scholarly journals Ets-dependent Regulation of Target Gene Expression during Megakaryopoiesis

2004 ◽  
Vol 279 (50) ◽  
pp. 52183-52190 ◽  
Author(s):  
Pascale Jackers ◽  
Gabor Szalai ◽  
Omar Moussa ◽  
Dennis K. Watson
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Hematopoietic Stem ◽  
Exogenous Expression ◽  
Direct Target ◽  
Transcriptional Complex

Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled by specific transcription factors. Fli-1 and GATA-1 transcription factors are required for development of megakaryocytes and promoter analysis has definedin vitrofunctional binding sites for these factors in several megakaryocytic genes, includingGPIIb,GPIX, andC-MPL. Herein, we utilize chromatin immunoprecipitation to examine the presence of Ets-1, Fli-1, and GATA-1 on these promotersin vivo. Fli-1 and Ets-1 occupy the promoters ofGPIIb,GPIX, andC-MPLgenes in both Meg-01 and CMK11-5 cells. WhereasGPIIbis expressed in both Meg-01 and CMK11-5 cells,GPIXandC-MPLare only expressed in the more differentiated CMK11–5 cells. Thus,in vivooccupancy by an Ets factor is not sufficient to promote transcription of some megakaryocytic genes. GATA-1 and Fli-1 are both expressed in CMK11-5 cells and co-occupy theGPIXandC-MPLpromoters. Transcription of all three megakaryocytic genes is correlated with the presence of acetylated histone H3 and phosphorylated RNA polymerase II on their promoters. We also show that exogenous expression of GATA-1 in Meg-01 cells leads to the expression of endogenous c-mpl and gpIX mRNA. WhereasGPIIb,GPIX, andC-MPLare direct target genes for Fli-1, both Fli-1 and GATA-1 are required for formation of an active transcriptional complex on theC-MPLandGPIXpromotersin vivo. In contrast,GPIIbexpression appears to be independent of GATA-1 in Meg-01 cells.

scholarly journals Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

2020 ◽  
Vol 295 (39) ◽  
pp. 13617-13629
Author(s):  
Clément Immarigeon ◽  
Sandra Bernat-Fabre ◽  
Emmanuelle Guillou ◽  
Alexis Verger ◽  
Elodie Prince ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Direct Interaction ◽  
Mediator Complex ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Rna Pol Ii ◽  
Evolutionarily Conserved

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

scholarly journals Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity

2020 ◽  
Vol 21 (24) ◽  
pp. 9401
Author(s):  
Antonio Bouthelier ◽  
Florinda Meléndez-Rodríguez ◽  
Andrés A. Urrutia ◽  
Julián Aragonés
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Cellular Response ◽  
Target Gene ◽  
Target Genes ◽  
Transactivation Domain ◽  
Transactivation Domains ◽  
Relative Contribution

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.

scholarly journals Investigation of Transcription Repression and Small-Molecule Responsiveness by TetR-Like Transcription Factors Using a Heterologous Escherichia coli-Based Assay

2007 ◽  
Vol 189 (18) ◽  
pp. 6655-6664 ◽  
Author(s):  
Sang Kyun Ahn ◽  
Kapil Tahlan ◽  
Zhou Yu ◽  
Justin Nodwell
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Small Molecule ◽  
Target Genes ◽  
Streptomyces Coelicolor ◽  
Targeted Mutagenesis ◽  
E Coli ◽  
Small Molecule Ligands

ABSTRACT The SCO7222 protein and ActR are two of ∼150 TetR-like transcription factors encoded in the Streptomyces coelicolor genome. Using bioluminescence as a readout, we have developed Escherichia coli-based biosensors that accurately report the regulatory activity of these proteins and used it to investigate their interactions with DNA and small-molecule ligands. We found that the SCO7222 protein and ActR repress the expression of their putative target genes, SCO7223 and actII-ORF2 (actA), respectively, by interacting with operator sequence in the promoters. The operators recognized by the two proteins are related such that O 7223 (an operator for SCO7223) could be bound by both the SCO7222 protein and ActR with similar affinities. In contrast, Oact (an operator for actII-ORF2) was bound tightly by ActR and more weakly by the SCO7222 protein. We demonstrated ligand specificity of these proteins by showing that while TetR (but not ActR or the SCO7222 protein) interacts with tetracyclines, ActR (but not TetR or the SCO7222 protein) interacts with actinorhodin and related molecules. Through operator-targeted mutagenesis, we found that at least two nucleotide changes in O 7223 were required to disrupt its interaction with SCO7222 protein, while ActR was more sensitive to changes on Oact . Most importantly, we found that the interaction of each protein with wild-type and mutant operator sequences in vivo and in vitro correlated perfectly. Our data suggest that E. coli-based biosensors of this type should be broadly applicable to TetR-like transcription factors.

scholarly journals A Multiplicity of Coactivators Is Required by Gcn4p at Individual Promoters In Vivo

