Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene

<p>Abstract</p> <p>Background</p> <p>Mono-ADP-ribosyltransferase (ART) 1 belongs to a family of mammalian ectoenzymes that catalyze the transfer of ADP-ribose from NAD⁺to a target protein. ART1 is predominantly expressed in skeletal and cardiac muscle. It ADP-ribosylates α7-integrin which together with β1-integrin forms a dimer and binds to laminin, a protein of the extracellular matrix involved in cell adhesion. This posttranslational modification leads to an increased laminin binding affinity.</p> <p>Results</p> <p>Using C2C12 and C3H-10T 1/2 cells as models of myogenesis, we found that ART1 expression was restricted to myotube formation. We identified a fragment spanning the gene 1.3 kb upstream of the transcriptional start site as the functional promoter of the <it>ART1 </it>gene. This region contains an E box and an A/T-rich element, two conserved binding sites for transcription factors found in the promoters of most skeletal muscle specific genes. Mutating the DNA consensus sequence of either the E box or the A/T-rich element resulted in a nearly complete loss of <it>ART1 </it>promoter inducibility, indicating a cooperative role of the transcription factors binding to those sites. Gel mobility shift analyses carried out with nuclear extracts from C2C12 and C3H-10T 1/2 cells revealed binding of myogenin to the E box and MEF-2 to the A/T-rich element, the binding being restricted to C2C12 and C3H-10T 1/2 myotubes.</p> <p>Conclusion</p> <p>Here we describe the molecular mechanism underlying the regulation of the <it>ART1 </it>gene expression in skeletal muscle cells. The differentiation-dependent upregulation of ART1 mRNA is induced by the binding of myogenin to an E box and of MEF-2 to an A/T-rich element in the proximal promoter region of the <it>ART1 </it>gene. Thus the transcriptional regulation involves molecular mechanisms similar to those used to activate muscle-specific genes.</p

The CHR site: definition and genome-wide identification of a cell cycle transcriptional element

The cell cycle genes homology region (CHR) has been identified as a DNA element with an important role in transcriptional regulation of late cell cycle genes. It has been shown that such genes are controlled by DREAM, MMB and FOXM1-MuvB and that these protein complexes can contact DNA via CHR sites. However, it has not been elucidated which sequence variations of the canonical CHR are functional and how frequent CHR-based regulation is utilized in mammalian genomes. Here, we define the spectrum of functional CHR elements. As the basis for a computational meta-analysis, we identify new CHR sequences and compile phylogenetic motif conservation as well as genome-wide protein-DNA binding and gene expression data. We identify CHR elements in most late cell cycle genes binding DREAM, MMB, or FOXM1-MuvB. In contrast, Myb- and forkhead-binding sites are underrepresented in both early and late cell cycle genes. Our findings support a general mechanism: sequential binding of DREAM, MMB and FOXM1-MuvB complexes to late cell cycle genes requires CHR elements. Taken together, we define the group of CHR-regulated genes in mammalian genomes and provide evidence that the CHR is the central promoter element in transcriptional regulation of late cell cycle genes by DREAM, MMB and FOXM1-MuvB

The Role of lncRNAs TAPIR-1 and -2 as Diagnostic Markers and Potential Therapeutic Targets in Prostate Cancer

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa

The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G0/G1. It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G0. Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G0/G1, but also for activation in S, G2 and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle

p53 activates the PANK1/miRNA-107 gene leading to downregulation of CDK6 and p130 cell cycle proteins

The tumor suppressor p53 is a central regulator of cell-cycle arrest and apoptosis by acting as a transcription factor to regulate numerous genes. We identified all human p53-regulated mRNAs by microarray analyses and searched for protein-coding genes which contain intronic miRNAs. Among others, this analysis yielded the panthothenate kinase 1 (PANK1) gene and its intronic miRNA-107. We showed that miRNA-107 and PANK1 are coregulated by p53 in different cell systems. The PANK1 protein, which catalyzes the rate-limiting step of coenzyme A biosynthesis, is also upregulated by p53. We observed that p53 directly activates PANK1 and miRNA-107 transcription through a binding site in the PANK1 promoter. Furthermore, p53 is recruited to the PANK1 promoter after DNA damage. In order to get more insight into miRNA-107 function we investigated its potential target genes. Cell-cycle regulators are significantly enriched among predicted miRNA-107 targets. We found miRNA-107-dependent regulation of two important regulators of G1/S progression, CDK6 and the RB-related 2 gene RBL2 (p130). CDK6 and p130 proteins are downregulated upon miRNA-107 expression. Our results uncover a novel miRNA-dependent signaling pathway which leads to downregulation of cell cycle proteins in the absence of transcriptional repression

Indirect p53-dependent transcriptional repression of Survivin, CDC25C, and PLK1 genes requires the cyclin-dependent kinase inhibitor p21/CDKN1A and CDE/CHR promoter sites binding the DREAM complex

The transcription factor p53 is central to cell cycle control by downregulation of cell cycle-promoting genes upon cell stress such as DNA damage. Survivin (BIRC5), CDC25C, and PLK1 encode important cell cycle regulators that are repressed following p53 activation. Here, we provide evidence that p53-dependent repression of these genes requires activation of p21 (CDKN1A, WAF1, CIP1). Chromatin immunoprecipitation (ChIP) data indicate that promoter binding of B-MYB switches to binding of E2F4 and p130 resulting in a replacement of the MMB (Myb-MuvB) by the DREAM complex. We demonstrate that this replacement depends on p21. Furthermore, transcriptional repression by p53 requires intact DREAM binding sites in the target promoters. The CDE and CHR cell cycle promoter elements are the sites for DREAM binding. These elements as well as the p53 response of Survivin, CDC25C, and PLK1 are evolutionarily conserved. No binding of p53 to these genes is detected by ChIP and mutation of proposed p53 binding sites does not alter the p53 response. Thus, a mechanism for direct p53-dependent transcriptional repression is not supported by the data. In contrast, repression by DREAM is consistent with most previous findings and unifies models based on p21-, E2F4-, p130-, and CDE/CHR-dependent repression by p53. In conclusion, the presented data suggest that the p53-p21-DREAM-CDE/CHR pathway regulates p53-dependent repression of Survivin, CDC25C, and PLK1

The transcription factor p53: Not a repressor, solely an activator

<div><p>The predominant function of the tumor suppressor p53 is transcriptional regulation. It is generally accepted that p53-dependent transcriptional activation occurs by binding to a specific recognition site in promoters of target genes. Additionally, several models for p53-dependent transcriptional repression have been postulated. Here, we evaluate these models based on a computational meta-analysis of genome-wide data. Surprisingly, several major models of p53-dependent gene regulation are implausible. Meta-analysis of large-scale data is unable to confirm reports on directly repressed p53 target genes and falsifies models of direct repression. This notion is supported by experimental re-analysis of representative genes reported as directly repressed by p53. Therefore, p53 is not a direct repressor of transcription, but solely activates its target genes. Moreover, models based on interference of p53 with activating transcription factors as well as models based on the function of ncRNAs are also not supported by the meta-analysis. As an alternative to models of direct repression, the meta-analysis leads to the conclusion that p53 represses transcription indirectly by activation of the p53-p21-DREAM/RB pathway.</p></div