Direct Examination of Histone Acetylation on Myc Target Genes Using Chromatin Immunoprecipitation
2000; Elsevier BV; Volume: 275; Issue: 43 Linguagem: Inglês
10.1074/jbc.m005154200
ISSN1083-351X
AutoresScott R. Eberhardy, Caroline A. D'Cunha, Peggy Farnham,
Tópico(s)Cancer-related Molecular Pathways
ResumoOverexpression of c-Myc can lead to altered transcriptional regulation of cellular genes and to neoplastic transformation. Although DNA binding is clearly required, the mechanism by which recruitment of c-Myc to target promoters results in transcriptional activation is highly debated. Much of this controversy comes from the difficulty in clearly defining a true Myc target gene. We have previously determined that cad is a bona fide Myc target gene and thus now use the cadpromoter as a model to study Myc function. Others have shown that Myc can interact indirectly with histone acetylases and have suggested that Myc mediates transcriptional activation by causing an increase in the levels of acetylated histones on target promoters. To directly test this model, we employed a chromatin immunoprecipitation assay to examine the levels of acetylated histones on the cadpromoter. Although Myc was bound to the cad promoter in S phase but not in G0 phase, we found high levels of acetylated histones on the promoter in both stages. We also examined acetylated histones on the cad promoter before and after differentiation of U937 cells. Although the levels of c-Myc bound to the cad promoter were greatly reduced after differentiation, we saw high levels of acetylated histones on thecad promoter both before and after differentiation. Finally, we found that a 30-fold change in binding of N-Myc to the telomerase promoter did not result in a concomitant change in histone acetylation. Thus, recruitment of a Myc family member to a target promoter does not necessarily influence the amount of acetylated histones at that promoter. Further investigations are in progress to define the role of Myc in transcriptional activation. Overexpression of c-Myc can lead to altered transcriptional regulation of cellular genes and to neoplastic transformation. Although DNA binding is clearly required, the mechanism by which recruitment of c-Myc to target promoters results in transcriptional activation is highly debated. Much of this controversy comes from the difficulty in clearly defining a true Myc target gene. We have previously determined that cad is a bona fide Myc target gene and thus now use the cadpromoter as a model to study Myc function. Others have shown that Myc can interact indirectly with histone acetylases and have suggested that Myc mediates transcriptional activation by causing an increase in the levels of acetylated histones on target promoters. To directly test this model, we employed a chromatin immunoprecipitation assay to examine the levels of acetylated histones on the cadpromoter. Although Myc was bound to the cad promoter in S phase but not in G0 phase, we found high levels of acetylated histones on the promoter in both stages. We also examined acetylated histones on the cad promoter before and after differentiation of U937 cells. Although the levels of c-Myc bound to the cad promoter were greatly reduced after differentiation, we saw high levels of acetylated histones on thecad promoter both before and after differentiation. Finally, we found that a 30-fold change in binding of N-Myc to the telomerase promoter did not result in a concomitant change in histone acetylation. Thus, recruitment of a Myc family member to a target promoter does not necessarily influence the amount of acetylated histones at that promoter. Further investigations are in progress to define the role of Myc in transcriptional activation. trichostatin A acetylated histone H3 acetylated histone H4 immunoprecipitation polymerase chain reaction histone deacetylase The c-Myc oncoprotein has been found to be deregulated in many different types of cancer, including breast, colon, and prostate cancers, as well as many types of leukemias and lymphomas. It is overexpressed in tumors by many different mechanisms, including gene amplification, translocation, retroviral insertion, and other means (1Henriksson M. Luscher B. Adv. Cancer