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PubMed2005

Coordinate transcriptional regulation of bile acid homeostasis and drug metabolism.

Drugs and bile acids are taken up into hepatocytes by specialized transport proteins localized at the basolateral membrane, e.g., organic anion transporting polypeptides . Following intracellular metabolism by cytochrome P450 (CYP) enzymes, drug metabolites are excreted into bile or urine via ATP-dependent multidrug resistance proteins (MDR1 and MRPs). Bile acids are excreted mainly via the bile salt export pump (BSEP, ABCB11). The genes coding for drug and bile acid transporters and CYP enzymes are regulated by a complex network of transcriptional cascades, notably by the ligand-activated nuclear receptors FXR, PXR, and CAR and by the ligand-independent nuclear receptor HNF-4alpha. The bile acid synthesizing enzymes CYP7A1, CYP8B1, and CYP27A1 are subject to negative feedback regulation by bile acids, which is partly mediated through the transcriptional repressor SHP. The role of transcriptional cofactors, such as SRC-1 and PGC-1, in mediating the gene-specific effects of individual nuclear receptors is becoming increasingly evident.

Animals↗
PubMed2002

Xist expression and macroH2A1.2 localisation in mouse primordial and pluripotent embryonic germ cells.

The molecular mechanism underlying X chromosome inactivation in female mammals involves the non-coding RNAs Xist and its antisense partner Tsix. Prior to X inactivation, these RNAs are transcribed in an unstable form from all X chromosomes, both in the early embryo and in undifferentiated embryonic stem (ES) cells. Upon differentiation, the expression of these unstable transcripts from all alleles is silenced, and Xist RNA becomes stabilised specifically on the inactivating X chromosome. This pattern of expression is then maintained throughout subsequent somatic cell divisions. Once established, the inactive state of the X chromosome is remarkably stable, the only natural case of reactivation occurring in XX primordial germ cells (PGCs) when they enter the genital ridge. To gain insight into the X reactivation process, we have analysed Xist gene expression using RNA FISH in PGCs and also in PGC-derived embryonic germ (EG) cells. XX EG cells were shown to express unstable Xist/Tsix from both X chromosomes. In contrast, no unstable Xist/Tsix transcripts were detected in XX PGCs at any stage. Instead, a proportion of XX PGCs isolated from the genital ridge between 11.5 and 13.5 dpc (the period during which X chromosome reactivation occurs) showed an accumulation of stable Xist RNA on one X. The number of these cells decreased progressively and was nearly extinguished by 13.5 dpc. As a late marker for the inactive state, we analysed localisation of the histone H2A variant macroH2A1.2. Although macroH2A1.2 expression was observed in PGCs, no significant localisation to the inactive X was detected at any stage. We discuss these results in the context of understanding X chromosome reactivation.

Animals↗
PubMed1999

Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2.

The proteins encoded by the SIR1, SIR2, SIR3 and SIR4 genes in yeast repress transcription at the mating type loci and telomeres. Among the SIR genes, SIR2 is the most evolutionarily conserved, and a number of genes with homology to SIR2 have been identified. In addition to transcriptional silencing, the product of SIR2 gene (Sir2p) has been shown to be involved in DNA repair and suppression of rDNA recombination. In the present study, the complete sequence of a human gene, SIR2L, with homology to the yeast SIR2 gene is presented. Comparison of the predicted sequence of the protein encoded by the SIR2L gene (SIR2Lp) with Sir2p or other proteins with homology to Sir2p reveals 20-33% overall identity and four highly conserved regions, the significance of which is unknown. SIR2L codes for a 2.1kb transcript which is expressed in various human tissues. The expression level of the transcript is found to be relatively high in the heart, brain and skeletal muscle tissues and low in lung and placenta. The intracellular location of SIR2Lp was visualized by fusion to the Green Fluorescent Protein or with a FLAG-tag. The results indicate that unlike Sir2p in yeast, SIR2Lp in human cells is found primarily in the cytoplasm. Using a mammalian inducible expression system, we also observed that unlike SIR2 in yeast, overexpression of SIR2L in human cancer cells has no effect on cell growth. Thus, although the human SIR2L gene appears to be related to the yeast SIR2 gene, it does not appear to have similar functions.

Amino Acid Sequence↗
PubMed2005

Psc3 cohesin of Schizosaccharomyces pombe: cell cycle analysis and identification of three distinct isoforms.

