Augmenting E Protein Activity Impairs cDC2 Differentiation at the Pre-cDC Stage

Dendritic cell (DC) specification and differentiation are controlled by a circuit of transcription factors, which regulate the expression of DC effector genes as well as the transcription factors themselves. E proteins are a widely expressed basic helix-loop-helix family of transcription factors whose activity is suppressed by their inhibitors, ID proteins. Loss-of-function studies have demonstrated the essential role of both E and ID proteins in different aspects of DC development. In this study, we employed a gain-of-function approach to illustrate the importance of the temporal control of E protein function in maintaining balanced differentiation of conventional DC (cDC) subsets, cDC1 and cDC2. We expressed an E protein mutant, ET2, which dimerizes with endogenous E proteins to overcome inhibition by ID proteins and activate the transcription of E protein targets. Induction of ET2 expression at the hematopoietic progenitor stage led to a dramatic reduction in cDC2 precursors (pre-cDC2s) with little impact on pre-cDC1s. Consequently, we observed decreased numbers of cDC2s in the spleen and lung, as well as in FLT3L-driven bone marrow-derived DC cultures. Furthermore, in mice bearing ET2, we detected increased expression of the IRF8 transcription factor in cDC2s, in which IRF8 is normally down-regulated and IRF4 up-regulated. This aberrant expression of IRF8 induced by ET2 may contribute to the impairment of cDC2 differentiation. In addition, analyses of the transcriptomes of splenic cDC1s and cDC2s revealed that ET2 expression led to a shift, at least in part, of the transcriptional profile characteristic of cDC2s to that of cDC1. Together, these results suggest that a precise control of E protein activity is crucial for balanced DC differentiation.

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Early B Cell Factor Promotes B Lymphopoiesis with Reduced Interleukin 7 Responsiveness in the Absence of E2A

Journal of Experimental Medicine ◽

10.1084/jem.20032202 ◽

2004 ◽

Vol 199 (12) ◽

pp. 1689-1700 ◽

Cited By ~ 119

Author(s):

Christopher S. Seet ◽

Rachel L. Brumbaugh ◽

Barbara L. Kee

Keyword(s):

Transcription Factors ◽

B Cell ◽

B Lymphopoiesis ◽

Protein Activity ◽

E Proteins ◽

Helix Loop Helix ◽

Interleukin 7 ◽

E Protein ◽

B Lineage ◽

Early B Cell Factor

The basic helix-loop-helix transcription factors encoded by the E2A gene function at the apex of a transcriptional hierarchy involving E2A, early B cell factor (EBF), and Pax5, which is essential for B lymphopoiesis. In committed B lineage progenitors, E2A proteins have also been shown to regulate many lineage-associated genes. Herein, we demonstrate that the block in B lymphopoiesis imposed by the absence of E2A can be overcome by expression of EBF, but not Pax5, indicating that EBF is the essential target of E2A required for development of B lineage progenitors. Our data demonstrate that EBF, in synergy with low levels of alternative E2A-related proteins (E proteins), is sufficient to promote expression of most B lineage genes. Remarkably, however, we find that E2A proteins are required for interleukin 7–dependent proliferation due, in part, to a role for E2A in optimal expression of N-myc. Therefore, high levels of E protein activity are essential for the activation of EBF and N-myc, whereas lower levels of E protein activity, in synergy with other B lineage transcription factors, are sufficient for expression of most B lineage genes.

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Integrative network analysis reveals USP7 haploinsufficiency inhibits E-protein activity in pediatric T-lineage acute lymphoblastic leukemia (T-ALL)

Scientific Reports ◽

10.1038/s41598-021-84647-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Timothy I. Shaw ◽

Li Dong ◽

Liqing Tian ◽

Chenxi Qian ◽

Yu Liu ◽

...

Keyword(s):

Network Analysis ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Cancer Therapeutics ◽

Physical Interaction ◽

Loss Of Function ◽

Protein Activity ◽

E Proteins ◽

Down Regulation ◽

E Protein

AbstractUSP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.

