Glucose Regulates Insulin Gene Transcription by Hyperacetylation of Histone H4
2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês
10.1074/jbc.m212375200
ISSN1083-351X
AutoresAmber L. Mosley, Sabire Özcan,
Tópico(s)Epigenetics and DNA Methylation
ResumoInduction of insulin gene expression in response to high blood glucose levels is essential for maintaining glucose homeostasis. Although several transcription factors including Beta-2, Ribe3b1, and Pdx-1 have been shown to play a role in glucose stimulation of insulin gene expression, the exact molecular mechanism(s) by which this regulation occurs is unknown. Previous data demonstrate that the transcription factors Beta-2/NeuroD1 and Pdx-1, which are involved in glucose-stimulated insulin gene expression, interact with the histone acetylase p300, suggesting a role for histone acetylation in glucose regulation of the insulin gene expression. We report that exposure of mouse insulinoma 6 cells to high concentrations of glucose results in hyperacetylation of histone H4 at the insulin gene promoter, which correlates with the increased level of insulin gene transcription. In addition, we demonstrate that hyperacetylation of histone H4 in response to high concentrations of glucose also occurs at the glucose transporter-2 gene promoter. Using histone deacetylase inhibitors, we show that increases in histone H4 acetylation cause stimulation of insulin gene transcription even in the absence of high concentrations of glucose. Furthermore, we show that fibroblasts, which lack insulin gene expression, also lack histone acetylation at the insulin gene promoter. In summary, our data support the idea that high concentrations of glucose stimulate insulin gene expression by causing hyperacetylation of histone H4 at the insulin gene promoter. Induction of insulin gene expression in response to high blood glucose levels is essential for maintaining glucose homeostasis. Although several transcription factors including Beta-2, Ribe3b1, and Pdx-1 have been shown to play a role in glucose stimulation of insulin gene expression, the exact molecular mechanism(s) by which this regulation occurs is unknown. Previous data demonstrate that the transcription factors Beta-2/NeuroD1 and Pdx-1, which are involved in glucose-stimulated insulin gene expression, interact with the histone acetylase p300, suggesting a role for histone acetylation in glucose regulation of the insulin gene expression. We report that exposure of mouse insulinoma 6 cells to high concentrations of glucose results in hyperacetylation of histone H4 at the insulin gene promoter, which correlates with the increased level of insulin gene transcription. In addition, we demonstrate that hyperacetylation of histone H4 in response to high concentrations of glucose also occurs at the glucose transporter-2 gene promoter. Using histone deacetylase inhibitors, we show that increases in histone H4 acetylation cause stimulation of insulin gene transcription even in the absence of high concentrations of glucose. Furthermore, we show that fibroblasts, which lack insulin gene expression, also lack histone acetylation at the insulin gene promoter. In summary, our data support the idea that high concentrations of glucose stimulate insulin gene expression by causing hyperacetylation of histone H4 at the insulin gene promoter. Type II diabetes is a multifactorial disease caused by a combination of defects in insulin production, insulin secretion, and insulin action. To maintain glucose homeostasis, it is imperative that insulin transcription, translation, and secretion are up-regulated in the β cells of the pancreas in response to high blood glucose levels (1LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus: A Fundamental and Clinical Text. 2nd Ed. Lippincott Williams and Wilkins, Philadelphia, Baltimore, New York, London, Buenos Aires, Hong Kong, Sydney, Tokyo2000Google Scholar). The pancreatic β cells respond to high blood glucose levels first by secreting insulin from the secretory granules followed by up-regulation of insulin gene transcription and translation as a more long term response (1LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus: A Fundamental and Clinical Text. 2nd Ed. Lippincott Williams and Wilkins, Philadelphia, Baltimore, New York, London, Buenos Aires, Hong Kong, Sydney, Tokyo2000Google Scholar). A number of other proteins have also been shown to be required for the glucose responsiveness of pancreatic β cells, including glucokinase and glucose transporter-2 (GLUT-2) 1The abbreviations used are: GLUT-2, glucose transporter-2; Ac, acetyl; TSA, trichostatin A; MIN6, mouse insulinoma 6; IP, immunoprecipitation; ChIP, chromatin IP; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcriptase; NHF1α, hepatocyte nuclear factor-1alpha. (2Newgard C.B. McGarry J.D. Ann. Rev. Biochem. 