Revisiting a selection of target genes for the hematopoietic transcription factor c-Myb using chromatin immunoprecipitation and c-Myb knockdown

Abstract Gfi-1 −/− mice generate abnormal immature myeloid cells exhibiting characteristics of both monocytes and granulocytes. One of Gfi-1’s critical functions is to downregulate monocyte specific genes in order for granulocytes to develop properly. Since the transcription factors C/EBP alpha and C/EBP epsilon are needed for granulocyte development we hypothesized that these factors may regulate Gfi-1 expression. The Gfi-1 promoter contains several putative C/EBP binding sites and we show by electrophoretic mobility shift and chromatin immunoprecipitation that C/EBP family members can bind to some of these sites. However we were unable to see activation of the Gfi-1 promoter by C/EBP proteins in transient transfection reporter assays. Other groups have shown that C/EBP proteins can synergize with the transcription factor c-myb. We observed that the Gfi-1 promoter contains sites for the hematopoietic transcription factor c-myb. Sevral of these c-myb binding sites are adjacent to C/EBP binding sites. In reporter assays in non-hematopoietic cells c-myb activated the Gfi-1 promoter by itself and this activity was enhanced when we included either C/EBP alpha or epsilon in the transfection. Our data suggests that C/EBP proteins and c-myb regulate the transcription of Gfi-1 in myeloid cells.

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Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.01344-08 ◽

2009 ◽

Vol 29 (9) ◽

pp. 2431-2442 ◽

Cited By ~ 49

Author(s):

Zhongmin Yuan ◽

Shoufang Gong ◽

Jingyan Luo ◽

Zhihao Zheng ◽

Bin Song ◽

...

Keyword(s):

Transcription Factor ◽

Neuronal Apoptosis ◽

Target Genes ◽

Dominant Negative ◽

Bimolecular Fluorescence Complementation ◽

Activator Protein 1 ◽

Fos Expression ◽

Activating Transcription Factor ◽

Living Neurons ◽

Factor C

ABSTRACT The activator protein 1 (AP-1) transcription factor c-Jun is crucial for neuronal apoptosis. However, c-Jun dimerization partners and the regulation of these proteins in neuronal apoptosis remain unknown. Here we report that c-Jun-mediated neuronal apoptosis requires the concomitant activation of activating transcription factor-2 (ATF2) and downregulation of c-Fos. Furthermore, we have observed that c-Jun predominantly heterodimerizes with ATF2 and that the c-Jun/ATF2 complex promotes apoptosis by triggering ATF activity. Inhibition of c-Jun/ATF2 heterodimerization using dominant negative mutants, small hairpin RNAs, or decoy oligonucleotides was able to rescue neurons from apoptosis, whereas constitutively active ATF2 and c-Jun mutants were found to synergistically stimulate apoptosis. Bimolecular fluorescence complementation analysis confirmed that, in living neurons, c-Fos downregulation facilitates c-Jun/ATF2 heterodimerization. A chromatin immunoprecipitation assay also revealed that c-Fos expression prevents the binding of c-Jun/ATF2 heterodimers to conserved ATF sites. Moreover, the presence of c-Fos is able to suppress the expression of c-Jun/ATF2-mediated target genes and, therefore, apoptosis. Taken together, our findings provide evidence that potassium deprivation-induced neuronal apoptosis is mediated by concurrent upregulation of c-Jun/ATF2 heterodimerization and downregulation of c-Fos expression. This paradigm demonstrates opposing roles for ATF2 and c-Fos in c-Jun-mediated neuronal apoptosis.

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Expresso: A database and web server for exploring the interaction of transcription factors and their target genes in Arabidopsis thaliana using ChIP-Seq peak data

F1000Research ◽

10.12688/f1000research.10041.1 ◽

2017 ◽

Vol 6 ◽

pp. 372 ◽

Cited By ~ 7

Author(s):

Delasa Aghamirzaie ◽

Karthik Raja Velmurugan ◽

Shuchi Wu ◽

Doaa Altarawy ◽

Lenwood S. Heath ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Chromatin Immunoprecipitation ◽

