FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2053606985> ?p ?o ?g. }

• W2053606985 endingPage "28016" @default.
• W2053606985 startingPage "28009" @default.
• W2053606985 abstract "ld;&.2qTo gain insight into the regulation of hmi-er1 expression, we cloned a human genomic DNA fragment containing one of the two hmi-er1 promoters and consisting of 1460 bp upstream of the translation initiation codon of hMI-ER1. Computer-assisted sequence analysis revealed that the hmi-er1 promoter region contains a CpG island but lacks an identifiable TATA element, initiator sequence and downstream promoter element. This genomic DNA was able to direct transcription of a luciferase reporter gene in a variety of human cell lines, and the minimal promoter was shown to be located within–68/+144 bp. Several putative Sp1 binding sites were identified, and we show that Sp1 can bind to the hmi-er1 minimal promoter and increase transcription, suggesting that the level of hmi-er1 expression may depend on the availability of Sp1 protein. Functional analysis revealed that hMI-ER1 represses Sp1-activated transcription from the minimal promoter by a histone deacetylase-independent mechanism. Chromatin immunoprecipitation analysis demonstrated that both Sp1 and hMI-ER1 are associated with the chromatin of the hmi-er1 promoter and that overexpression of hMI-ER1 in cell lines that allow Tet-On-inducible expression resulted in loss of detectable Sp1 from the endogenous hmi-er1 promoter. The mechanism by which this occurs does not involve binding of hMI-ER1 to cis-acting elements. Instead, we show that hMI-ER1 physically associates with Sp1 and that endogenous complexes containing the two proteins could be detected in vivo. Furthermore, hMI-ER1 specifically interferes with binding of Sp1 to the hmi-er1 minimal promoter as well as to an Sp1 consensus oligonucleotide. Deletion analysis revealed that this interaction occurs through a region containing the SANT domain of hMI-ER1. Together, these data reveal a functional role for the SANT domain in the action of co-repressor regulatory factors and suggest that the association of hMI-ER1 with Sp1 represents a novel mechanism for the negative regulation of Sp1 target promoters. ld;&.2qTo gain insight into the regulation of hmi-er1 expression, we cloned a human genomic DNA fragment containing one of the two hmi-er1 promoters and consisting of 1460 bp upstream of the translation initiation codon of hMI-ER1. Computer-assisted sequence analysis revealed that the hmi-er1 promoter region contains a CpG island but lacks an identifiable TATA element, initiator sequence and downstream promoter element. This genomic DNA was able to direct transcription of a luciferase reporter gene in a variety of human cell lines, and the minimal promoter was shown to be located within–68/+144 bp. Several putative Sp1 binding sites were identified, and we show that Sp1 can bind to the hmi-er1 minimal promoter and increase transcription, suggesting that the level of hmi-er1 expression may depend on the availability of Sp1 protein. Functional analysis revealed that hMI-ER1 represses Sp1-activated transcription from the minimal promoter by a histone deacetylase-independent mechanism. Chromatin immunoprecipitation analysis demonstrated that both Sp1 and hMI-ER1 are associated with the chromatin of the hmi-er1 promoter and that overexpression of hMI-ER1 in cell lines that allow Tet-On-inducible expression resulted in loss of detectable Sp1 from the endogenous hmi-er1 promoter. The mechanism by which this occurs does not involve binding of hMI-ER1 to cis-acting elements. Instead, we show that hMI-ER1 physically associates with Sp1 and that endogenous complexes containing the two proteins could be detected in vivo. Furthermore, hMI-ER1 specifically interferes with binding of Sp1 to the hmi-er1 minimal promoter as well as to an Sp1 consensus oligonucleotide. Deletion analysis revealed that this interaction occurs through a region containing the SANT domain of hMI-ER1. Together, these data reveal a functional role for the SANT domain in the action of co-repressor regulatory factors and suggest that the association of hMI-ER1 with Sp1 represents a novel mechanism for the negative regulation of Sp1 target promoters. hmi-er1 (human mesoderm induction-early response 1) is a growth factor-induced immediate early gene encoding a novel transcriptional regulator (1Paterno G.D. Li Y. Luchman H.A. Ryan P.J. Gillespie L.L. J. Biol. Chem. 1997; 272: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 2Ding Z. Gillespie L.L. Paterno G.D. Mol. Cell. Biol. 2003; 23: 250-258Crossref PubMed Scopus (53) Google Scholar) that is differentially expressed in breast carcinoma cell lines and tumors (3Paterno G.D. Mercer F.C. Chayter J.J. Yang X. Robb J.D. Gillespie L.L. Gene (Amst.). 