Within the adult mammalian brain, Neural Stem Cell (NSC)s are maintained in distinct neurogenic niches in a mostly quiescent state. Activation of quiescent NSCs first requires re-entry into the cell cycle in order for the pool to proliferate and eventually commit to a neural fate, giving rise to newborn neurons. The canonical Retinoblastoma (Rb)-E2 Promoter Binding Factor (E2f) pathway is not only key in overcoming the Gap 1 Phase (G1)/S-phase restriction, but novelly appears to be involved in adult neurogenesis and NSC activation. I hypothesized that activator transcription factors E2 Promoter Binding Factor 1 (E2f1) and E2 Promoter Binding Factor 3 (E2f3) are crucial for exit from a quiescent state in adult NSCs. The contribution of the activator E2fs in this transition was studied using a Nestin-driven Cre Recombinase-Estrogen Receptor Tamoxifen-2 Ligand Binding Domain (Cre-ERT2) system to induce targeted deletion of E2f1/3 within NSCs in adult mice. We show that loss of E2f1/3 causes significant neurogenic defects, including pro-neural activation and decreased pools of adult NSCs, that preferentially adopt a quiescent profile in the subventricular zone. We employed this model to further isolate subventricular zone-derived NSCs using a Rosa26:Yellow Fluorescent Protein (YFP) reporter and subsequently analysed transcriptional profiles by RNA sequencing. Loss of E2f1/3 shifts NSC transcriptomes towards one overlapping with quiescent neural stem cell signatures (Codega et al., 2014; Basak et al., 2018), further highlighting the requirement of these E2fs for initial activation. A significant portion of these differentially expressed genes are putative E2f targets. Transcriptionally, major pathways involving cell metabolism, cellular signaling, and neural development are perturbed without activator E2f expression. In effect, this combined approach based on in vivo data and bioinformatics analyses offers a method of prospective identification of novel regulators of adult neurogenesis that require the activator E2fs. Preliminary data suggests that AT-Hook Transcription Factor (Akna) is one such target worth pursuing. Cumulatively, this project describes a unique role for E2f1 and E2f3 during NSC exit from quiescence and subsequent activation towards differentiation. As ongoing maintenance of quiescent NSCs is a necessary prerequisite for lifelong neurogenesis, conclusions from this study could determine the therapeutic potential of targeting activator E2fs to combat the niche exhaustion associated with aging, injury, and neurodegenerative diseases.

Le contrôle du cycle cellulaire, processus fondamental pour la prolifération cellulaire, est souvent altéré au cours de la tumorigenèse. Les facteurs de transcription E2Fs sont des régulateurs majeurs de l'expression de gènes impliqués dans le cycle cellulaire, la réplication de l'ADN, la mort cellulaire programmée ou encore la différenciation cellulaire. La famille des facteurs E2Fs contient des membres qui agissent comme activateurs ou répresseurs de la transcription et dont l'activité est régulée par un grand nombre de corégulateurs transcriptionnels, incluant notamment les protéines à poche pRb, p107, p130. RIP140 (Receptor Interacting Protein of 140kDa) a été identifié comme un corépresseur de nombreux récepteurs nucléaires, une autre grande famille de facteurs de transcription qui, pour certains, régulent positivement l'expression du gène RIP140.Ce travail de thèse a permis d'identifier RIP140 comme un nouveau répresseur de l'activité transcriptionnelle du facteur E2F1, dans des expériences de transfection transitoire ainsi que sur l'expression de gènes endogènes. Nous avons également montré que l'expression ectopique de RIP140 bloque la progression des cellules dans le cycle cellulaire. Dans les cancers du sein, le niveau d'expression de RIP140 présente une corrélation inverse avec celui de différents gènes cibles des facteurs E2Fs et semble discriminer les tumeurs luminales des tumeurs basales. Nous avons également démontré que le niveau d'ARNm RIP140 est régulé au cours du cycle cellulaire et que le promoteur du gène RIP140 est une cible directe des facteurs E2Fs. Cette régulation implique des sites de liaison des facteurs E2Fs et Sp1 de la région proximale du promoteur. La régulation de ce gène par E2F1 a également été observée au cours du processus de différenciation adipocytaire en utilisant un modèle murin E2F1-/-.En conclusion, ce travail a permis d'identifier RIP140 comme un nouvel acteur de la voie de signalisation par les facteurs E2Fs. / Cell cycle control, a fundamental process which controls cell proliferation, is frequently altered during tumorigenesis. The E2F transcription factors are central regulators of target gene expression involved in cell cycle regulation, DNA replication, apoptosis and differentiation. The E2F transcription factors family encompasses members which act as activators or repressors. Their activities are