Gfi1b Association to Human Hematological Abnormalities.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3748-3748
Author(s):  
Ana Villegas ◽  
Fernando A. Gonzalez ◽  
Eduardo Anguita
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Acute Leukemia ◽  
Point Mutations ◽  
K562 Cells ◽  
Vector System ◽  
Luciferase Reporter ◽  
Human Pathology ◽  
Hematological Abnormalities

Abstract Lineage specific transcription factors play essential roles in regulation of hematopoietic development. Transcription factor abnormalities have been frequently described in acute leukemia, mostly through cytogenetic changes. Nevertheless, point mutations can be easily missed. Recently, mutations in the erythroid and megakaryocyte specific transcription factor GATA1 have been discovered in patients with dyserythropoietic anemia and acute megakaryoblastic leukemia (AML-M7) with Down syndrome. Besides GATA-1, located on the X-chromosome, point mutations have been described in biallelic genes. This is the case of AML1 (RUNX1). PU1 and C/EBPalpha also represent examples of transcription factors in which point mutations are found in leukemia. A new zinc finger transcription factor involved in erythropoiesis is Gfi1b. Gfi1b was recently identified by sequence homology with oncogene Gfi1. Gfi1b knockout has demonstrated that this gene is essential for development of both erythroid and megakaryocytic lineages, and in its absence no enucleated erythrocytes are produced. Several Gfi1b DNA and protein targets (GATA1, Gfi1, AML1, p21WAF1, IL-6 Socs1 and Socs2) have been described that might be involved in malignancy. These findings indicate that Gfi1b is at the centre of hematopoiesis and may be a good candidate gene to be involved in hematological abnormalities. We have searched for Gfi1b point mutations in 122 patients with acute leukemia of all FAB types at diagnosis or relapse and 9 cases of congenital dyserythropoietic anemia. We have amplified Gfi1b promoter, coding and non-coding exons (Nucleic Acids Res2004;32:3935–46, MN 004188) by high fidelity PCR and screen for point mutations through dHPLC (Wave, Transgenomic) followed by sequencing of the cases with abnormal pattern. SNIPs in the promoter and exons were further confirmed in at least another PCR, cloned in pGEM-T easy vector system (Promega) and sequenced. Alleles with promoter SNIPs were cloned in pGL3-Enhancer vector (Promega), and transiently cotransfected with pEGFP-C2 (Clontech) to K562 cells. Luciferase activity was determined with Dual-Luciferase Reporter Assay (Promega). We found two promoter SNIPs in sequences conserved from chicken to human, one of them affecting a GATA-1 site, reducing promoter in vitro activity by 60 and 50% respectively. We also discovered a congenital exonic SNIP causing a mammalian conserved serine change to leucine. We excluded these to be frequent polymorphisms by dHPLC analysis of 96 blood donors. Although we cannot at present establish a clear relation between the former SNIPS and leukemia, we will discuss the presence of other milder hematological abnormalities. So far this is the first report of Gfi1b mutations that can be related to human pathology.

scholarly journals The miR-203a Regulatory Network Affects the Proliferation of Chronic Myeloid Leukemia K562 Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Jinhua He ◽  
Zeping Han ◽  
Ziyi An ◽  
Yumin Li ◽  
Xingyi Xie ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Target Genes ◽  
Mononuclear Cells ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Upstream Promoter ◽  
Upstream Promoter Region

To study the molecular mechanism by which miR-203a affects the development of CML, bioinformatics software was used to predict the upstream transcription factors and downstream target genes of miR-203a. A 5’-rapid amplification of cDNA ends assay was performed to detect gene transcription initiation sites. A chromatin immunoprecipitation assay was used to verify the binding of transcription factors and promoter regions. A double luciferase reporter gene vector was constructed to demonstrate the regulatory effect of miR-203a on target genes. Real-time PCR and western blotting were used to detect the relative expression levels of genes and proteins, respectively. The results showed that there was a binding site for the transcription factor EGR1 in the upstream promoter region of miR-203a. WT1, BMI1, and XIAP were identified as target genes regulated by miR-203a. EGR1 and miR-203a were downregulated in human peripheral blood mononuclear cells and the CML K562 cell line, while WT1, BMI1, and XIAP were upregulated. The transcription initiation site of miR-203a was identified in the upstream promoter region (G nucleotide at −339 bp), and the transcription factor EGR1 could bind to the promoter region (at −268 bp) of miR-203a and increase its expression. Over expression of miR-203a inhibited the proliferation of K562 cells. A rescue assay showed that overexpression of WT1, BMI1, and XIAP offset the antitumor effect of miR-203a. Conclusion, EGR1 positively regulated the expression of miR-203a, thus relieving the inhibition of miR-203a on the translation of its target genes (WT1, BMI1, and XIAP) and affecting the proliferation of K562 cells.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chang Hyun Byon ◽  
Jay McDonald ◽  
Yabing Chen
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Molecular Mechanism ◽  
Luciferase Reporter ◽  
Rankl Expression ◽  
Reporter System ◽  
Mobility Shift ◽  
Atherosclerotic Lesions ◽  
Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

