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H Ge

Publications and source records attributed to H Ge.

At least 37 records · Page 2Linked to original sources
PubMed1999

The transcription factor Sp3 interacts with promoter elements of the lens specific MIP gene.

PURPOSE: To characterize the cis regulatory elements and their interaction with transcription factors responsible for the lens specific expression of the MIP gene, which encodes the Major Intrinsic Protein of the lens fiber membranes. METHODS: Study interaction of factors present in newborn mouse lens nuclear extracts with DNA fragments corresponding to mouse MIP gene 5' flanking sequence by electrophoresis mobility shift assay (EMSA) and DNase I footprinting. RESULTS: We found a high degree of identity in the first 100 bp of 5' flanking sequence of mice and humans, however, a lower degree of conservation is observed further upstream. We have found by DNase I footprinting analysis that lens specific factors may interact with the first 100 bp of 5' flanking sequence. A domain containing an E box, conserved in mouse and human, may interact with a lens specific factor. However, general factors may interact with a NF-1 binding site. An overlapping GC and CT box is present in the mouse MIP gene. In the human MIP gene GC and CT boxes are found in different domains of the MIP gene promoter. Both CT boxes interact with factors present in lens nuclear extracts including Sp3. They are able to interact with purified Sp1but not with Sp1 present in mouse lens nuclear extracts. CONCLUSIONS: The transcription factor Sp3 may play an important role in regulating MIP gene expression in the lens.

Animals↗
PubMed1999

Interferon consensus sequence-binding protein is constitutively expressed and differentially regulated in the ocular lens.

Interferon signaling is mediated by STATs and interferon regulatory factor (IRF) families of transcription factors. Ten distinct IRFs have been described and most are expressed in a variety of cells except for interferon consensus sequence-binding protein (ICSBP) and lymphoid-specific IRF/Pip that are thought to be exclusively expressed in lymphoid cells. We show here for the first time that ICSBP is constitutively and inducibly expressed in the mouse lens. In contrast to lymphoid cells with exclusive expression of ICSBP in the nucleus, ICSBP is present in both the cytoplasm and nucleus of the lens cell. However, ICSBP in the nucleus is of lower apparent molecular weight. We further show that the ICSBP promoter is constitutively bound by lens nuclear factors and that its activation requires binding of additional factors including STAT1. Furthermore, transcriptional activation of ICSBP gene by interferon gamma is accompanied by selective nuclear localization of ICSBP in proliferating epithelial cells but not in the nuclei of nondividing cells in the lens fiber compartment. Constitutive and inducible expression of ICSBP in the ocular lens and differential regulation of its subcellular localization in the developing lens suggest that ICSBP may have nonimmunity related functions and that the commonly held view that it is lymphoid-specific be modified.

Animals↗
PubMed1998

Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation.

Transcriptional activation in human cell-free systems containing RNA polymerase II and general initiation factors requires the action of one or more additional coactivators. Here, we report the isolation of cDNAs encoding two novel human transcriptional coactivators (p52 and p75) that are derived from alternatively spliced products of a single gene and share a region of 325 residues, but show distinct coactivator properties. p52 and p75 both show strong interactions with the VP16 activation domain and several components of the general transcriptional machinery. p52, like the previously described PC4, is a potent broad-specificity coactivator, whereas p75 is less active for most activation domains. These results suggest that p52 is a general transcriptional coactivator that mediates functional interactions between upstream sequence-specific activators and the general transcription apparatus, possibly through a novel mechanism.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Overlapping Sp1 and AP2 binding sites in a promoter element of the lens-specific MIP gene.