regulated by a large number of transcriptional coregulators, including in particular the pocket proteins pRb, p107, p130.The transcription coregulator RIP140 (Receptor Interacting Protein of 140kDa) has been identified as a partner of numerous nuclear receptors, another important transcription factor family. Some of these nuclear receptors positively regulate RIP140 gene expression.This work identified RIP140 as a new repressor of E2F1 transcriptional activity, both in transient transfection experiments and on the expression of endogenous target genes. We also showed that ectopic expression of RIP140 blocks cell cycle progression. In breast cancers, the level of RIP140 expression is inversely correlated with various target genes of E2Fs factors and seems to discriminate luminal from basal tumors. We also demonstrated that the RIP140 mRNA expression is regulated during cell cycle and that the RIP140 promoter is a direct target of E2F transcription factors. This regulation involves both E2F and Sp1 binding sites in the proximal region of the RIP140 promoter. The regulation of the RIP140 gene by E2F1 was also observed during adipocyte differentiation using an E2F1-/- mouse model.In conclusion, this study identified RIP140 as a new regulator of the E2F signalling pathway and as a novel E2F1 target gene. These results open new perspectives concerning the roles that this transcriptional coregulator might play in the control of cell proliferation and tumorigenesis.

Der pRB/E2F-Signalweg ist ein wichtiger Schlüsselpunkt für die Wachstumskontrolle in Säugerzellen und in vielen Tumoren sind Komponenten dieses Signalweges dereguliert. Durch die Nullmutation von E2F3 in Mausembryonalen Fibroblasten (MEFs) und Mäusen konnte gezeigt werden, dass E2F3 essentiell für das zelluläre Wachstum ist und in der Maus organspezifisch sowohl als Tumorsuppressor als auch Onkogen agieren kann. Jedoch sind dafür die zugrunde liegenden Mechanismen noch nicht genau geklärt. Möglicherweise tragen verschiedene Signalwege, die durch den Verlust von E2F3 dereguliert werden, zu den Defekten bei. In dieser Arbeit wurde TGFbeta1, ein wichtiger Wachstumsregulator, in den E2f3-/- MEFs untersucht und es konnte zum ersten Mal eine direkte Verbindung zwischen der E2F3-Expression und der TGFbeta1-Signalwirkung gezeigt werden. Durch den Verlust von E2F3 werden Tgfb1 und die TGFbeta1-regulierten Gene PAI-1, p21, Vimentin und Fibronectin in MEFs dereprimiert. Darüber hinaus werden MEFs und humane Lungenkarzinomzellen durch den Verlust von E2F3 gegenüber TGFbeta1 sensibilisiert und reagieren verstärkt auf TGFbeta1-induzierte Genexpression und Prozesse wie Wachstumsarrest und EMT. Somit wird E2F3 nicht nur durch TGFbeta1 reguliert, sondern kann auch auf TGFbeta1 und die TGFbeta1-Signalwirkung Einfluss nehmen, was für die Tumorprogression weit reichende Auswirkung haben kann. Um die tumorsuppressiven Eigenschaften von E2F3 besser zu verstehen, wurden im zweiten Teil dieser Arbeit murine medulläre Schilddrüsentumore mit unterschiedlichem metastatischen Potential miteinander verglichen und es konnten neue E2F-Zielgene identifiziert werden. Die Untersuchung von humanen Struma nodosa-Biopsien und metastatischen medullären Schilddrüsentumoren ergab, dass die in den Mäusen gefundenen Gene künftig auch als humane Metastasemarker Verwendung finden können. / The pRB/E2F-pathway plays a key role in growth control and it is deregulated in many tumors. Previously, by analysing E2f3 deficient mouse embryonic fibroblasts (MEFs) and mice it has been shown that E2F3, a key downstream target of pRB, is essential for cellular proliferation and can act either as an oncogene or tumorsuppressor in mice depending on the organ. However, the underlying mechanism is still unclear. We suggest that specific pathways which are deregulated due to the deletion of E2F3 contribute to these defects. TGFbeta1, which is one of the most potent growth regulators for mammalian cells was analysed in E2f3-/- MEFs. In this study, we could establish a direct link between E2F3 expression and TGFbeta1 signalling. Loss of E2F3 in MEFs leads to de-repression of Tgfb1 and TGFbeta1-regulated genes like PAI-1, p21, vimentin and fibronectin. Moreover, loss of E2F3 in MEFs or in human lung carcinoma cells results in an increased sensitivity to TGFbeta1-induced gene expression and processes like growth arrest and epithelial mesenchymal transition. These data suggest that not only TGFbeta1 can act on E2F3 but also E2F3 can affect TGFbeta1 and the outcome of TGFbeta1-induced signalling. In order to understand the tumor suppressive properties of E2F3, we compared gene expression profiles of murine medullary thyroid carcinomas (MTCs) of different metastatic potential and could identify novel E2F-target genes. Analysis of human struma nodosa biopsies and human metastatic medullary thyroid tumors showed that the genes identified in the mouse model can also be used as metastasis markers in human tumors.