Res. 1996; 68: 109-182Crossref PubMed Google Scholar). The role of c-Myc in cell proliferation has been documented by numerous studies. Increased expression of c-Myc is thought to be an important contributor to the neoplastic transformation of the tumor based on intentional overexpression of c-Myc in tissue culture cells and in transgenic mice. For example, cotransfection of Myc and Ras in rat embryo fibroblasts causes these cells to adopt a transformed phenotype (2Stone J. deLange T. Ramsay G. Jakobovits F. Bishop J.M. Varmus H. Lee W. Mol. Cell. Biol. 1987; 7: 1697-1709Crossref PubMed Scopus (381) Google Scholar). Overexpression of c-Myc in tissue culture causes increased proliferation of cells with a shortened G1 phase, whereas loss of c-Myc results in slow growth and a longer G1 phase (3Bush A. Mateyka M. Dugan K. Obaya A. Adachi S. Sedivy J. Cole M. Genes Dev. 1998; 12: 3797-3802Crossref PubMed Scopus (162) Google Scholar). Finally, studies of transgenic mice in which c-Myc overexpression is targeted to a specific tissue have also demonstrated the importance of c-Myc in tumorigenesis. Targeted overexpression of c-Myc in the breast, B lymphocytes, and liver in transgenic mice results in increased formation of tumors in these tissues, although tumor formation is greatly enhanced by coexpression of a cooperating oncogene, such as Ha-Ras (4Sinn E. Muller W. Pattengale P. Tepler I. Wallace R. Leder P. Cell. 1987; 49: 465-475Abstract Full Text PDF PubMed Scopus (648) Google Scholar, 5Adams J.M. Harris A.W. Pinkert C.A. Corcoran L.M. Alexander W.S. Cory S. Palmiter R.D. Brinster R.L. Nature. 1985; 318: 533-538Crossref PubMed Scopus (1406) Google Scholar, 6Harris A.W. Pinkert C.A. Crawford M. Langdon W.Y. Brinster R.L. Adams J.M. J. Exp. Med. 1988; 167: 353-371Crossref PubMed Scopus (342) Google Scholar, 7Sandgren E.P. Quaife C.J. Pinkert C.A. Palmiter R.D. Brinster R.L. Oncogene. 1989; 4: 715-724PubMed Google Scholar). The c-myc gene encodes a protein that is a member of the basic/helix-loop-helix/leucine zipper family of transcription factors. Myc heterodimerizes with the protein Max through its C-terminal helix-loop-helix and leucine zipper domains and binds to an E box motif, which is the hexanucleotide sequence CACGTG (8Blackwood E.M. Eisenman R.N. Science. 1991; 251: 1211-1217Crossref PubMed Scopus (1557) Google Scholar). Myc/Max heterodimers activate transcription because of a transactivation domain in the N terminus of the Myc protein (9Kato G.J. Barrett J. Villa-Garcia M. Dang C.V. Mol. Cell. Biol. 1990; 10: 5914-5920Crossref PubMed Scopus (316) Google Scholar). Max is also a heterodimerization partner for the Mad proteins; however, Mad/Max heterodimers bind to E boxes and repress transcription via a transcriptional repression domain in the Mad protein (10Ayer D.E. Kretzner L. Eisenman R.N. Cell. 1993; 72: 211-222Abstract Full Text PDF PubMed Scopus (646) Google Scholar). A commonly accepted model of Myc-mediated transcriptional regulation invokes an exchange of co-repressors and co-activators on Myc target genes that occurs as Myc/Max heterodimers replace Mad/Max heterodimers (Fig. 1). Specifically, it is thought that binding of Mad/Max heterodimers to promoter DNA results in transcriptional repression via histone deacetylation because of the recruitment of mSin3A or mSin3B and subsequent recruitment of HDAC1 or HDAC2 (11Ayer D.E. Lawrence Q.A. Eisenman R.N. Cell. 1995; 80: 767-776Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 12Sommer A. Hilfenhaus S. Menkel A. Kremmer E. Seiser C. Loidl P. Luscher B. Curr. Biol. 1997; 7: 357-365Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). These transcriptional repressor complexes are thought to bind to Myc target genes in quiescent or differentiated cells, i.e. in cells that contain very low amounts of Myc protein. However, proliferating cells contain increased amounts of Myc, and Myc/Max heterodimers replace Mad/Max heterodimers on target promoters. Because Myc can interact with TRRAP, which in turn interacts with the histone acetyltransferase hGCN5, it has been suggested that replacement of Mad/Max with Myc/Max results in increased amounts of acetylated histones on Myc target genes (13McMahon S.B. Wood M.A. Cole