Suppression of GATA3 Binding Drives Selective Abrogation of NOTCH1-MYC Enhancer Activity By Nucleosome Invasion in Thymocyte Development and Leukemia

Abstract Long range enhancers play critical roles in the control of gene expression during development and have emerged as key regulators of lineage commitment and oncogenic programs in hematopoiesis and leukemia. The MYC oncogene is dynamically regulated in the hematopoietic system under the control of a network of clustered distal enhancers, which provide modular regulation of MYC expression during lymphoid and myeloid development. In thymocyte development MYC transcription critically depends on the activity of N-Me, a distinct T-cell specific enhancer controlled by NOTCH1 signaling and located 1.4 Mb telomeric to the MYC transcription start site. Yet, the specific mechanisms governing N-Me enhancer activity and lineage specific control of MYC expression remain rudimentarily understood. Analysis of chromatin looping by 4C and chromatin accessibility by ATACseq revealed an unanticipated high density of chromatin contacts between N-Me and additional regulatory elements in the Myc locus and showed a distinct pattern of N-Me chromatin accessibility -opening as progenitors mature into T cell committed CD4 CD8 double negative (DN) 2b cells and returning to a closed configuration in CD4 CD8 double positive (DP) thymocytes-. To explore potential regulators of N-Me activity we performed Mass Spectrometry proteomic profiling of N-Me binding proteins and ChIPseq analyses identifying numerous factors involved in hematopoietic and lymphoid development (ERG, ETS1, GATA3, RUNX1, TCF3 and TCF12) and transcription factor oncogenes with prominent roles in the pathogenesis of T-ALL (HOXA9, MYB, MYC, LMO1, LMO2, TAL1 and TLX1). Moreover, phylogenetic footprinting analyses across vertebrate species identified two ultraconserved elements matching GATA factor binding motifs (GS1 and GS2). To test the functionality of these elements we introduced targeted mutations in the N-Me sequence at these sites using CRISPR/CAS9 directed mutagenesis. Mice homozygous for combined N-Me GS1 and GS2 mutations (GS1+2mut) revealed a marked defect in thymus cellularity with characteristic accumulation of DN and intermediate single positive (ISP) thymocytes and decreased numbers of more mature populations. Mechanistically, immunohistochemical, flow cytometry and single cell RNaseq analyses revealed decreased Myc protein levels in thymocyte poulations of GS1+2 mutant animals. In this context, we hypothesized that GATA3, a prominent N-Me binding transcription factor in our ChIP and proteomic analyses critically implicated in T-cell commitment, could play a major role in N-Me regulation via interaction with the GS1 and GS2 N-Me GATA sites. Consistent with this hypothesis analysis of Gata3 ChIPs from heterozygous GS1+2 mutant mice recovered only the N-Me wild type sequence, formally demonstrating the strict requirement of these sites for N-Me Gata3 binding. Mechanistically, ATACseq analysis revealed a marked reduction in chromatin accessibility and nucleosome invasion in thymocytes from GS1+2 mutant mice in support of a critical pioneering activity for GATA3 in the control of N-Me activity. Finally, given the important role of NOTCH1 induced MYC upregulation in the pathogenesis of T-ALL, we hypothesized that disruption of N-Me activity via targeted mutation of N-Me GATA sites could effectively impair the development of NOTCH1-driven T-ALL in N-Me GS1+2 mutant mice. To test this possibility we infected hematopoietic progenitors from N-Me wild type and N-Me GS1+2 homozygous mice with retroviruses driving the expression of an oncogenic constitutively active form of NOTCH1 (DE-NOTCH1) and transplanted them into sublethally irradiated recipients. In these experiments, mice transplanted with DE-NOTCH1 infected N-Me wild type cells developed overt T-ALL 6 weeks postransplant with 100% penetrance. In contrast, mice transplanted with DE-NOTCH1-expressing N-Me GS1+2 homozygous cells showed complete protection from NOTCH1 induced T-ALL (P <0.001). In all these results identify GATA3 binding to the N-Me enhancer as a critical driver of nucleosome eviction and enhancer activation strictly required for thymocyte development and NOTCH1-induced T-cell transformation. Disclosures No relevant conflicts of interest to declare.