1998; 222: 77-82Crossref PubMed Scopus (20) Google Scholar). hmi-er1 is transcribed from two distinct promoters P1 and P2 (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar), and transcripts undergo alternative splicing to produce six protein isoforms differing in their N and C termini (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar). Two of the N-terminal variants differ in the sequence of their 5′-UTR, 1The abbreviations used are: UTR, untranslated region; aa, amino acid; ChIP, chromatin immunoprecipitation; DTBP, 3–3′ dithiobispropionimidate-2HCl; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; HDAC, histone deacetylase; TSA, trichostatin A; RLU, relative luciferase units; IP, immunoprecipitation; dox, doxycycline. whereas the third contains an additional exon that encodes 25 amino acids (aa). The C-terminal variants, hMI-ER1α and -β, differ both in the size and sequence of their C-terminal domains: the α C terminus consists of 23 aa and includes an LXXLL motif, a domain known to be important for interaction with nuclear hormone receptors (5Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1778) Google Scholar). In this regard, the α isoform mRNA is only detectable in endocrine tissues (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar). The β C terminus contains 102 aa and includes the only functional nuclear localization signal (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar, 6Post J.N. Gillespie L.L. Paterno G.D. FEBS Lett. 2001; 502: 41-45Crossref PubMed Scopus (9) Google Scholar). Whereas the divergent C-terminal amino acid sequences would suggest that these two isoforms have distinct functions, so far no difference in function has been determined. Instead, both isoforms can act as transcriptional repressors, and this repression was shown to involve recruitment of histone deacetylase 1 (HDAC1) (2Ding Z. Gillespie L.L. Paterno G.D. Mol. Cell. Biol. 2003; 23: 250-258Crossref PubMed Scopus (53) Google Scholar). The common internal sequence of hMI-ER1 contains conserved domains found in a number of transcriptional regulators, including an acid activation domain (1Paterno G.D. Li Y. Luchman H.A. Ryan P.J. Gillespie L.L. J. Biol. Chem. 1997; 272: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), an ELM2 domain (7Solari F. Bateman A. Ahringer J. Development. 1999; 126: 2483-2494Crossref PubMed Google Scholar), and a signature SANT domain (8Aasland R. Stewart A.F. Gibson T. Trends Biochem. Sci. 1996; 21: 87-88Abstract Full Text PDF PubMed Scopus (294) Google Scholar). In a recent report, we showed that the ELM2 domain functions in the recruitment of HDAC1 and transcriptional repression (2Ding Z. Gillespie L.L. Paterno G.D. Mol. Cell. Biol. 2003; 23: 250-258Crossref PubMed Scopus (53) Google Scholar). The SANT domain is located immediately downstream of the ELM2 domain and, in other proteins, has been implicated in DNA binding as well as in protein-protein interactions (8Aasland R. Stewart A.F. Gibson T. Trends Biochem. Sci. 1996; 21: 87-88Abstract Full Text PDF PubMed Scopus (294) Google Scholar), including interactions with histone deacetylase 3 (HDAC3)- and histone acetyltransferase-containing complexes (9Guenther M.G. Barak O. Lazar M.A. Mol. Cell. Biol. 2001; 21: 6091-6101Crossref PubMed Scopus (492) Google Scholar, 10You A. Tong J.K. Grozinger C.M. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1454-1458Crossref PubMed Scopus (396) Google Scholar, 11Sterner D.E. Wang X. Bloom M.H. Simon G.M. Berger S.L. J. Biol. Chem. 2002; 277: 8178-8186Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), as well as nonacetylated histones (12Yu J. Li Y. Ishizuka T. Guenther M.G. Lazar M.A. EMBO J. 2003; 22: 3403-3410Crossref PubMed Scopus (145) Google Scholar). Recently, the SANT domain of the nuclear receptor corepressor SMRT has been implicated in the ability of this protein to target specific promoters through interpretation of the so-called histone code (12Yu J. Li Y. Ishizuka T. Guenther M.G. Lazar M.A. EMBO J. 2003; 22: 3403-3410Crossref PubMed Scopus (145) Google Scholar). To date, no function has been ascribed to the SANT domain of hMI-ER1. In this report, we investigate mechanisms of transcriptional regulation of the hmi-er1 P2 promoter and demonstrate that promoter activity is regulated by Sp1 protein and that this activation is repressed in a dose-dependent manner by hMI-ER1 protein. This autorepression does not depend upon HDAC activity but involves interference of Sp1 binding to the chromatin surrounding the hmi-er1 P2 promoter and from the cognate binding sites by physical association with a region containing the hMI-ER1 SANT domain. This represents a novel mechanism for negative regulation of Sp1 target promoters and a functional role for the SANT domain in the activity of co-repressor regulatory factors. Cell Lines—All cell lines were obtained from the American Tissue Culture Collection and cultured at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Plasmids and Constructs—A 1460-bp sequence, containing 144 bp of hmi-er1 5′-UTR and 1316 bp of 5′-flanking genomic sequence, was generated by PCR from human genomic DNA, using the primer pairs listed in Table I. The PCR product (–1316) was cloned into the pCR2.1 vector (pCR(–1316)), using the TOPO-TA cloning kit (Invitrogen) and sequenced on both strands. The sequence was verified by comparison to previously reported hmi-er1 sequences (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar) and to the human genome sequence data (Sanger Centre). The pGL3(–1316) plasmid was generated by digestion of pCR(–1316) and subcloning into the XhoI/HindIII sites of the promoterless pGL3-Basic vector (Promega Corp.). The pGL3(AS) plasmid was generated by digestion of pCR(–1316) and subcloning into the KpnI/XhoI sites of pGL3-Basic.Table IPrimer pairs used for preparing hmi-er1 promoter constructs in pGL3-Basic and hMI-ER1 constructs in pGEX-4T1-1 vectorConstructaDeletion constructs were named according to the encoded aa residues of the hMI-ER1α or -β proteinForward primerReverse primerPGL3(-1316)5′-GACTGTCTGTAGACTCTTTTCC-3′5′-CGTACTGCCGGGTCACATCTCC-3′PGL3(-945)5′-TGAAGATTAGGAAAAAAATCCCAGTC-3′5′-CGTACTGCCGGGTCACATCTCC-3′PGL3(-469)5′-GATATAGAATTTTACATTTCCTGTCG-3′5′-CGTACTGCCGGGTCACATCTCC-3′PGL3(-312)5′-ACGTATTTTTCCTCTGCTGTGTCA-3′5′-CGTACTGCCGGGTCACATCTCC-3′PGL3(-68)5′-TTTCCCTCCAGTCCAGCCCAGCCG-3′5′-CGTACTGCCGGGTCACATCTCC-3′PGL3(+28)5′-AGTGGCGGCGGGAGCGGCAGAGA-3′5′-CGTACTGCCGGGTCACATCTCC-3′hmi-er1αaa 1-4335′-CACCATGGCGACATCTGTTGAATC-3′5′-CGGGATCCAAAACAAGACCACAGAAGC-3′hmi-er1βaa 1-5125′-CACCATGGCGACATCTGTTGAATC-3′5′-CGGGATCCTTAGTCATCTGTGTTTTCAAG-3′aa 1-2835′-CACCATGGCGACATCTGTTGAATC-3′5′-ATCCTCTCTAGCTGCTTTTACA-3′aa 287-4335′-CACCATGGTTTGGACAGAGGAAGAGTGTA-3′5′-CGGGATCCAAAACAAGACCACAGAAGC-3′aa 325-4335′-CACCATGGCATTCTATTACATGTGGAAAAAATCT-3′5′-CGGGATCCAAAACAAGACCACAGAAGC-3′aa 287-4105′-CACCATGGTTTGGACAGAGGAAGAGTGTA-3′5′-CTGGTCCATTAGATGACACTCCA-3′aa 287-3575′-CACCATGGTTTGGACAGAGGAAGAGTGTA-3′5′-CCGTTACACCAGGATGAAGATT-3′aa 287-3295′-CACCATGGTTTGGACAGAGGAAGAGTGTA-3′5′-CCACATGTAATAGAATGCTACA-3′aa 287-5125′-CACCATGGTTTGGACAGAGGAAGAGTGTA-3′5′-CGGGATCCTTAGTCATCTGTGTTTTCAAG-3′a Deletion constructs were named according to the encoded aa residues of the hMI-ER1α or -β protein Open table in a new tab The pGL3(–133) construct was generated by SmaI digestion of pGL3(–1316) followed by religation; the pGL3(–657) construct was generated by SmaI/BglII digestion of pCR(–1316) and subcloning into the SmaI/BamHI sites of pGL3-Basic. The other deletion mutants were constructed by PCR, using the primer pairs listed in Table I; the PCR products were cloned into pCR2.1 and then subcloned into pGL3-Basic, using either the XhoI/HindIII sites (pGL3(–469), pGL3(–312), and pGL3(+28)) or the KpnI/XhoI sites (pGL3(–945) and pGL3(–68)). Myc-tagged plasmids (CS3+MT) containing either full-length hmi-er1α (pMyc-α) or β (pMyc-β) have been previously described (2Ding