scholarly journals Graded PU.1 Levels Regulate Granulocyte Vs. Macrophage Genes Via Multiple Enhancer Elements

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 403-403
Author(s):  
Vitek Pospisil ◽  
Pavle Krsmanovic ◽  
Jan Valecka ◽  
Kamila Chramostová ◽  
Vojtech Kulvait ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Binding Sites ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Dna Looping ◽  
Proximal Promoter ◽  
Regulatory Sequences ◽  
3D Interaction ◽  
Chromosome Conformation

Abstract PU.1 is a transcription factor absolutely required for normal hematopoiesis. Cumulating evidence indicates that precise levels of PU.1 expression are critical for differentiation to distinct blood lineages, and if perturbed, even modest decreases in PU.1 can lead to leukemogenesis. In contrast to extensive knowledge of regulation of PU.1 gene itself, the mechanism of how target genes senses different PU.1 levels remain largely unknown. To address this, we used PU.1-/- mouse myeloid progenitors encoding inducible PU.1 transgene (PU.1ER, PUER, Walsh 2002) that allows tight control of PU.1 activity. Interestingly, intermediate PU.1 activity induced differentiation of PUER progenitors into granulocyte like cells, while high PU.1 produced macrophages, supporting the model that different PU.1 expression is not a consequence but a driver of cell fate choice. Global expression analysis using 4 different levels of PU.1 at 8 time points (2-96 hrs) revealed that granulocyte specific genes were activated exclusively by intermediate PU.1 levels in 3 distinct modes: 1. not expressed in progenitors while strongly induced at intermediate PU.1 (e.g. Gelatinase B (Mmp9) and Neutrophil collagenase (NC) 2. moderately expressed in progenitors while strongly activated at intermediate PU.1 and repressed at high PU.1 (e.g. Myeloperaxidase (Mpo) 3. highly expressed in unstimulated progenitors with expression maintained at intermediate PU.1 but strongly repressed at high PU.1 (e.g. Neutrophil elastase (NE), Proteinase 3 (primary granule proteins), Cebpe and Gfi1 (Growth factor independent1) Majority of macrophage genes (incl. CD14, Csf1R, Egr2) were regulated as early PU.1 target genes; being gradually activated by high PU.1 activity within 8hrs. However, most granulocyte genes (NE, Mmp9, Mpo, NC but not Cebpe and GFI1) were late activated PU.1 targets (48 and 96hrs) indicating that these genes are coregulated by additional factor(s), likely an early PU.1 target. Next we analyzed the regulatory sequences (+-50kb) of two genes activated exclusively by intermediate PU.1, Mpo and Mmp9, using own and public ChIP(seq) data of transcription factors (TFs) (PU.1, GFI.1), DNAseI hypersensitive sites, histone modifications (H3K4Me, H3K27Ac, H3K9Ac) and expression of enhancer specific bidirectional ncRNAs (eRNA) (CAGE). 14 Mpo and 16 Mmp9 putative enhancers, selected by above mentioned criteria, were cloned into luciferase vector containing their proximal promoter (PP) and were tested for functional activity in response to PU.1 levels. Interestingly, the PU.1 binding motifs within these regions have a low to intermediate affinity (log of score, Jaspar) and are often present in multiples and/or enriched for binding sites of other lineage determining transcription factors. Although PU.1 bound to all of these DNA regions resembling superenhancer, just a small fraction of PU.1 binding was functionally responsive. Specifically, we identified novel enhancer elements at -3.4 kb and -15kb of MPO which were activated by intermediate (but not high) PU.1 levels. Interestingly, activity of -3.4 kb enhancer required presence of PP, while the -15kb element required presence of both PP and the -3.4kb element. Similar phenomenon was observed at -5kb and +4.6kb (intronic) MMP9 enhancers. Collectively, these observations suggest that a cooperative assembly of several cell type-specific enhancers is required for optimal Mpo and Mmp9 activation. This model is supported by our Chromosome conformation capture (3C) data identifying 3D interaction of these enhancer elements at intermediate PU.1 levels suggesting that PU.1 binding mediates DNA looping that allows enhancer cooperation. In addition, activity of these enhancers at intermediate PU.1 levels was associated with expression of bidirectional noncoding enhancer RNAs, confirming functionality of these elements. In conclusion, our data support the model that PU.1 at intermediate concentration binds to low and intermediate affinity binding sites in several enhancers of granulocyte genes, causing their successive looping and interaction with proximal promoter that leads to transcription activation. The role of cooperating TFs, mechanisms of how granulocyte genes are switched off at high PU.1 concentration and deregulation of these mechanisms in AML are being further studied. Grants 16-05649S P305/12/1033 16-31586A 16-27790A 16-31586A UNCE 204021 PRVOUK P24 Disclosures No relevant conflicts of interest to declare.