1995; 64: 689-719Crossref PubMed Scopus (496) Google Scholar, 3Unger R.H. Science. 1991; 251: 1200-1205Crossref PubMed Scopus (253) Google Scholar). The expression of GLUT-2 has been shown to be up-regulated by glucose in pancreatic β cells (4Waeber G. Thompson N. Haefliger J.A. Nicod P. J. Biol. Chem. 1994; 269: 26912-26919Abstract Full Text PDF PubMed Google Scholar, 5Bonny C. Roduit R. Gremlich S. Nicod P. Thorens B. Waeber G. Mol. Cell. 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Commun. 2001; 287: 229-235Crossref PubMed Scopus (24) Google Scholar), the exact mechanism(s) by which they stimulate insulin gene expression in response to high blood glucose levels are unknown. Transcriptional regulation of eukaryotic genes is a very complex process that requires the cooperation of a number of transcription factors, as well as various co-activator and co-repressor proteins, which modulate histone structure (28Berger S.L. Curr. Opin. Genet. Dev. 2002; 12: 142-148Crossref PubMed Scopus (991) Google Scholar, 29Cheung P. Allis C.D. Sassone-Corsi P. Cell. 2000; 103: 263-271Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar). Changes in histone modification have been shown to increase or decrease the accessibility of promoters to the transcription machinery, thereby leading to repression or activation of gene expression (28Berger S.L. Curr. Opin. Genet. Dev. 2002; 12: 142-148Crossref PubMed Scopus (991) Google Scholar, 29Cheung P. Allis C.D. 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Previous data indicate that two of the insulin gene transcription factors required for glucose-regulated expression, Beta-2/NeuroD1 and Pdx-1, interact with the histone acetylase p300 (17Sharma A. Moore M. Marcora E. Lee J.E. Qiu Y. Samaras S. Stein R. Mol. Cell. Biol. 1999; 19: 704-713Crossref PubMed Scopus (82) Google Scholar, 26Qiu Y. Guo M. Huang S. Stein R. Mol. Cell. Biol. 2002; 22: 412-420Crossref PubMed Scopus (156) Google Scholar, 40Qiu Y. Sharma A. Stein R. Mol. Cell. Biol. 1998; 18: 2957-2964Crossref PubMed Scopus (123) Google Scholar). This prompted us to investigate whether changes in histone acetylation levels play a role in regulation of insulin gene expression by glucose. We report that high concentrations of glucose stimulate insulin gene transcription by mediating hyperacetylation of histone H4 at the insulin gene promoter in the insulinoma cell line MIN6. Cell Culture—MIN6 cells of passage 20 to 24 were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 25 mm glucose, 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin, 2 mm glutamine, and 100 μm β-mercaptoethanol (41Miyazaki J. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K. Endocrinology. 1990; 127: 126-132Crossref PubMed Scopus (1060) Google Scholar). All experiments were carried out with MIN6 cells of passage less than 30. NIH-3T3 fibroblasts (ATCC) were maintained in DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin. For glucose regulation experiments, cells were washed three times with 1× phosphate-buffered saline and grown overnight, unless otherwise indicated, in DMEM without fetal bovine serum containing the indicated glucose concentration(s). RNA Isolation and RT-PCR—poly(A) RNA from total RNA was isolated using the GenElute Direct mRNA Miniprep kit (Sigma) according to the manufacturer's instructions. After treatment with DNaseI (Sigma), the poly(A) RNA was reverse-transcribed using enhanced avian myeloblastosis virus reverse transcriptase (Sigma). The resulting cDNAs were used as template for PCR with oligonucleotides to amplify the insulin and β-actin genes (42Roderigo-Milne H. Hauge-Evans A.C. Persaud S.J. Jones P.M. Biochem. Biophys. Res. Commun. 2002; 296: 589-595Crossref PubMed Scopus (37) Google Scholar). The oligonucleotide primers used are listed in Table I. The primers for the β-actin gene were designed to cross an intron so that contamination with genomic DNA can be detected, which would result in a PCR product of 330 bp versus 243 bp from the cDNA (43Rout U.K. Armant D.R. Reprod. Toxicol. 