Z. Gillespie L.L. Paterno G.D. Mol. Cell. Biol. 2003; 23: 250-258Crossref PubMed Scopus (53) Google Scholar). Human Sp1 cDNA was obtained from Dr. Robert Tjian (University of California, Berkeley) and subcloned into pCR3.1, using 5′-caagatcactccatggatgaaatgacag-3′ and 5′-tgcctgatctcagaagccattgcca-3′ as forward and reverse primers, respectively. To obtain GST-hmi-er1α or β fusion constructs, cDNA representing the appropriate isoform was subcloned in-frame into the pGEX-4T-1 vector (Amersham Biosciences). A series of hMI-ER1 deletion mutations was generated by first amplifying fragments encoding the appropriate amino acid residues of hMI-ER1α or -β, using the primer pairs listed in Table I. PCR products were cloned into pCR3.1, and EcoRI fragments were then inserted into the complementary sites of the pGEX-4T-1 plasmid. Deletion constructs were named according to the encoded amino acid residues of the hMI-ER1α or -β proteins. The GST-Sp1 fusion was constructed by subcloning an Sp1 EcoRI fragment from Sp1-pCR3.1 in-frame into pGEX-4T-2. Computer Analysis of the hmi-er1 Promoter Region—Computer-assisted analysis was performed using the following programs: 1) for promoter prediction: Promoter Scan (PROSCAN) available on the World Wide Web at bimas.dcrt.nih.gov/molbio/proscan/ and Neural Network PromoterPrediction (NNPP) available at www.fruitfly.org/seq_tools/promoter.html; 2) for CpG islands: Webgene available on the World Wide Web at www.itba.mi.cnr.it/webgene; 3) for transcription factor binding sites: Transcription Factor Binding Site (TFSEARCH) available on the World Wide Web at www.cbrc.jp/research/db/TFSEARCHJ.html, Transcription Element Search System (TESS) at www.cbil.upenn.edu/tess/, and PROSCAN. Transfection and Reporter Assays—All transfections and TSA treatments were performed as previously described (2Ding Z. Gillespie L.L. Paterno G.D. Mol. Cell. Biol. 2003; 23: 250-258Crossref PubMed Scopus (53) Google Scholar), in duplicate in 6-well plates, using the indicated amount of plasmid DNA, and cells were harvested after 48 h in culture. Luciferase assays were performed on cell lysates using a Monolight 2010 Luminometer (Analytical Luminescence Laboratory) and a luciferase assay reagent (Promega), according to the manufacturer's directions. The values obtained, in relative luciferase units (RLU), were normalized to the amount of cellular protein in each sample and plotted either as a -fold increase over that obtained with the control vector or as a percentage of control activity. Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays (EMSAs) were performed as in (13Kinzler K.W. Vogelstein B. Mol. Cell. Biol. 1990; 10: 634-642Crossref PubMed Scopus (420) Google Scholar). Briefly, the double-stranded consensus Sp1 oligonucleotides (Promega) or the minimal functional promoter fragment (–68 to +144) was labeled with [λ-32P]ATP and T4 polynucleotide kinase and purified on NucTrap probe purification columns (Stratagene, Inc.). The labeled double-stranded oligonucleotides were incubated with 2 μl of HeLa nuclear extract (Promega) or GST fusion protein at room temperature for 20 min in 20 μl of reaction buffer containing 5% glycerol, 5 mm MgCl2, 1 mm dithiothreitol, 50 mm KCl, 10 μm ZnSO4, 85 μg/ml bovine serum albumin, and 50 mm HEPES (pH 7.5). Poly(dI-dC) was used as a heterologous competitor in the reaction (2 μg/reaction). Where indicated, a 20-fold molar excess of unlabeled probe was included in the binding reaction. For antibody supershift assays, the extract was incubated for 30 min at room temperature with Sp1-specific antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Bound and free probes were resolved by nondenaturing electrophoresis on 4% polyacrylamide gels and analyzed by autoradiography. GST Fusion Protein Production—GST fusion proteins were expressed in Escherichia coli BL21 and purified according to the instructions supplied with the pGEX-4T-1 and pGEX-4T-2 vectors. GST fusion protein level and purity were determined by SDS-PAGE. Co-immunoprecipitation (Co-IP), GST Pull-down Assays and Western Blot Analysis—Coupled transcription-translation (TNT; Promega) reactions and in vitro co-IP assays were performed as in Ding et al. (2Ding Z. Gillespie L.L. Paterno G.D. Mol. Cell. Biol. 