Defining Essential Enhancer for Pluripotent stem cells using Features Oriented CRISPR-Cas9 Screen

ABSTRACTCis Regulatory Elements (CREs) regulate the expression of the genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there remain major gaps in the functional characterization of CREs and the identification of its target genes in the cellular native environment. In this study, we performed a Features Oriented CRISPR Utilized Systematic (FOCUS) screen of OCT4-bound CREs using CRISPR/Cas9 to identify functional enhancers important for pluripotency maintenance in mouse ES cells. From the initial 235 candidates tested, 16 CREs were identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncovered a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway.

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Runx proteins regulate Foxp3 expression

Journal of Experimental Medicine ◽

10.1084/jem.20090226 ◽

2009 ◽

Vol 206 (11) ◽

pp. 2329-2337 ◽

Cited By ~ 66

Author(s):

Ludovica Bruno ◽

Luca Mazzarella ◽

Maarten Hoogenkamp ◽

Arnulf Hertweck ◽

Bradley S. Cobb ◽

...

Keyword(s):

T Cells ◽

Dna Binding ◽

Cd4 T Cells ◽

Target Genes ◽

De Novo ◽

Foxp3 Expression ◽

Regulatory Elements ◽

Dominant Negative ◽

Downstream Target ◽

Runx Proteins

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes.

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Abstract 356: Scl Represses Cardiogenesis via Distant Enhancers during Hemogenic Endothelium Specification

Circulation Research ◽

10.1161/res.113.suppl_1.a356 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Tonis Org ◽

Dan Duan ◽

Roberto Ferrari ◽

Amelie Montel-Hagen ◽

Ben Van Handel ◽

...

Keyword(s):

Target Genes ◽

Es Cells ◽

Bhlh Transcription Factor ◽

Hemogenic Endothelium ◽

Master Regulator ◽

Critical Function ◽

Hematopoietic Stem ◽

Mesoderm Development ◽

Chip Sequencing ◽

Mouse Es Cells

Understanding the mechanisms directing mesoderm specification holds a great potential to advance the development of cell-based therapies for cardiovascular and blood disorders. The bHLH transcription factor Scl is known as the master regulator of the hematopoietic fate. We recently discovered that, in addition to its critical function in promoting the establishment of hemogenic endothelium during hematopoietic stem/progenitor cell (HS/PC) development, Scl is also required to repress cardiomyogenesis in endothelium in hematopoietic tissues and endocardium in the heart. However, the mechanisms for the cardiac repression have remained unknown. Using ChIP-sequencing and microarray analysis of Flk+ mesoderm differentiated from mouse ES cells, we show that Scl both directly activates a broad gene regulatory network required for hemogenic endothelium and HS/PC development (e.g. Runx1, cMyb, Lyl1, Mef2C, Sox7 etc.), and directly represses transcriptional regulators required for cardiogenesis (e.g. Gata4, Gata6, Myocd, etc.) and mesoderm development (Eomes, Mixl1, Etv2, etc.). Repression of cardiac and mesodermal programs occurs during a short developmental window through Scl binding to distant enhancers, while binding to hematopoietic regulators extends throughout HS/PC and red blood cell development and encompasses both distant and proximal binding sites. We also discovered that, surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification and Scl binding to majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors that facilitate HS/PC emergence from hemogenic endothelium. These results denote Scl as a true master regulator of hematopoietic vs. cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates.

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Functional characterization of human Polycomb-like 3 isoforms identifies them as components of distinct EZH2 protein complexes

Biochemical Journal ◽

10.1042/bj20100944 ◽

2011 ◽

Vol 434 (2) ◽

pp. 333-342 ◽

Cited By ~ 26

Author(s):

Gaylor Boulay ◽

Claire Rosnoblet ◽

Cateline Guérardel ◽

Pierre-Olivier Angrand ◽

Dominique Leprince

Keyword(s):