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The temporal transcription factor E93 controls dynamic enhancer activity and chromatin accessibility during development

10.1101/679753 ◽

2019 ◽

Author(s):

Spencer L. Nystrom ◽

Matthew J. Niederhuber ◽

Daniel J. McKay

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Developmental Time ◽

Chromatin Accessibility ◽

Wild Type ◽

Spatial Cues ◽

Enhancer Activity ◽

Temporal Gene Expression ◽

Developmental Program ◽

Temporal Cues

ABSTRACTHow temporal cues combine with spatial inputs to control gene expression during development is poorly understood. Here, we test the hypothesis that the Drosophila transcription factor E93 controls temporal gene expression by regulating chromatin accessibility. Precocious expression of E93 early in wing development reveals that it can simultaneously activate and deactivate different target enhancers. Notably, the precocious patterns of enhancer activity resemble the wild-type patterns that occur later in development, suggesting that provision of E93 alters the competence of enhancers to respond to spatial cues. Genomic profiling reveals that precocious E93 expression is sufficient to regulate chromatin accessibility at a subset of its targets. These accessibility changes mimic those that normally occur later in development, indicating that precocious E93 accelerates the wild-type developmental program. Further, we find that target enhancers that do not respond to precocious E93 in early wings become responsive after a developmental transition, suggesting that parallel temporal pathways work alongside E93. These findings support a model wherein E93 expression functions as an instructive cue that defines a broad window of developmental time through control of chromatin accessibility.

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Regulation of the T-cell receptor delta enhancer by functional cooperation between c-Myb and core-binding factors.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.473 ◽

1994 ◽

Vol 14 (1) ◽

pp. 473-483 ◽

Cited By ~ 98

Author(s):

C Hernandez-Munain ◽

M S Krangel

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Transcriptional Activation ◽

Cell Receptor ◽

Enhancer Activity ◽

Control Of Gene Expression ◽

Enhancer Element ◽

The Core ◽

Core Site ◽

Factor C

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor (TCR) delta gene. The 30-bp minimal enhancer element denoted delta E3 carries a core sequence (TGTGGTTT) that binds a T-cell-specific factor, and that is necessary but not sufficient for transcriptional activation. Here we demonstrate that the transcription factor c-Myb regulates TCR delta enhancer activity through a binding site in delta E3 that is adjacent to the core site. Both v-Myb and c-Myb bind specifically to delta E3. The Myb site is necessary for enhancer activity, because a mutation that eliminates Myb binding abolishes transcriptional activation by the delta E3 element and by the 370-bp TCR delta enhancer. Transfection of cells with a c-Myb expression construct upregulates delta E3 enhancer activity, whereas treatment of cells with an antisense c-myb oligonucleotide inhibits delta E3 enhancer activity. Since intact Myb and core sites are both required for delta E3 function, our data argue that c-Myb and core binding factors must cooperate to mediate transcriptional activation through delta E3. Efficient cooperation depends on the relative positioning of the Myb and core sites, since only one of two overlapping Myb sites within delta E3 is functional and alterations of the distance between this site and the core site disrupt enhancer activity. Cooperative regulation by c-Myb and core-binding factors is likely to play an important role in the control of gene expression during T-cell development.

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GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.

Blood ◽

10.1182/blood.v110.11.374.374 ◽

2007 ◽

Vol 110 (11) ◽

pp. 374-374 ◽

Cited By ~ 2

Author(s):

Zhong-fa Yang ◽

Karen Drumea ◽

Alan G. Rosmarin

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

T Cell ◽

Myeloid Cells ◽

Myeloid Cell ◽

Cell Development ◽

Wild Type ◽

Ets Domain

Abstract GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.

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The transcription factor Gli3 regulates differentiation of fetal CD4–CD8– double-negative thymocytes

Blood ◽

10.1182/blood-2005-03-0998 ◽

2005 ◽

Vol 106 (4) ◽

pp. 1296-1304 ◽

Cited By ~ 32

Author(s):

Ariadne L. Hager-Theodorides ◽

Johannes T. Dessens ◽

Susan V. Outram ◽

Tessa Crompton

Keyword(s):

Transcription Factor ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

T Cell Differentiation ◽

Double Negative ◽

Thymocyte Development ◽

Tcr Signaling ◽

Double Positive ◽

Dose Dependent

AbstractGlioblastoma 3 (Gli3) is a transcription factor involved in patterning and oncogenesis. Here, we demonstrate a role for Gli3 in thymocyte development. Gli3 is differentially expressed in fetal CD4–CD8– double-negative (DN) thymocytes and is most highly expressed at the CD44+ CD25– DN (DN1) and CD44–CD25– (DN4) stages of development but was not detected in adult thymocytes. Analysis of null mutants showed that Gli3 is involved at the transitions from DN1 to CD44+ CD25+ DN (DN2) cell and from DN to CD4+CD8+ double-positive (DP) cell. Gli3 is required for differentiation from DN to DP thymocyte, after pre–T-cell receptor (TCR) signaling but is not necessary for pre-TCR–induced proliferation or survival. The effect of Gli3 was dose dependent, suggesting its direct involvement in the transcriptional regulation of genes controlling T-cell differentiation during fetal development.