2003; 23: 250-258Crossref PubMed Scopus (53) Google Scholar); the antibody used was either an anti-Sp1 antibody (Santa Cruz Biotechnology) or the anti-Myc monoclonal antibody, 9E10 (a kind gift from Dr. K. Kao, Memorial University). Pull-down assays with GST fusion proteins were performed as described in Ref. 14Routledge E.J. White R. Parker M.G. Sumpter J.P. J. Biol. Chem. 2000; 275: 35986-35993Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, using 1 μg of GST fusion protein, 50 μl of glutathione-Sepharose beads (Amersham Biosciences) and 50,000 cpm of 35S-labeled TNT product. Bound proteins were analyzed by SDS-PAGE and autoradiography. For all assays, one-twentieth volume of the indicated TNT was loaded into the input lanes. For the in vivo co-IP assays, either nontransfected HeLa cells or cells transfected with pMyc, pMyc-α, or pMyc-β were used. Immunoprecipitation using anti-Sp1 or anti-pan-hMI-ER1 (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar) antibodies and Western blot analysis using anti-Myc or anti-Sp1 were performed as described (2Ding Z. Gillespie L.L. Paterno G.D. Mol. Cell. Biol. 2003; 23: 250-258Crossref PubMed Scopus (53) Google Scholar). Input lanes contained one-third of the volume used for immunoprecipitation. Establishment of Stable hMI-ER1α and -β Tet-On HeLa Cell Lines and Doxycycline Induction—hMI-ER1α and β Tet-On HeLa cell lines were generated using the hmi-er1α or -β coding region inserted into the pTRE2 vector and a Tet-On gene expression system (Clontech), according to the manufacturer's protocol. Control cell lines were generated by transfection with the pTRE2 empty vector. Stable clones were induced to express hMI-ER1α or -β using 2 μg/ml doxycycline (dox), and expression was verified by Western blot analysis of whole cell extracts, using an anti-hMI-ER1 antibody (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar). Quantitative Real Time PCR and Semiquantitative PCR Analysis— RNA was extracted from uninduced and dox-induced Tet-On cell lines and reverse transcribed as in Ref. 1Paterno G.D. Li Y. Luchman H.A. Ryan P.J. Gillespie L.L. J. Biol. Chem. 1997; 272: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar. Quantitative real time PCR was performed using the Syber Green PCR Master Mix and the ABI Prism 7000 SDS (Applied Biosystems), according to the manufacturer's protocol. The 5′-UTR of hmi-er1 was amplified using 5′-AGTGGCGGCGGGAGCGGCAGAGA-3′ (forward) and 5′-CGTACTGCCGGGTCACATCTCC-3′ (reverse), whereas β-actin was amplified using 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ (F) and 5′-ATGGCTGGGGTGTTGAAGGTCTC-3′ (R). Data analysis and the ratio of expression in induced cells relative to uninduced cells (relative expression ratio) was calculated as described in Ref. 15Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25871) Google Scholar. Semiquantitative PCR was performed as in Ref. 4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar using the primers listed above to amplify β-actin and the 5′-UTR of hmi-er1, as well as 5′-CAAGGGCTGAAGGCCTATGG-3′ (forward) and 5′-CCAAATCGTGTTTGCTGAGC-3′ (reverse) to amplify the coding region of hmi-er1. Chromatin Immunoprecipitation Assays—Chromatin from HeLa cells or dox-induced Tet-On cell lines was cross-linked with formaldehyde, sheared, and then subjected to immunoprecipitation as described by Weinmann et al. (16Weinmann A.S. Mitchell D.M. Sanjabi S. Bradley M.N. Hoffmann A. Liou H.C. Smale S.T. Nat. Immunol. 2001; 2: 51-57Crossref PubMed Scopus (146) Google Scholar), with the following modifications. Anti-Sp1 (Santa Cruz) or anti-pan-hMI-ER1 (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar) antibodies were used for immunoprecipitation, and 50 μl of protein A-Sepharose (Amersham Biosciences) was used instead of Staph A cells. Immunoprecipitated chromatin was eluted, treated with RNase A, and then treated with proteinase K as in Ref. 16Weinmann A.S. Mitchell D.M. Sanjabi S. Bradley M.N. Hoffmann A. Liou H.C. Smale S.T. Nat. Immunol. 