Cell Fate ◽

Target Genes ◽

Protein Complexes ◽

Functional Characterization ◽

Polycomb Group ◽

Phd Finger ◽

Reverse Transcription Pcr ◽

Tudor Domain ◽

Self Association ◽

Polycomb Repressive Complex 1

PcG (Polycomb group) proteins are conserved transcriptional repressors essential to regulate cell fate and to maintain epigenetic cellular memory. They work in concert through two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2–4. In Drosophila, PRC2 contains the H3K27 histone methyltransferase E(Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (Polycomb-like). In the present study, we have examined hPCL3, one of its three human paralogues. Through alternative splicing, hPCL3 encodes a long isoform, hPCL3L, containing an N-terminal TUDOR domain and two PHDs (plant homeodomains) and a smaller isoform, hPCL3S, lacking the second PHD finger (PHD2). By quantitative reverse transcription–PCR analyses, we showed that both isoforms are widely co-expressed at high levels in medulloblastoma. By co-immunoprecipitation analyses, we demonstrated that both isoforms interact with EZH2 through their common TUDOR domain. However, the hPCL3L-specific PHD2 domain, which is better conserved than PHD1 in the PCL family, is also involved in this interaction and implicated in the self-association of hPCL3L. Finally, we have demonstrated that both hPCL3 isoforms are physically associated with EZH2, but in different complexes. Our results provide the first evidence that the two hPCL3 isoforms belong to different complexes and raise important questions about their relative functions, particularly in tumorigenesis.

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Antagonistic BMP–cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1701607114 ◽

2017 ◽

Vol 114 (28) ◽

pp. E5608-E5615 ◽

Cited By ~ 16

Author(s):

Naveen Wijesena ◽

David K. Simmons ◽

Mark Q. Martindale

Keyword(s):

Cell Fate ◽

Target Genes ◽

Bmp Signaling ◽

Nematostella Vectensis ◽

Beta Catenin ◽

Molecular Signaling ◽

Downstream Target ◽

Evolutionary Innovation ◽

Morphogenetic Protein ◽

Cnidarian Nematostella Vectensis

Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta bone morphogenetic protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans, but there has been no direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (an anthozoan cnidarian). Using forward transcriptomics, we show that beta-catenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network (GRN) at the onset of gastrulation (24 h postfertilization) in N. vectensis. Surprisingly, beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oral–aboral axis in Nematostella. We also show functionally that the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in N. vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesodermal tissue.

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Key Mechanisms of Early Lung Development

Pediatric and Developmental Pathology ◽

10.2350/07-06-0290.1 ◽

2007 ◽

Vol 10 (5) ◽

pp. 335-347 ◽

Cited By ~ 54

Author(s):

Jun Kimura ◽

Gail H. Deutsch

Keyword(s):

Respiratory Tract ◽

Cell Fate ◽

Target Genes ◽

Critical Role ◽

Hierarchical Classification ◽

Branching Morphogenesis ◽

Loss Of Function ◽

Innovative Strategies ◽

Downstream Target ◽

Molecular Events

Lung morphogenesis requires the integration of multiple regulatory factors, which results in a functional air-blood interface required for gas exchange at birth. The respiratory tract is composed of endodermally derived epithelium surrounded by cells of mesodermal origin. Inductive signaling between these 2 tissue compartments plays a critical role in formation and differentiation of the lung, which is mediated by evolutionarily conserved signaling families used reiteratively during lung formation, including the fibroblast growth factor, hedgehog, retinoic acid, bone morphogenetic protein, and Wnt signaling pathways. Cells coordinate their response to these signaling proteins largely through transcription factors, which determine respiratory cell fate and pattern formation via the activation and repression of downstream target genes. Gain- and loss-of-function studies in null mutant and transgenic mice models have greatly facilitated the identification and hierarchical classification of these molecular programs. In this review, we highlight select molecular events that drive key phases of pulmonary development, including specification of a lung cell fate, primary lung bud formation, tracheoesophageal septation, branching morphogenesis, and proximal-distal epithelial patterning. Understanding the genetic pathways that regulate respiratory tract development is essential to provide insight into the pathogenesis of congenital anomalies and to develop innovative strategies to treat inherited and acquired lung disease.

