The pluripotent state of embryonic stem cells (ESCs) provides a unique perspective on regulatory programs that govern self-renewal and differentiation and somatic cell reprogramming. Here, we review ...the highly connected protein and transcriptional networks that maintain pluripotency and how they are intertwined with factors that affect chromatin structure and function. The complex interrelationships between pluripotency and chromatin factors are illustrated by X chromosome inactivation, regulatory control by noncoding RNAs, and environmental influences on cell states. Manipulation of cell state through the process of transdifferentiation suggests that environmental cues may direct transcriptional programs as cells enter a transiently “plastic” state during reprogramming.
Long noncoding RNAs (lncRNAs) have been implicated in controlling various aspects of embryonic stem cell (ESC) biology, although the functions of specific lncRNAs, and the molecular mechanisms ...through which they act, remain unclear. Here, we demonstrate discrete and opposing roles for the lncRNA transcript Haunt and its genomic locus in regulating the HOXA gene cluster during ESC differentiation. Reducing or enhancing Haunt expression, with minimal disruption of the Haunt locus, led to upregulation or downregulation of HOXA genes, respectively. In contrast, increasingly large genomic deletions within the Haunt locus attenuated HOXA activation. The Haunt DNA locus contains potential enhancers of HOXA activation, whereas Haunt RNA acts to prevent aberrant HOXA expression. This work reveals a multifaceted model of lncRNA-mediated transcriptional regulation of the HOXA cluster, with distinct roles for a lncRNA transcript and its genomic locus, while illustrating the power of rapid CRISPR/Cas9-based genome editing for assigning lncRNA functions.
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•The lncRNA Haunt and its genomic locus play distinct roles in HOXA gene activation•The Haunt DNA locus contains potential HOXA enhancers•Haunt RNA binds to chromatin and acts to prevent aberrant HOXA expression•Haunt orchestrates proper ESC differentiation
Dissection of the precise contribution of RNA transcripts versus genomic DNA sequences is important for functional assessment of long noncoding RNAs. Here, Shen and colleagues demonstrate discrete and opposing roles for the lncRNA transcript Haunt and its genomic locus in regulating the HOXA gene cluster during embryonic stem cell differentiation.
The genetic basis of sickle cell disease (SCD) was elucidated >60 years ago, yet current therapy does not rely on this knowledge. Recent advances raise prospects for improved, and perhaps curative, ...treatment. First, transcription factors, BCL11A and LRF/ZBTB7A, that mediate silencing of the β-like fetal (γ-) globin gene after birth have been identified and demonstrated to act at the γ-globin promoters, precisely at recognition sequences disrupted in rare individuals with hereditary persistence of fetal hemoglobin. Second, transformative advances in gene editing and progress in lentiviral gene therapy provide diverse opportunities for genetic strategies to cure SCD. Approaches include hematopoietic gene therapy by globin gene addition, gene editing to correct the SCD mutation, and genetic manipulations to enhance fetal hemoglobin production, a potent modifier of the clinical phenotype. Clinical trials may soon identify efficacious and safe genetic approaches to the ultimate goal of cure for SCD.
Much attention has focused on a small set of transcription factors that maintain human or mouse embryonic stem (ES) cells in a pluripotent state. To gain a more complete understanding of the ...regulatory network that maintains this state, we identified target promoters of nine transcription factors, including somatic cell reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) and others (Nanog, Dax1, Rex1, Zpf281, and Nac1), on a global scale in mouse ES cells. We found that target genes fall into two classes: promoters bound by few factors tend to be inactive or repressed, whereas promoters bound by more than four factors are largely active in the pluripotent state and become repressed upon differentiation. Furthermore, we propose a transcriptional hierarchy for reprogramming factors and broadly distinguish targets of c-Myc versus other factors. Our data provide a resource for exploration of the complex network maintaining pluripotency.
The major disorders of β-globin, sickle cell disease and β-thalassemia, may be ameliorated by expression of the fetal gene paralog γ-globin. Uncertainty regarding the mechanisms repressing fetal ...hemoglobin in the adult stage has served as a puzzle of developmental gene regulation as well as a barrier to rational therapeutic design. Recent genome-wide association studies implicated the zinc-finger transcriptional repressor BCL11A in fetal hemoglobin regulation. Extensive genetic analyses have validated BCL11A as a potent repressor of fetal hemoglobin level. Studies of BCL11A exemplify how contextual gene regulation may often be the substrate for trait-associated common genetic variation. These discoveries have suggested novel rational approaches for the β-hemoglobin disorders including therapeutic genome editing.
Recent studies point to a pivotal role of Polycomb repressive complex 2 (PRC2) in stem cell function and cancer. Loss-of-function approaches targeting individual PRC2 subunits have, however, ...generated findings that are difficult to reconcile. Here, we prevent assembly of both Ezh1- and Ezh2-containing PRC2 complexes by conditional deletion of Eed, a core subunit, and assess hematopoiesis. We find that deletion of Eed exhausts adult bone marrow hematopoietic stem cells (HSCs), although fetal liver HSCs are produced in normal numbers. Eed-null neonatal HSCs express HSC signature genes but are defective in maintenance and differentiation. Comparative gene expression profiling revealed that neonatal and adult HSCs lacking Eed upregulated gene sets of conflicting pathways. Deletion of Cdkn2a, a PRC2 target gene, in Eed-null mice enhances hematopoietic stem/progenitor cell (HSPC) survival but fails to restore HSC functions. Taken together, our findings define developmental-stage-specific requirements for canonical PRC2 complexes in normal HSC function.
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•Ezh2 is dispensable for HSC formation and maintenance•Loss of Eed leads to HSC exhaustion in adults•PRC2 suppresses diverse classes of genes with conflicting functions•Deletion of Cdkn2a partially rescues HSC defects of Eed loss
Orkin and colleagues show that canonical PRC2 activity is required for hematopoietic stem cell differentiation during development and maintenance during adulthood, as revealed by conditional deletion of Eed.
Establishment and maintenance of the blood system relies on self-renewing hematopoietic stem cells (HSCs) that normally reside in small numbers in the bone marrow niche of adult mammals. This Review ...describes the developmental origins of HSCs and the molecular mechanisms that regulate lineage-specific differentiation. Studies of hematopoiesis provide critical insights of general relevance to other areas of stem cell biology including the role of cellular interactions in development and tissue homeostasis, lineage programming and reprogramming by transcription factors, and stage- and age-specific differences in cellular phenotypes.
Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for ...complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based “single-cell MCA analysis” pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.
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•Development of Microwell-seq, a high-throughput and low-cost scRNA-seq platform•Construction of a single-cell mouse cell atlas (scMCA) covering major cell types•Characterization of cellular heterogeneity with minimal batch effect•Characterization of cross-tissue cellular network at the single-cell level
Development of Microwell-seq allows construction of a mouse cell atlas at the single-cell level with a high-throughput and low-cost platform.
Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal ...in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.
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•CARLIN is a stable, genetically defined mouse line for CRISPR-based lineage tracing•Can be activated at any point to generate 44,000 transcribed barcodes across tissues•Sequential, pulsed induction can be used to determine cellular phylogeny in vivo•Heterogeneity in HSC proliferation following myeloablation revealed
CARLIN is a mouse model that allows for simultaneous analysis of lineage and transcriptomic information of single cells in vivo.
We introduce CUT&RUNTools as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN ...primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short-read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. CUT&RUNTools is available at https://bitbucket.org/qzhudfci/cutruntools/ .
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