The MIP gene, the founder of the MIP family of channel proteins, is specifically expressed in fiber cells of the ocular lens and expression is regulated temporally and spatially during development. We previously found that a DNA fragment containing 253 bp of 5'-flanking sequence and 42 bp of exon 1 of the human MIP gene contains regulatory elements responsible for lens-specific expression of the MIP gene. In this report we have analyzed the function of overlapping Sp1 and AP2 binding sites present in the MIP promoter. Using DNase I footprinting analysis we found that purified Sp1 and AP2 transcription factors interact with several domains of the human MIP promoter sequence -253/+42. Furthermore, addition of purified Sp1 to Drosophila nuclear extracts activates in vitro transcription from the MIP promoter -253/+42. This promoter activity is competed by oligonucleotides containing domains footprinted with Sp1. Using promoter-reporter gene ( CAT ) constructs we found that the sequence -39/-70 contains a cis regulatory element essential for promoter activity in transient assays in lens cells. EMSA analysis showed that lens nuclear extracts contain factors that bind to the MIP 5'-flanking sequence containing overlapping Sp1 and AP2 binding domains at positions -37/-65. Supershift experiments with lens nuclear extracts indicated that Sp3 is also able to interact with this regulatory element, suggesting that Sp1 and Sp3 may be involved in regulation of transcription of the MIP gene in the lens.

Amino Acid Sequence↗
PubMed1998

A novel transcriptional coactivator, p52, functionally interacts with the essential splicing factor ASF/SF2.

Increasing evidence suggests that pre-mRNA splicing can take place cotranscriptionally in vivo. However, insight into how these two processes are linked has been lacking. Here, we describe that a novel transcriptional coactivator, p52, interacts not only with transcriptional activators and general transcription factors to enhance activated transcription but also with the essential splicing factor ASF/SF2 both in vitro and in vivo to modulate ASF/SF2-mediated pre-mRNA splicing. Furthermore, immunofluorescence studies indicate that the majority of endogenous p52 is colocalized with ASF/SF2 in the nucleoplasm of HeLa cells. Together, these observations suggest that, in addition to functioning as a transcriptional coactivator, p52 may also act as an adaptor to coordinate pre-mRNA splicing and transcriptional activation of class II genes.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Novel cofactors and TFIIA mediate functional core promoter selectivity by the human TAFII150-containing TFIID complex.

TATA-binding protein-associated factors (TAFIIs) within TFIID control differential gene transcription through interactions with both activators and core promoter elements. In particular, TAFII150 contributes to initiator-dependent transcription through an unknown mechanism. Here, we address whether TAFIIs within TFIID are sufficient, in conjunction with highly purified general transcription factors (GTFs), for differential core promoter-dependent transcription by RNA polymerase II and whether additional cofactors are required. We identify the human homologue of Drosophila TAFII150 through cognate cDNA cloning and show that it is a tightly associated component of human TFIID. More importantly, we demonstrate that the human TAFII150-containing TFIID complex is not sufficient, in the context of all purified GTFs and RNA polymerase II, to mediate transcription synergism between TATA and initiator elements and initiator-directed transcription from a TAFII-dependent TATA-less promoter. Therefore, TAFII-promoter interactions are not sufficient for the productive core promoter-selective functions of TFIID. Consistent with this finding, we have partially purified novel cofactor activities (TICs) that potentiate the TAFII-mediated synergism between TATA and initiator elements (TIC-1) and TAFII-dependent transcription from TATA-less promoters (TIC-2 and -3). Furthermore, we demonstrate an essential function for TFIIA in TIC- and TAFII-dependent basal transcription from a TATA-less promoter. Our results reveal a parallel between the basal transcription activity of TAFIIs through core promoter elements and TAFII-dependent activator function.

ARNTL Transcription Factors↗
PubMed1998

Coactivation by OCA-B: definition of critical regions and synergism with general cofactors.

Molecular dissection of the B-cell-specific transcription coactivator OCA-B has revealed distinct regions important, respectively, for recruitment to immunoglobulin promoters through interaction with octamer-bound Oct-1 and for subsequent coactivator function. Further analysis of general coactivator requirements showed that selective removal of PC4 from the essential USA fraction severely impairs Oct-1 and OCA-B function in a cell-free system reconstituted with partially purified factors. Full activity can be restored by the combined action of recombinant PC4 and the PC4-depleted USA fraction, thus suggesting a joint requirement for PC4 and another, USA-derived component(s) for optimal function of Oct-1/OCA-B in the reconstituted system. Indeed, USA-derived PC2 was found to act synergistically with PC4 in reproducing the function of intact USA in the assay system. Consistent with the requirement for PC4 in the reconstituted system, OCA-B was found to interact directly with PC4. Surprisingly, however, removal of PC4 from the unfractionated nuclear extract has no detrimental effect on OCA-B/Oct-1-dependent transcription. These results lead to a general model for the synergistic function of activation domains in Oct-1 and OCA-B (mediated by the combined action of the multiple USA components) and, further, suggest a functional redundancy in general coactivators.