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TERRA regulate the transcriptional landscape of pluripotent cells through TRF1-dependent recruitment of PRC2

eLife ◽

10.7554/elife.44656 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Rosa María Marión ◽

Juan J Montero ◽

Isabel López de Silanes ◽

Osvaldo Graña-Castro ◽

Paula Martínez ◽

...

Keyword(s):

Cell Fate ◽

Es Cells ◽

Epigenetic Changes ◽

Pluripotent Cells ◽

Telomere Repeat ◽

Mouse Es Cells ◽

Genome Wide ◽

Binding Factor ◽

Transcriptional Landscape ◽

Pluripotency Genes

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

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Identification of the Regulatory Elements and Target Genes of Megakaryopoietic Transcription Factor MEF2C

Thrombosis and Haemostasis ◽

10.1055/s-0039-1678694 ◽

2019 ◽

Vol 119 (05) ◽

pp. 716-725 ◽

Cited By ~ 2

Author(s):

Xianguo Kong ◽

Lin Ma ◽

Edward Chen ◽

Chad Shaw ◽

Leonard Edelstein

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Genetic Variation ◽

Transcription Factors ◽

Cell Fate ◽

Target Genes ◽

Regulatory Elements ◽

Haematopoietic Stem Cells ◽

Platelet Production ◽

Induced Pluripotent

AbstractMegakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: MOV10, AGO3, HDAC1, RBBP5 and WASF2. To study expression of these genes, we silenced MEF2C gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down MEF2C expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced MEF2C expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in MEF2C alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.

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Inhibition of BET Proteins and Histone Deacetylase (HDACs): Crossing Roads in Cancer Therapy

Cancers ◽

10.3390/cancers11030304 ◽

2019 ◽

Vol 11 (3) ◽

pp. 304 ◽

Cited By ~ 18

Author(s):

Gloria Manzotti ◽

Alessia Ciarrocchi ◽

Valentina Sancisi

Keyword(s):

Cancer Therapy ◽

Histone Deacetylases ◽

Target Genes ◽

State Of The Art ◽

Regulatory Elements ◽

Functional Interaction ◽

Cellular Processes ◽

Silent Genes ◽

Dual Inhibition ◽

Chromatin Acetylation

Histone DeACetylases (HDACs) are enzymes that remove acetyl groups from histones and other proteins, regulating the expression of target genes. Pharmacological inhibition of these enzymes re-shapes chromatin acetylation status, confusing boundaries between transcriptionally active and quiescent chromatin. This results in reinducing expression of silent genes while repressing highly transcribed genes. Bromodomain and Extraterminal domain (BET) proteins are readers of acetylated chromatin status and accumulate on transcriptionally active regulatory elements where they serve as scaffold for the building of transcription-promoting complexes. The expression of many well-known oncogenes relies on BET proteins function, indicating BET inhibition as a strategy to counteract their activity. BETi and HDACi share many common targets and affect similar cellular processes to the point that combined inhibition of both these classes of proteins is regarded as a strategy to improve the effectiveness of these drugs in cancer. In this work, we aim to discuss the molecular basis of the interplay between HDAC and BET proteins, pointing at chromatin acetylation as a crucial node of their functional interaction. We will also describe the state of the art of their dual inhibition in cancer therapy. Finally, starting from their mechanism of action we will provide a speculative perspective on how these drugs may be employed in combination with standard therapies to improve effectiveness and/or overcome resistance.

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Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation

Development ◽

10.1242/dev.126.20.4557 ◽

1999 ◽

Vol 126 (20) ◽

pp. 4557-4568 ◽

Cited By ~ 37

Author(s):

R. DasGupta ◽

E. Fuchs

Keyword(s):