Der Transkriptionsfaktor CCAAT/Enhancer-Binding Protein alpha (C/EBPa) koordiniert Proliferationshemmung und Differenzierung von myeloiden VorlŠuferzellen und Adipozyten. C/EBPa ist ein transkriptioneller Aktivator von abstammungspezifischen Genen und blockiert den Zellzyklus durch Repression von proliferationsfšrdernden E2F Zielgenen. Die hier gezeigten Daten zeigen, dass auch umgekehrt E2F die transkriptionelle und differenzierungsfšrdernde AktivitŠt von C/EBPa entgegenwirkt. Somit besitzen E2F-C/EBPa eine zentrale Schalterfunktion zwischen Proliferation und Differenzierung. Der Repressionsmechanismus durch E2F ist in mehreren Aspekten neuartig: Zum erstenmal wurde gezeigt, dass E2F einen anderen Transkriptionsfaktor reprimieren kann. E2F reprimiert die transkriptionelle AktivitŠt von C/EBPa ohne Bindung an cis-regulatorischen Elemente, sondern durch direkte Protein-Protein Interaktionen, die die Bindung von C/EBPa an DNA verhindern. Diese Form der transkriptionellen Repression geschieht unabhŠngig von "Pocket-Proteinen''". Patienten mit Akuter Myeloiden LeukŠmie (AML) weisen hŠufig eine gestšrte DNA Bindung von C/EBPa auf, welche ursachlich fŸr granulozitŠren Funktionsstšrungen sein kšnnte. Daher wŠre es wichtig zu analysieren ob E2F die DNA Bindung von C/EBPa in AML Patienten beeintrŠchtigt und ob auf E2F gerichtete Therapien granulozitŠre Reifung wiederherstellen. C/EBPa blockiert Zellproliferation durch vielseitigen Mechanismen. Hier wurde gezeigt, dass C/EBPa mit UBF1, dem Co-Aktivator der RNA Polymerase I, an chromosomalen Foci positioniert wird. Eine €hnlichkeit zu anderen fokalen Strukturen suggeriert, dass C/EBPa die Transkription von Polymerase I regulierten rRNA Gene reprimieren und somit ribosomale Biogenese beeintrŠchtigen kšnnte. Die Assoziation zwischen C/EBPa und UBF1 wird durch die Histon-Methyltransferase SUV39H1 stimuliert. Demnach kšnnte die antiproliferative Funktion von C/EBPa nicht nur auf der Regulierung von RNA Pol II-abhŠngiger Transkription, sondern auch auf der Repression von RNA Pol I regulierter rRNA Synthese basieren. / The transcription factor CCAAT/Enhancer-Binding Protein alpha (C/EBPa) coordinates proliferation arrest and differentiation of myeloid progenitors and adipocytes. C/EBPa acts as a transcriptional activator of lineage specific genes and blocks the cell cycle by repressing transcription of E2F-regulated genes. Data presented here suggest that also inversely E2F interferes with the transcriptional activity of C/EBPa, counteracting C/EBPa-mediated differentiation processes. Thus, E2F-C/EBPa are part of a switch mechanism between proliferation and differentiation. The mechanism by which E2F suppresses C/EBPa-mediated transactivation is novel in several aspects. E2F acts as a co-repressor of another transcription factor, C/EBPa, without binding to cis-regulatory elements, but by direct protein-protein interactions that abolish the binding of C/EBPa to DNA. This mechanism of transcriptional repression occurs independent of pocket proteins. Disturbed DNA binding of C/EBPa is often observed in AML patients suggesting a causative role in granulocytic disorders. Thus, it would be of main interest to analyze whether E2F mediates disruption of C/EBPa''s DNA-binding in AML patients and whether therapies directed against E2F could restore granulocytic maturation. Despite the extensive knowledge of mechanisms involved in the inhibitory function of C/EBPa, it has not been addressed whether C/EBPa may impinge on cell proliferation by affecting the ribosomal biogenesis of a cell. This work demonstrates an association of C/EBPa to the RNA Pol I transcription factor UBF1, both proteins retained in large chromosomal foci. Similarities to other focal structures associated to UBF1, suggest that C/EBPa may repress transcription of Pol I-transcribed rRNA genes, and thus affect ribosomal biogenesis. The enrichment of C/EBPa at sites of UBF1 is induced by the histone methyltransferase SUV39H1. Thus, C/EBPa may not only control lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also may act as a repressor of RNA Pol I mediated rRNA synthesis.

