The human RAP250 gene: genomic structure and promoter analysis.
Journal - Gene (Netherlands )
Ligand-induced gene activation by nuclear receptors involves the recruitment of coactivators to hormone bound receptors. Recent results have shown that RAP250, also termed as ASC-2/PRIP/TRBP/NRC/AIB3, plays a critical role as a coactivator of nuclear receptors. In this study, we have determined the genomic organization of the human RAP250 gene in order to identify the promoter region. By searching the GenBank database for EST sequences, we could identify two previously unknown exons in the 5'-end of the gene. Our results show that the RAP250 gene consists of 15 exons spanning 111 kb. All sequences of the splice donor and acceptor sites fit the GT-AG role for splicing. We also show using linkage analysis that the mouse Rap250 gene is located on chromosome 2 close to the agouti gene. The RAP250 promoter is GC-rich and TATA-less, and contains multiple Sp1 binding sites and a MYC binding site. A reporter plasmid containing the 5' flanking region of the RAP250 gene showed significant activity compared to pGL3-basic and minimal thymidine kinase (TK) reporter plasmids in transfection experiments using luciferase reporter genes. Our data show that the RAP250 has a complex genomic structure with a promoter that is regulated by multiple transcription factors for its basal expression.
|ISSN : ||0378-1119|
|Mesh Heading : ||5' Flanking Region Agouti Signaling Protein Animals Base Sequence Binding Sites Carrier Proteins Cell Line, Tumor Chromosome Mapping Chromosomes, Human, Pair 20 Chromosomes, Mammalian Exons Genes Humans Intercellular Signaling Peptides and Proteins Introns Linkage (Genetics) Luciferases Mice Mice, Inbred C57BL Molecular Sequence Data Promoter Regions, Genetic Recombinant Fusion Proteins Sequence Analysis, DNA Synteny genetics genetics genetics genetics genetics genetics genetics metabolism genetics metabolism|
|Mesh Heading Relevant : ||Intracellular Signaling Peptides and Proteins genetics genetics|
A novel human CCAAT/enhancer binding protein gene, C/EBPepsilon, is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to the T-cell receptor alpha/delta locus.
Journal - Genomics (UNITED STATES )
Members of the CsolidusEBP family of transcriptional factors have been implicated in the regulation of genes in a variety of tissues. We report here the isolation and characterization of the human C/EBPepsilon gene (CEBPE). By using low-stringency hybridization conditions and probes derived from the C/EBPalpha and C/EBPdelta genes, we have isolated overlapping genomic clones that cover almost 25 kb of the C/EBPepsilon gene locus and corresponding cDNA clones. DNA sequence analysis reveals that the gene encodes a protein highly homologous to rat CRP1. The gene was assigned to chromosome 14q11.2 by fluorescence in situ hybridization and was physically linked to the genetic marker D14S990. Based on linkage data derived from this marker, we positioned the CEBPE gene between the T-cell receptor alpha/delta locus and a cluster of four serine proteases expressed exclusively in hematopoietic cells. Expression of C/EBPepsilon was detected in Jurkat T-cell and in HL 60 promyelocytic cell lines. From a variety of normal human tissues studied, expression of mRNA was monitored only in peripheral blood mononuclear cells, tissues involved in the immune system, and ovaries. These data demonstrate that the C/EBPepsilon gene shows a restricted pattern of expression, has an intriguing chromosomal location, and suggest a possible role for the regulation of certain genes in cells of myeloid and lymphoid lineages.
|ISSN : ||0888-7543|
|Mesh Heading : ||Amino Acid Sequence Animals Base Sequence Bone Marrow Bone Marrow Cells Chromosomes, Human, Pair 14 Female Gene Expression HL-60 Cells Humans Leukemia-Lymphoma, Adult T-Cell Lymphoid Tissue Male Molecular Sequence Data Multigene Family Neoplasm Proteins Organ Specificity RNA, Messenger RNA, Neoplasm Rats Receptors, Antigen, T-Cell, alpha-beta Receptors, Antigen, T-Cell, gamma-delta Sequence Alignment Sequence Homology, Amino Acid Species Specificity Transcription Factors Tumor Cells, Cultured metabolism pathology cytology biosynthesis genetics biosynthesis genetics biosynthesis genetics genetics genetics biosynthesis|
|Mesh Heading Relevant : ||CCAAT-Enhancer-Binding Proteins Genes metabolism genetics metabolism genetics|
Molecular cloning, sequence, and expression patterns of the human gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha).
Journal - Biochemical and biophysical research communications (UNITED STATES )
The human gene encoding the transcription factor C/EBP alpha was isolated from an umbilical cord genomic library screened by low stringency hybridization. Two overlapping clones were characterized by restriction enzyme analysis and included 13.2 kb of the C/EBP alpha locus. The entire gene and 471 bp of the promoter were sequenced. The human C/EBP alpha gene is 2783 bp long and encodes a 356 amino acid long protein, which is the same in length as for rat C/EBP alpha. Compared to rat C/EBP alpha, there are two insertions of two amino acids and one deletion of four. The amino acid similarity between the two proteins is over 92%. The human C/EBP alpha gene was found to be expressed at the highest levels in placenta. High expression was also found in liver, lung, skeletal muscle, pancreas, small intestine, colon and in peripheral blood leukocytes. However, the expression was undetectable or very low in brain, kidney, thymus, testis and ovary. These results show that the human C/EBP alpha gene is expressed in a tissue restricted manner.
|ISSN : ||0006-291X|
|Mesh Heading : ||Amino Acid Sequence Animals Base Sequence Blotting, Northern CCAAT-Enhancer-Binding Proteins Humans In Situ Hybridization, Fluorescence Molecular Sequence Data Organ Specificity Promoter Regions, Genetic RNA, Messenger Rats Restriction Mapping TATA Box Transcription Factors analysis|
|Mesh Heading Relevant : ||Cloning, Molecular Gene Expression Sequence Analysis, DNA genetics|
Inactivation of the Nuclear Receptor Coactivator RAP250 in Mice Results in Placental Vascular Dysfunction
Journal - Molecular and Cellular Biology
Coactivators constitute a diverse group of proteins that are essential for optimal transcriptional activity of nuclear receptors. In the past few years many coactivators have been identified but it is still unclear whether these proteins interact indiscriminately with all nuclear receptors and whether there is some redundancy in their functions. We have previously cloned and characterized RAP250 (ASC-2/PRIP/TRBP/NRC), an LXXLL-containing coactivator for nuclear receptors. In order to study its biological role, Rap250 null mice were generated by gene targeting. Here we show that genetic disruption of Rap250 results in embryonic lethality at embryonic day (E) 13.5. Histological examination of placentas revealed a dramatically reduced spongiotrophoblast layer, a collapse of blood vessels in the region bordering the spongiotrophoblast, and labyrinthine layers in placentas from Rap250-/- embryos. These findings suggest that the lethality of Rap250-/- embryos is the result of obstructed placental blood circulation. Moreover, the transcriptional activity of PPAR? is reduced in fibroblasts derived from Rap250-/- embryos, suggesting that RAP250 is an essential coactivator for this nuclear receptor in the placenta. Our results demonstrate that RAP250 is necessary for placental development and thus essential for embryonic development.