M.D. Mol. Cell. Biol. 2000; 20: 556-562Crossref PubMed Scopus (381) Google Scholar). It is believed that acetylation of lysines of the N-terminal tails of histones in core nucleosomes promotes chromatin remodeling, which then allows site specific transcription factors and the RNA polymerase II basal transcriptional machinery access to the start site of transcription (for review see Ref. 14Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1570) Google Scholar).Figure 1Current model for Myc-mediated transcriptional activation. Transcriptional repression mediated by Mad/Max is believed to be due to recruitment of the mSin3 repressor complex. mSin3 binds to HDACs, which remove acetyl groups from histone tails and maintain chromatin in a repressive configuration. In contrast, Myc is thought to activate target gene transcription by recruiting proteins with histone acetyltransferase activity through TRRAP or other means. The addition of acetyl groups onto histone tails would result in a more open chromatin configuration allowing RNA polymerase II to bind the promoter and begin transcription.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A direct test of the model described above requires examination of promoter occupancy by acetylated histones on a Myc target gene. However, there is great deal of uncertainty as to which genes are actually regulated by c-Myc. A recent study found that only 5% of the 6000 genes tested using DNA microchips responded to overexpression of c-Myc (15Coller H.A. Grandori C. Tamayo P. Colbert T. Lander E.S. Eisenman R.N. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3260-3265Crossref PubMed Scopus (722) Google Scholar). A number of candidate genes have been proposed to be targets of Myc regulation, including odc(ornithine decarboxylase) (16Bello-Fernandez C. Packham G. Cleveland J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7804-7808Crossref PubMed Scopus (693) Google Scholar), the translation initiation factor eIF4E (17Jones R.M. Branda J. Johnston K.A. Polymenis M. Gadd M. Rustgi A. Callanan L. Schmidt E.V. Mol. Cell. Biol. 1996; 16: 4754-4764Crossref PubMed Scopus (205) Google Scholar), the catalytic subunit of telomerase (tert) (18Wang J.L. Xie L.Y. Allan S. Beach D. G. J., H. Genes Dev. 1998; 12: 1769-1774Crossref PubMed Scopus (578) Google Scholar), and cdk4 (19Hermeking H. Rago C. Schuhmacher M. Li Q. Barrett J.F. Obaya A.J. O'Connell B.C. Mateyak M.K. Tam W. Kohlhuber F. Dang C.V. Sedivy J.M. Eick D. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2229-2234Crossref PubMed Scopus (395) Google Scholar). Another complicating factor in the study of Myc target genes is the modest transcriptional activity mediated by the Myc protein. Most genes that respond to increased levels of c-Myc change by only 2–5-fold. Some of the difficulty in identifying a Myc target gene may be due to the presence of other, more abundant, proteins in the cell (such as USF) that can bind to E boxes. Therefore, identification of Myc target genes requires demonstrating that binding of Myc to the promoter region is required for transcriptional regulation. We have recently shown a correlation between promoter occupancy by c-Myc and transcriptional activation of the cad(carbamoyl phosphate synthase/aspartate transcarbamoylase/dihydroorotase) gene. The cadgene encodes the trifunctional enzyme carbamoyl phosphate synthase/aspartate carbamoyltransferase/dihydroorotase, which catalyzes the first three rate-limiting steps of pyrimidine biosynthesis.cad expression is minimal in quiescent cells, and expression increases as cells enter the cell cycle, with peak expression occurring at the G1/S phase boundary. The cad gene contains an E box downstream from the transcription start site that is conserved in the mouse, rat, hamster, and human homologues of the gene. Chromatin immunoprecipitation studies have shown that both Myc and USF bind to the cad promoter (20Boyd 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 (247) Google Scholar). However, mutations flanking the E box that disrupt Myc binding to the cad promoter but do not affect USF binding result in a loss of growth regulation (21Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1999; 19: 8389-8399Crossref