Hair Follicle ◽

Cell Fate ◽

Target Genes ◽

Inducible Promoter ◽

Hair Shaft ◽

Precursor Cells ◽

Beta Catenin ◽

Hair Cycle ◽

Outer Root Sheath ◽

Downstream Target

LEF/TCF DNA-binding proteins act in concert with activated beta -catenin, the product of Wnt signaling, to transactivate downstream target genes. To probe the role of activated LEF/TCF transcription factor complexes in hair follicle morphogenesis and differentiation, we engineered mice harboring TOPGAL, a beta -galactosidase gene under the control of a LEF/TCF and beta -catenin inducible promoter. In mice, TOPGAL expression was directly stimulated by a stabilized form of beta -catenin, but was also dependent upon LEF1/TCF3 in skin. During embryogenesis, TOPGAL activation occurred transiently in a subset of LEF1-positive cells of pluripotent ectoderm and underlying mesenchyme. Downgrowth of initiated follicles proceeded in the absence of detectable TOPGAL expression, even though LEF1 was still expressed. While proliferative matrix cells expressed the highest levels of Lef1 mRNAs, LEF1 concentrated in the precursor cells to the hair shaft, where TOPGAL expression was co-induced with hair-specific keratin genes containing LEF/TCF-binding motifs. LEF1 and TOPGAL expression ceased during catagen and telogen, but reappeared at the start of the postnatal hair cycle, concomitant with precortex formation. In contrast to hair shaft precursor cells, postnatal outer root sheath expressed TCF3, but not TOPGAL. TCF3 was also expressed in the putative follicle stem cells, and while TOPGAL was generally silent in this compartment, it was stimulated at the start of the hair cycle in a fashion that appeared to be dependent upon stabilization of beta -catenin. Taken together, our findings demonstrate that LEF1/TCF3 is necessary but not sufficient for TOPGAL activation, revealing the existence of positive and negative regulators of these factors in the skin. Furthermore, our findings unveil the importance of activated LEF/TCF complexes at distinct times in hair development and cycling when changes in cell fate and differentiation commitments take place.

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Identification of a Mouse Homolog of the Human BTEB2Transcription Factor as a β-Catenin-Independent Wnt-1-Responsive Gene

Molecular and Cellular Biology ◽

10.1128/mcb.21.2.562-574.2001 ◽

2001 ◽

Vol 21 (2) ◽

pp. 562-574 ◽

Cited By ~ 40

Author(s):

Lisa Taneyhill Ziemer ◽

Diane Pennica ◽

Arnold J. Levine

Keyword(s):

Cell Line ◽

Cell Fate ◽

Target Genes ◽

Wild Type Mouse ◽

Subtractive Hybridization ◽

Parental Cell ◽

Mammary Tissue ◽

Parental Cell Line ◽

Mouse Homolog ◽

Downstream Target

ABSTRACT The Wnt/Wg signaling pathway functions during development to regulate cell fate determination and patterning in various organisms. Two pathways are reported to lie downstream of Wnt signaling in vertebrates. The canonical pathway relies on the activation of target genes through the β-catenin–Lef/TCF complex, while the noncanonical pathway employs the activation of protein kinase C (PKC) and increases in intracellular calcium to induce target gene expression. cDNA subtractive hybridization between a cell line that overexpresses Wnt-1 (C57MG/Wnt-1) and the parental cell line (C57MG) was performed to identify downstream target genes of Wnt-1 signaling. Among the putative Wnt-1 target genes, we have identified a mouse homolog of the gene encoding human transcription factor basic transcription element binding protein 2 (mBTEB2). ThemBTEB2 transcript is found at high levels in mammary tissue taken from a transgenic mouse overexpressing Wnt-1 (both tissue prior to active proliferation and tumor tissue) but is barely detectable in wild-type mouse mammary glands. The regulation of mBTEB2 by Wnt-1 signaling in tissue culture occurs through a β-catenin–Lef/TCF-independent mechanism, as it is instead partially regulated by PKC. The Wnt-1-induced, PKC-dependent activation of mouse BTEB2 in C57MG cells, as well as the ability of Wnt-1 to stabilize β-catenin in these cells, is consistent with the hypothesis that both the noncanonical and canonical Wnt pathways are activated concomitantly in the same cell. These results suggest that mBTEB2 is a biologically relevant target of Wnt-1 signaling that is activated through a β-catenin-independent, PKC-sensitive pathway in response to Wnt-1.

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