2001; 2: 51-57Crossref PubMed Scopus (146) Google Scholar. PCR was performed as in Ref. 1Paterno G.D. Li Y. Luchman H.A. Ryan P.J. Gillespie L.L. J. Biol. Chem. 1997; 272: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar using 5 μl of resuspended DNA and 30 cycles, with 5′-TTTCCTCTGCTGTGTCAATGC-3′ and 5′-GAGATGTGACCCGGCAGTAC-3′ as forward and reverse primers, respectively. For experiments using 3,3′-dithiobispropionimidate-2HCl (DTBP), cells were treated for 30 min with 5 mm DTBP, as described in Ref. 17Fujita N. Jaye D.L. Kajita M. Geigerman C. Moreno C.S. Wade P.A. Cell. 2003; 113: 207-219Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, prior to cross-linking with formaldehyde. Cloning of hmi-er1 5′ Regulatory Sequence and Identification of the Minimal Functional Promoter—A genomic DNA fragment encompassing the promoter region, P2 (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar), and containing 1460 bp upstream of the ATG translational start codon of hmi-er1 was isolated by PCR from human genomic DNA. This sequence contains 144 bp of 5′-UTR (+1 to +144) and 1316 bp (–1 to –1316) upstream of the start of transcription (4Paterno G.D. Ding Z. Lew Y.-Y. Nash G.N. Mercer F.C. Gillespie L.L. Gene (Amst.). 2002; 295: 79-88Crossref PubMed Scopus (15) Google Scholar). Computer-assisted analysis of the 1460-bp fragment to identify potential cis-regulatory elements predicted a CpG island located between nucleotide position –389 and the start of translation (Fig. 1A). Further analysis revealed that, like many other GC-rich promoters, the upstream sequence does not contain a TATA box; nor does it contain an initiator (18Smale S.T. Baltimore D. Cell. 1989; 57: 103-113Abstract Full Text PDF PubMed Scopus (1151) Google Scholar) or a downstream promoter element (19Burke T.W. Kadonaga J.T. Genes Dev. 1996; 10: 711-724Crossref PubMed Scopus (329) Google Scholar). A number of potential transcription factor binding sites, including multiple Sp1 binding sites, were identified using TFSEARCH, PROSCAN, and TESS (Fig. 1A). The 1460-bp fragment was subcloned into the promoterless luciferase reporter vector, pGL3-Basic, in sense and antisense orientations, and its promoter activity was analyzed in HeLa cervical carcinoma cells. As shown in Fig. 1B, luciferase activity was 8–10-fold higher when expression was driven by the 1460-bp fragment, compared with the pGL3-Basic vector and was markedly lower when the 1460-bp fragment was in the antisense orientation. We tested the cellular specificity of the hmi-er1 promoter in six different cell lines: C33A, HEK293, BT-20, MCF-7, SK-OV-3, and U87. Luciferase activity was high in all transfected cell lines and ranged from 10- to 34-fold higher than control levels, demonstrating that the hmi-er1 promoter can function in a variety of cell types (data not shown). To identify the minimal functional region of the hmi-er1 promoter, a series of 5′ deletions was constructed in pGL3 and transiently transfected into HeLa cells. Deletion of nucleotides –1316 to –133 resulted in little change in luciferase activity (Fig. 1B). Further deletion to nucleotide –68 reduced but did not abolish luciferase activity, whereas deletion to nucleotide +28 completely abolished activity (Fig. 1B). These data demonstrate that the minimal functional promoter is located within the sequence –68/+144. The hmi-er1 minimal promoter is predicted to contain four Sp1 binding sites (Fig. 1A); therefore, we investigated whether Sp1 binds to this sequence. EMSAs were performed using a HeLa cell nuclear extract and a probe consisting of the minimal promoter sequence (–68/+144). Two bands representing DNA-protein complexes appeared in samples containing nuclear extract (Fig. 1C, lane 2); however, only the larger complex was specific, as revealed by competition with excess unlabeled probe (Fig. 1C, lane 3). Supershift assays, using an Sp1 antibody that does not cross-react with Sp2, Sp3, or Sp4, resulted in the appearance of a higher mobility band that disappeared in the presence of excess unlabeled probe (Fig. 1C, lanes 4 and 5), thus confirming the presence of Sp1 in the DNA-protein complexes. hMI-ER1α and -β Repress Activation of the Minimal Promoter by Sp1—The ability of Sp1 to regulate transcription from the hmi-er1 promoter was investigated in vivo using luciferase reporter assays. HeLa cells were co-transfected with a luciferase reporter construct containing the minimal promoter sequence –68/+144 (pGL3(–68)), along