Epigenetic Control of C/EBPa by Distant Synergic Regulatory Elements.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1470-1470
Author(s):  
Alexander K Ebralidze ◽  
Annalisa Di Ruscio ◽  
Sanghoon Lee ◽  
Karen O'Brien ◽  
Daniel G. Tenen
Keyword(s):  
Conflicts Of Interest ◽  
Transgenic Animals ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Regulatory Sequences ◽  
Dna Hypermethylation ◽  
Chromosome Conformation ◽  
Hematopoietic Stem ◽  
Factor C

Abstract Abstract 1470 Poster Board I-493 The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo. In this study, we demonstrate synergetic regulation of C/EBPa by two distant cis-elemets located 5' and 3' to the gene and their effect on chromatin architecture. Previous studies have indicated that as much as 4.8 kb of 5' upstream C/EBPa regulatory sequences were unable to express significant levels of reporter gene activity in transgenic mice. Therefore, we initiated a search for important distal elements in the C/EBPa locus. We have applied a combination of 1) comparative analysis of human and mouse genomic sequences; 2) DNase I hypersensitive studies; 3) chromosome conformation capture (3C); 4) analysis of reporter constructs in stable cells lines; and 5) generation and analysis of transgenic mouse lines. This let us to identify the regulatory role of two distal conserved homology elements located at ∼38 kb 5' of the transcription start site (TSS) of murine C/EBPa (corresponding to ∼45 kb 5' of the TSS of human C/EBPa) and at ∼33 kb 3' to TSS of both murine and human C/EBPa. We show that the constructs lacking both distal elements were unable to express C/EBPa mRNA, while addition of each region resulted in detectable (by Northern blot analysis) expression in transgenic animals. We have observed a cooperative effect of these two regions on C/EBPa expression, a construct carrying both elements expresses ∼2.5-fold level over constructs carrying either one element alone. We have investigated the mechanism for the increased expression by these distal elements by using deletion constructs. Our results suggest that lack of these elements results in aberrant gene expression due to proximal promoter DNA hypermethylation and point to a novel mechanism in establishment of critical epigenetic marks. Disclosures: No relevant conflicts of interest to declare.

Mechanisms of Transcriptional Regulation and Transformation by HOXA9

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-30-SCI-30
Author(s):  
Jay L. Hess ◽  
Cailin Collins ◽  
Joel Bronstein ◽  
Yuqing Sun ◽  
Surya Nagaraja
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
A Genome ◽  
The Impact

