E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family.

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells

Beyond microarrays: Finding key transcription factors controlling signal transduction pathways

BACKGROUND: Massive gene expression changes in different cellular states measured by microarrays, in fact, reflect just an "echo" of real molecular processes in the cells. Transcription factors constitute a class of the regulatory molecules that typically require posttranscriptional modifications or ligand binding in order to exert their function. Therefore, such important functional changes of transcription factors are not directly visible in the microarray experiments. RESULTS: We developed a novel approach to find key transcription factors that may explain concerted expression changes of specific components of the signal transduction network. The approach aims at revealing evidence of positive feedback loops in the signal transduction circuits through activation of pathway-specific transcription factors. We demonstrate that promoters of genes encoding components of many known signal transduction pathways are enriched by binding sites of those transcription factors that are endpoints of the considered pathways. Application of the approach to the microarray gene expression data on TNF-alpha stimulated primary human endothelial cells helped to reveal novel key transcription factors potentially involved in the regulation of the signal transduction pathways of the cells. CONCLUSION: We developed a novel computational approach for revealing key transcription factors by knowledge-based analysis of gene expression data with the help of databases on gene regulatory networks (TRANSFAC(® )and TRANSPATH(®)). The corresponding software and databases are available at

The RIP140 Gene Is a Transcriptional Target of E2F1

RIP140 is a transcriptional coregulator involved in energy homeostasis and ovulation which is controlled at the transcriptional level by several nuclear receptors. We demonstrate here that RIP140 is a novel target gene of the E2F1 transcription factor. Bioinformatics analysis, gel shift assay, and chromatin immunoprecipitation demonstrate that the RIP140 promoter contains bona fide E2F response elements. In transiently transfected MCF-7 breast cancer cells, the RIP140 promoter is transactivated by overexpression of E2F1/DP1. Interestingly, RIP140 mRNA is finely regulated during cell cycle progression (5-fold increase at the G1/S and G2/M transitions). The positive regulation by E2F1 requires sequences located in the proximal region of the promoter (−73/+167), involves Sp1 transcription factors, and undergoes a negative feedback control by RIP140. Finally, we show that E2F1 participates in the induction of RIP140 expression during adipocyte differentiation. Altogether, this work identifies the RIP140 gene as a new transcriptional target of E2F1 which may explain some of the effect of E2F1 in both cancer and metabolic diseases

Regulation of Cell Cycle by E2F1 in Primary Cells

Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of cancer, and in the absence of other genetic alterations, results in lack of differentiation, hyperproliferation and apoptosis. pRB acts as a transcriptional repressor by targeting the E2F transcription factors whose functions are required for S phase entry. Increased E2F activity can induce S phase in quiescent cells and this fact is a central element of most models for the development of cancer. I provide evidence that E2F1 alone is not sufficient to induce S phase in diploid mouse and human fibroblasts. However, increased E2F1 activity can result in S phase entry in diploid fibroblasts in which the p53-mediated Gl checkpoint is suppressed. Furthermore, I show that E2F1 can induce S phase in primary mouse fibroblasts lacking pRB. These results demonstrate that in addition to working as an E2F-dependent transcriptional repressor, pRB is also required for retaining the Gl checkpoint in response to unprogrammed proliferative signals. The role of E2F in cell proliferation is not completely understood because it is not known if the E2Fs mainly function as transcriptional repressors or activators. E2Fs need dimerisation with a DP protein to give rise to functional E2F activity and to regulate promoters containing E2F binding sites. I inactivated endogenous DP in tissue culture by RNA interference providing evidence that loss of DPI abrogates E2F DNA binding activity. DP is required for tumour and normal cell growth. In addition, the expression of E2F target genes is severely impaired. These results define a crucial role for DPI in cell proliferation

Involvement of CDP/Cux in the Regulation of Histone H4 Gene Expression, Proliferation and Differentiation: a Dissertation