with an Sp1 expression vector (pCR-Sp1) or control empty vector (pCR). Co-transfection with Sp1 resulted in a dose-dependent increase in luciferase activity to 4-fold that of control (Fig. 2A), demonstrating that Sp1 can activate transcription from the hmi-er1 minimal promoter, either directly or indirectly. We have shown previously that hMI-ER1 functions as a HDAC-dependent transcriptional repressor (2Ding Z. Gillespie L.L. Paterno G.D. Mol. Cell. Biol. 2003; 23: 250-258Crossref PubMed Scopus (53) Google Scholar); therefore, we investigated whether hMI-ER1 could regulate transcription from its own promoter. HeLa cells were co-transfected with pGL3(–68) along with a plasmid expressing Myc tag alone (pMyc) or fused to hMI-ERα (pMyc-α) or hMI-ER1β (pMyc-β). Myc-α and Myc-β repressed the activity of the hmi-er1 minimal promoter to 40 and 33% of control, respectively (Fig. 2B). The HDAC dependence of this repression was determined by assaying in the presence of TSA. Fig. 2C revealed that repression was not relieved by TSA, demonstrating that hMI-ER1 represses its own promoter by a HDAC-independent mechanism. An obvious question is whether hMI-ER1α and -β can affect transcriptional activation by Sp1. To investigate this, HeLa cells were co-transfected with pGL3(–68), pCR-Sp1, and increasing concentrations of pMyc, pMyc-α, or pMyc-β. Promoter activation by Sp1 was repressed in a dose-dependent manner by both hMI-ER1α and -β (Fig. 3), and repression resulted in a 50% reduction in luciferase activity at the highest hMI-ER1 levels. This repression was not due to a down-regulation of Sp1 protein, which remained constant at all hMI-ER1 levels (Fig. 3). We have confirmed that this repression also occurs at the level of the endogenous hmi-er1 gene, using dox-inducible hMI-ER1 HeLa cell lines (Fig. 4A). We treated hMI-ER1α-expressing (HTα222), hMI-ER1β-expressing (HTβ53), and control (HTC314) cell lines with 2 μg/ml dox to induce hMI-ER1 expression and then extracted RNA for real time RT-PCR and for semiquantitative RT-PCR analysis. We compared changes in the steady state levels of endogenous hmi-er1 mRNA using primers in the noncoding region of hmi-er1 that are absent in the transfected construct. Fig. 4B shows that dox-induced expression of hMI-ER1 reduced steady state levels of endogenous hmi-er1 mRNA by 50% in HTα222 cells and 42% in HTβ53 cells, relative to the expression level in HTC314 cells. This level of repression is consistent with the level of repression by hMI-ER1 observed in transient transfection approaches reported in Fig. 3A. A similar reduction in the expression level of endogenous hmi-er1 mRNA was observed using semiquantitative PCR (Fig. 4C). As expected, amplification using primers in the coding region revealed an increase in the total hmi-er1 mRNA expression level, due to dox induction of the transfected hmi-er1 cDNA, whereas no discernable effect of the steady state levels of endogenous β-actin was observed (Fig. 4C). hMI-ER1 Is Associated with the Chromatin of Its Own Promoter and Interferes with Sp1 Binding—Although we have demonstrated that Sp1 can bind to recognition sequences in the hmi-er1 promoter, it is important to demonstrate that endogenous Sp1 can associate with the hmi-er1 promoter in vivo. Therefore, we performed ChIP assays using HeLa cells to investigate this possibility. Formaldehyde-cross-linked, sheared chromatin" @default.
• W2053606985 created "2016-06-24" @default.
• W2053606985 creator A5047654654 @default.
• W2053606985 creator A5055024074 @default.
• W2053606985 creator A5068701513 @default.
• W2053606985 creator A5079372704 @default.
• W2053606985 date "2004-07-01" @default.
• W2053606985 modified "2023-09-29" @default.
• W2053606985 title "The SANT Domain of Human MI-ER1 Interacts with Sp1 to Interfere with GC Box Recognition and Repress Transcription from Its Own Promoter" @default.
• W2053606985 cites W1501286679 @default.
• W2053606985 cites W1579961340 @default.
• W2053606985 cites W1753047181 @default.
• W2053606985 cites W1945773609 @default.
• W2053606985 cites W1974545855 @default.
• W2053606985 cites W1979028385 @default.
• W2053606985 cites W1991293300 @default.
• W2053606985 cites W1993063708 @default.
• W2053606985 cites W2011263116 @default.