Amino Acid Sequence↗
PubMed1997

High-performance liquid chromatography of selenoamino acids and organo selenium compounds. Speciation by inductively coupled plasma mass spectrometry.

As part of an ongoing study to identify selenium compounds with cancer chemopreventive activity, extracts of selenium-enriched samples were analyzed by HPLC-inductively coupled plasma (ICP)-MS. Ion-exchange, ion pair and derivatization methods for reversed-phase HPLC were considered and advantages and disadvantages for each compared. Anion exchange allows separation of selenite and selenate, but otherwise provides poor separation. Pre-column derivatization and reversed-phase chromatography provides separation of compounds with terminal amine functionalities, but many other species elute in the void volume. The ion pair method gave optimal separation and was compatible with standard ICP-MS operating conditions.

Amino Acids↗
PubMed1997

Acetylation of general transcription factors by histone acetyltransferases.

The acetylation of histones increases the accessibility of nucleosomal DNA to transcription factors [1,2], relieving transcriptional repression [3] and correlating with the potential for transcriptional activity in vivo [4 - 7]. The characterization of several novel histone acetyltransferases - including the human GCN5 homolog PCAF (p300/CBP-associated factor) [8], the transcription coactivator p300/CBP [9], and TAFII250 [10] - has provided a potential explanation for the relationship between histone acetylation and transcriptional activation. In addition to histones, however, other components of the basal transcription machinery might be acetylated by these enzymes and directly affect transcription. Here, we examine the acetylation of the basal transcriptional machinery for RNA polymerase II by PCAF, p300 and TAFII250. We find that all three acetyltransferases can direct the acetylation of TFIIEbetaand TFIIF, and we identify a preferred site of acetylation in TFIIEbeta. Human TFIIE consists of two subunits, alpha(p56) and beta(p34), which form a heterotetramer (alpha2 beta2) in solution ([11], reviewed in [12]). TFIIE enters the preinitiation complex after RNA polymerase II and TFIIF, suggesting that TFIIE may interact directly with RNA polymerase II and/or TFIIF [13,14]. In addition, TFIIE can facilitate promoter melting either in the presence or absence of TFIIH and can stimulate TFIIH-dependent phosphorylation of the carboxy-terminal domain of RNA polymerase II [15-18]. TFIIF has an essential role in both transcription initiation and elongation ([19,20], for review see [21]). We discuss the implications of the acetylation of TFIIEbetaand TFIIF for transcriptional control by PCAF, p300 and TAFII250.

Acetyl Coenzyme A↗
PubMed1996

Transcription-positive cofactor 4 forms complexes with HSSB (RPA) on single-stranded DNA and influences HSSB-dependent enzymatic synthesis of simian virus 40 DNA.

The replication of simian virus 40 (SV40) DNA in vitro requires a trimeric single-stranded DNA (ssDNA)-binding protein called HSSB or RPA. HSSB supports the unwinding of DNA containing the SV40 origin in the presence of the viral-encoded T antigen and is required for the initiation of RNA primer synthesis as well as processive elongation of DNA catalyzed by the DNA polymerase delta holoenzyme. In this report we show that the transcription positive cofactor 4 (PC4), a ssDNA-binding protein, forms complexes with HSSB on ssDNA and markedly affects the replication functions of HSSB. PC4 supports T antigen-catalyzed unwinding of SV40 origins in lieu of HSSB but inhibits both RNA primer synthesis and polymerase delta-catalyzed DNA chain elongation reactions. These inhibitory effects can be reversed by the addition of excess HSSB. Depending on the concentration of HSSB, PC4 is capable of either inhibiting or activating SV40 DNA replication measured in both mono- and dipolymerase systems. The possible role of PC4 in the initiation of DNA replication is discussed.