scholarly journals Expression of Adhesive Pili and the Collagen-Binding Adhesin Ace Is Activated by ArgR Family Transcription Factors inEnterococcus faecalis

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Dawn A. Manias ◽  
Gary M. Dunny
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Enterococcus Faecalis ◽  
Opportunistic Infections ◽  
Structural Genes ◽  
Gene Encoding ◽  
Collagen Binding ◽  
Content Type ◽  
Surface Adhesins

ABSTRACTIt was shown previously that the disruption of theahrCgene encoding a predicted ArgR family transcription factor results in a severe defect in biofilm formationin vitro, as well as a significant attenuation of virulence ofEnterococcus faecalisstrain OG1RF in multiple experimental infection models. Using transcriptome sequencing (RNA-seq), we observedahrC-dependent changes in the expression of more than 20 genes. AhrC-repressed genes included predicted determinants of arginine catabolism and several other metabolic genes and predicted transporters, while AhrC-activated genes included determinants involved in the production of surface protein adhesins. Most notably, the structural and regulatory genes of theebplocus encoding adhesive pili were positively regulated, as well as theacegene, encoding a collagen-binding adhesin. UsinglacZtranscription reporter fusions, we determined thatahrCand a secondargRtranscription factor gene,argR2, both function to activate the expression ofebpR, which directly activates the transcription of the pilus structural genes. Our data suggest that in the wild-typeE. faecalis, the low levels of EbpR limit the expression of pili and that biofilm biomass is also limited by the amount of pili expressed by the bacteria. The expression ofaceis similarly enhanced by AhrC and ArgR2, butaceexpression is not dependent on EbpR. Our results demonstrate the existence of novel regulatory cascades controlled by a pair of ArgR family transcription factors that might function as a heteromeric protein complex.IMPORTANCECell surface adhesins play critical roles in the formation of biofilms, host colonization, and the pathogenesis of opportunistic infections byEnterococcus faecalis. Here, we present new results showing that the expression of two major enterococcal surface adhesins,ebppili, and the collagen-binding protein Ace is positively regulated at the transcription level by twoargRfamily transcription factors, AhrC and ArgR2. In the case of pili, the direct target of regulation is theebpRgene, previously shown to activate the transcription of the pilus structural genes, while the activation ofacetranscription appears to be directly impacted by the two ArgR proteins. These transcription factors may represent new targets for blocking enterococcal infections.

scholarly journals The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor.

1994 ◽  
Vol 14 (1) ◽  
pp. 373-381 ◽  
Author(s):  
D E Zhang ◽  
C J Hetherington ◽  
H M Chen ◽  
D G Tenen
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transcription Initiation ◽  
Colony Stimulating Factor ◽  
Specific Expression ◽  
Tissue Specific ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The macrophage colony-stimulating factor (M-CSF) receptor is expressed in a tissue-specific fashion from two distinct promoters in monocytes/macrophages and the placenta. In order to further understand the transcription factors which play a role in the commitment of multipotential progenitors to the monocyte/macrophage lineage, we have initiated an investigation of the factors which activate the M-CSF receptor very early during the monocyte differentiation process. Here we demonstrate that the human monocytic M-CSF receptor promoter directs reporter gene activity in a tissue-specific fashion. Since one of the few transcription factors which have been implicated in the regulation of monocyte genes is the macrophage- and B-cell-specific PU.1 transcription factor, we investigated whether PU.1 binds and activates the M-CSF receptor promoter. Here we demonstrate that both in vitro-translated PU.1 and PU.1 from nuclear extracts bind to a specific site in the M-CSF receptor promoter just upstream from the major transcription initiation site. Mutations in this site which eliminate PU.1 binding decrease M-CSF receptor promoter activity significantly in macrophage cell lines only. Furthermore, PU.1 transactivates the M-CSF receptor promoter in nonmacrophage cells. These results suggest that PU.1 plays a major role in macrophage gene regulation and development by directing the expression of a receptor for a key macrophage growth factor.