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Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1424104112 ◽

2015 ◽

Vol 112 (25) ◽

pp. 7773-7778 ◽

Cited By ~ 9

Author(s):

Hyung-Ok Lee ◽

Xiao He ◽

Jayati Mookerjee-Basu ◽

Dai Zhongping ◽

Xiang Hua ◽

...

Keyword(s):

Transcription Factor ◽

T Cell ◽

Lymphoblastic Leukemia ◽

T Cell Development ◽

Cell Receptor ◽

Cell Development ◽

Thymocyte Development ◽

Tcr Signaling ◽

Double Positive ◽

Human T Cell

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell–specific ThPOK transgene (ThPOKconst mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOKconst RAG-deficient mice, which promotes development to the CD4+8+ (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

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Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by compacting super-enhancers

10.21203/rs.3.rs-1069634/v1 ◽

2021 ◽

Author(s):

Delong Feng ◽

Yanhong Chen ◽

Ranran Dai ◽

Shasha Bian ◽

Wei Xue ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Receptor Gene ◽

Cell Receptor ◽

Regulation Mechanism ◽

Thymocyte Development ◽

Double Positive ◽

Enhancer Activity ◽

Cell Identity ◽

Single Positive

Abstract CD4+ and CD8+ double-positive (DP) thymocytes are at a crucial stage during the T cell development in the thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCRβ. Then DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, Regulatory T cells, or invariant nature kill T cells (iNKT) according to the TCR signal. Chromatin organizer SATB1 is highly expressed in DP cells and plays an essential role in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing assay of SATB1-deficient thymocytes showed that the cell identity of DP thymocytes was changed, and the genes specifically highly expressed in DP cells were down-regulated. The super-enhancers regulate the expressions of the DP-specific genes, and the SATB1 deficiency reduced the super-enhancer activity. Hi-C data showed that interactions in super-enhancers and between super-enhancers and promoters decreased in SATB1 deficient thymocytes. We further explored the regulation mechanism of two SATB1-regulating genes, Ets2 and Bcl6, in DP cells and found that the knockout of the super-enhancers of these two genes impaired the development of DP cells. Our research reveals that SATB1 globally regulates super-enhancers of DP cells and promotes the establishment of DP cell identity, which helps understand the role of SATB1 in thymocyte development.

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Transcription Factor Gene Fluorescent Reporters Track Lineage Fate in Lymphoid Commitment

Blood ◽

10.1182/blood.v128.22.sci-3.sci-3 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-3-SCI-3

Author(s):

Ellen Rothenberg

Keyword(s):

T Cells ◽

Transcription Factor ◽

T Cell ◽

Conflicts Of Interest ◽

Epigenetic Modification ◽

Single Cells ◽

Transcriptional Level ◽

Regulatory State ◽

Cell Commitment

Abstract The transition from multipotency to lineage commitment can be followed with particular clarity for T cell precursors. In this lineage, the role of environmental signals can be clearly separated from the role of intrinsic fate programming in individual cells and the cells' developmental responses to changing conditions and can be tracked in real time. T cell precursors are still multipotent when they first enter the thymus, and if they are removed from the thymic microenvironment at this stage they can give rise to non-T cells including dendritic cells and myeloid cells. For multiple cell divisions, they preserve this multipotency and are only kept in line to become T cells conditionally, by Notch signaling from the thymic stroma. Then at a specific point of no return, the cells become unable to give rise to anything except T cells regardless of environment, and this is the point of commitment. Commitment is clearly the readout of a change in internal transcriptional regulatory state. To determine how this is controlled, we and others have charted transcription factor expression changes across this interval and changes in chromatin modification and DNA accessibility that accompany the transition, and we have been able to use functional perturbation tests to narrow down the key regulators that catalyze and enforce this transition. A particularly important commitment factor is encoded by the Bcl11b gene, which is released from previously repressed chromatin and sharply activated at the transcriptional level just as the cells become committed. The Bcl11b gene product is required in all alpha beta and most gamma delta T cells to enable the commitment process to occur. These properties make it highly illuminating as an indicator of the regulatory state in individual differentiating T-cell precursors. We have generated a series of knock-in Bcl11b fluorescent reporter alleles to probe the correlation of Bcl11b expression with changes in specific target genes, to determine the transcription factor requirements for Bcl11b gene activation in the gene regulatory network controlling commitment in single cells, and to measure the role of epigenetic modification of the Bcl11b locus on the kinetics of transition from uncommitted to committed states. These results and their implications will be presented. Importantly, the use of live-cell reporters reveals a level of all-or-none, stochastic regulation in the responses of individual cells to combinatorial transcription factor action at this developmental watershed1. Reference: 1. Kueh HY, Yui MA, Ng KKH, et al. Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment. Nature Immunology. 2016.17, 956-965. Disclosures No relevant conflicts of interest to declare.