Scopus (147) Google Scholar). Taken together, these results suggest that cad is abona fide Myc target gene, and thus in the studies described below we have used the cad promoter to study Myc function. We have used a chromatin immunoprecipitation assay to determine whether changes in histone acetylation correlate with Myc-mediated changes incad expression. We found only modest differences in histone acetylation on the cad promoter as NIH3T3 cells enter the cell cycle from quiescence or as U937 cells exit the cell cycle into a differentiated state. Furthermore, our studies indicate that high levels of histone acetylation are present at the cadpromoter in G0 and in S phase. This high degree of acetylation is not seen in the transcribed regions of thecad gene or at a promoter that is never active in NIH3T3 cells. Additionally, we have examined an N-Myc responsive promoter in neuroblastoma cells, and we do not see changes in histone acetylation that correlate with N-Myc binding. Our results suggest that although high levels of histone acetylation can identify a promoter that has transcriptional potential in a certain cell type, histone acetylation does not appear to be the mechanism by which Myc regulates transcription. NIH3T3 cell cultures were maintained and synchronized as described previously (22Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1997; 17: 2529-2537Crossref PubMed Scopus (141) Google Scholar). Briefly, 6–8 × 106 cells were seeded into 500-cm2 tissue culture dishes (1–2 dishes were used per antibody per time point in the formaldehyde cross-linking experiments) and incubated in starvation medium for 48–72 h. Cells were then either stimulated to enter the cell cycle by the addition of stimulation medium for 4 h (early G1 phase) or 12 h (G1/S phase) prior to cross-linking. Progression of the cells through the cell cycle was measured by flow cytometric analysis of propidium iodide-stained cells as described previously (23Means A.L. Slansky J.E. McMahon S.L. Knuth M.W. Farnham P.J. Mol. Cell. Biol. 1992; 12: 1054-1063Crossref PubMed Scopus (132) Google Scholar). Data were acquired on a FACScan flow cytometer (Becton Dickinson) using CellQuest acquisition and analysis software. The treatment of G0 phase cells with trichostatin A (TSA)1 was performed as follows. 4 × 106 cells were seeded into 225-cm2 tissue culture flasks (5 flasks/experimental condition; combined prior to cell lysis) and incubated in starvation medium for 48–52 h to induce quiescence. Trichostatin A (Sigma) (1 mg/ml, solubilized in 100% ethanol) was diluted in medium and added to the cells at a final concentration of 1 μg/ml of medium (3.30 μm). Cells were then incubated for an additional 16–20 h prior to harvesting. Untreated cells received an equal volume of ethanol without TSA, whereas S phase cells received only stimulation medium 12 h prior to harvesting of RNA and protein. U937 cells were maintained in spinner flasks containing RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C and 5% CO2. Differentiation of U937 cells was performed by adding all-trans-retinoic acid (Sigma) to a final concentration of 1 μm. Cells were then incubated for an additional 5 days prior to harvesting; cell cycle analysis of U937 cells was performed as described above for 3T3 cells. Tet21N cells were maintained at 37 °C and 5% CO2 in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 4 mm l-glutamine. Tetracycline was added to a final concentration of 1 μg/ml for 2 weeks to repress N-Myc transcription. RNA preparation and RNase protection assays were performed as described previously (20Boyd 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 (247) Google Scholar, 24Slansky J.E. Li Y. Kaelin W.G. Farnham P.J. Mol. Cell. Biol. 1993; 13 (; Correction (1993) Mol. Cell. Biol.13, 7201): 1610-1618Crossref PubMed Scopus (264) Google Scholar). For analysis ofcad mRNA, 35–40 μg of cytoplasmic RNA from serum-starved, serum-stimulated, or TSA-treated cells was incubated with 1 × 105 cpm of probe at 65 °C for 3 h. For analysis of glyceraldehyde-phosphate dehydrogenase mRNA, 5–10 μg of cytoplasmic RNA was incubated with 1.7 × 104cpm of probe at 52 °C for 3 h. Unhybridized RNA was digested by the addition of 10 μg of RNase