Abstract Abstract SCI-30 HOXA9 plays important roles in both development and hematopoiesis and is overexpressed in more than 50 percent of acute myeloid leukemias (AML). Nearly all cases of AML with mixed lineage leukemia (MLL) translocations show increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. In most cases, upregulation of HOXA9 is accompanied by upregulation of its homeodomain-containing cofactor MEIS1, which directly interacts with HOXA9. While HOXA9 alone is sufficient for transformation of hematopoietic stem cells in culture, the addition of MEIS1 increases the transformation efficiency and results in rapidly fatal leukemias in transplanted animals. Despite the crucial role that HOXA9 plays in development, hematopoiesis, and leukemia, its transcriptional targets and mechanisms of action are poorly understood. We have used ChIP-seq to identify Hoxa9 and Meis1 binding sites on a genome-wide level in myeloblastic cells, profiled their associated epigenetic modifications, identified the target genes regulated by HOXA9 and identified HOXA9 interacting proteins. HOXA9 and MEIS1 cobind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/P300 binding. These include many proleukemogenic gene loci, such as Erg, Flt3, Myb, Lmo2, and Sox4. In addition, HOXA9 binding sites overlap a subset of enhancers previously implicated in myeloid differentiation and inflammation. HOXA9 binding at enhancers stabilizes association of MEIS1 and lineage-restricted transcription factors, including C/EBPα, PU.1, and STAT5A/B thereby promoting CBP/p300 recruitment, histone acetylation, and transcriptional activation. Current efforts are focused on using both biochemical and genetic approaches to assess the role of HOXA9 “enhanceosome” components C/EBPα, PU.1, and STAT5A/B in transcriptional regulation and leukemogenesis. Studies to date suggest that C/EBPα and PU.1 binding can occur in the absence of HOXA9/MEIS1, supporting a model in which these proteins act as pioneer transcription factors for establishment of poised, but not activated, HOXA9-regulated enhancers. Work is under way to assess the impact of high-level HOXA9 and MEIS1 on enhanceosome assembly and the role of recruitment of transcriptional coactivators involved in target gene up- or downregulation, including histone acetyltransferases and chromatin remodeling complexes. Collectively, our findings suggest that HOXA9-regulated enhancers are a fundamental mechanism of HOX-mediated transcription in normal development that is deregulated in leukemia. Disclosures: No relevant conflicts of interest to declare.

Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (8) ◽  
pp. 3517-3523
Author(s):  
D P McDonnell ◽  
J W Pike ◽  
D J Drutz ◽  
T R Butt ◽  
B W O'Malley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vitamin D ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Transcription Unit ◽  
Proximal Promoter ◽  
Regulatory Sequences ◽  
Cis Acting ◽  
Osteocalcin Gene ◽  
Definition Of

The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms. The sequences responsible for this activity have been mapped to within the -1339 region of the gene. We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor. When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity. This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor. This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors.

scholarly journals The PARP Inhibitor Olaparib Modulates the Transcriptional Regulatory Networks of Long Non-Coding RNAs during Vasculogenic Mimicry

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2690
Author(s):  
Mónica Fernández-Cortés ◽  
Eduardo Andrés-León ◽  
Francisco Javier Oliver
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Regulatory Networks ◽  
Target Genes ◽  
Parp Inhibitor ◽  
Vasculogenic Mimicry ◽  
Transcriptome Profiling ◽  
Key Species ◽  
Non Coding Rnas ◽  
The Impact

In highly metastatic tumors, vasculogenic mimicry (VM) involves the acquisition by tumor cells of endothelial-like traits. Poly-(ADP-ribose) polymerase (PARP) inhibitors are currently used against tumors displaying BRCA1/2-dependent deficient homologous recombination, and they may have antimetastatic activity. Long non-coding RNAs (lncRNAs) are emerging as key species-specific regulators of cellular and disease processes. To evaluate the impact of olaparib treatment in the context of non-coding RNA, we have analyzed the expression of lncRNA after performing unbiased whole-transcriptome profiling of human uveal melanoma cells cultured to form VM. RNAseq revealed that the non-coding transcriptomic landscape differed between olaparib-treated and non-treated cells: olaparib significantly modulated the expression of 20 lncRNAs, 11 lncRNAs being upregulated, and 9 downregulated. We subjected the data to different bioinformatics tools and analysis in public databases. We found that copy-number variation alterations in some olaparib-modulated lncRNAs had a statistically significant correlation with alterations in some key tumor suppressor genes. Furthermore, the lncRNAs that were modulated by olaparib appeared to be regulated by common transcription factors: ETS1 had high-score binding sites in the promoters of all olaparib upregulated lncRNAs, while MZF1, RHOXF1 and NR2C2 had high-score binding sites in the promoters of all olaparib downregulated lncRNAs. Finally, we predicted that olaparib-modulated lncRNAs could further regulate several transcription factors and their subsequent target genes in melanoma, suggesting that olaparib may trigger a major shift in gene expression mediated by the regulation lncRNA. Globally, olaparib changed the lncRNA expression landscape during VM affecting angiogenesis-related genes.