2002; 16: 253-258Crossref PubMed Scopus (15) Google Scholar). PCR reactions (20-μl volume) contained 20 ng of cDNA, 300 μm dNTPs, 2.5 pmol of appropriate oligonucleotide primers, and 1.5 units of JumpStart AccuTaq LA DNA polymerase (Sigma). PCR amplification conditions were as follows: 5 min at 95 °C followed by 25 cycles of 95 °C for 30 s, 58 °C for 1 min, and 72 °C for 30 s. The PCR products were separated on 8% non-denaturing polyacrylamide gels and stained with ethidium bromide (Sigma). The bands were visualized using a ChemiDoc System BioRad Imager (Bio-Rad) and quantified using Quantity One imaging software (Bio-Rad) as a function of both band size and band intensity (intensity/mm2).Table IList of sequences of oligonucleotide primers used in this studyGeneAmplified regionaWhere indicated the numbers represent the position of the amplified fragment relative to the known transcriptional start site for the gene.ReferencesOligonucleotids sequenceMouse insulin I gene42Roderigo-Milne H. Hauge-Evans A.C. Persaud S.J. Jones P.M. Biochem. Biophys. Res. Commun. 2002; 296: 589-595Crossref PubMed Scopus (37) Google Scholar5′-CCTGTTGGTGCACTTCCTAC-3′5′-TGCAGTAGTTCTCCAGCTGG-3′β-Actin gene43Rout U.K. Armant D.R. Reprod. Toxicol. 2002; 16: 253-258Crossref PubMed Scopus (15) Google Scholar5′-CGTGGGCCGCCCTAGGCAACC-3′5′-TTGGCCTTAGGGTTCAGGGGGG-3′Mouse insulin I promoter-10 to -28145Steiner D.F. Chan S.J. Welsh J.M. Kwok S.C. Annu. Rev. Genet. 1985; 19: 463-484Crossref PubMed Scopus (197) Google Scholar5′-GAAGGTCTCACCTTCTGG-3′5′-GGGGGTTACTGGATGCC-3′cad promoter-105 to +25431Eberhardy S.R. D'Cunha C.A. Farnham P.J. J. Biol. Chem. 2000; 275: 33798-33805Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 46Boyd K.E. Wells J. Gutman J. Bartley S.M. Farnham P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13887-13892Crossref PubMed Scopus (246) Google Scholar5′-TGACTAGCGGTACCGGGGTTGCTGCTGTGGAACC-3′5′-CGGGCTTGCTTACCCACCTTCCCCAGCAGTCGACAC-3′GLUT-2 promoter-523 to -73847Chakrabarti S.K. James J.C. Mirmira R.G. J. Biol. Chem. 2002; 277: 13286-13293Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar5′-ATCTGGCTCCGCACTCTCATTCTTG-3′5′-CCCTGTGACTTTTCTGTGTTCTTAGG-3′a Where indicated the numbers represent the position of the amplified fragment relative to the known transcriptional start site for the gene. Open table in a new tab Chromatin Immunoprecipitation (ChIP)—Chromatin isolation was performed as published previously (31Eberhardy S.R. D'Cunha C.A. Farnham P.J. J. Biol. Chem. 2000; 275: 33798-33805Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 44Wells J. Farnham P.J. Methods. 2002; 26: 48-56Crossref PubMed Scopus (208) Google Scholar). Approximately 3 × 107 MIN6 or NIH-3T3 cells were cross-linked with formaldehyde (1% final concentration). After lysis of the cells, the nuclear extracts were sonicated with glass beads (0.1 g) for five 10-s pulses at 60% power using a Tekmar Sonic Disruptor. One-third of the sample was used for immunoprecipitation with acetyl-histone H3 (K9, K14) or acetyl-histone H4 (K5, K8, K12, K16) antibodies (Upstate Biotechnology, Inc.). The samples were pre-cleared with 20 μl of blocked Pansorbin Staph A cells (Calbiochem). After 4-fold dilution of the samples in IP buffer (1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.0, 150 mm NaCl) and incubation with 2 μg of specific antibodies or rabbit IgG (Sigma) overnight at 4 °C, the immunocomplexes were recovered by incubation with blocked Staph A cells. After washing twice in dialysis buffer (2 mm EDTA, 50 mm Tris-Cl, pH 8.0, and 0.2% Sarkosyl) and four times with IP wash buffer (1% Nonidet P-40, 100 mm Tris-HCl, pH 8.0, 500 mm LiCl, and 1% deoxycholic acid), the immunocomplexes were eluted twice from the Staph A cells (with 150 μl of 1% SDS in 50 mm NaHCO3). The cross-links were reversed by adding 20 μl of 5 m NaCl and 1 μl of 10 mg/ml RNase A and by incubating at 65 °C for 8 h. After treating with 1.5 μl of proteinase K (10 μg/μl) the samples were extracted with phenol/chloroform and subsequently ethanol-precipitated using 20 μg of glycogen as a carrier. PCR Analysis of Immunoprecipitated DNA—All PCR reactions were performed on a Robocycler Gradient 96 (Stratagene) in a 20-μl reaction volume containing 50 mm KCl, 10 mm Tris-HCl, pH 8.3, 1.5 mm MgCl, 200 μm dNTPs, and 2 μl of primers (2.5 pmol/μl). The linear range for each primer pair was determined empirically, using different amounts of MIN6 and NIH-3T3 genomic DNA. The PCR reactions and the quantification of the obtained bands were carried out as described above. The PCR products obtained with the immunoprecipitated DNA were normalized to the products obtained with the total input DNA. The primers used for PCR are listed in Table I. A detailed PCR protocol is available upon request. All of the PCR products obtained had the expected size. The identity of the PCR products was confirmed by sequencing. Statistical Analysis—Comparison of the histone acetylation or insulin mRNA levels from MIN6 cells grown on 3 or 30 mm glucose were performed using the two-tailed, unpaired Student's t test. A p value less than 0.05 was considered statistically significant. Data are expressed as means ± S.D. Glucose Mediates Hyperacetylation of Histone H4 at the Insulin Gene Promoter—To test whether high concentrations of glucose mediate changes in histone acetylation at the insulin gene promoter in the insulinoma cell line MIN6, we utilized the ChIP assay with acetyl histone H3 or acetyl histone H4 antibodies. To quantify the amount of insulin gene promoter associated with acetylated histone H3 or histone H4, the total input and the immunoprecipitated DNA were used as template for PCR with primers against the mouse insulin I gene promoter (covers the promoter region from –10 to –281). The results shown in Fig. 1A indicate that exposure of MIN6 cells to high concentrations of glucose (30 mm) causes an increase in histone H4 acetylation at the insulin gene promoter. However, there is no significant change in acetyl histone H3 levels in response to high levels of glucose. MIN6 cells incubated with 30 mm glucose displayed an ∼4- to 5-fold increase in acetylated histone H4 levels compared with cells incubated with 3 mm glucose in five independent experiments (Fig. 1B). Although in this experiment the MIN6 cells were incubated overnight with low and high glucose media, we observed the increase in histone H4 acetylation even after a 2-h incubation period with 30 mm glucose (data not shown). To verify that the observed increase in histone H4 acetylation levels in response to high glucose concentrations correlated with increases in insulin gene transcription, we quantified the insulin mRNA levels in MIN6 cells grown on low or high glucose media. RT-PCR analysis performed using cDNA from low or high glucose-incubated MIN6 cells indicate a 2.5-fold increase in insulin mRNA levels in response to high glucose (30 mm) compared with the β-actin levels used as control (Fig. 1, C and D). As a control for contamination of the cDNA with genomic DNA, we employed actin primers that give an additional larger PCR product when the sample is contaminated with genomic DNA (Fig. 1, C and D). The acetyl histone H3 and histone H4 antibodies used in this study specifically recognize acetylated histones in Western blots with MIN6 cell extracts (Fig. 1E). Hyperacetylation of Histone H4 at the GLUT-2 Promoter in Response to Glucose—To test whether glucose causes increases in histone acetylation at other β cell-specific promoters, we used the same immunoprecipitated and total DNA samples as template in PCR analysis with primers against the GLUT-2 promoter (Fig. 2). In the presence of low concentrations of glucose (3 mm), the level of acetylated histone H4 associated with the GLUT-2 promoter was minimal; however, at high concentrations of glucose (30 mm) the acetylated histone H4 levels at the GLUT-2 promoter increased drastically (Fig. 2). The level of acetylated histone H3 at the GLUT-2 promoter remained the same on low and high glucose (Fig. 2). This indicates that high levels of glucose (30 mm) cause hyperacetylation of histone H4 at both insulin and GLUT-2 gene promoters in MIN6 cells. Histone H4 Acetylation at the Insulin Gene Promoter Is Not Increased at High Concentrations ofl-Glucose—Activation of insulin gene transcription is regulated by cellular stress, as well as glucose (48Macfarlane W.M. McKinnon C.M. Felton-Edkins Z.A. Cragg H. James R.F. Docherty K. J. Biol. Chem. 1999; 274: 1011-1016Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). To test that the observed increase in histone H4 acetylation is not because of secondary effects such osmotic stress caused by the high concentrations of glucose (30 mm) used in this experiment, we repeated the ChIP assay with acetyl histone H3 or histone H4 antibodies using l-glucose. Because l-glucose is not taken up by glucose transporters and thus is not metabolized, it should mimic the osmotic stress caused by high concentrations of extracellular d-glucose. The analysis of acetylated histone H3 and histone H4 levels associated with the insulin gene promoter in MIN6 cells grown on low (3 mm) or high (30 mm) l-glucose in the presence of 3 mm d-glucose indicates that there is no increase in histone H4 acetylation levels in response to high concentrations of l-glucose (Fig. 3). In summary, these data indicate that glucose causes hyperacetylation of histone H4 at the insulin gene promoter and that this effect is specific and is not caused by osmotic stress. Histone H4 Hyperacetylation at the Insulin and GLUT-2 Gene Promoters Increases in a Glucose Concentration-dependent Manner—It has been shown previously (1LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus: A Fundamental and Clinical Text. 2nd Ed. Lippincott Williams and Wilkins, Philadelphia, Baltimore, New York, London, Buenos Aires, Hong Kong, Sydney, Tokyo2000Google Scholar, 49Macfarlane W.M. Shepherd R.M. Cosgrove K.E. James R.F. Dunne M.J. Docherty K. Diabetes. 