ETS variant transcription factor 5 and c-Myc cooperate in derepressing the human telomerase gene promoter via composite ETS/E-box motifs

The human telomerase gene (hTERT) is repressed in most somatic cells. How transcription factors activate the hTERT promoter in its repressive chromatin environment is unknown. Here, we report that the ETS family protein ETS variant transcription factor 5 (ETV5) mediates epidermal growth factor (EGF)-induced hTERT expression in MCF10A cells. This activation required MYC proto-oncogene bHLH transcription factor (c-Myc) and depended on the chromatin state of the hTERT promoter. Using chromatinized bacterial artificial chromosome (BAC) reporters in human fibroblasts, we found that ETV5 and c-Myc/MYC-associated factor X (MAX) synergistically activate the hTERT promoter via two identical, but inverted, composite Ets/E-box motifs enclosing the core promoter. Mutations of Ets or E-box sites in either DNA motif abolished the activation and reduced or eliminated the synergism. ETV5 and c-Myc facilitated each other's binding to the hTERT promoter. ETV5 bound to the hTERT promoter in both telomerase-negative and -positive cells, but it activated the repressed hTERT promoter and altered histone modifications only in telomerase-negative cells. The synergistic ETV5/c-Myc activation disappeared when hTERT promoter repression became relieved because of the loss of distal regulatory elements in chimeric human/mouse BAC reporters. Our results suggest that the binding of c-Myc and ETS family proteins to the Ets/E-box motifs derepresses the hTERT promoter by inducing an active promoter configuration, providing a mechanistic insight into hTERT activation during tumorigenesis.

Download Full-text

Function of AP-1 in Transcription of the Telomerase Reverse Transcriptase Gene (TERT) in Human and Mouse Cells

Molecular and Cellular Biology ◽

10.1128/mcb.25.18.8037-8043.2005 ◽

2005 ◽

Vol 25 (18) ◽

pp. 8037-8043 ◽

Cited By ~ 80

Author(s):

Masahiro Takakura ◽

Satoru Kyo ◽

Masaki Inoue ◽

Woodring E. Wright ◽

Jerry W. Shay

Keyword(s):

Promoter Activity ◽

Cellular Proliferation ◽

Core Promoter ◽

Critical Role ◽

Regulatory Elements ◽

Telomerase Activity ◽

Activator Protein 1 ◽

Mouse Cells ◽

Human Telomerase ◽

Human And Mouse

ABSTRACT The transcriptional regulation of the human telomerase catalytic subunit (hTERT) plays a critical role in telomerase activity. Approximately 200 bp of the proximal core promoter is responsible for basic hTERT expression; however, the function of the distal regulatory elements remains unclear. The transcription factor activator protein 1 (AP-1) is involved in cellular proliferation, differentiation, carcinogenesis, and apoptosis and is expressed broadly in both cancer and normal cells. There are several putative AP-1 sites in the hTERT promoter, but their functions are unknown. The present study examined the regulatory role of AP-1 in hTERT gene transcription. Overexpression of AP-1 leads to transcriptional suppression of hTERT in cancer cells. The combination of c-Fos and c-Jun or c-Fos and JunD strongly suppresses hTERT promoter activity in transient-expression analyses. The hTERT promoter region between −2000 and −378 is responsible for this function. Gel shift and supershift analyses, as well as ChIP, show binding of JunD and c-Jun on two putative AP-1 sites within this region. Mutations in the AP-1 binding sites rescued suppressions caused by AP-1, suggesting this is a direct regulation of the hTERT promoter. In contrast, there was no effect on mTERT expression or mTERT promoter activity by AP-1 overexpression in mouse fibroblasts. The species-specific function of AP-1 in TERT expression may in part help explain the difference in telomerase activity between normal human and mouse cells.

Download Full-text

Genomic Characterization of Endothelial Enhancers Reveals a Multifunctional Role for NR2F2 in Regulation of Arteriovenous Gene Expression

Circulation Research ◽

10.1161/circresaha.119.316075 ◽

2020 ◽

Vol 126 (7) ◽

pp. 875-888 ◽

Cited By ~ 6

Author(s):

Samir Sissaoui ◽

Jun Yu ◽

Aimin Yan ◽

Rui Li ◽

Onur Yukselen ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Transcription Factor ◽

Deep Sequencing ◽

Chromatin Immunoprecipitation ◽

Regulatory Elements ◽

Bhlh Transcription Factor ◽

Genome Wide ◽

A Genome

Rationale: Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap. Objective: To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated. Methods and Results: Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation. Conclusions: By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

Download Full-text

Identification of the Novel Tooth-Specific Transcription Factor AmeloD

Journal of Dental Research ◽

10.1177/0022034518808254 ◽

2018 ◽

Vol 98 (2) ◽

pp. 234-241 ◽

Cited By ~ 7

Author(s):

B. He ◽

Y. Chiba ◽

H. Li ◽

S. de Vega ◽

K. Tanaka ◽

...