Target Genes ◽

Regulation Of Gene Expression ◽

Data Sets ◽

Motif Analysis ◽

Chromatin Immunoprecipitation Sequencing

Motivation: The increasing availability of chromatin immunoprecipitation sequencing (ChIP-Seq) data enables us to learn more about the action of transcription factors in the regulation of gene expression. Even though in vivo transcriptional regulation often involves the concerted action of more than one transcription factor, the format of each individual ChIP-Seq dataset usually represents the action of a single transcription factor. Therefore, a relational database in which available ChIP-Seq datasets are curated is essential. Results: We present Expresso (database and webserver) as a tool for the collection and integration of available Arabidopsis ChIP-Seq peak data, which in turn can be linked to a user’s gene expression data. Known target genes of transcription factors were identified by motif analysis of publicly available GEO ChIP-Seq data sets. Expresso currently provides three services: 1) Identification of target genes of a given transcription factor; 2) Identification of transcription factors that regulate a gene of interest; 3) Computation of correlation between the gene expression of transcription factors and their target genes. Availability: Expresso is freely available at http://bioinformatics.cs.vt.edu/expresso/

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Bag1-L Is a Phosphorylation-Dependent Coactivator of c-Jun during Neuronal Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.01610-09 ◽

2010 ◽

Vol 30 (15) ◽

pp. 3842-3852 ◽

Cited By ~ 7

Author(s):

Clive R. Da Costa ◽

Javier Villadiego ◽

Rocio Sancho ◽

Xavier Fontana ◽

Graham Packham ◽

...

Keyword(s):

Cell Death ◽

Transcriptional Activation ◽

Chromatin Immunoprecipitation ◽

Neuronal Apoptosis ◽

Target Genes ◽

Critical Role ◽

Transactivation Domain ◽

Multifunctional Protein ◽

System Cell ◽

Factor C

ABSTRACT In the nervous system, cell death by apoptosis plays a critical role during normal development and pathological neurodegeneration. Jun N-terminal kinases (JNKs) are essential regulators of neuronal apoptosis. The AP-1 transcription factor c-Jun is phosphorylated at multiple sites within its transactivation domain by the JNKs, and c-Jun phosphorylation is required for JNK-induced neurotoxicity. While the importance of c-Jun as a mediator of apoptotic JNK signaling in neurons is firmly established, the molecular mechanism underlying the requirement for c-Jun N-terminal phosphorylation is enigmatic. Here we identify the multifunctional protein Bag1-L as a coactivator of phosphorylated c-Jun. Bag1-L preferentially interacts with N-terminally phosphorylated c-Jun, and Bag1-L greatly augments transcriptional activation by phosphorylated c-Jun. Chromatin immunoprecipitation experiments revealed binding of Bag1-L to the promoters of proapoptotic AP-1 target genes, and overexpression of Bag1-L augmented cell death in primary neurons. Therefore, Bag1-L functions as a coactivator regulating neurotoxicity mediated by phosphorylated c-Jun.

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GSK3β inactivation induces apoptosis of leukemia cells by repressing the function of c-Myb

Molecular Biology of the Cell ◽

10.1091/mbc.e11-06-0483 ◽

2011 ◽

Vol 22 (18) ◽

pp. 3533-3540 ◽

Cited By ~ 34

Author(s):

Fangfang Zhou ◽

Long Zhang ◽

Theo van Laar ◽

Hans van Dam ◽

Peter ten Dijke

Keyword(s):

Transcription Factor ◽

Cell Survival ◽

Chromatin Immunoprecipitation ◽

Kinase Activity ◽

Glycogen Synthase ◽

Leukemia Cells ◽

Physiological Processes ◽

Different Types ◽

Reporter Assays ◽

Factor C

Glycogen synthase kinase 3β (GSK3β) regulates diverse physiological processes, including metabolism, development, oncogenesis, and neuroprotection. GSK3β kinase activity has been reported to be critical for various types of cancer cells, but the mechanism has remained elusive. In this study we examine the mechanism by which GSK3β regulates the survival of leukemia cells. We demonstrate that upon GSK3β kinase inhibition different types of leukemia cells show severe proliferation defects as a result of apoptosis. The transcription factor c-Myb is found to be the main target of GSK3β inhibition in cell survival. GSK3β inactivation reduces the expression of c-Myb by promoting its ubiquitination-mediated degradation, thereby inhibiting the expression of c-Myb–dependent antiapoptotic genes Bcl2 and survivin. Coimmunoprecipitation, reporter assays, chromatin immunoprecipitation, and knockdown studies show that c-Myb needs to interact and cooperate with transcription factor LEF-1 in the activation of Bcl2 and survivin and that both transcription factors are required for cell survival. These data reveal an as-yet-unknown mechanism by which GSK3β controls cell survival.