Proliferation and differentiation are essential processes for the growth and development of higher eukaryotic organisms. Regulation of gene expression is essential for control of cell division and differentiation. Normal eukaryotic cells have a limited proliferative capacity, and ultimately undergo cellular senescence and apoptosis. Terminal differentiation of cells is associated with loss of proliferative capacity and acquisition of specialized functions. Proliferation and differentiation are processes required for the creation and maintenance of diverse tissues both during embryonic development and postnatal life. The cell cycle is the process by which cells reproduce, and requires duplication and segregation of hereditary material. Loss of cell cycle control leads to genetic instability and cancer. Expression of replication-dependent histone genes is tightly coupled to DNA synthesis, thus making histone genes a good model for studying cell cycle regulation. The HiNF-D complex interacts with all five classes (H1, H2A, H2B, H3 and H4) of histone genes in a cell cycle-dependent manner. The CCAAT displacement protein (CDP)/Cux and the tumor suppressor pRB are key components of the HiNF-D complex. However, the molecular interactions that enable CDP/Cux and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters are poorly understood. Transient transfection assays show that CDP/Cux represses the histone H4 promoter and that the pRB large pocket domain functions with CDP/Cux as a co-repressor. Direct interaction between CDP/Cux C-terminus and the pRB pocket domain was observed in GST pull-down assays. Furthermore, co-immunoprecipitation assays and immunofluorescence microscopy established that CDP/Cux and pRB form complexes in vivo and associate in situ. pRB interaction and co-repression with CDP/Cux is independent of pRB phosphosphorylation sites, as revealed by GST pull-down assays and transient transfection assays using a series of pRB mutant proteins. Thus, several converging lines of evidence indicate that complexes between CDP/Cux and pRB repress cell cycle-regulated histone gene promoters. CDP/Cux is regulated by phosphorylation and acetylation at the C-terminus, which contains two repressor domains and interacts with histone deacetylase HDAC1. In vivo function of the CDP/Cux C-terminus in development and gene regulation was assessed in genetically targeted mice (Cutl1tm2Ejn, referred to as Cutl1ΔC). The mice express a mutant CDP/Cux protein with a deletion of the C-terminus including the homeodomain. Indirect immunofluorescence microscopy showed that the mutant protein exhibited significantly reduced nuclear localization in comparison to the wildtype protein. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Cutl1 ΔC -/-) mice, indicating the functional loss of CDP/Cux in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wildtype and Cutl1 ΔC -/- MEFs in culture. However, the histone H4.1 (murine FO108) gene containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice exhibit stunted growth (20-50% weight reduction), a high postnatal death rate of 60-70%, sparse abnormal coat hair and severely reduced fertility. Hair follicle deformities and severely diminished fertility in Cutl1 ΔC -/- mice suggest that CDP/Cux is required for normal development of dermal tissues and reproductive functions. Together the data presented in this dissertation provide new insight into the in vivo functions of CDP/Cux in the regulation of histone gene expression, growth control and differentiation

Transcriptional Targeting in Cancer Gene Therapy

Cancer gene therapy has been one of the most exciting areas of therapeutic research in the past decade. In this review, we discuss strategies to restrict transcription of transgenes to tumour cells. A range of promoters which are tissue-specific, tumour-specific, or inducible by exogenous agents are presented. Transcriptional targeting should prevent normal tissue toxicities associated with other cancer treatments, such as radiation and chemotherapy. In addition, the specificity of these strategies should provide improved targeting of metastatic tumours following systemic gene delivery. Rapid progress in the ability to specifically control transgenes will allow systemic gene delivery for cancer therapy to become a real possibility in the near future

Expression of a Neuroendocrine Gene Signature in Gastric Tumor Cells from CEA 424-SV40 Large T Antigen-Transgenic Mice Depends on SV40 Large T Antigen

A large fraction of murine tumors induced by transgenic expression of SV40 large T antigen (SV40 TAg) exhibits a neuroendocrine phenotype. It is unclear whether SV40 TAg induces the neuroendocrine phenotype by preferential transformation of progenitor cells committed to the neuroendocrine lineage or by transcriptional activation of neuroendocrine genes. To address this question we analyzed CEA424-SV40 TAg-transgenic mice that develop spontaneous tumors in the antral stomach region. Immunohistology revealed expression of the neuroendocrine marker chromogranin A in tumor cells. By ELISA an 18-fold higher level of serotonin could be detected in the blood of tumor-bearing mice in comparison to nontransgenic littermates. Transcriptome analyses of antral tumors combined with gene set enrichment analysis showed significant enrichment of genes considered relevant for human neuroendocrine tumor biology. This neuroendocrine gene signature was also expressed in 424GC, a cell line derived from a CEA424-SV40 TAg tumor, indicating that the tumor cells exhibit a similar neuroendocrine phenotype also in vitro. Treatment of 424GC cells with SV40 TAg-specific siRNA downregulated expression of the neuroendocrine gene signature. SV40 TAg thus appears to directly induce a neuroendocrine gene signature in gastric carcinomas of CEA424-SV40 TAg-transgenic mice. This might explain the high incidence of neuroendocrine tumors in other murine SV40 TAg tumor models. Since the oncogenic effect of SV40 TAg is caused by inactivation of the tumor suppressor proteins p53 and RB1 and loss of function of these proteins is commonly observed in human neuroendocrine tumors, a similar mechanism might cause neuroendocrine phenotypes in human tumors