Chromatin Architecture and Transcription Factor Occupancy of Erythrocyte Membrane Genes Studied by Chromatin Immunoprecipitation on Microarrays (ChIP-chip)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2436-2436
Author(s):  
Laurie A Steiner ◽  
Yelena Maksimova ◽  
Clara Wong ◽  
Vincent Schulz ◽  
Patrick G. Gallagher
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Genomic Dna ◽  
K562 Cells ◽  
Erythrocyte Membrane Protein ◽  
Factor Binding ◽  
Chromatin Architecture ◽  
Chip Chip

Abstract Erythrocyte membrane protein genes serve as excellent models of complex gene loci structure and function, as most encode multiple tissue-, cell-, developmental-, and stagespecific isoforms. Dynamic chromatin modifications participate in the regulatory control of many gene loci. We hypothesize that specific DNA sequences, transcription factors, and chromatin architecture (epigenetic modifications) regulate the tissue-specific expression of erythrocyte membrane protein genes. Advances in genomics technology have permitted rapid identification of DNA sequences bound by transcription factors and other DNAassociated proteins on a genome-wide scale. One technique available for mapping protein-DNA interactions in vivo couples chromatin immunoprecipitation to microarrays that contain regions of genomic DNA (ChIP-chip). We are using DNA obtained from chromatin immunoprecipitations performed with histone and erythroid transcription factor antibodies hybridized to genomic DNA microarray chips (ChIP-chip) to study the regulation of membrane protein genes in erythroid and nonerythroid cells. Chromatin immunoprecipitations (ChIP) were done in erythroid (K562) and non-erythroid (HeLa) cell lines using antibodies against H3 tri-methyl lysine 4 (H3K4me3, a marker of active chromatin) and the erythroid transcription factors GATA-1 and NF-E2. The chromatin resulting from these ChIPs was hybridized to a custom made NimbleGen high density human genomic DNA microarray (chip) focused on 15 genes critical to the erythrocyte membrane: ankyrin (ANK1), α-spectrin (SPTA1), β-spectrin (SPTB), band 3 (SLC4A1), β-adducin (ADD2), α-adducin (ADD1), γ-adducin (ADD3), ICAM-4, Erythroid Associated Membrane Protein (ERMAP), Protein 4.1 (EPB41), Protein 4.2 (EPB42), Dematin (ERPB49), β-Actin (ACTB), tropomodulin (TMOD1), and tropomyosin (TPM3). Probes for the chip were ~50bp in length with Tm ≥ 76°C, tiled every 65bp. From 50–100kb of flanking DNA was included on the chip for each locus. The Tamalpais peak calling algorithm using L1–L3 level of stringency (Genom Res16:595, 2006) was used to analyze the resulting data and identify regions of epigenetic modifications and transcription factor binding. Fourteen of 15 genes were enriched for H3K4me3 at promoter and transcriptional start sites (TSS) in K562 cells, with one gene, TMOD1, demonstrating a large peak of enrichment 5′ of the currently identified TSS, but not at the promoter. Two compact genes, β-actin and ICAM4, had H3K4me3 enrichment at the promoter and throughout gene. A total of 19 GATA-1 sites and 18 NF-E2 sites were identified. GATA-1 sites were found in 8 of 15 genes or in their flanking DNA. Three sites were in the 5′ flanking DNA, 1 site was at the promoter (~500bp from transcription start site, TSS), 12 sites were in introns, and 3 sites were in the 3′ flanking DNA. NF-E2 sites were found in 10 of 15 genes or their flanking DNA. 6 sites were in the 5′ flanking DNA, 1 site was at the promoter (~200bp from TSS), 8 sites were in introns, and 3 sites were in the 3′ flanking DNA. 18 of 19 GATA-1 sites (95%) and 13 of 18 NF-E2 sites (72%) were validated using qPCR-based quantitative ChIP. In K562 cells, 15 of 19 (79%) validated GATA-1 sites were associated with regions of chromatin enriched for H3K4me3, suggesting that ~a fifth of GATA-1 sites were in regions of inactive chromatin, consistent with a repressor function for GATA-1 at these sites. Eleven of 13 validated NF-E2 sites (85%) were associated with regions of K562 chromatin enriched for H3K4me3. In HeLa cells, the sites of GATA-1 and NF-E2 occupancy identified in K562 cells were almost never associated with H3K4me3 enrichment. GATA-1 and NF-E2 sites identified by Tamalpais and validated in K562 cells were analyzed in CD71-bright, glycophorin A-bright cultured primary erythroid cells using conventional quantitative ChIP analyses. Of the 13 NF-E2 sites identified in K562 cells, all 13 were also occupied in primary erythroid cells. ChIP-chip is a powerful tool for studying chromatin architecture and identifying transcription factor binding sites in complex genetic loci such as the erythrocyte membrane protein genes. It will be useful in constructing a comprehensive catalogue of chromatin architecture and transcription factor binding of genes expressed in erythroid cells.