Der Transkriptionsfaktor CCAAT/enhancer-binding protein α (C/EBPα) kontrolliert Zellzyklusarrest und terminale Differenzierung von neutrophilen Granulozyten und Adipozyten. Mutationen von C/EBPα treten häufig im Zusammenhang mit akuter myeloischer Leukämie auf. Massenspektrometrische Untersuchungen zeigten, dass C/EBPα an mehreren konservierten Argininen citrunilliert ist, einschließlich R297 in der C-terminalen basischen Region von C/EBPα. Mutationen von C/EBPα R297 wurden bereits beschrieben, weshalb der Schwerpunkt dieser Arbeit auf die Analyse der Modifikation dieses Aminosäurerestes gelegt wurde. Die Ergebnisse zeigen, dass die Peptidyl-Arginin-Deaminase (PADI4) mit C/EBPα interagiert und an mehreren Aminosäureresten citrunilliert. Citrunillierung oder Mutation von R297 beeinflusst die Aktivität von C/EBPα, einschließlich DNA-Bindung und Interaktion mit Partnerproteinen. Mutationsanalysen legen nahe, dass die positive Ladung des Aminosäurerestes R297 für die Bindung an cis-regulatorische DNA-Elemente, Protein Interaktionen, Genaktivierung, Fettzelldifferenzierung und Zellzyklusarrest ausschlaggebend ist. Knock-down von PADI4 in der myeloischen Vorläufer-Zelllinie 32D oder in der leukämischen U937 Zelllinie induziert Granulozyten-Differenzierung, möglicherweise durch Blockierung der PADI4-vermittelten Citrunillierung und Inaktivierung von C/EBPα. Zusammengefasst ergibt sich aus den Daten, dass PADI4 die positiv-geladene Seitenkette von C/EBPα R297 in eine ungeladene, citrunillierte Form umwandelt, die die Assoziation mit DNA destabilisiert und die C/EBPα-E2F-Interaktion beeinflusst, was wiederum das Gleichgewicht zwischen Proliferation und Differenzierung bestimmt. / The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) coordinates cell cycle arrest and terminal differentiation of neutrophil granulocytes and adipocytes. Mutations in C/EBPα are frequently associated with acute myeloid leukemia. Mass spectrometric analysis revealed that citrullination occurred on multiple conserved C/EBPα arginine residues including R297 in the C/EBPα basic region. C/EBPα R297 was previously reported to be mutated in acute myeloid leukemia and we therefore focused on the modification this residue. Data presented here show that peptidylarginine deiminase 4 (PADI4) interacts with and citrullinates C/EBPα at several sites. Citrullination or mutation of R297 dramatically changed C/EBPα activities, including DNA binding and interaction with protein partners. Mutational analysis demonstrated that the positive charge of residue R297 was critical for binding to cis-regulatory sites on DNA, gene activation, adipocytic differentiation, and cell cycle arrest. Knock down of PADI4 in the myeloid precursor cell line 32D or U937 leukemia cells induced granulocyte differentiation, potentially through relieving PADI4 mediated citrullination and inactivation of C/EBPα. Taken together, the data suggest that PADI4 converts the positive C/EBPα R297 side chain to the non-charged citrulline side chain which destabilizes the association with DNA and affects C/EBPα - E2F interaction that determines the balance between proliferation and differentiation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Erythropoiesis, the process of producing mature erythrocytes from the haematopoietic stem cells (HSCs) that reside in the bone marrow, is tightly regulated at both the cell and molecular level by a well defined set of extracellular cytokine signals and intracellular transcription factors. Diseases affecting erythropoiesis are among the most commonly inherited conditions and result from disturbances to the cellular and molecular events that normally regulate this process. Erythroid Kruppel-like factor (EKLF/KLF1) is a transcription factor that is essential for erythropoiesis. EKLF is the founding member of the Kruppel-like factor family of transcription factors that bind to GC rich CACC-Box elements within gene promoters