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Transcriptional network orchestrating regional patterning of cortical progenitors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2024795118 ◽

2021 ◽

Vol 118 (51) ◽

pp. e2024795118

Author(s):

Athéna R. Ypsilanti ◽

Kartik Pattabiraman ◽

Rinaldo Catta-Preta ◽

Olga Golonzhka ◽

Susan Lindtner ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Ventricular Zone ◽

Integrated Approach ◽

Mutant Mice ◽

Gene Interactions ◽

Wild Type ◽

Dna Accessibility ◽

Mouse Cortex ◽

Radial Glial

We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer–gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

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High expression of the transcription factor ERG predicts unfavorable outcome in acute T-lymphoblastic leukemia (T-ALL) in adults

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.6505 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 6505-6505

Author(s):

C. D. Baldus ◽

T. Burmeister ◽

P. Martus ◽

S. Schwartz ◽

N. Goekbuget ◽

...

Keyword(s):

Transcription Factor ◽

T Cell ◽

Real Time ◽

Lymphoblastic Leukemia ◽

High Expression ◽

Prognostic Impact ◽

Rt Pcr ◽

Term Survival ◽

Cell Commitment

6505 Background: In adult T-ALL long-term survival remains limited to 32–46%. Transcription factors are frequently targeted by chromosomal translocations resulting in disruption of hematopoietic proliferation and differentiation. The oncogenic ETS transcription factor ERG is expressed during early T-cell development and shut off once T-cell commitment is complete. We hypothesized that due to its specific involvement in T-cell maturation and oncogenic potential, ERG might contribute to leukemogenesis. Thus we have determined the prognostic impact of ERG expression in T-ALL. Patients and Methods: ERG mRNA expression was analyzed by real-time RT-PCR in pretreatment marrow samples of 105 adults with T-ALL treated on German ALL protocols. Patients (pts) were dichotomized at ERG’s median expression into low (n=52) and high (n=53) expressers. HOX11 and HOX11L2 expression was determined by real-time RT-PCR. Immunophenotyping was performed differentiating T-ALL into 3 subtypes: pre-T (CD2-), thymic (CD1a+), and mature (sCD3+). Results: High ERG expression was associated with a higher relapse rate (45%) compared to pts with low ERG expression (20%; P=0.01). High ERG expressers compared to low ERG expressers had an inferior overall survival (OS, P=0.02; 5-year OS: high ERG 26% vs low ERG 58%) and relapse-free survival (RFS, P=0.003; 5-year RFS: high ERG 34% vs low ERG 72%). On multivariable analysis high ERG expression (P=0.005), immunophenotypic subgroups (pre-T vs mature vs thymic; overall P=0.04), HOX11L2 positivity (P=0.055) and absence of HOX11 expression (P=0.017) were independent adverse risk factors predicting RFS. Patients with high ERG expression had a hazard ratio (HR) for relapse of 3.2. Within the good prognostic subgroup of thymic T-ALL (n=57) high ERG (HR 4.1; P=0.02) and presence of HOX11L2 (HR 6.6; P=0.008) were independent adverse factors for RFS. Conclusion: High expression of the oncogene ERG is an adverse factor in adult T-ALL. Within thymic T-ALL otherwise classified as standard risk, high ERG expression identified pts that were more than four times likely to fail long-term RFS. The prognostic impact of ERG may assist treatment stratification and suggest the need of more intensive regimens for these high risk thymic T-ALL pts. No significant financial relationships to disclose.

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