Cohesins are a group of proteins that function to mediate correct chromosome segregation, DNA repair and meiotic recombination. This report presents the amino acid sequence for the Schizosaccharomyces pombe cohesin Psc3 based on the translation of the cDNA sequence, showing that the protein is smaller than previously predicted. Interestingly, comparison of the amino acid and DNA coding sequences of Psc3 with fission yeast Rec11 meiotic region-specific recombination activator shows that both intron positioning within the genes and the amino-terminal half of the two proteins are highly conserved. We demonstrate that although the intergenic region upstream of the psc3+ start codon contains a consensus sequence for the cell-cycle regulatory MluI cell-cycle box, psc3+ transcription is not differentially regulated during the mitotic cell cycle. Finally, we demonstrate that an epitope-tagged version of Psc3 undergoes no major changes during the mitotic cell cycle. However, instead we identify at least three distinct isoforms of Psc3, suggesting that post-translational modification of Psc3 contributes to the regulation of cohesion function.

Cell Cycle↗
PubMed2002

Mutation analysis of MECP2 and clinical characterization in Korean patients with Rett syndrome.

Rett syndrome is a progressive neurodevelopmental disorder occurring predominantly in females. Recently, mutations in the MECP2 gene on Xq28, which encodes methyl-CpG binding protein 2, were identified as responsible for some cases of Rett syndrome. In the present study, we analyzed the entire coding sequence of the MECP2 gene in 20 sporadic cases of Rett syndrome in Korea. Of the 20 patients, 14 (70%) had pathogenic mutations, which included 10 different mutations. Altogether, there were five missense mutations (D97Y, L100V, R133C, T158M, R306C), four nonsense mutations (R168X, R255X, R270X, R294X), and one frameshift mutation (a 41-bp deletion at 1157-1197). Two of these were novel mutations (D97Y, L100V). Most of the nucleotide substitutions involved C to T transitions at CpG hotspots. We could find no clear phenotype-genotype correlation according to the type of mutation. However, there was a tendency for patients with no MECP2 mutation (30%) to show more severe symptoms and more rapid clinical progression than patients with mutations. Further studies are necessary to identify the other possible genetic causes of Rett syndrome.

Adolescent↗
PubMed1999

N-myc-dependent repression of ndr1, a gene identified by direct subtraction of whole mouse embryo cDNAs between wild type and N-myc mutant.

To identify genes regulated by N-myc, subtraction of whole embryo cDNA was carried out between wild type and N-myc-deficient mutant mice. Six cDNA clones were isolated as representing genes expressed higher in the mutant embryos and two as those expressed lower. One of them, Ndr1, coding for 43 kDa cytoplasmic protein was studied in detail. The Ndr1 gene was augmented 20-fold in the mutant embryos at 10.5 days post coitus which is indicative of repression by N-myc. An inverse relationship actually existed between the expression of N-myc and Ndr1 in various developing tissues of the wild type embryos. In the early stage of differentiation of these tissues when N-myc expression was high Ndr1 expression was low or undetectable, and later when N-myc activity diminished Ndr1 expression was augmented concomitantly with the occurrence of terminal differentiation. To establish the direct link between N-myc activity and the Ndr1 regulation, the Ndr1 gene was cloned and analyzed. The Ndr1 promoter activity was down-regulated by N-myc, and more strongly by the combination of N-myc and Max in the cotransfection assay. This repressive effect was mediated by the promoter region within 52 base pairs from the transcription start site but direct binding of N-myc:Max to the promoter sequence was not demonstrated, which is analogous to the cases recently reported for transcriptional repression by c-myc. c-myc also repressed Ndr1 promoter activity similarly to N-myc. The effect of N-myc:Max was sensitive to Trichostatin A, indicating involvement of histone deacetylase activity in repression of the Ndr1 promoter. The strategy we adopted in identifying target genes of a transcription factor should prove widely applicable when mutant animals are available.

Amino Acid Sequence↗
PubMed2000

Steroid receptor co-activator-1 mediates 1,25-dihydroxyvitamin D(3)-stimulated alkaline phosphatase in human osteosarcoma cells.