and activate transcription. The β-like globin genes are critical targets of EKLF through its binding at sites within the proximal promoters and the upstream locus control region (LCR) enhancer. Mice lacking EKLF die prior to birth by E16 with a phenotype that closely resembles the human disease thalassaemia. Thalassaemia is due to mutations in the α or β-globin genes, leading to globin chain imbalance, red cell destruction and ineffective erythropoiesis. However, restoration of expression of γ-globin (a β-like gene) failed to prevent embryonic lethality in EKLF knockout mice and suggested that additional target genes were critical to erythropoiesis. This thesis describes investigation into the transcriptional network of EKLF and an in depth analysis of previously uncharacterised phenotypes present in the EKLF knockout mouse. I have identified a suite of target genes for EKLF that include critical components of the cells cycle. I have also tested the hypothesis that EKLF is able to function in vivo as a tumour suppressor gene. Additionally, I report a role for EKLF in the determination of cell fate within the haematopoietic system and describe the development of a new approach to globally understanding erythroid transcription factor function. A previously performed microarray transcriptional profiling study provided a set of potential target genes for EKLF. I have expanded on this study by identifying that the cell cycle genes p18INK4c, and E2f2 are direct transcriptional targets of EKLF, where binding of EKLF occurs at the promoter and a novel intronic enhancer region, respectively. I have also described a previously undiscovered cell cycle phenotype of aberrant entry into S-phase in EKLF -/- erythroid cells that is directly related to abrogated expression of E2f2. The Kruppel-like factor family of genes have been implicated as players in the tumour process. By constructing a model for the loss of EKLF within HSCs in vivo, I have tested whether EKLF is functional as a tumour suppressor. The loss of EKLF in vivo was found to be insufficient to generate erythroleukaemia, however did result in erythroid hyperplasia, extramedullary haematopoieis and a mild macrocytic anaemia. In addition to regulation of erythropoiesis, EKLF performs a critical role in the lineage choice for a megakaryocyte-erythroid progenitor (MEP) between the megakaryocytic and erythroid lineages. This thesis describes that in the absence of EKLF, MEPs fail to commit properly to either lineage and proceed along a promiscuous pathway sharing the hallmarks of both megakaryocytes and erythroid cells. A detailed molecular mechanism for this phenotype remains undetermined, but is likely to involve interactions with the megakaryocyte transcription factor Fli1 and other members of the Kruppel-like factor family, such as BKLF (KLF3). While the transcriptional mechanisms that drive erythropoiesis have been slowly discovered, the development of chromatin immunoprecipitation (ChIP) assays and next generation DNA sequencing technology has presented the potential to rapidly enhance the progression of these studies. In this thesis I describe the development of ChIP-seq using Applied Biosystems SOLiD technology, an approach to rapidly identify binding sites for erythroid transcription factors in an unbiased genome wide approach. The work described in this thesis has expanded the transcriptional network of EKLF to include critical components of the cell cycle and has suggested many additional target genes from ChIP-seq requiring validation. As one of the major transcription factor players during erythropoiesis, EKLF performs many critical functions that include the regulation of the cell cycle, lineage selection and erythroid development. I suggest that current and future studies of EKLF function will influence our understanding of erythropoiesis and refine our understanding of human conditions such as thalassaemia and erythroleukaemia.