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Multivalent Binding of the ETO Corepressor to E Proteins Facilitates Dual Repression Controls Targeting Chromatin and the Basal Transcription Machinery

Molecular and Cellular Biology ◽

10.1128/mcb.00073-09 ◽

2009 ◽

Vol 29 (10) ◽

pp. 2644-2657 ◽

Cited By ~ 23

Author(s):

Chun Guo ◽

Qiande Hu ◽

Chunxia Yan ◽

Jinsong Zhang

Keyword(s):

Fusion Protein ◽

Protein Fusion ◽

Protein Activity ◽

E Proteins ◽

Basal Transcription ◽

Activation Domains ◽

Basal Transcription Machinery ◽

Transcription Machinery ◽

Helix Loop Helix ◽

E Protein

ABSTRACT E proteins are a family of helix-loop-helix transcription factors that play important roles in cell differentiation and homeostasis. They contain at least two activation domains, AD1 and AD2. ETO family proteins and the leukemogenic AML1-ETO fusion protein are corepressors of E proteins. It is thought that ETO represses E-protein activity by interacting with AD1, which competes away p300/CBP histone acetyltransferases. Here we report that E proteins contain another conserved ETO-interacting region, termed DES, and that differential associations with AD1 and DES allow ETO to repress transcription through both chromatin-dependent and chromatin-independent mechanisms. At the chromatin level, AD1 and AD2 cooperatively recruit p300. ETO interacts with AD1 to abolish p300 recruitment and to allow HDAC-dependent silencing. At the post-chromatin-remodeling level, binding to DES enables ETO to directly inhibit activation of the basal transcription machinery. This novel repression mechanism is conserved in ETO family proteins and in the AML1-ETO fusion protein. In addition, the repression capacity exerted by each mechanism is differentially modulated by cross talk among various ETO domains and the AML1 domain of AML1-ETO. In particular, the oligomerization domain of ETO plays a major role in targeting ETO to the DES region and independently potentiates the TAFH domain-mediated AD1 interaction. The ability to exert repression at different levels not only may allow these corepressors to impose robust inhibition of signal-independent transcription but may also allow a rapid response to signals. In addition, our newly defined domain interactions and their interplays have important implications in effectively targeting both E-protein fusion proteins and AML1-ETO found in cancers.

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E proteins orchestrate dynamic transcriptional cascades to suppress ILC2 differentiation

10.1101/2020.03.26.008037 ◽

2020 ◽

Cited By ~ 1

Author(s):

Vincent Peng ◽

Constantin Georgescu ◽

Anna Bakowska ◽

Liangyue Qian ◽

Jonathan D Wren ◽

...

Keyword(s):

Transcription Factors ◽

Molecular Mechanisms ◽

Innate Lymphoid Cells ◽

Transcriptional Network ◽

Lymphoid Cells ◽

Loss Of Function ◽

Sequencing Data ◽

E Proteins ◽

Down Regulation ◽

E Protein

AbstractThe basic helix-loop-helix transcription factors collectively called E proteins powerfully suppress the differentiation of group2 innate lymphoid cells from bone marrow and thymic progenitors. Here we investigated the underlying molecular mechanisms using inducible gain and loss of function approaches in ILC2s and their precursors, respectively. Cross-examination of RNA sequencing and ATAC sequencing data obtained at different time points reveals a set of genes which are likely direct targets of E proteins. Consequently, a widespread down-regulation of chromatin accessibility occurs at a later time point, possibly due to the activation of transcriptional repressor genes such as Cbfa2t3 and Jdp2. The large number of genes repressed by gain of E protein function leads to the down-regulation of a transcriptional network important for ILC2 differentiation.SummaryDifferentiation of group 2 innate lymphoid cells is forcefully repressed by E protein transcription factors. This report elucidates how E proteins repress a transcriptional network important for ILC2 differentiation by up-regulating the expression of transcriptional repressors.

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E proteins orchestrate dynamic transcriptional cascades implicated in the suppression of the differentiation of group 2 innate lymphoid cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013806 ◽

2020 ◽

Vol 295 (44) ◽

pp. 14866-14877

Author(s):

Vincent Peng ◽

Constantin Georgescu ◽

Anna Bakowska ◽

Aneta Pankow ◽

Liangyue Qian ◽

...