Antigens, Viral, Tumor↗
PubMed1996

Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex.

Transcriptional regulation by nuclear hormone receptors is thought to involve interactions with putative cofactors that may potentiate receptor function. Here we show that human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3) is associated with a group of distinct nuclear proteins termed thyroid hormone receptor-associated proteins (TRAPs). In an in vitro system reconstituted with general initiation factors and cofactors (and in the absence of added T3), the "liganded" thyroid hormone receptor (TR)/TRAP complex markedly activates transcription from a promoter template containing T3-response elements. Moreover, whereas the retinoid X receptor is not detected in the TR/TRAP complex, its presence is required for the function of the complex. In contrast, human thyroid hormone receptor alpha purified from cells grown in the absence of T3 lacks the TRAPs and effects only a low level of activation that is dependent on added ligand. These findings demonstrate the ligand-dependent in vivo formation of a transcriptionally active TR-multisubunit protein complex and suggest a role for TRAPs as positive coactivators for gene-specific transcriptional activation.

DNA-Binding Proteins↗
PubMed1996

Isolation and characterization of two human transcription factor IIH (TFIIH)-related complexes: ERCC2/CAK and TFIIH.

Transcription factor IIH (TFIIH) is a multisubunit protein complex essential for both the initiation of RNA polymerase class II (pol II)-catalyzed transcription and nucleotide excision repair of DNA. Recent studies have shown that TFIIH copurifies with the cyclin-dependent kinase (cdk)-activating kinase complex (CAK) that includes cdk7, cyclin H, and p36/MAT1. Here we report the isolation of two TFIIH-related complexes: TFIIH* and ERCC2/CAK. TFIIH* consists of a subset of the TFIIH complex proteins including ERCC3 (XPB), p62, p44, p41, and p34 but is devoid of detectable levels of ERCC2 (XPD) and CAK. ERCC2/CAK was isolated as a complex that exhibits CAK activity that cosediments with the three CAK components (cdk7, cyclin H, and p36/MAT1) as well as the ERCC2 (XPD) protein. TFIIH* can support pol II-catalyzed transcription in vitro with lower efficiency compared with TFIIH. This TFIIH*-dependent transcription reaction was stimulated by ERCC2/CAK. The ERCC2/CAK and TFIIH* complexes are each active in DNA repair as shown by their ability to complement extracts prepared from ERCC2 (XPD)- and ERCC3 (XPB)-deficient cells, respectively, in supporting the excision of DNA containing a cholesterol lesion. These data suggest that TFIIH* and ERCC2/CAK interact to form the TFIIH holoenzyme capable of efficiently assembling the pol II transcription initiation complex and directly participating in excision repair reactions.

Animals↗
PubMed1996

Detection and evaluation of p53 intron 2 polymorphism in lung carcinomas in Hong Kong.

A polymorphism in intron 2 of the p53 gene, which gives rise to 2 alleles, A1 and A2, was analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct DNA-sequencing techniques. The distribution of this allele in the peripheral blood in the Chinese population comprising 27 healthy individuals, 30 bronchiectasis patients, 34 non-small-cell lung cancer (NSCLC) patients and 27 SCLC patients was analyzed. The genotypic distributions for this marker were significantly different between the blood of healthy individuals and SCLC patients. There was no significant difference between genotypes of Caucasians and Chinese. Tumors, normal lungs and peripheral blood of 83 adenocarcinoma and 10 squamous cell carcinoma patients were also studied. There was a significant difference in the distribution of the genotypes detected in tumor tissues vs. blood of adenocarcinoma patients. The frequency of detection of the A1/A1 genotype in the tumor tissues was increased in adenocarcinoma patients as compared with the blood of adenocarcinoma patients and was decreased in the blood of SCLC patients as compared with the blood of healthy individuals. Survival rates in Hong Kong adenocarcinoma patients with the A1/A1 genotype were lower than those in patients with A1/A2 and A2/A2 genotypes up to 30 months post-operation. Point mutations were detected at the p53 intron 2 polymorphic locus in NSCLC specimens, with a mutation rate of 15.4% (8/52). All mutations were GC transversions. The significance of this instability in p53 intron 2 remains to be elucidated.