2000; 49: 418-423Crossref PubMed Scopus (61) Google Scholar) that insulin mRNA levels increase in a dose-dependent fashion in response to increasing glucose concentrations. To analyze the effects of increasing concentrations of glucose on the level of histone H4 acetylation at the insulin gene promoter, MIN6 cells were incubated in media containing 3, 5, 10, or 20 mm glucose for 3 h. Analysis of histone H3 and histone H4 acetylation levels using the ChIP assay demonstrated that histone H3 acetylation did not change significantly at the insulin and GLUT-2 gene promoters by increasing the glucose concentration (Fig. 4). However, the acetylation level of histone H4 at both promoters increased in parallel with increasing the glucose concentration (Fig. 4). As a control for this experiment we amplified the cad (carbamoyl phosphate synthase/aspartate transcarbamoylase/dihydroorotase) gene promoter using the same DNA immunoprecipitated with acetyl histone H3 or histone H4 antibodies and total DNA as template. We found that the levels of histone H3 acetylation at the cad promoter, whose expression is not glucose-regulated, did not change in response to increases in glucose concentration (Fig. 4, third panel). This experiment again confirms that the observed hyperacetylation of histone H4 at the insulin and GLUT-2 promoters is glucose-specific. The Decrease in Histone H4 Acetylation on Low Glucose Is Mediated by the Recruitment of Histone Deacetylases to the Insulin Gene Promoter—Histone deacetylases such as HDAC1 and HDAC2 have been shown to repress gene expression by decreasing the acetylation status of histones at specific promoters (28Berger S.L. Curr. Opin. Genet. Dev. 2002; 12: 142-148Crossref PubMed Scopus (991) Google Scholar). Therefore, it was possible that the decrease in histone H4 acetylation on low levels of glucose (3 mm) was because of the recruitment of histone deacetylases to the insulin gene promoter. To address this question, we carried out the ChIP assay using acetyl histone H3 or histone H4 antibodies in MIN6 cells grown on media containing low or high glucose, in the presence or absence of the histone deacetylase inhibitors trichostatin A (TSA) or sodium butyrate. The inhibition of histone deacetylases in MIN6 cells resulted in equal levels of both histone H3 and H4 acetylation at the insulin gene promoter on low and high concentrations of glucose (Fig. 5A). We obtained similar results with both inhibitors; however, the acetylation levels of both histone H3 and histone H4 were consistently lower with sodium butyrate-treated samples in three independent experiments, which is likely due to additional effects that sodium butyrate has on cultured cells (50Candido E.P. Reeves R. Davie J.R. Cell. 1978; 14: 105-113Abstract Full Text PDF PubMed Scopus (809) Google Scholar). The level of histone H4 acetylation at the insulin gene promoter on low levels of glucose was very similar to that of high concentrations of glucose. These data suggest that the reduced level of histone H4 acetylation on low concentrations of glucose is likely because of the active recruitment of deacetylases to the insulin gene promoter. To test whether the increase in histone H4 acetylation on low concentrations of glucose as observed with TSA treatment causes increased insulin gene transcription, we quantified the expression level of the insulin gene in MIN6 cells grown on media containing low or high concentrations of glucose treated with TSA by RT-PCR analysis. As shown in Fig. 6, the levels of insulin mRNA in MIN6 cells grown on low glucose-containing media was equal to that of high glucose-grown cells following TSA treatment. The obtained data are consistent with the idea that the decrease in histone H4 acetylation levels at the insulin gene promoter on low levels of glucose is because of the action of deacetylases and that increases in histone H4 acetylation levels correlate with increased insulin gene expression. Lack of Insulin Gene Expression in Fibroblasts Is Associated with a Lack of Histone Acetylation at the Insulin Gene Promoter—It has been shown that acetylated histones are normally associated with promoters of actively transcribed genes (28Berger S.L. Curr. Opin. Genet. Dev. 2002; 12: 142-148Crossref PubMed Scopus (991) Google Scholar, 29Cheung P. Allis C.D. Sassone-Corsi P. Cell. 2000; 103: 263-271Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar). Silent genes appear to either lack or have only minimal levels of histone acetylation at their promoter regions. T
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