A. The products were resolved on an 8% denaturing polyacrylamide gel and visualized by autoradiography. Signals were collected using a PhosphorImager and quantitated using ImageQuant v4.2a (Molecular Dynamics). Acid-soluble protein was prepared from the nuclei of cells that were harvested for cytoplasmic RNA. The nuclear pellets were thawed on ice and resuspended in 10 volumes of protein extraction buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 1.5 mm phenylmethylsulfonyl fluoride, 0.5 mmdithiothreitol). Sulfuric acid was added dropwise to a final concentration of 0.4 n (0.2 m) while vortexing gently, and the nuclear lysates were incubated on ice for 1 h with vigorous vortexing every 10 min. The lysates were centrifuged at 14,000 rpm in an Eppendorf 5415C centrifuge for 10 min at 4 °C. The supernatant was precipitated with 3 volumes of 20% trichloroacetic acid on ice for 1 h with vigorous vortexing every 10 min and centrifuged at 14,000 rpm for 10 min at 4 °C. The precipitate was washed twice with acidified acetone (0.1% HCl) and twice with acetone, dried completely by desiccation for 30 min, resuspended in 200 μl of H2O, and stored at 4 °C overnight. The concentration and purity of each acid-soluble histone protein preparation was determined using: 1) the Bio-Rad protein assay followed by measurement of A595 using a Shimadzu UV16OU Spectrophotometer and 2) SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining. Protein preparations were stored at −80 °C. For analysis of histone H3 and H4 acetylation levels, 15 μg of acid-soluble protein was used for SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis with anti-acetylated histone H3 06–599 (Upstate Biotechnology) or anti-acetylated histone H4 06–598 (Upstate Biotechnology) antibodies. The formaldehyde cross-linking and chromatin immunoprecipitation assays of tissue culture cells were performed as described previously with the following modifications (20Boyd 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 (247) Google Scholar, 21Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1999; 19: 8389-8399Crossref Scopus (147) Google Scholar). Immunoprecipitations (IPs) were performed overnight at 4 °C using 1 μg of anti-c-Myc sc-764-X (Santa Cruz), anti-acetylated histone H3 06–599 (Upstate Biotech.), anti-acetylated histone H4 06-598 or 06-866 (Upstate Biotechnology), anti-acetyl-lysine (Upstate Biotechnology), anti-phospho-H3 (Ser-10, a gift from C. David Allis), anti-N-Myc sc-791 (Santa Cruz), anti-Max sc-765-X (Santa Cruz), or no antibody, as indicated. Before the first wash, 20% of the supernatant from the IP without antibody for each condition was saved as total input chromatin and was processed with the eluted IPs beginning with the cross-linking reversal step. After the final ethanol precipitation, each IP sample was resuspended in 30 μl of PCR grade TE (10 mmTris (pH 7.5), 1 mm EDTA). Total input chromatin samples were resuspended in 30 μl of TE. For formaldehyde cross-linking and immunoprecipitation of chromatin from mouse livers, livers were harvested from newborn and adult C57BL/6J mice. After harvesting, livers were minced with a razor blade, brought up in 20 ml of medium, and cross-linked for 10 min with 1% formaldehyde. The cross-linking reaction was stopped by addition of 0.125 m glycine. Cross-linked livers were then homogenized to individual cells using a Medi-Machine (BD Bioscience). Cells were transferred to 1.5-ml Eppendorf tubes, centrifuged at 1000 rpm in an Eppendorf 541C Centrifuge for 10 min at 4 °C to pellet the cells, which were then resuspended in cell lysis buffer, and processed for chromatin immunoprecipitation as described above. PCR reactions contained 2 μl of IP sample or 2 μl of a 1:100 dilution of input sample, 1.2 mm MgCl2, 50 ng of each primer, 200 μm each dATP, dGTP, dCTP, and dTTP, 4 μl of 5 m betaine (Sigma), 1× Thermophilic buffer (Promega), and 1.25 units of Taq DNA polymerase (Promega) in 20 μl of total volume. After 34–36 cycles of amplification, 8 μl of the PCR products was electrophoresed on a 1.5% agarose gel, and DNA was stained with ethidium bromide and visualized under UV light. Signals were quantitated using ImageQuant v4.2 (Molecular Dynamics). Fold changes