Keyword(s):

Protein Function ◽

Molecular Mechanisms ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Microbial Pathogens ◽

Loss Of Function ◽

Sequencing Data ◽

E Proteins ◽

Down Regulation ◽

Group 2

Group 2 innate lymphoid cells (ILC2s) represent a subset of newly discovered immune cells that are involved in immune reactions against microbial pathogens, host allergic reactions, as well as tissue repair. The basic helix-loop-helix transcription factors collectively called E proteins powerfully suppress the differentiation of ILC2s from bone marrow and thymic progenitors while promoting the development of B and T lymphocytes. How E proteins exert the suppression is not well understood. Here we investigated the underlying molecular mechanisms using inducible gain and loss of function approaches in ILC2s and their precursors, respectively. Cross-examination of RNA-seq and ATAC sequencing data obtained at different time points reveals a set of genes that are likely direct targets of E proteins. Consequently, a widespread down-regulation of chromatin accessibility occurs at a later time point, possibly due to the activation of transcriptional repressor genes such as Cbfa2t3 and Jdp2. The large number of genes repressed by gain of E protein function leads to the down-regulation of a transcriptional network important for ILC2 differentiation.

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Balance between Id and E proteins regulates myeloid-versus-lymphoid lineage decisions

Blood ◽

10.1182/blood-2008-06-164996 ◽

2009 ◽

Vol 113 (5) ◽

pp. 1016-1026 ◽

Cited By ~ 42

Author(s):

Shawn W. Cochrane ◽

Ying Zhao ◽

Robert S. Welner ◽

Xiao-Hong Sun

Keyword(s):

Transcription Factors ◽

B Cell ◽

Cell Fate ◽

Intracellular Signaling ◽

Specific Gene ◽

B Cell Differentiation ◽

E Proteins ◽

Lymphoid Lineage ◽

E Protein ◽

Multipotent Progenitors

Abstract Hematopoiesis consists of a series of lineage decisions controlled by specific gene expression that is regulated by transcription factors and intracellular signaling events in response to environmental cues. Here, we demonstrate that the balance between E-protein transcription factors and their inhibitors, Id proteins, is important for the myeloid-versus-lymphoid fate choice. Using Id1-GFP knockin mice, we show that transcription of the Id1 gene begins to be up-regulated at the granulocyte-macrophage progenitor stage and continues throughout myelopoiesis. Id1 expression is also stimulated by cytokines favoring myeloid differentiation. Forced expression of Id1 in multipotent progenitors promotes myeloid development and suppresses B-cell formation. Conversely, enhancing E-protein activity by expressing a variant of E47 resistant to Id-mediated inhibition prevents the myeloid cell fate while driving B-cell differentiation from lymphoid-primed multipotent progenitors. Together, these results suggest a crucial function for E proteins in the myeloid-versus-lymphoid lineage decision.

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Differential Interactions of Id Proteins with Basic-Helix-Loop-Helix Transcription Factors

Journal of Biological Chemistry ◽

10.1074/jbc.272.32.19785 ◽

1997 ◽

Vol 272 (32) ◽

pp. 19785-19793 ◽

Cited By ~ 159

Author(s):

Kenneth Langlands ◽

Xiaoying Yin ◽

Geetha Anand ◽

Edward V. Prochownik

Keyword(s):

Transcription Factors ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Id Proteins

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SCL Assembles a Multifactorial Complex That Determines Glycophorin A Expression

Molecular and Cellular Biology ◽

10.1128/mcb.24.4.1439-1452.2004 ◽

2004 ◽

Vol 24 (4) ◽

pp. 1439-1452 ◽

Cited By ~ 112

Author(s):

Rachid Lahlil ◽

Eric Lécuyer ◽

Sabine Herblot ◽

Trang Hoang

Keyword(s):

Transcription Factors ◽

Hematopoietic Cells ◽

Inhibitory Effect ◽

Erythroid Cell ◽

Glycophorin A ◽

Protein Activity ◽

Protein Levels ◽

Helix Loop Helix

ABSTRACT SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.

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MTG16 (CBFA2T3) represses E protein-dependent transcription to regulate colonic secretory cell differentiation, epithelial regeneration, and tumorigenesis

10.1101/2021.11.03.467178 ◽

2021 ◽

Author(s):

Rachel E. Brown ◽

Justin Jacobse ◽

Shruti A. Anant ◽

Koral M. Blunt ◽

Bob Chen ◽

...