scholarly journals Graded repression of PU.1/Sfpi1 gene transcription by GATA factors regulates hematopoietic cell fate

Blood ◽  
2009 ◽  
Vol 114 (5) ◽  
pp. 983-994 ◽  
Author(s):  
Stella T. Chou ◽  
Eugene Khandros ◽  
L. Charles Bailey ◽  
Kim E. Nichols ◽  
Christopher R. Vakoc ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Gene Transcription ◽  
Target Genes ◽  
Hematopoietic Cell ◽  
Physical Interaction ◽  
Related Factor ◽  
Gata Factors ◽  
Undifferentiated State

GATA-1 and PU.1 are essential hematopoietic transcription factors that control erythromegakaryocytic and myelolymphoid differentiation, respectively. These proteins antagonize each other through direct physical interaction to repress alternate lineage programs. We used immortalized Gata1− erythromegakaryocytic progenitor cells to study how PU.1/Sfpi1 expression is regulated by GATA-1 and GATA-2, a related factor that is normally expressed at earlier stages of hematopoiesis. Both GATA factors bind the PU.1/Sfpi1 gene at 2 highly conserved regions. In the absence of GATA-1, GATA-2 binding is associated with an undifferentiated state, intermediate level PU.1/Sfpi1 expression, and low-level expression of its downstream myeloid target genes. Restoration of GATA-1 function induces erythromegakaryocytic differentiation. Concomitantly, GATA-1 replaces GATA-2 at the PU.1/Sfpi1 locus and PU.1/Sfpi1 expression is extinguished. In contrast, when GATA-1 is not present, shRNA knockdown of GATA-2 increases PU.1/Sfpi1 expression by 3-fold and reprograms the cells to become macrophages. Our findings indicate that GATA factors act sequentially to regulate lineage determination during hematopoiesis, in part by exerting variable repressive effects at the PU.1/Sfpi1 locus.

C/ebpα and Pu.1 Are Members of a Critical Regulatory Network Required for Hoxa9-Mediated Immortalization

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 650-650
Author(s):  
Cailin Collins ◽  
Jingya Wang ◽  
Joel Bronstein ◽  
Jay L. Hess
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Target Genes ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modifications ◽  
Conditional Knockout ◽  
Motif Analysis ◽  
Myeloid Leukemias