• W2053606985 cites W2037954261 @default.
• W2053606985 cites W2042226550 @default.
• W2053606985 cites W2047553717 @default.
• W2053606985 cites W2049862742 @default.
• W2053606985 cites W2055413599 @default.
• W2053606985 cites W2065319970 @default.
• W2053606985 cites W2066664309 @default.
• W2053606985 cites W2068663793 @default.
• W2053606985 cites W2069169033 @default.
• W2053606985 cites W2074351361 @default.
• W2053606985 cites W2075654456 @default.
• W2053606985 cites W2079891060 @default.
• W2053606985 cites W2089306515 @default.
• W2053606985 cites W2094105923 @default.
• W2053606985 cites W2096130703 @default.
• W2053606985 cites W2104187492 @default.
• W2053606985 cites W2108244474 @default.
• W2053606985 cites W2110328765 @default.
• W2053606985 cites W2112425896 @default.
• W2053606985 cites W2113210499 @default.
• W2053606985 cites W2118650392 @default.
• W2053606985 cites W2142459043 @default.
• W2053606985 cites W2148896458 @default.
• W2053606985 cites W2149462291 @default.
• W2053606985 cites W2156058796 @default.
• W2053606985 cites W2171027260 @default.
• W2053606985 cites W2293440579 @default.
• W2053606985 doi "https://doi.org/10.1074/jbc.m403793200" @default.
• W2053606985 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15117948" @default.
• W2053606985 hasPublicationYear "2004" @default.
• W2053606985 type Work @default.
• W2053606985 sameAs 2053606985 @default.
• W2053606985 citedByCount "37" @default.
• W2053606985 countsByYear W20536069852012 @default.
• W2053606985 countsByYear W20536069852013 @default.
• W2053606985 countsByYear W20536069852015 @default.
• W2053606985 countsByYear W20536069852016 @default.
• W2053606985 countsByYear W20536069852017 @default.
• W2053606985 countsByYear W20536069852019 @default.
• W2053606985 countsByYear W20536069852020 @default.
• W2053606985 countsByYear W20536069852021 @default.
• W2053606985 countsByYear W20536069852022 @default.
• W2053606985 crossrefType "journal-article" @default.
• W2053606985 hasAuthorship W2053606985A5047654654 @default.
• W2053606985 hasAuthorship W2053606985A5055024074 @default.
• W2053606985 hasAuthorship W2053606985A5068701513 @default.
• W2053606985 hasAuthorship W2053606985A5079372704 @default.
• W2053606985 hasBestOaLocation W20536069851 @default.
• W2053606985 hasConcept C101762097 @default.
• W2053606985 hasConcept C104317684 @default.
• W2053606985 hasConcept C134306372 @default.
• W2053606985 hasConcept C138885662 @default.
• W2053606985 hasConcept C150194340 @default.
• W2053606985 hasConcept C153911025 @default.
• W2053606985 hasConcept C179716448 @default.
• W2053606985 hasConcept C179926584 @default.
• W2053606985 hasConcept C33923547 @default.
• W2053606985 hasConcept C36503486 @default.
• W2053606985 hasConcept C41895202 @default.
• W2053606985 hasConcept C54355233 @default.
• W2053606985 hasConcept C57467964 @default.
• W2053606985 hasConcept C86339819 @default.
• W2053606985 hasConcept C86803240 @default.
• W2053606985 hasConcept C95444343 @default.
• W2053606985 hasConceptScore W2053606985C101762097 @default.
• W2053606985 hasConceptScore W2053606985C104317684 @default.
• W2053606985 hasConceptScore W2053606985C134306372 @default.
• W2053606985 hasConceptScore W2053606985C138885662 @default.
• W2053606985 hasConceptScore W2053606985C150194340 @default.
• W2053606985 hasConceptScore W2053606985C153911025 @default.
• W2053606985 hasConceptScore W2053606985C179716448 @default.
• W2053606985 hasConceptScore W2053606985C179926584 @default.
• W2053606985 hasConceptScore W2053606985C33923547 @default.
• W2053606985 hasConceptScore W2053606985C36503486 @default.
• W2053606985 hasConceptScore W2053606985C41895202 @default.
• W2053606985 hasConceptScore W2053606985C54355233 @default.
• W2053606985 hasConceptScore W2053606985C57467964 @default.
• W2053606985 hasConceptScore W2053606985C86339819 @default.
• W2053606985 hasConceptScore W2053606985C86803240 @default.
• W2053606985 hasConceptScore W2053606985C95444343 @default.

• next