For steroid hormone function to occur, nuclear receptors interact with a series of coactivators including steroid receptor coactivator-1 (SRC-1). The SRC-1 binds the vitamin D receptor (VDR) in the presence of ligand in an activation function 2 (AF-2)-dependent manner. In order to understand the role of this interaction in 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]-mediated gene expression, the level of SRC-1 expression was altered in MG-63 cells. Previous studies had demonstrated that MG-63 cells express the VDR and that 1,25(OH)(2)D(3) regulates expression of alkaline phosphatase (ALP). Analysis of MG-63 cells demonstrated that SRC-1 is expressed. A full-length cDNA coding for SRC-1 was inserted in antisense orientation into an expression vector (anti-SRC-1). The MG-63 cells were transfected with anti-SRC-1 or mock vector and stable transformants were selected. Western blot analysis showed a 95% reduction in SRC-1 protein as compared with mock cells. We determined the effect of normal and reduced SRC-1 expression in MG-63 cells on 1,25(OH)(2)D(3)-mediated stimulation of ALP. Whereas 10(-8) M 1,25(OH)(2)D(3) produced a 3.6-fold stimulation in ALP in mock cells expressing normal levels of SRC-1, it did not alter ALP in cells expressing reduced levels of SRC-1. Thus, SRC-1 is required for 1,25(OH)(2)D(3)-mediated gene expression of ALP by human MG-63 cells.

Alkaline Phosphatase↗
PubMed2002

A Rad26-Def1 complex coordinates repair and RNA pol II proteolysis in response to DNA damage.

Eukaryotic cells use multiple, highly conserved mechanisms to contend with ultraviolet-light-induced DNA damage. One important response mechanism is transcription-coupled repair (TCR), during which DNA lesions in the transcribed strand of an active gene are repaired much faster than in the genome overall. In mammalian cells, defective TCR gives rise to the severe human disorder Cockayne's syndrome (CS). The best-studied CS gene, CSB, codes for a Swi/Snf-like DNA-dependent ATPase, whose yeast homologue is called Rad26 (ref. 4). Here we identify a yeast protein, termed Def1, which forms a complex with Rad26 in chromatin. The phenotypes of cells lacking DEF1 are consistent with a role for this factor in the DNA damage response, but Def1 is not required for TCR. Rather, def1 cells are compromised for transcript elongation, and are unable to degrade RNA polymerase II (RNAPII) in response to DNA damage. Our data suggest that RNAPII stalled at a DNA lesion triggers a coordinated rescue mechanism that requires the Rad26-Def1 complex, and that Def1 enables ubiquitination and proteolysis of RNAPII when the lesion cannot be rapidly removed by Rad26-promoted DNA repair.

Cell Cycle Proteins↗
PubMed1999

PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes.

The phaC1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of Pseudomonas oleovorans GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant P. oleovorans strain carrying a phaC1::lacZ reporter system, that the phaC1 gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon source. Moreover, a P. oleovorans GPo1 mutant (strain GPG-Tc6) expressing higher levels of the reporter gene than the wild-type strain in the presence of glucose or citrate has been generated by mini-Tn5 insertional mutagenesis. Characterization of this mutant allowed us to conclude that phaF, a gene located downstream of the pha gene cluster, was knocked out in this strain. P. oleovorans GPG-Tc6 regained the ability to control phaC1 gene expression when complemented with the phaF wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 and phaI and phaF genes. The amino acid sequences of the phaI gene product and the N-terminal half of the PhaF protein showed a significant degree of similarity. Furthermore, the primary structure of the PhaF C terminus identifies this protein as a member of the histone H1-like group of proteins. Northern blot analysis showed two transcription units containing phaF, i.e., phaF and phaIF transcripts. Expression of the phaIF operon is more efficient in the presence of octanoic acid and is enhanced by the lack of the PhaF protein. In addition, it has also been demonstrated that both PhaF and PhaI proteins are bound to PHA granules produced by P. oleovorans. A model for the role of PhaF in regulating PHA synthesis is presented.

Acyltransferases↗
PubMed1998

Expression, purification, and characterization of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit nepsilon-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) Nepsilon-methyltransferase (Rubisco LSMT, EC 2.1.1.127) catalyzes methylation of the LS of Rubisco. A pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT cDNA was expressed in Escherichia coli, but most of the expressed protein was found in the insoluble fraction as an inclusion body. Expression at lower temperatures increased the level of soluble Rubisco LSMT and the associated enzymatic activity. However, the soluble form of Rubisco LSMT occurred as two molecular mass forms with the lower molecular mass suggestive of N-terminal processing at Ser-37. Deletion of 108 nucleotides from the 5' end encoding the N-terminal 36 amino acids of Rubisco LSMT resulted in a 10-fold increase in solubility and activity. Further addition of a 3' nucleotide sequence coding for a hexahistidyl carboxy-terminal peptide enabled purification of the N-terminally truncated Rubisco LSMT to homogeneity. Five milligrams of pure recombinant Rubisco LSMT was obtained from a 1-liter E. coli cell culture. The apparent kinetic constants for recombinant Rubisco LSMT for spinach Rubisco and AdoMet were only slightly different from the constants determined using affinity-purified native Rubisco LSMT from pea chloroplasts. However, there was a 6- to 7-fold reduction in the kcat for Rubisco LSMT, which was apparently a consequence of catalytic inactivation due to exposure to NiSO4 during purification. The availability of larger quantities of purified Rubisco LSMT should enable studies of the structure-function relationships in Rubisco LSMT and moreover its interaction with Rubisco.