Keyword(s):

Transcription Factor ◽

Epithelial Cell ◽

Tooth Development ◽

Tooth Germ ◽

Regulatory Elements ◽

Proximal Promoter ◽

Epithelial Cell Line ◽

Bhlh Transcription Factor ◽

Specific Cell ◽

E Cadherin

Basic-helix-loop-helix (bHLH) transcription factors play an important role in various organs’ development; however, a tooth-specific bHLH factor has not been reported. In this study, we identified a novel tooth-specific bHLH transcription factor, which we named AmeloD, by screening a tooth germ complementary DNA (cDNA) library using a yeast 2-hybrid system. AmeloD was mapped onto the mouse chromosome 1q32. Phylogenetic analysis showed that AmeloD belongs to the achaete-scute complex-like ( ASCL) gene family and is a homologue of ASCL5. AmeloD was uniquely expressed in the inner enamel epithelium (IEE), but its expression was suppressed after IEE cell differentiation into ameloblasts. Furthermore, AmeloD expression showed an inverse expression pattern with the epithelial cell-specific cell–cell adhesion molecule E-cadherin in the dental epithelium. Overexpression of AmeloD in dental epithelial cell line CLDE cells resulted in E-cadherin suppression. We found that AmeloD bound to E-box cis-regulatory elements in the proximal promoter region of the E-cadherin gene. These results reveal that AmeloD functions as a suppressor of E-cadherin transcription in IEE cells. Our study demonstrated that AmeloD is a novel tooth-specific bHLH transcription factor that may regulate tooth development through the suppression of E-cadherin in IEE cells.

Download Full-text

Cell-Restricted Immortalization by Human Papillomavirus Correlates with Telomerase Activation and Engagement of the hTERT Promoter by Myc

Journal of Virology ◽

10.1128/jvi.01318-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11568-11576 ◽

Cited By ~ 37

Author(s):

Xuefeng Liu ◽

Aleksandra Dakic ◽

Renxiang Chen ◽

Gary L. Disbrow ◽

Yiyu Zhang ◽

...

Keyword(s):

High Risk ◽

Core Promoter ◽

Human Fibroblasts ◽

Human Keratinocytes ◽

Telomerase Activity ◽

Human Papillomaviruses ◽

Htert Promoter ◽

Causative Agents ◽

E6 And E7 ◽

Interfering Rna

ABSTRACT The high-risk human papillomaviruses (HPVs) are the causative agents of nearly all cervical cancers and are etiologically linked to additional human cancers, including those of anal, oral, and laryngeal origin. The main transforming genes of the high-risk HPVs are E6 and E7. E6, in addition to its role in p53 degradation, induces hTERT mRNA transcription in genital keratinocytes via interactions with Myc protein, thereby increasing cellular telomerase activity. While the HPV type 16 E6 and E7 genes efficiently immortalize human keratinocytes, they appear to only prolong the life span of human fibroblasts. To examine the molecular basis for this cell-type dependency, we examined the correlation between the ability of E6 to transactivate endogenous and exogenous hTERT promoters and to immortalize genital keratinocytes and fibroblasts. Confirming earlier studies, the E6 and E7 genes were incapable of immortalizing human fibroblasts but did delay senescence. Despite the lack of immortalization, E6 was functional in the fibroblasts, mediating p53 degradation and strongly transactivating an exogenous hTERT promoter. However, E6 failed to transactivate the endogenous hTERT promoter. Coordinately with this failure, we observed that Myc protein was not associated with the endogenous hTERT promoter, most likely due to the extremely low level of Myc expression in these cells and/or to differences in chromatin structure, in contrast with hTERT promoters that we found to be activated by E6 (i.e., the endogenous hTERT promoter in primary keratinoctyes and the exogenous hTERT core promoter in fibroblasts), where Myc is associated with the promoter in either a quiescent or an E6-induced state. These findings are consistent with those of our previous studies on mutagenesis and the knockdown of small interfering RNA, which demonstrated a requirement for Myc in the induction of the hTERT promoter by E6 and suggested that occupancy of the promoter by Myc determines the responsiveness of E6 and the downstream induction of telomerase and cell immortalization.

Download Full-text

The MNT transcription factor autoregulates its expression and supports proliferation in MYC-associated factor X (MAX)-deficient cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010389 ◽

2020 ◽

Vol 295 (7) ◽

pp. 2001-2017 ◽

Cited By ~ 5

Author(s):

M. Carmen Lafita-Navarro ◽

Judit Liaño-Pons ◽

Andrea Quintanilla ◽

Ignacio Varela ◽

Rosa Blanco ◽

...