in acetylation were calculated by first dividing the IP signal intensity by the input signal intensity (to control for slightly different cells numbers at each time point) for both the G1/S sample and the G0 sample. Then, the G1/S value was divided by the G0 value. The primers used for PCR analysis are shown in Table I.Table IPrimers used in chromatin immunoprecipitation experimentsGenePrimer sequenceMurinecadpromotermcadA5′-TGACTAGCGGTACCGGGGTTGCTGCTGTGGAACC-3′3′CAD5′-CGGGCTTGCTTACCCACCTTCCCCAGCAGTCGACAC-3′cad coding sequenceCadF5′-CGGGATCCGGTCAGTTCATCCTCACTCCCC-3′CadR5′-CGGAATTCGGATGTACATGCCGTTCTCAGC-3′Humancad promoterhucadUS5′-CCAGTTCCCATTGGTGTTGTTGCC-3′hucadDS5′-GAGAGGCGCATCACAGAGTGGGATAA-3′Murineodc genemODC-c5′-CATGACGACGTGCTCGGCGTATAAGTA-3′mODC-d 5′-AGGTCCAGGAGCAGCTGCCTTCAG-3′Humantert gene5tel15-CCTTCACGTCCGGCATTCGTGG-33tel13-AAGGTGAAGGGGCAGGACGGGT-3Murinealbumin genemalbA5′-GGACACAAGACTTCTGAAAGTCCTC-3′malbB5′-TTCCTACCCCATTACAAAATCATA-3′Murinecdc2 genecdc2 3585′-GTGGACTGTCACTTTGGTGGCTGGC-3′cdc2 205′-GGTAAAGCTCCCGGGATCCGCCAAT-3′For each primer set, the top primer is the upstream primer, and the bottom primer is the downstream primer. Open table in a new tab For each primer set, the top primer is the upstream primer, and the bottom primer is the downstream primer. To test the hypothesis that Myc target genes are regulated via changes in histone acetylation, we first determined whether increased histone acetylation could substitute for promoter-bound Myc to activate transcription of the cad promoter. Quiescent NIH3T3 cells, which contain very low levels of Myc protein, were treated with the drug TSA, an inhibitor of histone deacetylases. Treatment of cells with TSA results in genome-wide increases in the levels of histone acetylation and has been shown to alter the expression of a number of genes (25Van Lint C. Emiliani S. Verdin E. Gene Expr. 1996; 5: 245-253PubMed Google Scholar). Cells were treated with increasing amounts of TSA for 16 h and harvested for isolation of histones and RNA. As shown in Fig.2 A, TSA treatment of NIH3T3 cells causes a large increase in the amount of acetylated histone H3 and acetylated histone H4. We then examined cad mRNA levels using an RNase protection assay in which a radiolabeled probe is hybridized to the cad message and unprotected RNA is digested with RNase A (Fig. 2 B). We found that treatment of quiescent NIH3T3 cells with even low levels of TSA increased transcription of cad mRNA to approximately S phase levels. This result suggests that forced histone acetylation can bypass regulation of target genes by Myc and supports the model that recruitment of Myc may result in increased acetylation of target promoter histones. The results obtained from treatment of NIH3T3 cells with TSA suggest that histone acetylation may play a role in activation of cadtranscription. To test this hypothesis, we first determined whether the level of acetylation of bulk histones increased when quiescent cells re-entered the cell cycle. NIH3T3 cells were synchronized by starvation in low serum medium for 48 h and then made to progress through the cell cycle through the addition of serum. Flow cytometry was performed to ensure that cells were synchronized and progressing through the cell cycle (Fig. 3 A). Western analysis shows that there was no increase in acetylated histone H3 or acetylated histone H4 in S phase, as compared with quiescent cells (Fig. 2 A), although the antibodies clearly had the ability to detect acetylated histones. However, it was possible that histones bound to specific promoter regions would show a difference in acetylation. Therefore, we performed a formaldehyde cross-linking and chromatin immunoprecipitation assay to determine the levels of histone acetylation at the cad promoter as cells progress from G0 to S phase. Cells were cross-linked with formaldehyde and, after sonicating the chromatin to a length of about 500 base pairs, DNA was immunoprecipitated with antibodies that recognize histones acetylated at their N-terminal tails. After immunoprecipitation, proteins were digested, and purified DNA was analyzed by PCR. We found easily detectable levels of acetylated histone H3 on thecad promoter in quiescent cells when the promoter