2003 ◽  
Vol 23 (8) ◽  
pp. 2800-2820 ◽  
Author(s):  
Mark J. Swanson ◽  
Hongfang Qiu ◽  
Laarni Sumibcay ◽  
Anna Krueger ◽  
Soon-ja Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chromatin Immunoprecipitation ◽  
Target Gene ◽  
Target Genes ◽  
Yeast Cells ◽  
Histone Acetyltransferases ◽  
Vitro Binding ◽  
Paf1 Complex

ABSTRACT Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes. Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p. We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo. The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation. Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p. We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex. In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED. Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo. We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.

scholarly journals A New Role for Sterol Regulatory Element Binding Protein 1 Transcription Factors in the Regulation of Muscle Mass and Muscle Cell Differentiation

2009 ◽  
Vol 30 (5) ◽  
pp. 1182-1198 ◽  
Author(s):  
Virginie Lecomte ◽  
Emmanuelle Meugnier ◽  
Vanessa Euthine ◽  
Christine Durand ◽  
Damien Freyssenet ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Muscle Mass ◽  
Target Genes ◽  
Binding Protein ◽  
Regulatory Element ◽  
Element Binding Protein ◽  
Sterol Regulatory Element ◽  
Myogenic Program

ABSTRACT The role of the transcription factors sterol regulatory element binding protein 1a (SREBP-1a) and SREBP-1c in the regulation of cholesterol and fatty acid metabolism has been well studied; however, little is known about their specific function in muscle. In the present study, analysis of recent microarray data from muscle cells overexpressing SREBP1 suggested that they may play a role in the regulation of myogenesis. We then demonstrated that SREBP-1a and -1c inhibit myoblast-to-myotube differentiation and also induce in vivo and in vitro muscle atrophy. Furthermore, we have identified the transcriptional repressors BHLHB2 and BHLHB3 as mediators of these effects of SREBP-1a and -1c in muscle. Both repressors are SREBP-1 target genes, and they affect the expression of numerous genes involved in the myogenic program. Our findings identify a new role for SREBP-1 transcription factors in muscle, thus linking the control of muscle mass to metabolic pathways.

PU.1 Dose-Dependently Induces Granulocyte or Macrophage Commitment by Targeting Lineage Restricted Genes and by Regulating Transcription Factors Egr2, Nab2, Cebpa and Gfi1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 661-661
Author(s):  
Vit Pospisil ◽  
Juraj Kokavec ◽  
Pavel Burda ◽  
Nikola Curik ◽  
Arthur I. Skoultchi ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Fetal Liver ◽  
Regulatory Regions ◽  
Target Mrnas ◽  
Interleukin 7 ◽  
Ets Family ◽  
Maximum Expression

Abstract PU.1 (Sfpi1) is an ets family transcription factor required for the proper generation of both myeloid (macrophages and neutrophils) and lymphoid lineages (B and T lymphocytes)(Scott 1994, McKercher 1996). Graded expression of exogenous PU.1 in murine PU.1-deficient fetal liver hematopoietic progenitors demonstrated that increased levels of PU.1 are required to initiate development of macrophages (DeKoter, 2000). We have studied the effects of graded expression of PU.1 on its occupancy in chromatin and on the development of myeloid cells in vitro. We measured changes in gene expression, PU.1 occupancy and histone modifications in PU.1-null hematopoietic progenitor cells stably expressing PU.1 fused to the ligand-binding domain of the estrogen receptor (PU.1-ER) (Walsh 2002). The level of active PU.1-ER was regulated with graded levels of the ER inducer tamoxifen. In vitro, intermediate levels of tamoxifen produced cells with granulocyte characteristics in the suspension cell fraction and macrophage-like characteristics in the attached fraction, whereas high levels of PU.1 produced mostly attached macrophage-like cells. Expression of granulocyte-specific PU.1 target mRNAs including gelatinase B (Mmp9) and myeloperoxidase (Mpo) were observed to be expressed only with intermediate levels of tamoxifen. In contrast, expression of macrophage PU.1 target mRNAs including Cd14, F4/80 and Cd68 mRNAs were observed to be gradually upregulated upon PU.1-ER activation, with the maximum expression at the highest levels of tamoxifen. Thus, the expression levels of PU.1 target genes and phenotypic characteristics of the cells are dependent on PU.1 levels. Interestingly, macrophage-like cells can be produced from granulocytic-like cells by changing tamoxifen levels and vice versa. Chromatin immunoprecipitation analysis revealed specific PU.1 occupancy within regulatory regions of the genes predominantly expressed in macrophages including Cd14 and Cd11b after treatment with high levels of tamoxifen. Specific PU.1 occupancy within regulatory regions of the granulocyte specific genes including MMP9 was observed at intermediate levels of tamoxifen. Suprisingly, chromatin immunoprecipitation analysis revealed specific PU.1 occupancy within regulatory regions of the lymphocytic PU.1 target genes including Interleukin-7 receptor (Il-7r) and RAG1 at intermediate levels of tamoxifen even though expression of these genes was not detected. Accumulation of acetylated K9 and methylated K4 of histone H3 in gene loci of macrophage and granulocytic markers such as Cd14, Cd11b, and Mmp9 correlated with their mRNA expression. However, lymphocyte-specific regulatory regions including that of Il-7r gene were hypoacetylated in H3K9 despite a marked PU.1 recruitment suggesting additional factors may be required for PU.1 mediated transactivation. To identify these molecules we have tested PU.1-dependent transcription factors: Egr2, Nab2, Cebpa and Gfi-1 and found that upon increasing PU.1 levels, expression of Egr2/Nab2 and Gfi-1/Cebpa changed in a reciprocal manner and these changes preceded expression of the lineage specific markers. We are currently testing if PU.1 directly regulates expression of Egr2, Nab2, Cebpa and Gfi-1 during granulocytic/macrophage differentiation.