Keyword(s):

Cell Proliferation ◽

Transcription Factor ◽

Transcriptional Regulator ◽

Bhlh Transcription Factor ◽

Factor X ◽

Associated Factor ◽

Helix Loop Helix ◽

E Box ◽

E Boxes ◽

Cycle Regulation

The MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the basic helix-loop-helix (bHLH) family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E-boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC–MAX dimer activates gene expression. MNT also binds to the MAX dimerization protein MLX (MLX), and MNT–MLX and MNT–MAX dimers co-exist. However, all MNT functions have been attributed to MNT–MAX dimers, and no functions of the MNT–MLX dimer have been described. MNT's biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT–MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E-boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-sequencing experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT–MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.

Download Full-text

Putative cis-regulatory elements predict iron deficiency responses in Arabidopsis roots

10.1101/603290 ◽

2019 ◽

Author(s):

Birte Schwarz ◽

Christina B. Azodi ◽

Shin-Han Shiu ◽

Petra Bauer

Keyword(s):

Transcription Factor ◽

Iron Deficiency ◽

Large Scale ◽

Computational Models ◽

Regulatory Elements ◽

Transport Systems ◽

Fe Deficiency ◽

Grass Species ◽

Bhlh Transcription Factor ◽

Fe Uptake

AbstractIron (Fe) is a key cofactor in many cellular redox processes, including respiration and photosynthesis. Plant Fe deficiency (-Fe) activates a complex regulatory network which coordinates root Fe uptake and distribution to sink tissues, while avoiding over-accumulation of Fe and other metals to toxic levels. In Arabidopsis (Arabidopsis thaliana), FIT (FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR), a bHLH transcription factor (TF), is required for up-regulation of root Fe acquisition genes. However, other root and shoot -Fe-induced genes involved in Fe allocation and signaling are FIT-independent. The cis-regulatory code, i.e. the cis-regulatory elements (CREs) and their combinations that regulate plant -Fe-responses, remains largely elusive. Using Arabidopsis genome and transcriptome data, we identified over 100 putative CREs (pCREs) that were predictive of -Fe-induced up-regulation of genes in root tissue. We used large-scale in vitro TF binding data, association with FIT-dependent or FIT-independent co-expression clusters, positional bias, and evolutionary conservation to assess pCRE properties and possible functions. In addition to bHLH and MYB TFs, also B3, NAC, bZIP, and TCP TFs might be important regulators for -Fe responses. Our approach uncovered IDE1 (Iron Deficiency-responsive Element 1), a -Fe response CRE in grass species, to be conserved in regulating genes for biosynthesis of Fe-chelating compounds also in Arabidopsis. Our findings provide a comprehensive source of cis-regulatory information for -Fe-responsive genes, that advances our mechanistic understanding and informs future efforts in engineering plants with more efficient Fe uptake or transport systems.One sentence summary>100 putative cis-regulatory elements robustly predict Arabidopsis root Fe deficiency-responses in computational models, and shed light on the mechanisms of transcriptional regulation.

Download Full-text

The scl +18/19 Stem Cell Enhancer Is Not Required for Hematopoiesis: Identification of a 5′ Bifunctional Hematopoietic-Endothelial Enhancer Bound by Fli-1 and Elf-1

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.1870-1883.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 1870-1883 ◽

Cited By ~ 61

Author(s):

Berthold Göttgens ◽

Cyril Broccardo ◽

Maria-Jose Sanchez ◽

Sophie Deveaux ◽

George Murphy ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Transcription Factor ◽

Cell Formation ◽

Regulatory Elements ◽

General Strategy ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Ets Family ◽

Endothelial Development

ABSTRACT Analysis of cis-regulatory elements is central to understanding the genomic program for development. The scl/tal-1 transcription factor is essential for lineage commitment to blood cell formation and previous studies identified an scl enhancer (the +18/19 element) which was sufficient to target the vast majority of hematopoietic stem cells, together with hematopoietic progenitors and endothelium. Moreover, expression of scl under control of the +18/19 enhancer rescued blood progenitor formation in scl−/− embryos. However, here we demonstrate by using a knockout approach that, within the endogenous scl locus, the +18/19 enhancer is not necessary for the initiation of scl transcription or for the formation of hematopoietic cells. These results led to the identification of a bifunctional 5′ enhancer (−3.8 element), which targets expression to hematopoietic progenitors and endothelium, contains conserved critical Ets sites, and is bound by Ets family transcription factors, including Fli-1 and Elf-1. These data demonstrate that two geographically distinct but functionally related enhancers regulate scl transcription in hematopoietic progenitors and endothelial cells and suggest that enhancers with dual hematopoietic-endothelial activity may represent a general strategy for regulating blood and endothelial development.