The Acetyl-Transferase Tip60 Interacts with c-Myb and Inactivates Its Transcriptional Activity in Human Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3981-3981
Author(s):  
Huiwu Zhao ◽  
Shenghao Jin ◽  
Alan M. Gewirtz
Keyword(s):  
Histone Deacetylases ◽  
Transcriptional Activity ◽  
Hematopoietic Cells ◽  
Adaptor Protein ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Transactivation Domain ◽  
Interacting Protein ◽  
Factor C

Abstract Abstract 3981 Poster Board III-917 The acetyl-transferaseTip60 (Tat interacting protein, 60 kDa; also known as K(lysine) acetyltransferase 5:KAT5) is a co-regulator of transcription factors and is implicated in tumorigenesis. The protooncogene c-myb encodes a transcription factor, c-Myb, which is essential for normal hematopoisis and promotes hematologic malignancies. In this study, we explored potential regulatory relationships between Tip60 and c-Myb in hematopoietic cells. We first sought to detect any direct physical interactions by performing co-immunoprecitation (co-IP) assays. These revealed that Tip60 associated with c-Myb in Jurkat (T leukemia) and K562 (CML) cells. In vitro translated, epitope-tagged c-Myb and Tip60 also interacted with each other, suggesting that the Tip60, c-Myb interaction did not require an adaptor protein or co-factor. We then sought to define the interacting protein domains. Deletion analysis studies revealed that the interaction between Tip60 and c-Myb was dependent on the Tip60 MYST acetytransferase domain and transactivation domain of c-Myb. We then determined whether Tip60 could acetylate c-Myb, a post-translational event known to modulate c-Myb activity. Interestingly, an in vitro acetyltransferase assay showed that c-Myb was not a substrate of Tip60, even though Tip60 acetylated itself in the same assay. We then examined the effect of Tip60 on the ability of c-Myb to transcriptionally activate known target genes. In HEK293T cells, co-expressing Tip60 with c-Myb decreased c-Myb's ability to activate a luciferase reporter driven by the cim-1 promoter, a verified c-Myb target, by ∼60% compared to activation in the absence of Tip60. The physiologic significance of this observation was then explored. A chromatin immunoprecipiation (ChIP) assay revealed that Tip60 bound to the c-myc promoter, another known c-Myb target gene, in K562 cells. Furthermore, inactivation of endogenous c-Myb in K562 cells stably expressing an inducible c-Myb DNA binding domain reduced the occupancy of Tip60 in the c-myc promoter, suggesting that Tip60 utilizes c-Myb to bind its preferred site in the c-myc promoter. Using c-Myb, Tip60, and appropriate control siRNAs we achieved specific knockdowns of c-Myb, and Tip60 (∼80-90%, and ∼70-80% respectively compared to controls). Consistent with prior reports, c-myc expression decreased ∼60% when c-Myb was targeted, and ∼50% increased when Tip60 was targeted. A mechanistic explanation was sought to explain this finding. Tip60 is represses transcription when associated with histone deacetylases (HDAC), including HDAC1, HDAC2 and HDAC7. Co-IP of Jurkat cell lysates revealed that c-Myb is associated with HDAC1 and HDAC2. Altogether, these data suggest that Tip60 directly associates with c-Myb, and may inhibit its transcriptional activity by recruiting histone deacetylases(HDAC1 and HDAC2) to the activation complex. Finally, we compared Tip60 expression in 6 primary AML samples, with 3 normal CD34+ cell samples using QRT-PCR. Tip60 expression was significantly (∼60%) lower in the AML samples. In summary, these studies demonstrate that Tip60 modulates c-Myb transcriptional activity in human hematopoietic cells leading us to hypothesize that Tip60 is a normal regulator of c-Myb function and that dysregulated or mutated Tip60 may contribute to c-Myb driven leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

A T-Cell Specific Element Activates the TAL1 Oncogene Via an Interchromosomal Interaction During Leukemogenesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3507-3507
Author(s):  
Yuanyuan Kang ◽  
Bhavita Patel ◽  
Kairong Cui ◽  
Keji Zhao ◽  
Yi Qiu ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
K562 Cells ◽  
Chromosome Conformation Capture ◽  
Luciferase Reporter ◽  
Erythroid Progenitor ◽  
Chromosome Conformation

