integrative analysis of genomic and epigenomic data reveal underlying superenhancer-mediated microRNA regulatory network for human bone mineral density.

Bone mineral density (BMD) is a highly heritable complex trait and is a key indicator for diagnosis and treatment for osteoporosis. In the last decade, numerous susceptibility loci for BMD and fracture have been identified by genome-wide association studies (GWAS); however, fine mapping of these loci is challengeable. Here, we proposed a new long-range fine-mapping approach that combined superenhancers (SEs) and microRNAs (miRNAs) data, which were two important factors in control of cell identity and specific differentiation, with the GWAS summary datasets in cell-type-restricted way. Genome-wide SE-based analysis found that the BMD-related variants were significantly enriched in the osteoblast SE regions, indicative of potential long-range effects of such SNPs. With the SNP-mapped SEs (mSEs), 13 accessible long-range mSE-interacted miRNAs (mSE-miRNAs) were identified by integrating osteoblast Hi-C and ATAC-seq data, including three known bone-related miRNAs (miR-132-3p, miR-212-3p and miR-125b-5p). The putative targets of the two newly identified mSE-miRNAs (miR-548aj-3p and miR-190a-3p) were found largely enriched in osteogenic-related pathway and processes, suggesting that these mSE-miRNAs could be functional in the regulation of osteoblast differentiation. Furthermore, we identified 54 genes with the long-range ‘mSE-miRNA’ approach, and 24 of them were previously reported to be related to skeletal development. Besides, enrichment analysis found that these genes were specifically enriched in the post-transcriptional regulation and bone formation processes. This study provided a new insight into the approach of fine-mapping of GWAS loci. A tool was provided for the genome-wide SE-based analysis and the detection of long-range osteoblast-restricted mSE-miRNAs (https://github.com/Zheng-Lab-Westlake/Osteo-Fine-Mapp-SNP2SE2miRNA).

Tlx3 Exerts Direct Control in Specifying Excitatory Over Inhibitory Neurons in the Dorsal Spinal Cord.

The spinal cord dorsal horn is a major station for integration and relay of somatosensory information and comprises both excitatory and inhibitory neuronal populations. The homeobox gene Tlx3 acts as a selector gene to control the development of late-born excitatory (dILB) neurons by specifying glutamatergic transmitter fate in dorsal spinal cord. However, since Tlx3 direct transcriptional targets remain largely unknown, it remains to be uncovered how Tlx3 functions to promote excitatory cell fate. Here we combined a genomics approach based on chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and expression profiling, with validation experiments in Tlx3 null embryos, to characterize the transcriptional program of Tlx3 in mouse embryonic dorsal spinal cord. We found most dILB neuron specific genes previously identified to be directly activated by Tlx3. Surprisingly, we found Tlx3 also directly represses many genes associated with the alternative inhibitory dILA neuronal fate. In both cases, direct targets include transcription factors and terminal differentiation genes, showing that Tlx3 directly controls cell identity at distinct levels. Our findings provide a molecular frame for the master regulatory role of Tlx3 in developing glutamatergic dILB neurons. In addition, they suggest a novel function for Tlx3 as direct repressor of GABAergic dILA identity, pointing to how generation of the two alternative cell fates being tightly coupled.