Keyword(s):

Cell Differentiation ◽

Mouse Model ◽

Cell Fate ◽

Protein Interactions ◽

Protein Function ◽

Colonic Epithelium ◽

E Proteins ◽

Cell Fate Decisions ◽

Epithelial Regeneration ◽

E Protein

Aberrant epithelial differentiation and regeneration pathways contribute to colon pathologies including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). MTG16 (also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 interaction partners include E box-binding basic helix-loop-helix transcription factors (E proteins). MTG16-deficient mice exhibit worse colitis and increased tumor burden in inflammatory carcinogenesis. In this study, we sought to understand the role of MTG16 in colonic epithelial homeostasis and the mechanisms by which MTG16 protects the epithelium in colitis and CAC. We demonstrated that MTG16 deficiency enabled enteroendocrine cell differentiation from secretory precursor cells at the expense of goblet cells. Transcriptomic analysis implicated dysregulated E protein function in MTG16-deficient colon crypts. Using a novel mouse model with a point mutation that disrupts MTG16:E protein complex formation (Mtg16P209T), we established that enteroendocrine:goblet cell balance was dependent on MTG16:E protein interactions and that the shift in lineage allocation was associated with enhanced expression of Neurog3, the central driver of enteroendocrine lineage specification. Furthermore, Mtg16 was upregulated in the previously described Ascl2+, de-differentiating cells that replenish the stem cell compartment in response to colon injury. Mtg16 expression was also increased in dextran sulfate sodium (DSS)-treated mouse colon crypts and in IBD patients compared to unaffected controls. We determined that the effects of MTG16 in regeneration are also dependent on its repression of E proteins, as the colonic epithelium failed to regenerate following DSS-induced injury in our novel mutant mouse model. Finally, we revealed that uncoupling MTG16:E protein interactions contributes to the enhanced tumorigenicity in Mtg16-/- colon in the azoxymethane(AOM)/DSS-induced model of CAC. Collectively, our results demonstrate that MTG16, via its repression of E protein targets, is a key regulator of cell fate decisions during colonic differentiation and regeneration.

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Mtg16-dependent repression of E protein activity is required for early lymphopoiesis

10.1101/2020.07.24.220525 ◽

2020 ◽

Author(s):

Pankaj Acharya ◽

Shilpa Sampathi ◽

David K. Flaherty ◽

Brittany K. Matlock ◽

Christopher S. Williams ◽

...

Keyword(s):

Stem Cell ◽

Germ Line ◽

Adult Stem Cell ◽

The Cancer Genome Atlas ◽

Single Nucleotide Variants ◽

Protein Activity ◽

E Proteins ◽

Cell Functions ◽

E Protein ◽

Cancer Genome Atlas

AbstractThe ETO/MTG family of transcriptional co-repressors play a key role in adult stem cell functions in various tissues. These factors are commonly found in complex with E proteins such as E2A, HEB, and Lyl1 as well as PRDM14 and BTB/POZ domain factors. Structural studies identified a region in the first domain of MTGs that is conserved in the Drosophila homologue Nervy (Nervy Homology Domain-1, or NHR1) that is essential for ETO/MTG8 to inhibit E protein-dependent transcription. The Cancer Genome Atlas (TCGA) identified cancer associated single nucleotide variants (SNVs) near the MTG16:E protein contact site. We tested these SNVs using sensitive yeast two-hybrid association assays, which suggested that only P209T significantly affected E protein binding. We then used CRISPR-Cas9 and homology directed DNA repair to insert P209T and a known inactivating mutation, F210A, into NHR1 of Mtg16 in the germ line of mice. These mice developed normally, but in competitive bone marrow transplantation assays, the F210A-containing stem cells failed to contribute to lymphopoiesis, while P209T mutant cells were reduced in mature T cell populations. High content fluorescent activated analytical flow cytometry assays identified a defect in the multi-potent progenitor to common lymphoid progenitor transition during lymphopoiesis. These data indicate that the cancer associated changes are likely benign polymorphisms, and the MTG:E protein association is required for lymphopoiesis, but less important for myelopoiesis and stem cell functions.

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