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First-in-human, first-in-class phase I study of MTL-CEBPA, a small activating RNA (saRNA) targeting the transcription factor C/EBP-α in patients with advanced liver cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.tps2612 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. TPS2612-TPS2612

Author(s):

Debashis Sarker ◽

Elizabeth R. Plummer ◽

Bristi Basu ◽

Tim Meyer ◽

Kai-Wen Huang ◽

...

Keyword(s):

Transcription Factor ◽

Liver Cancer ◽

Liver Function ◽

Target Genes ◽

White Blood Cells ◽

Primary Objective ◽

Mrna Levels ◽

Dose Escalation Study ◽

Advanced Hcc ◽

Factor C

TPS2612 Background: saRNAs are small oligonucleotide drugs designed to selectively upregulate therapeutic proteins by recruiting endogenous transcriptional complexes to a target gene, leading to increased expression of naturally processed mRNA. Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) is a leucine zipper protein which acts as a master regulator of liver homeostasis and multiple oncogenic processes including cell cycle control, proliferation and angiogenesis. MTL-CEBPA comprises a double stranded RNA payload formulated inside a SMARTICLES liposomal nanoparticle to specifically target the CEBPA gene and has been shown to improve liver function and inhibit hepatocellular cancer (HCC) tumor growth in preclinical models (Reebye et al, Hepatology, 2014). MTL-CEBPA is the first saRNA and the first drug targeting C/EBP-α to enter clinical trials. Methods: Pts with advanced HCC (Child-Pugh A or B7 only) or secondary liver cancer refractory to or ineligible for standard treatment, ECOG PS 0-1, acceptable haematologic, liver and renal function, are currently being enrolled in a standard 3+3 dose escalation study. Once the RP2D is defined, 12-15 patients with advanced HCC will be evaluated further in a dose expansion cohort. MTL-CEBPA is administered as a 1-hr IV infusion on Day 1, 8 and 15 of a 28-day cycle. RECIST tumor response is assessed after every 2 cycles. The primary objective is to determine safety and tolerability; secondary objectives include PK, liver function improvement and anti-tumor activity. Correlative studies include C/EBP-α mRNA levels in PBMCs and optional tumor tissue, evaluation of C/EBP-α downstream target genes (e.g.TGFβ) and distal target engagement in white blood cells (e.g.IL-6, NF-κB, IFN-γ). Recruitment to cohort 2 is shortly to be completed, with no DLTs reported to date. Clinical trial information: NCT02716012.

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The transcription factor C/EBPβ orchestrates dendritic cell maturation and functionality under homeostatic and malignant conditions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008883117 ◽

2020 ◽

Vol 117 (42) ◽

pp. 26328-26339

Author(s):

Florian Scholz ◽

Michael Grau ◽

Lutz Menzel ◽

Annika Graband ◽

Myroslav Zapukhlyak ◽

...

Keyword(s):