Abstract Abstract 650 HOXA9 is a homeodomain-containing transcription factor that plays important roles in both development and hematopoiesis. Deregulation of HOXA9 occurs in a variety of acute lymphoid and myeloid leukemias and plays a key role in their pathogenesis. More than 50% of acute myeloid leukemia (AML) cases show up-regulation of HOXA9, which correlates strongly with poor prognosis. Nearly all cases of AML with mixed lineage leukemia (MLL) translocations have increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. Despite the crucial role that HOXA9 plays in development, hematopoiesis and leukemia, its transcriptional targets and mechanisms of action are poorly understood. Previously we identified Hoxa9 and Meis1 binding sites in myeloblastic cells, profiled their epigenetic modifications, and identified the target genes regulated by Hoxa9. Hoxa9 and Meis1 co-bind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/p300 binding characteristic of enhancers. Hoxa9 association at these sites correlates strongly with increases in histone H3K27 acetylation and activation of downstream target genes, including many proleukemic gene loci. De novo motif analysis of Hoxa9 binding sites shows a marked enrichment of motifs for the transcription factors in the C/EBP and ETS families, and C/ebpα and the ETS transcription factor Pu.1 were found to cobind at Hoxa9-regulated enhancers. Both C/ebpα and Pu.1 are known to play critical roles in the establishment of functional enhancers during normal myeloid development and are mutated or otherwise deregulated in various myeloid leukemias. To determine the importance of co-association of Hoxa9, C/ebpα and Pu.1 at myeloid enhancers, we generated cell lines from C/ebpα and Pu.1 conditional knockout mice (kindly provided by Dr. Daniel Tenen, Harvard University) by immortalization with Hoxa9 and Meis1. In addition we transformed bone marrow with a tamoxifen-regulated form of Hoxa9. Strikingly, loss of C/ebpα or Pu.1, or inactivation of Hoxa9, blocks proliferation and leads to myeloid differentiation. ChIP experiments show that both C/ebpα and Pu.1 remain bound to Hoxa9 binding sites in the absence of Hoxa9. After the loss of Pu.1, both Hoxa9 and C/ebpα dissociate from Hoxa9 binding sites with a corresponding decrease in target gene expression. In contrast, loss of C/ebpα does not lead to an immediate decrease in either Hoxa9 or Pu.1 binding, suggesting that C/ebpα may be playing a regulatory as opposed to a scaffolding role at enhancers. Current work focuses on performing ChIP-seq analysis to assess how C/ebpα and Pu.1 affect Hoxa9 and Meis1 binding and epigenetic modifications genome-wide, and in vivo leukemogenesis assays to confirm the requirement of both Pu.1 and C/ebpα in the establishment and maintenance of leukemias with high levels of Hoxa9. Collectively, our findings implicate C/ebpα and Pu.1 as members of a critical transcription factor network required for Hoxa9-mediated transcriptional regulation in leukemia. Disclosures: No relevant conflicts of interest to declare.

Enhancer Landscape Dynamics and the Role of IRF8 in Mononuclear Phagocyte Development

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2384-2384
Author(s):  
Daisuke Kurotaki ◽  
Jun Nakabayashi ◽  
Akira Nishiyama ◽  
Haruka Sasaki ◽  
Naofumi Kaneko ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Mononuclear Phagocyte ◽  
Landscape Dynamics ◽  
Mononuclear Phagocytes ◽  
Motif Analysis ◽  
Hematopoietic Stem ◽  
Dna Motif ◽  
Fate Decision

Abstract Monocytes and dendritic cells (DCs) are critical mononuclear phagocytes that regulate innate and adaptive immune responses. Hematopoietic stem cells give rise to monocytes and DCs via intermediate myeloid progenitor populations, such as granulocyte-monocyte progenitors (GMPs), monocyte-DC progenitors (MDPs), and common monocyte progenitors (cMoPs) or common DC progenitors (CDPs). However, the molecular mechanism underlying their lineage determination is poorly understood. Recently, promoter-distal enhancers have been found to be key for cell fate decision. In this study, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) analysis of three histone modifications (H3K4me1, H3K27ac, and H3K4me3, representing primed enhancers, active enhancers, and transcriptional intiation, respectively) in seven myeloid populations (GMPs, MDPs, cMoPs, CDPs, monocytes, DCs, and neutrophils), and identified approximately sixty thousand putative enhancer regions. We found that a majority of monocyte- and DC-specific active enhancers were gradually established at progenitor stages prior to gene expression and terminal differentiation. Computational DNA motif analysis implicated that these enhancers were regulated by combinations of lineage-determining transcription factors such as PU.1, RUNX, C/EBP, and IRF. Indeed, ChIP-seq of PU.1 confirmed that the myeloid master regulator PU.1 was the common transcription factors bound to enhancers at all the stages examined. IRF binding motifs were enriched at the enhancer regions in MDPs, cMoPs and CDPs, but not GMPs. Among IRFs, IRF8, a partner of PU.1, has been shown to be highly expressed in MDPs and their descendants, and to be required for MDP-to-CDP and cMoP-to-monocyte transitions. Indeed, ChIP-seq analysis in Irf8-/- progenitors demonstrated that the enhancer landscapes of Irf8-/- GMPs, MDPs, and cMoPs all remained similar to that of wild-type GMPs. Moreover, ChIP-seq for IRF8 binding revealed that IRF8 directly promoted the priming and activation of many enhancers in MDPs and cMoPs. These results contribute to a comprehensive understanding of how transcription factors govern the enhancer landscape dynamics during mononuclear phagocyte development. Disclosures No relevant conflicts of interest to declare.

Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors

2003 ◽  
Vol 31 (1) ◽  
pp. 292-297 ◽  
Author(s):  
K.U. Birkenkamp ◽  
P.J. Coffer
Keyword(s):  
Transcription Factors ◽  
Cell Survival ◽  
Cell Fate ◽  
Nuclear Export ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Acute Lymphocytic Leukaemia ◽  
Cell Fate Decisions ◽  
Forkhead Transcription Factors ◽  
Forkhead Box

Recently, the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors has been identified as direct targets of phosphoinositide 3-kinase-mediated signal transduction. The AFX (acute-lymphocytic-leukaemia-1 fused gene from chromosome X), FKHR (Forkhead in rhabdomyosarcoma) and FKHR-L1 (FKHR-like 1) transcription factors are directly phosphorylated by protein kinase B, resulting in nuclear export and inhibition of transcription. This signalling pathway was first identified in the nematode worm Caenorhabditis elegans, where it has a role in regulation of the life span of the organism. Studies have shown that this evolutionarily conserved signalling module has a role in regulation of both cell-cycle progression and cell survival in higher eukaryotes. These effects are co-ordinated by FOXO-mediated induction of a variety of specific target genes that are only now beginning to be identified. Interestingly, FOXO transcription factors appear to be able to regulate transcription through both DNA-binding-dependent and -independent mechanisms. Our understanding of the regulation of FOXO activity, and defining specific transcriptional targets, may provide clues to the molecular mechanisms controlling cell fate decisions to divide, differentiate or die.

scholarly journals The Proneural Proteins Atonal and Scute Regulate Neural Target Genes through Different E-Box Binding Sites

2004 ◽  
Vol 24 (21) ◽  
pp. 9517-9526 ◽  
Author(s):  
Lynn M. Powell ◽  
Petra I. zur Lage ◽  
David R. A. Prentice ◽  
Biruntha Senthinathan ◽  
Andrew P. Jarman
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Tandem Repeats ◽  
Target Genes ◽  
Expression Patterns ◽  
Sense Organ ◽  
Ample Evidence ◽  
E Box ◽  
Bhlh Transcription Factors

ABSTRACT For a particular functional family of basic helix-loop-helix (bHLH) transcription factors, there is ample evidence that different factors regulate different target genes but little idea of how these different target genes are distinguished. We investigated the contribution of DNA binding site differences to the specificities of two functionally related proneural bHLH transcription factors required for the genesis of Drosophila sense organ precursors (Atonal and Scute). We show that the proneural target gene, Bearded, is regulated by both Scute and Atonal via distinct E-box consensus binding sites. By comparing with other Ato-dependent enhancer sequences, we define an Ato-specific binding consensus that differs from the previously defined Scute-specific E-box consensus, thereby defining distinct EAto and ESc sites. These E-box variants are crucial for function. First, tandem repeats of 20-bp sequences containing EAto and ESc sites are sufficient to confer Atonal- and Scute-specific expression patterns, respectively, on a reporter gene in vivo. Second, interchanging EAto and ESc sites within enhancers almost abolishes enhancer activity. While the latter finding shows that enhancer context is also important in defining how proneural proteins interact with these sites, it is clear that differential utilization of DNA binding sites underlies proneural protein specificity.

scholarly journals PRDM1 Binds an Extensive Network of Genes to Regulate Human Natural Killer Cell Homeostasis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2536-2536
Author(s):  
Xuxiang Liu ◽  
Yunfei Shi ◽  
Yuping Li ◽  
Ru Chen ◽  
Sheng Pan ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nk Cells ◽  
Binding Sites ◽  
Cell Activation ◽  
Target Genes ◽  
Plasma Cells ◽  
Nk Cell ◽  
Feeder Cells ◽  
Cell Homeostasis ◽  
T Box