The RbµE2F transcriptional regulatory pathway plays a critical role in the cell cycle. Rb is inactivated through multiple waves of phosphorylation, mediated mainly by cyclin D and cyclin E associated kinases. Once Rb is inactivated, cells can enter Sµphase. Collectively, three Rb family members and ten E2F proteins coordinate every additional stage of the cell cycle, from quiescence to mitosis. However the RbµE2F pathway is frequently altered in cancer. Aside from cell proliferation, the RbµE2F pathway regulates other essential cellular processes including apoptosis, cell differentiation, angiogenesis and DNA damage repair pathways, but its role in invasion and cancer progression is less clear. We demonstrate here that matrix metalloproteinases genes (MMPs), which regulate the invasion, migration and collagen degradation activities of cancer cells during metastasis are transcriptionally regulated by the RbµE2F pathway. Unlike E2F target genes involved in cell proliferation, which are solely regulated by the E2F activators (E2F1µ3), additional E2F family members can regulate MMP9, MMP14, and MMP15. While we had previously shown that Rafµ1 kinase physically interacts with Rb, and that disruption of this interaction with a small molecule inhibitor of the RbµRafµ1 interaction (RRDµ251) can inhibit cell proliferation, angiogenesis, and growth of tumors in mouse models, we now show RRDµ251 inhibits the expression of MMPs and the biological functions mediated by MMPs as well—including invasion, migration, and collagen degradation. RRDµ251 also inhibits metastatic foci development in a tail vein lung colonization model in mice. These results suggest that E2F transcription factors may play a role in promoting metastasis through regulation of MMP genes. Conversely, another MMP gene connected to metastasis, MMP2, is transcriptionally repressed by E2F1 in lung cancer cells through a p53µKAP1µHDAC1µmediated mechanism. However, E2F1 cannot repress the MMP2 promoter in cells that are lacking any component of this complex, such as p53 mutant breast cancer cells. Therefore the role of the RbµE2F pathway in MMP transcription and metastasis is cell type dependent. In addition to growth factors, nicotine can also induce cell proliferation, angiogenesis, EMT, and progression of lung cancer. In our studies, nicotine induced invasion, collagen degradation, and transcription of MMP2, MMP9, MMP14, and MMP15 required nAChRs, and multiple E2F family members. Our studies also show that nicotine not only promotes tumor growth in vivo through the nAChRµE2F pathway—it also results in metastasis to the liver and brain. Taken together, these studies link the RbµE2F pathway to the regulation of many facets of cancer.

Apoptotic cell death of cardiac myocytes plays an important pathological role after a myocardial infarction and during heart failure. Apoptotic myocytes are not regenerated because of the restricted ability of terminally differentiated cardiac myocytes to undergo cell division. Because ventricular function is directly related to the number of active muscle cells, the inappropriate loss or premature death of cardiac myocytes results in reduced cardiac performance. Bnip3 was previously identified by Dr. Lorrie Kirshenbaum’s laboratory as a critical mediator of hypoxia-induced apoptosis in the heart. Importantly, his lab established that the cytoprotective actions of NF-κB during hypoxia included the transcriptional repression of Bnip3. However, the mechanism by which NF-κB acted as a transcriptional repressor was undefined. The present work strongly supports the hypothesis that NF-κB-mediated inhibition of Bnip3 transcription is dependent on the recruitment of the corepressor protein HDAC1. Immunoprecipitation experiments revealed that HDAC1 and p65 NF-κB formed protein-protein interactions. ChIP assays demonstrated that HDAC1 and p65 NF-κB associated with the Bnip3 promoter. HDAC1-mediated repression of Bnip3 was lost in cells deficient for p65 NF-κB, and restored upon repletion of p65. A second avenue of investigation described in this work demonstrated that the cell cycle factor E2F-1 directly activated Bnip3 transcription. Earlier work by Dr. Kirshenbaum found that adenovirus-mediated overexpression of E2F-1 in ventricular myocytes induced apoptosis. Herein, it is shown that E2F-1-mediated cell death is largely Bnip3-dependent because functional loss of Bnip3 inhibited E2F-1-induced cell death. Concerning hypoxia, Bnip3 expression is dependent upon the loss of p65/HDAC1-mediated repression, and on the presence of transcriptionally active E2F-1. During hypoxia, overexpression of p65, HDAC1, or Rb, an endogenous inhibitor of E2F-1-dependent transcription, attenuated hypoxia-induced Bnip3 transcription. Based on these findings, future therapies may be designed to repress Bnip3 gene expression after a myocardial infarction, thereby averting cardiac cell death and preserving cardiac function post-infarction.