Transcription Factor ◽

Dendritic Cell ◽

Cell Fate ◽

Target Genes ◽

Expression Profiles ◽

B Cell Lymphoma ◽

Negative Regulator ◽

Cell Fate Decisions ◽

Factor C ◽

Dc Maturation

Dendritic cell (DC) maturation is a prerequisite for the induction of adaptive immune responses against pathogens and cancer. Transcription factor (TF) networks control differential aspects of early DC progenitor versus late-stage DC cell fate decisions. Here, we identified the TF C/EBPβ as a key regulator for DC maturation and immunogenic functionality under homeostatic and lymphoma-transformed conditions. Upon cell-specific deletion of C/EBPβ in CD11c+MHCIIhiDCs, gene expression profiles of splenic C/EBPβ−/−DCs showed a down-regulation of E2F cell cycle target genes and associated proliferation signaling pathways, whereas maturation signatures were enriched. Total splenic DC cell numbers were modestly increased but differentiation into cDC1 and cDC2 subsets were unaltered. The splenic CD11c+MHCIIhiCD64+DC compartment was also increased, suggesting that C/EBPβ deficiency favors the expansion of monocytic-derived DCs. Expression of C/EBPβ could be mimicked in LAP/LAP* isoform knockin DCs, whereas the short isoform LIP supported a differentiation program similar to deletion of the full-length TF. In accordance with E2F1 being a negative regulator of DC maturation, C/EBPβ−/−bone marrow-derived DCs matured much faster enabling them to activate and polarize T cells stronger. In contrast to a homeostatic condition, lymphoma-exposed DCs exhibited an up-regulation of the E2F transcriptional pathways and an impaired maturation. Pharmacological blockade of C/EBPβ/mTOR signaling in human DCs abrogated their protumorigenic function in primary B cell lymphoma cocultures. Thus, C/EBPβ plays a unique role in DC maturation and immunostimulatory functionality and emerges as a key factor of the tumor microenvironment that promotes lymphomagenesis.

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LSD1 defines erythroleukemia metabolism by controlling the lineage-specific transcription factors GATA1 and C/EBPα

Blood Advances ◽

10.1182/bloodadvances.2020003521 ◽

2021 ◽

Vol 5 (9) ◽

pp. 2305-2318

Author(s):

Kensaku Kohrogi ◽

Shinjiro Hino ◽

Akihisa Sakamoto ◽

Kotaro Anan ◽

Ryuta Takase ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Aerobic Glycolysis ◽

Clinical Samples ◽

Gene Encoding ◽

Heme Synthesis ◽

Metabolic Remodeling ◽

Chromatin Immunoprecipitation Sequencing ◽

Factor C

Abstract Acute myeloid leukemia (AML) is a heterogenous malignancy characterized by distinct lineage subtypes and various genetic/epigenetic alterations. As with other neoplasms, AML cells have well-known aerobic glycolysis, but metabolic variations depending on cellular lineages also exist. Lysine-specific demethylase-1 (LSD1) has been reported to be crucial for human leukemogenesis, which is currently one of the emerging therapeutic targets. However, metabolic roles of LSD1 and lineage-dependent factors remain to be elucidated in AML cells. Here, we show that LSD1 directs a hematopoietic lineage-specific metabolic program in AML subtypes. Erythroid leukemia (EL) cells particularly showed activated glycolysis and high expression of LSD1 in both AML cell lines and clinical samples. Transcriptome, chromatin immunoprecipitation–sequencing, and metabolomic analyses revealed that LSD1 was essential not only for glycolysis but also for heme synthesis, the most characteristic metabolic pathway of erythroid origin. Notably, LSD1 stabilized the erythroid transcription factor GATA1, which directly enhanced the expression of glycolysis and heme synthesis genes. In contrast, LSD1 epigenetically downregulated the granulo-monocytic transcription factor C/EBPα. Thus, the use of LSD1 knockdown or chemical inhibitor dominated C/EBPα instead of GATA1 in EL cells, resulting in metabolic shifts and growth arrest. Furthermore, GATA1 suppressed the gene encoding C/EBPα that then acted as a repressor of GATA1 target genes. Collectively, we conclude that LSD1 shapes metabolic phenotypes in EL cells by balancing these lineage-specific transcription factors and that LSD1 inhibitors pharmacologically cause lineage-dependent metabolic remodeling.

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Reduced Binding of C/EBPα to Myeloid Specific Promoters with Altered Gene Expression in the Presence of PML/RARα.

Blood ◽

10.1182/blood.v106.11.2999.2999 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2999-2999

Author(s):

Katharina Wagner ◽

Jürgen Krauter ◽

Kerstin Görlich ◽

Bettina Drescher ◽

Lisa M. Johansen ◽

...