New Approaches for Mapping Epigenome Dynamics

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-21-SCI-21
Author(s):  
Steven Henikoff
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Conflicts Of Interest ◽  
Regulation Of Transcription ◽  
Base Pairs ◽  
Simple Method ◽  
Genome Wide ◽  
Resolution Mapping

Abstract The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely mapping RNAPII genome-wide. We have used this method to show exactly where RNAPII stalls as it unwraps a nucleosome in vivo, surprisingly in a different place in vivo from where it stalls in vitro. We also have discovered that a variant histone, H2A.Z, which is found in essentially all eukaryotes, helps to reduce the nucleosome barrier to transcription, and in this way may modulate transcription. Other protein components of the epigenome involved in dynamic processes are nucleosome remodelers, which use the energy of ATP to slide or even evict nucleosomes from DNA. Some remodelers help RNAPII get started and others help it overcome the nucleosome barrier to transcription, and by mapping them at base-pair resolution, we can gain insight into how they act. We have also applied our high-resolution mapping tools to transcription factors, which bind DNA at specific sites to regulate transcription and other processes. Our ability to achieve high spatial and temporal resolution mapping of the binding and action of nucleosomes, transcription factors, remodelers and RNAPII provides us with a detailed picture of epigenome dynamics. By using these tools we are beginning to understand how DNA sequence and conformation are recognized for regulation of transcription and other epigenomic processes. Disclosures No relevant conflicts of interest to declare.

scholarly journals c-Jun Homodimers Can Function as a Context-Specific Coactivator

2007 ◽  
Vol 27 (8) ◽  
pp. 2919-2933 ◽  
Author(s):  
Benoit Grondin ◽  
Martin Lefrancois ◽  
Mathieu Tremblay ◽  
Marianne Saint-Denis ◽  
André Haman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Protein Interactions ◽  
Expression Profiles ◽  
Basic Domain ◽  
Interaction Mode

ABSTRACT Transcription factors can function as DNA-binding-specific activators or as coactivators. c-Jun drives gene expression via binding to AP-1 sequences or as a cofactor for PU.1 in macrophages. c-Jun heterodimers bind AP-1 sequences with higher affinity than homodimers, but how c-Jun works as a coactivator is unknown. Here, we provide in vitro and in vivo evidence that c-Jun homodimers are recruited to the interleukin-1β (IL-1β) promoter in the absence of direct DNA binding via protein-protein interactions with DNA-anchored PU.1 and CCAAT/enhancer-binding protein β (C/EBPβ). Unexpectedly, the interaction interface with PU.1 and C/EBPβ involves four of the residues within the basic domain of c-Jun that contact DNA, indicating that the capacities of c-Jun to function as a coactivator or as a DNA-bound transcription factor are mutually exclusive. Our observations indicate that the IL-1β locus is occupied by PU.1 and C/EBPβ and poised for expression and that c-Jun enhances transcription by facilitating a rate-limiting step, the assembly of the RNA polymerase II preinitiation complex, with minimal effect on the local chromatin status. We propose that the basic domain of other transcription factors may also be redirected from a DNA interaction mode to a protein-protein interaction mode and that this switch represents a novel mechanism regulating gene expression profiles.

scholarly journals GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

2011 ◽  
Vol 286 (41) ◽  
pp. 35553-35561 ◽  
Author(s):  
Lidija Staresincic ◽  
Jane Walker ◽  
A. Barbara Dirac-Svejstrup ◽  
Richard Mitter ◽  
Jesper Q. Svejstrup
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Import ◽  
Chromatin Immunoprecipitation ◽  
Nuclear Transport ◽  
Proteomic Screen ◽  
Genome Wide ◽  
Importin Α

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/β pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.

scholarly journals In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

1992 ◽  
Vol 12 (4) ◽  
pp. 1639-1651 ◽  
Author(s):  
S C Batson ◽  
R Sundseth ◽  
C V Heath ◽  
M Samuels ◽  
U Hansen
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Viral Dna ◽  
Dna Templates ◽  
Initiation Of Transcription ◽  
Transcription In Vitro ◽  
Alpha Amanitin

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.

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