Amino Acid Sequence↗
PubMed1989

Cloning of cDNAs coding for human HMG I and HMG Y proteins: both are capable of binding to the octamer sequence motif.

In human B lymphocytes and placenta HMG I and its smaller isoform HMG Y are encoded by two distinct but structurally highly similar mRNAs which arise most likely by alternative splicing of a single primary transcript. Both have been cloned as cDNAs. On Northern blots an abundant mRNA species 2000 nucleotides in length was detected in all cell lines examined. Exclusively in erythroid cells an additional rare 3800 nucleotides long mRNA species was noted. In quiescent cells the mRNA levels of HMG I/Y were not significantly down-regulated. Southern blot analysis indicated that at least four genes are present per haploid human genome. Both proteins when expressed in bacteria bind specifically to A-T rich stretches of DNA suggesting that no posttranslational modifications are necessary for specific DNA binding. Interestingly, HMG I as well as HMG Y are capable of binding to the octamer transcriptional regulatory sequence motif.

Amino Acid Sequence↗
PubMed1998

The cenpB gene is not essential in mice.

Centromere protein B (CENP-B) is a centromeric DNA-binding protein that binds to alpha-satellite DNA at the 17 bp CENP-B box sequence. The binding of CENP-B, along with other proteins, to alpha-satellite DNA sequences at the centromere, is thought to package the DNA into heterochromatin subjacent to the kinetochore of mitotic chromosomes. To determine the importance of CENP-B to kinetochore assembly and function, we generated a mouse null for the cenpB gene. The deletion removed part of the promoter and the entire coding sequence except for the carboxyl-terminal 35 amino acids of the CENP-B polypeptide. Mice heterozygous or homozygous for the cenpB null mutation are viable and healthy, with no apparent defect in growth and morphology. We have established mouse embryo fibroblasts from heterozygous and homozygous cenpB null littermates. Microscopic analysis, using immunofluorescence and electron microscopy of the cultured cells, indicated that the centromere-kinetochore complex was intact and identical to control cells. Mitosis was identical in fibroblasts derived from cenpB wild-type, heterozygous and null animals. Our studies demonstrate that CENP-B is not required for the assembly of heterochromatin or the kinetochore, or for completion of mitosis.

Animals↗
PubMed1995

Characterization of cis-acting sequences and decay intermediates involved in nonsense-mediated mRNA turnover.

Several lines of evidence indicate that the processes of mRNA turnover and translation are intimately linked and that understanding this relationship is critical to elucidating the mechanism of mRNA decay. One clear example of this relationship is the observation that nonsense mutations can accelerate the decay of mRNAs in a process that we term nonsense-mediated mRNA decay. The experiments described here demonstrate that in the yeast Saccharomyces cerevisiae premature translational termination within the initial two-thirds of the PGK1 coding region accelerates decay of that transcript regardless of which of the stop codons is used. Nonsense mutations within the last quarter of the coding region have no effect on PGK1 mRNA decay. The sequences required for nonsense-mediated mRNA decay include a termination codon and specific sequences 3' to the nonsense mutation. Translation of two-thirds of the PGK1 coding region inactivates the nonsense-mediated mRNA decay pathway. This observation explains why carboxyl-terminal nonsense mutations are resistant to accelerated decay. Characterization of the decay of nonsense-containing HIS4 transcripts yielded results mirroring those described above, suggesting that the sequence requirements described for the PGK1 transcript are likely to be a general characteristic of this decay pathway. In addition, an analysis of the decay intermediates of nonsense-containing mRNAs indicates that nonsense-mediated mRNA decay flows through a pathway similar to that described for a class of wild-type transcripts. The initial cleavage event occurs near the 5' terminus of the nonsense-containing transcript and is followed by 5'-->3' exonucleolytic digestion. A model for nonsense-mediated mRNA decay based on these results is discussed.

Alleles↗
PubMed1997

A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins.

Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity. Proteins fused in frame with CBP can be purified from crude E. coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992). Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications. To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n. The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E. coli. The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7(lac)O promoter. A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest. The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E. coli cells at 15-20 mg/1 culture. CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin. Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin. We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase. Hence, the purified fusion proteins can be labeled directly with [gamma-32P]ATP and used to probe protein-protein or protein-nucleic acid interactions.

Adenosine Triphosphate↗
PubMed2005

Association of NCOA3 polymorphisms with breast cancer risk.

The nuclear receptor coactivator 3 (NCOA3, also known as AIB1) is a coactivator of nuclear receptors like the estrogen receptor. NCOA3 is overexpressed in approximately 60% of primary human breast tumors, and high levels of NCOA3 expression are associated with tamoxifen resistance and worse survival rate. In contrast, NCOA3 deficiency suppresses v-Ha-ras-induced breast cancer initiation and progression in mice. Here, we analyzed the influence of NCOA3 coding single nucleotide polymorphisms on breast cancer risk by performing a case-control study using a German and a Polish study population and identified an association between NCOA3 polymorphisms and breast cancer. A joint analysis of the German and the Polish study population revealed a significant protective effect for the 1758G>C (Q586H) and 2880A>G (T960T) variants. In addition, haplotype analysis showed a protective effect of the 1758C-2880A and 1758G-2880G haplotypes (odds ratio 0.79; 95% confidence interval, 0.67-0.93; P = 0.004). Because of the impact of NCOA3 in antiestrogen therapy resistance, these polymorphisms might also influence therapy outcome in breast cancer.

Acetyltransferases↗
PubMed2001

Mutation analysis of the methyl-CpG-binding protein 2 gene (MECP2) in Rett patients with preserved speech.

Genomic DNAs from 35 Japanese sporadic patients with Rett syndrome (RTT) were screened for DNA mutations in the entire coding region and exon-intron boundaries of methyl-CpG-binding protein 2 (MECP2). We detected mutations in 30 (85.7%) of 35 patients. Among these 35 RTT patients, five patients (14%) had the preserved speech variant of this disease. Four respective mutations (R133C, R306C, R294X, 2 base pair (bp) deletion) were found in these five patients. Two patients had the same missense mutation, R133C. The patients with the R133C mutation and one with frameshift mutation presented the relatively mild clinical presentation, and the R133C mutation was not found in any other patient without preserved speech. We confirmed that the preserved speech variant is one of the clinical phenotypes of RTT and is also caused by MECP2 mutation. We speculated that the clinical phenotype of patients with the R133C missense mutation might be mild.

Adult↗
PubMed1993

Chromosomal structure and expression of the human OTF1 locus encoding the Oct-1 protein.

The genomic structure of the POU domain containing oct-1 gene (OTF1 locus) coding region has been determined using human DNA recombinant bacteriophage and a yeast artificial chromosome clone. The gene is encoded by 16 exons spanning over 150 kb, and the Oct-1 protein reading frame has been extended to 766 amino acids. The exonic structure has been compared to the mouse Oct-2 protein and reveals a conservation of exon-intron boundaries as well as protein sequence similarity. To provide insight into Oct-1 control of transcriptional regulation during the cell-cycle the expression of the oct-1 gene was examined during cellular DNA replication and shows that the steady-state level of the oct-1 mRNA is not S-phase regulated.

Amino Acid Sequence↗
PubMed1983

The terminal RNA stem-loop structure and 80 bp of spacer DNA are required for the formation of 3' termini of sea urchin H2A mRNA.

We have studied the exact sequence requirement for the formation of 3' termini of the sea urchin H2A mRNA in frog oocyte injection experiments. Point mutations destroying the symmetry of the inverted DNA repeat in the mRNA trailer coding sequences prevent the generation of genuine 3' termini. Mutants containing complementary base changes are pseudorevertants and allow the production of H2A mRNA with faithful 3' termini at wild-type levels. Our transcription analyses show that it is primarily the sequence of the transcribed strand that decides whether or not true 3' mRNA termini are produced. This is evidence that an RNA stem-loop structure, rather than a DNA cruciform, is essential for this process. Spacer sequences are absolutely required, because in their absence only H2A mRNA with spacer transcript extensions are found. Once the canonical CAAGAAAGA and flanking sequences are linked to the H2A gene, H2A messenger is synthesized at a suboptimal rate, which can be increased to wild-type levels by the addition of 80 bp of the spacer immediately adjacent to the H2A gene.

Animals↗