Keyword(s):

Gene Expression ◽

Chromatin Immunoprecipitation ◽

Target Genes ◽

Fold Increase ◽

Surface Expression ◽

Rna Expression ◽

Altered Gene Expression ◽

Myeloid Development ◽

Altered Gene ◽

Factor C

Abstract The hallmark of acute promyelocytic leukemia is the reciprocal translocation t(15;17) resulting in the fusion protein PML/RARα . According to the current concept of the pathogenesis of APL, PML/RARα interferes with RARα target gene expression. Until now, the crucial target genes have not been found. The transcription factor C/EBPα is essential for myeloid development and mutations have been found in 10% of AML patients. No mutations of C/EBPα in APL have been described. However, using the U937 cell line with a Zinc-inducible PML/RARα (U937PR9), a decrease of C/EBPα binding to the G-CSF-R promoter in the presence of PML/RARα was detected in EMSA as well as chromatin immunoprecipitation. This finding indicates, that the function of C/EBPα might be impaired in APL. To test whether the effect is limited to the G-CSF-R promoter, C/EBPα binding to other myeloid promoters was analyzed upon Zn-induction of PML/RARα . In those experiments decreased binding of C/EBPα to other promoters of myeloid specific genes, such as neutrophil elastase, was detected. In contrast, no effect of Zn-induction on the binding of Sp3 to the thymidine-kinase promotor was found. Next, the functional importance of the alteration in binding was demonstrated. In the presence of PML/RARα , decreased G-CSF-R RNA expression as well as G-CSF-R surface expression was found in U937PR9 cells. In addition, the RNA expression of elastase was reduced after Zn-induction. To investigate the effect of PML/RARα on C/EBPα function in t(15;17) positive cells, a knockdown of PML/RARα by lentivirally delivered shRNAs in NB4 cells was used. After lentiviral transduction, a marked decrease of PML/RARα RNA and protein was found. Consistent with the results in U937PR9 cells a 5–6 fold increase of elastase RNA expression and a 2 fold increase in G-CSF-R-RNA was observed after PML/RARα knockdown. Taken together, decreased binding of C/EBPα to promoters of myeloid specific genes was found in the presence of PML/RARα . This alteration in binding was associated with a reduction of RNA expression. Decreasing PML/RARα with siRNA had the opposite effects. Currently, the mechanism for this effect is being investigated.

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The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

Molecular Biology of the Cell ◽

10.1091/mbc.e08-04-0359 ◽

2008 ◽

Vol 19 (8) ◽

pp. 3263-3271 ◽

Cited By ~ 73

Author(s):

Jennifer L. Marlowe ◽

Yunxia Fan ◽

Xiaoqing Chang ◽

Li Peng ◽

Erik S. Knudsen ◽

...

Keyword(s):

Transcription Factor ◽

Chromatin Immunoprecipitation ◽

Expression Vector ◽

Target Genes ◽

Ah Receptor ◽

Aryl Hydrocarbon ◽

Mrna Induction ◽

Induced Apoptosis ◽

Interfering Rna ◽

Transcriptional Induction

Cellular stress by DNA damage induces checkpoint kinase-2 (CHK2)-mediated phosphorylation and stabilization of the E2F1 transcription factor, leading to induction of apoptosis by activation of a subset of proapoptotic E2F1 target genes, including Apaf1 and p73. This report characterizes an interaction between the aryl hydrocarbon (Ah) receptor (AHR), a ligand-activated transcription factor, and E2F1 that results in the attenuation of E2F1-mediated apoptosis. In Ahr−/− fibroblasts stably transfected with a doxycycline-regulated AHR expression vector, inhibition of AHR expression causes a significant elevation of oxidative stress, γH2A.X histone phosphorylation, and E2F1-dependent apoptosis, which can be blocked by small interfering RNA-mediated knockdown of E2F1 expression. In contrast, ligand-dependent AHR activation protects these cells from etoposide-induced cell death. In cells expressing both proteins, AHR and E2F1 interact independently of the retinoblastoma protein (RB), because AHR and E2F1 coimmunoprecipitate from extracts of RB-negative cells. Additionally, chromatin immunoprecipitation assays indicate that AHR and E2F1 bind to the Apaf1 promoter at a region containing a consensus E2F1 binding site but no AHR binding sites. AHR activation represses Apaf1 and TAp73 mRNA induction by a constitutively active CHK2 expression vector. Furthermore, AHR overexpression blocks the transcriptional induction of Apaf1 and p73 and the accumulation of sub-G0/G1 cells resulting from ectopic overexpression of E2F1. These results point to a proproliferative, antiapoptotic function of the Ah receptor that likely plays a role in tumor progression.

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