Natural killer (NK) cells are innate lymphocytes responsible for early defense against infections and malignant cells. Specific transcription factors crucial for NK cell development and function include PRDM1, which also regulates T cell homeostasis and is essential for the terminal differentiation of B cells into plasma cells. Importantly, PRDM1 deletion, methylation and loss-of-function mutations were commonly detected in NK cell malignancies. However, the detailed mechanisms through which PRDM1 regulates NK cell homeostasis are still largely undefined. Here, we employed an in vitro culture system of human NK cells isolated from healthy donors, in which the NK cells were cultured in the presence of IL-2 with or without an engineered feeder cell line, K562-Cl9-mb21, expressing membrane-bound IL-21, 4-1BBL and CD86. The NK cells were able to expand for months when co-cultured with feeder cells, whereas IL-2 alone could only maintain NK cell survival with limited proliferation for one week. We performed ChIP-seq to compare the genome-wide binding profiles of PRDM1 in NK cells grown with or without feeder cells. We found that PRDM1 bound much fewer target genes (802) in NK cells with feeder than in NK cells with IL-2 alone (2880), and 98.5% (790/802) of the binding sites in feeder-stimulated NK cells overlapped with those found in NK cells without feeder. The PRDM1 consensus motifs were almost identical in both conditions. MEME analyses also identified motifs of other transcription factors enriched in the PRDM1 binding sites, such as the RUNX and T-Box families. Interestingly, the RUNX and T-Box motifs, among others, were more enriched in the PRDM1 binding sites lacking the PRDM1 motif than in those with the PRDM1 motif, which indicates that PRDM1 may often bind to DNA indirectly through other transcription factors. PRDM1 bound a large number of genes in the NK or T cell receptor signaling pathway, which are employed during NK cell activation. In addition, several genes encoding immune checkpoints that may restrict NK cell activation such as TIGIT, HAVCR2 (TIM3), and IL-1R8 were targeted by PRDM1. Many genes encoding NK cell inhibitory and activating receptors were also bound by PRDM1. Moreover, PRDM1 was found to target transcription factors that are important for NK cell development and homeostasis, such as the RUNX family, TBX21, MAF, and PRDM1 itself. Despite an extensive overlap of PRDM1 binding sites detected in NK cells grown with or without feeder cells, the most enriched pathways were not exactly the same. Importantly, our RNA-seq data on PRDM1-knockout NK cells validated the regulatory role of PRDM1 on a fraction of these PRDM1-bound genes and interestingly, PRDM1 appeared to be an activator for some of the genes, including those encoding immune checkpoint molecules TIGIT, HAVCR2, and IL-1R8. We also utilized ATAC-seq to examine the chromatin accessibility of NK cells grown with feeder cells or with IL-2 alone. We identified differentially enriched pathways for the NK cells cultured under different conditions. When compared with the PRDM1 ChIP-seq, we found that those PRDM1 binding sites that contain a consensus PRDM1 motif were less likely to be accessible to transposase than those without the PRDM1 motif, thereby confirming the transcriptional repressor role of PRDM1. To further understand how PRDM1 regulates its target genes in NK cells, we performed mass spectrometric analysis on the protein complexes associated with PRDM1. We were able to identify the interaction of PRDM1 with the corepressor Groucho (TLE3) and components from the SIN3, NCoR, and NuRD complex, which have been previously reported in mouse plasma cells. Surprisingly, the protein compositions of the PRDM1-associated complexes from the two NK cell populations were very different. Although PRDM1 is generally associated with transcriptional repression, we also detected the association of PRDM1 with transcriptional activators or coactivators, such as the RUNX-binding protein CBFβ and the T-Box family member EOMES, which may thereby upregulate some of the PRDM1 target genes. In summary, we found that PRDM1 binds and regulates an extensive network of genes responsible for NK cell activation and function. Extrinsic stimuli, as provided by the feeder cells, can alter the extent and profile of PRDM1 binding as well as the associated protein complexes and hence alter its regulatory function in NK cells. Disclosures No relevant conflicts of interest to declare.

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