Adenocarcinoma↗
PubMed1996

Analysis of L-myc and GSTM1 genotypes in Chinese non-small cell lung carcinoma patients.

The genotypes of L-myc and GSTM1 genes were studied in normal lung tissues of 98 non-small cell lung carcinoma (NSCLC) patients from Hong Kong using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) techniques. Results showed a statistical difference in L-myc genotypes between Chinese and African Americans (P = 0.02). A significant deficit in heterozygotes resulting in the departure from Hardy-Weinberg equilibrium in lung cancer female patients was detected (0.01 < P < 0.02). There were significant differences in survival times in patients having L-L and S-S genotypes, with shorter survival times in the patients with L-L genotypes (0.01 < P < 0.05). Data on age, size of tumor, histological types, and lymph node metastasis showed no significant association with L-myc genotype. The survival time in the GSTM1-negative (null gene) group was significantly different from the GSTM1 positive group between 16 and 24 months after operation (0.01 < P < 0.05). There was no significant difference in the distribution of GSTM1 genotypes between Chinese and Caucasian Americans.

Biomarkers, Tumor↗
PubMed1996

[Human melanoma-specific peptide antigens recognized by HLA-A2 restricted tumor-infiltrating lymphocytes].

OBJECTIVE: HLA-A2-associated peptides were extracted from human melanoma cell lines and used to study human melanoma-specific peptide antigens for HLA-A2-restricted tumor-infiltrating lymphocyte (TIL). METHODS: TILS were derived from the peripheral lymph nodes of HLA-A2+ human melanoma patients, HLA-A2 molecules were purified from the melanoma cell lines by immunoaffinity column chromatography of detergent-solubilized cell pellets. Peptides bound to the HLA-A2 molecules were acid eluted and fractionated by reversed phase RP-HPLC. Individual fractions were assessed for their ability to reconstitute melanoma-specific epitopes by adding to the HLA-A2+ Ag-processing mutant cell, T2. RESULTS: These TIL lysed HLA-A2+ autologous and allogeneic melanomas, but not the HLA-A2- melanomas. They also did not lyse the HLA-A2+ non-melanoma cell lines. The RP-HPLC separations of reconstituting fractions revealed three peaks (fractions) of reconstitution. CONCLUSION: These results showed that peptides derived from three active fractions were related to human melanoma-specific peptide antigens recognized by HLA-A2-restricted TIL.

Antigens, Neoplasm↗
PubMed1995

TATA-binding protein residues implicated in a functional interplay between negative cofactor NC2 (Dr1) and general factors TFIIA and TFIIB.

The TATA-binding protein (TBP) plays a key role in transcription initiation. Several negative cofactors (NC1, NC2, and Dr1) are known to interact with TBP in a manner that prevents productive interactions of transcription factors TFIIA and TFIIB with promoter-bound TBP. To gain insights into the regulatory interplay on the surface of TBP, we have employed mutant forms of TBP to identify amino acid residues important for interactions with the negative regulatory cofactor NC2 and the general factor TFIIB. The results show the involvement of distinct domains of TBP in these interactions. Residues (Lys-133, Lys-145, and Lys-151) in the basic repeat region are important for interactions with NC2, as well as with TFIIA (Buratowski, S., and Zhou, H. (1992) Science 255, 1130-1132; Lee, D. K., DeJong, J., Hashimoto, S., Horikoshi, M., and Roeder, R. G. (1992) Mol. Cell. Biol. 12, 5189-5196), whereas a residue (Leu-189) in the second stirrup-like loop spanning S2' and S3' is required for interaction with TFIIB. In addition, we demonstrate that NC2 is identical to the previously cloned negative cofactor Dr1. The implications of these results for TBP structure and function are discussed.

Binding Sites↗