Position and orientation-selective silencer in protein-coding sequences of the rat osteocalcin gene
Aronow, Michael A.
Stein, Janet L.
Lian, Jane B.
Stein, Gary S.
UMass Chan AffiliationsDepartment of Orthopedic Surgery
Department of Cell Biology
Graduate School of Biomedical Sciences
Document TypeJournal Article
KeywordsAnimals; Base Sequence; Cells, Cultured; Chloramphenicol O-Acetyltransferase; Embryo, Mammalian; Exons; Genes, Reporter; Molecular Sequence Data; Osteoblasts; Osteocalcin; Promoter Regions (Genetics); RNA; Rats; *Regulatory Sequences, Nucleic Acid; Repetitive Sequences, Nucleic Acid; Transfection
Medicine and Health Sciences
MetadataShow full item record
AbstractOsteocalcin (OC) is a bone-specific protein which is expressed postproliferatively by osteoblasts during late stages of differentiation. We have found that a silencer element is present within the rat OC gene (between nt +39 and +104), overlapping the OC signal prepropeptide-coding sequence. The presence of this sequence in OC promoter-CAT reporter constructs suppresses promoter activity in transiently transfected proliferating osteoblasts, which do not express OC, by up to 50-fold. This is the first demonstration of contribution from protein-coding sequences to silencing of animal genes. The element appears to be bipartite; silencer activity requires both the protein-coding sequence +39 to +63 and the +93 to +104 exon 1/intron 1 border region. Both of these domains contain sequences highly similar to silencer motifs in several other genes, including chicken lysozyme as well as rat collagen type II, insulin, and growth hormone. OC silencer activity is fully retained when the element is placed outside the RNA-coding region, 3' but not 5' of the OC-CAT fusion gene. Repression activity is orientation independent in the native position but requires the native orientation when located in 3' extragenic positions. The silencer does not inhibit the activity of the heterologous SV40 early promoter. These results suggest interaction between the transcribed silencer and specific OC promoter element(s) residing farther upstream. The OC transcribed silencer may contribute to developmental control of OC expression.
Biochemistry. 1993 Dec 14;32(49):13636-43.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/33691
Showing items related by title, author, creator and subject.
Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagellaPazour, Gregory J.; Dickert, Bethany L.; Vucica, Yvonne; Seeley, E. Scott; Rosenbaum, Joel L.; Witman, George B.; Cole, Douglas G. (2000-11-04)Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.
ATF1 and CREB trans-activate a cell cycle regulated histone H4 gene at a distal nuclear matrix associated promoter elementGuo, Bo; Stein, Janet L.; Van Wijnen, Andre J.; Stein, Gary S. (1997-12-16)Proteins of the ATF/CREB class of transcription factors stimulate gene expression of several cell growth-related genes through protein kinase A-related cAMP response elements. The promoter activity of cell cycle regulated histone H4 genes is regulated by at least four principal cis-acting elements which mediate G1/S phase control and/or enhancement of transcription during the cell cycle. Using protein-DNA interaction assays we show that the H4 promoter contains two ATF/CREB recognition motifs which interact with CREB, ATF1, and ATF2 but not with ATF4/CREB2. One ATF/CRE motif is located in the distal promoter at the nuclear matrix-associated Site IV, and the second motif is present in the proximal promoter at Site I. Both ATF/CRE motifs overlap binding sequences for the multifunctional YY1 transcription factor, which has previously been shown to be nuclear matrix associated. Subnuclear fractionation reveals that there are two ATF1 isoforms which appear to differ with respect to DNA binding activity and partition selectively between nuclear matrix and nonmatrix compartments, consistent with the role of the nuclear matrix in regulating gene expression. Site-directed mutational studies demonstrate that Site I and Site IV together support ATF1- and CREB-induced trans-activation of the H4 promoter. Thus, our data establish that ATF/CREB factors functionally modulate histone H4 gene transcription at distal and proximal promoter elements.
The unique catalytic subunit of sperm cAMP-dependent protein kinase is the product of an alternative Calpha mRNA expressed specifically in spermatogenic cellsSan Agustin, Jovenal T.; Wilkerson, Curtis G.; Witman, George B. (2000-09-12)cAMP-dependent protein kinase has a central role in the control of mammalian sperm capacitation and motility. Previous protein biochemical studies indicated that the only cAMP-dependent protein kinase catalytic subunit (C) in ovine sperm is an unusual isoform, termed C(s), whose amino terminus differs from those of published C isoforms of other species. Isolation and sequencing of cDNA clones encoding ovine C(s) and Calpha1 (the predominant somatic isoform) now reveal that C(s) is the product of an alternative transcript of the Calpha gene. C(s) cDNA clones from murine and human testes also were isolated and sequenced, indicating that C(s) is of ancient origin and widespread in mammals. In the mouse, C(s) transcripts were detected only in testis and not in any other tissue examined, including ciliated tissues and ovaries. Finally, immunohistochemistry of the testis shows that C(s) first appears in pachytene spermatocytes. This is the first demonstration of a cell type-specific expression for any C isoform. The conservation of C(s) throughout mammalian evolution suggests that the unique structure of C(s) is important in the subunit's localization or function within the sperm.