Download Full-text

Distinct c-Myb Regulation by HoxA9, Meis1 and Pbx1 in Haemopoietic and Leukaemic-Like Stem Cells.

Blood ◽

10.1182/blood.v114.22.1431.1431 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1431-1431

Author(s):

Emilie Dassé ◽

Giacomo Volpe ◽

Walter del Pozzo ◽

Jonathan Frampton ◽

Stephanie Dumon

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Stem Cell ◽

Regulatory Elements ◽

Proximal Promoter ◽

Cell Leukaemia ◽

Mouse Bone Marrow ◽

First Intron ◽

Hypersensitive Sites ◽

Factor C

Abstract Abstract 1431 Poster Board I-454 The transcription factor c-Myb is an essential regulator of haemopoiesis and its expression is deregulated in several types of leukaemia. Although some c-Myb functions have been defined, the mechanisms involved in the control of its expression have yet to be elucidated. Previous studies have suggested that transcription initiation at the c-myb gene is constitutive, and that the level of mRNA is regulated by an elongation-blocking mechanism operating in its first intron. Here, we define and compare mechanisms influencing c-myb expression in haemopoietic stem cells (HSCs) versus leukaemic stem cell (LSC)-like cells. Using a nuclease sensitivity assay we have defined several potential regulatory elements in both HSC and LSC-like model cell lines. These hypersensitive sites are in the proximal promoter and the first intron, the latter correlating with the position of the putative transcription elongation regulatory region. Moreover, the hypersensitive sites are located in regions of sequence conservation and encompass a number of potential binding sites for homeodomain (HD)-containing proteins. In this study, we were able to demonstrate that the HD-containing transcription factors HoxA9 and Meis1, which are highly expressed in HSCs and whose co-expression in mouse bone marrow leads to rapid development of acute myeloid leukaemia (AML), are necessary but not sufficient for c-myb expression. In addition, we show that the pre-B-cell leukaemia transcription factor-1 (Pbx1), known to be a key binding partner of HD-containing factors, is indispensable in the regulation of c-myb expression. Comparing the effects of altered levels of HoxA9, Meis1 and Pbx1 in HSCs versus LSCs suggests that distinct mechanisms involving dimeric or trimeric complexes operate to regulate c-myb expression in these two stem cell types. Disclosures No relevant conflicts of interest to declare.

Download Full-text

GmPTF1 Modifies Root Architecture Responses to Phosphate Starvation in Soybean

10.1101/830612 ◽

2019 ◽

Author(s):

Zhaojun Yang ◽

Ying He ◽

Yanxing Liu ◽

Yelin Lai ◽

Jiakun Zheng ◽

...

Keyword(s):

Transcription Factor ◽

Root Architecture ◽

Bhlh Transcription Factor ◽

Dependent Manner ◽

Regulatory Factor ◽

P Efficiency ◽

Soybean Yield ◽

Pi Starvation ◽

Promoter Deletion Analysis ◽

E Box

ABSTRACTThough root architecture modifications may be critically important for improving phosphorus (P) efficiency in crops, the regulatory mechanisms triggering these changes remain unclear. In this study, we demonstrate that genotypic variation in GmEXPB2 expression is strongly correlated with root elongation and P acquisition efficiency, and enhancing its transcription significantly improves soybean yield in the field. Promoter deletion analysis was performed using six 5’ truncation fragments (P1-P6) of GmEXPB2 fused with the GUS reporter gene in transgenic hairy roots, which revealed that the P1 segment containing 3 E-box elements significantly enhances induction of gene expression in response to phosphate (Pi) starvation. Further experimentation demonstrated that GmPTF1, a bHLH transcription factor, is the regulatory factor responsible for the induction of GmEXPB2 expression in response to Pi starvation. In short, Pi starvation induced expression of GmPTF1, with the GmPTF1 product not only directly binding the E-box motif in the P1 region of the GmEXPB2 promoter, but also activating GUS expression in a dosage dependent manner. Further work with soybean transgenic composite plants showed that, altering GmPTF1 expression significantly impacted GmEXPB2 transcription, and thereby affected root growth, biomass and P uptake. Taken together, this work identifies a novel regulatory factor, GmPTF1, involved in changing soybean root architecture through regulation the expression of GmEXPB2. These findings contribute to understanding the molecular basis of root architecture modifications in response to P deficiency, and, in the process, suggest candidate genes and a promoter region to target for improving soybean yield through molecular breeding of P efficiency.One Sentence SummaryThe bHLH transcription factor GmPTF1 regulates the expression of β-expansin gene GmEXPB2 to modify root architecture, and thus promote phosphate acquisition, and biomass in soybean.

Download Full-text