is inactive, but there was no increase in the amount of acetylated H3 bound to the cad promoter as cells progressed to the G1/S phase boundary (Fig. 3 B). However, the expected increase in binding of Myc to the cad promoter did occur. We also used an antibody that recognized phosphorylated H3 because it has been previously shown that histone H3 phosphorylation levels change throughout the cell cycle at immediate early gene promoters (26Chadee D.N. Hendzel M.J. Tylipski C.P. Allis C.D. Bazett-Jones D.P. Wright J.A. Davie J.R. J. Biol. Chem. 1999; 274: 24914-24920Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), However, we found very low levels of histone H3 phosphorylation at the cad promoter in either G0or S phase. To ensure that the results of the first experiment were valid, we performed a second time course and chromatin immunoprecipitation experiment. This time, we used the antibody that recognizes acetylated histone H3 and two different antibodies that recognize acetylated histone H4. As before, the amount of Myc bound to the cadpromoter increased about 7-fold by the G1/S phase boundary. However, we saw only modest increases in the amount of acetylated histone H3. The two different acetylated histone H4 antibodies gave slightly different results. One antibody showed no increase, whereas the other antibody showed a 3.8-fold increase in acetylation. We note that the Western blotting results shown above demonstrate the specificity of the antibodies. We have repeated the chromatin immunoprecipitation a number of times, and although we have seen varying changes in Myc binding as NIH3T3 cells progress through the cell cycle, we do not see a correlation between the increase in Myc binding and the change in histone acetylation at the cadpromoter. The average change in acetylation at the cadpromoter from G0 to G1/S is only about 1.4-fold for histone H3 and 1.4-fold for histone H4 (Fig. 3 D). We also used a different set of primers to monitor changes in acetylation of histones H3 and H4 within the first 500 nucleotides of transcribed sequences of the cad gene; similar to the results shown above for the cad promoter, all changes observed were 2-fold or less (data not shown). Although we did see modest changes in histone acetylation on the cad promoter, it was unclear whether these changes were linked to transcription or whether they were simply due to cell cycle stage-specific changes in histone abundance or to general changes in chromatin structure that occur irrespective of transcription. With the goal of distinguishing between these possibilities, we turned to a second system in which Myc is known to mediate changes in gene expression. In general, differentiated cells express very low levels of Myc protein. The decrease in transcription of Myc target genes because of differentiation is a long term change, and Myc may regulate transcription during an irreversible withdrawal from the cell cycle in a different manner than it does at the G1/S phase boundary. For example, maintaining high levels of histone acetylation on the cad promoter throughout the cell cycle may be necessary for the cyclical regulation of the gene. However, it is possible that histone acetylation on the cadpromoter may be decreased when cells are terminally differentiated. To examine histone acetylation on the cad promoter in a differentiation system we chose to use U937 cells, which are a human monocyte cell line. U937 cells can be made to differentiate into granulocytes with retinoic acid, and it has been previously shown that Myc mRNA and protein levels are greatly decreased after differentiation of these cells (27Larsson L.G. Bahram F. Burkhardt H. Luscher B. Oncogene. 1997; 15: 737-748Crossref PubMed Scopus (34) Google Scholar). U937 cells were differentiated for 5 days with 1 μm all-trans-retinoic acid, treated and untreated cells were cross-linked with formaldehyde, and chromatin immunoprecipitation was performed with antibodies recognizing Myc and acetylated histones H3 and H4. To ensure that cells treated with retinoic acid had exited the cell cycle, we examined the DNA content of the cells using flow cytometry and found that almost all the
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