Abstract Abstract 3507 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant disease of thymocytes that mainly affects children and has very poor prognosis with high rates of relapse. A prominent feature observed in 60% of T-ALL childhood patients is the ectopic expression of a key hematopoietic transcription factor TAL1/SCL. Although several enhancers has been identified to play an important role in normal hematopoietic differentiation, the histone modification patterns and chromatin organization over the whole TAL1 locus reveled that none of them is active in T-ALL cell lines such as Jurkat and Rex cells. It remains currently unknown how TAL1 is activated in the majority of T-ALL patients lacking the TAL1 locus rearrangements. To understand the molecular mechanism underlying regulation of the TAL1 oncogene in leukemic T-cells, we employed circularized chromosome conformation capture (4C) methodology to identify new regulatory elements that activate TAL1 specifically in T-ALL leukemia. Using the TAL1 promoter 1a as the bait, we discovered that the TAL1 promoter 1a interacts with the TIL16 element (TAL1 interacting locus in chromosome 16) that is located at ∼15 Kb downstream of T-cell specific CD2BP2 gene in T-ALL cell line Jurkat, but not in erythroid progenitor K562 cells. The CD2BP2 protein is a cellular adapter protein that was originally identified as a binding partner of the T cell adhesion protein CD2 in the context of T cell signaling. The TIL16 element contains the bind sites for several transcription factors that are important for hematopoiesis such as C-Maf, Pax5, HoxA7 and USF2. The inter-chromosomal interaction between the TIL16 and the TAL1 promoter 1a was further confirmed by chromosome conformation capture (3C) assay in three TAL1 over-expressing T-ALL cell lines, Jurkat, REX and Molt4, but not in K562 cells. Recent genome wide study has correlates H3K4 mono- or dimethyl marks with distal enhancers while trimethyl H3K4 is enriched in promoters of active genes. To further test if the TIL16 acts as T-cell specific enhancer for TAL1 activation in T-ALL cells, we carried out ChIP-seq and ChIP analysis in CD4 T cells, Jurkat, and K562 cells. We found that the TIL16 element is specifically marked by H3K4me1 in Jurkat and CD4+ T-cells but not in K562 cells. The enrichment of H3K4me1 is correlated with the binding of c-Maf, a T-cell specific transcription factor. To further test whether the TIL16 element contributes to transcription activity, a DNA fragments containing the TIL16 element were cloneed into SV40 minimal promoter driven luciferase reporter and introduced into K562 and several T-ALL cell lines. Compared to the pGL3-SV40 vector that showed only minimal luciferase activity, the 1 Kb TIL element specifically activated transcription of the luciferase reporter in T-ALL cells, but not in erythroid progenitor K562 cells suggesting that the TIL16 element functions as a T-cell specific TAL1 enhancer. Thus, our data revealed a novel epigenetic mechanism by which the TAL1 oncogene is ectopically activated in T-cell leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regulation of E2F1-Dependent Gene Transcription and Apoptosis by the ETS-Related Transcription Factor GABPγ1

2002 ◽  
Vol 22 (7) ◽  
pp. 2147-2158 ◽  
Author(s):  
Ludger Hauck ◽  
Rudolf G. Kaba ◽  
Martin Lipp ◽  
Rainer Dietz ◽  
Rüdiger von Harsdorf
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Cell Fate ◽  
Binding Domain ◽  
Pocket Proteins ◽  
Related Transcription Factor ◽  
Terminal Amino ◽  
Fate Decision

ABSTRACT The E2F family of transcription factors comprises six related members which are involved in the control of the coordinated progression through the G1/S-phase transition of cell cycle or in cell fate decision. Their activity is regulated by pocket proteins, including pRb, p107, and p130. Here we show that E2F1 directly interacts with the ETS-related transcription factor GABPγ1 in vitro and in vivo. The binding domain interacting with GABPγ1 was mapped to the C-terminal amino acids 310 to 437 of E2F1, which include its transactivation and pRb binding domain. Among the E2F family of transcription factors, the interaction with GABPγ1 is restricted to E2F1. DNA-binding E2F1 complexes containing GABPγ1 are characterized by enhanced E2F1-dependent transcriptional activity. Moreover, GABPγ1 suppresses E2F1-dependent apoptosis by mechanisms other than the inhibition of the transactivation capacity of E2F1. In summary, our results provide evidence for a novel pRb-independent mechanism regulating E2F1-dependent transcription and apoptosis.

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