Identification of novel regulatory modules in dicotyledonous plants using expression data and comparative genomics

BACKGROUND: Transcriptional regulation plays an important role in the control of many biological processes. Transcription factor binding sites (TFBSs) are the functional elements that determine transcriptional activity and are organized into separable cis-regulatory modules, each defining the cooperation of several transcription factors required for a specific spatio-temporal expression pattern. Consequently, the discovery of novel TFBSs in promoter sequences is an important step to improve our understanding of gene regulation. RESULTS: Here, we applied a detection strategy that combines features of classic motif overrepresentation approaches in co-regulated genes with general comparative footprinting principles for the identification of biologically relevant regulatory elements and modules in Arabidopsis thaliana, a model system for plant biology. In total, we identified 80 TFBSs and 139 regulatory modules, most of which are novel, and primarily consist of two or three regulatory elements that could be linked to different important biological processes, such as protein biosynthesis, cell cycle control, photosynthesis and embryonic development. Moreover, studying the physical properties of some specific regulatory modules revealed that Arabidopsis promoters have a compact nature, with cooperative TFBSs located in close proximity of each other. CONCLUSION: These results create a starting point to unravel regulatory networks in plants and to study the regulation of biological processes from a systems biology point of view

Gene programs regulated by MEF2 transcription factors in rodent striated muscle cells

Transcriptional programs regulating myogenesis are multi-layered, requiring carefully orchestrated temporal activation of a wide range of myogenic transcription factors for proper muscle formation. The MEF2 transcription factor family is required for muscle differentiation, however the roles of individual mammalian MEF2 isoforms, MEF2A, -B, -C, and -D, in this process has not been thoroughly investigated. Acute knockdown of individual MEF2 isoforms in skeletal myoblasts revealed that MEF2A is required for myogenesis in vitro, whereas MEF2B, -C, and -D are dispensable for this process. Microarray analysis performed on myotubes depleted of each MEF2 isoform revealed that MEF2 factors regulate distinct gene programs in skeletal muscle. Moreover, computational analysis of the upstream regulatory regions of MEF2 isoform-dependent genes uncovered a distinct complement of transcription factor binding sites suggesting potential co-factor interactions in muscle gene regulation. Whereas all four MEF2 family members are expressed in adult skeletal muscle, MEF2A and MEF2D are the major isoforms expressed in the post-natal heart. Previous studies in cardiomyocytes have demonstrated that MEF2A regulates genes encoding proteins localized to the costamere, an essential macromolecular complex required for proper muscle contraction. By contrast, genome-wide expression analysis suggests a role for MEF2D in cardiomyocyte cell cycle regulation. MEF2D- deficient cardiomyocytes up-regulate a subset of positive cell cycle regulators and display activation of the PI3K/AKT signaling pathway. Furthermore, MEF2D-depleted cardiomyocytes have increased levels of cytoplasmic FOXO3a, a cell cycle inhibitor and direct AKT target. Along these lines, MEF2D-depleted cardiomyocytes have decreased levels of the PI3K/AKT repressor PTEN. Analysis of the Pten promoter revealed a highly conserved MEF2 site, which is required for activation of this promoter by MEF2D. Taken together, these findings demonstrate that MEF2D modulates PI3K/AKT activation through transcriptional regulation of the tumor suppressor PTEN. In the absence of MEF2D, aberrant activation of the cell cycle ultimately results in cardiomyocyte cell death. These results demonstrate that MEF2 family members regulate distinct gene programs required for proper skeletal and cardiac muscle function

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

© 2014 The Authors. Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene