Hepatocyte Nuclear Factor 3 Relieves Chromatin-mediated Repression of the α-Fetoprotein Gene
1999; Elsevier BV; Volume: 274; Issue: 35 Linguagem: Inglês
10.1074/jbc.274.35.25113
ISSN1083-351X
AutoresAlison J. Crowe, Ling Sang, Kelly Ke Li, Kathleen C. Lee, Brett T. Spear, Michelle Barton,
Tópico(s)Kruppel-like factors research
ResumoThe α-fetoprotein gene (AFP) is tightly regulated at the tissue-specific level, with expression confined to endoderm-derived cells. We have reconstituted AFP transcription on chromatin-assembled DNA templates in vitro. Our studies show that chromatin assembly is essential for hepatic-specific expression of the AFP gene. While nucleosome-free AFP DNA is robustly transcribed in vitro by both cervical (HeLa) and hepatocellular (HepG2) carcinoma extracts, the general transcription factors and transactivators present in HeLa extract cannot relieve chromatin-mediated repression of AFP. In contrast, preincubation with either HepG2 extract or HeLa extract supplemented with recombinant hepatocyte nuclear factor 3 α (HNF3α), a hepatic-enriched factor expressed very early during liver development, is sufficient to confer transcriptional activation on a chromatin-repressed AFP template. Transient transfection studies illustrate that HNF3α can activate AFP expression in a non-liver cellular environment, confirming a pivotal role for HNF3α in establishing hepatic-specific gene expression. Restriction enzyme accessibility assays reveal that HNF3α promotes the assembly of an open chromatin structure at the AFP promoter. Combined, these functional and structural data suggest that chromatin assembly establishes a barrier to block inappropriate expression of AFP in non-hepatic tissues and that tissue-specific factors, such as HNF3α, are required to alleviate the chromatin-mediated repression. The α-fetoprotein gene (AFP) is tightly regulated at the tissue-specific level, with expression confined to endoderm-derived cells. We have reconstituted AFP transcription on chromatin-assembled DNA templates in vitro. Our studies show that chromatin assembly is essential for hepatic-specific expression of the AFP gene. While nucleosome-free AFP DNA is robustly transcribed in vitro by both cervical (HeLa) and hepatocellular (HepG2) carcinoma extracts, the general transcription factors and transactivators present in HeLa extract cannot relieve chromatin-mediated repression of AFP. In contrast, preincubation with either HepG2 extract or HeLa extract supplemented with recombinant hepatocyte nuclear factor 3 α (HNF3α), a hepatic-enriched factor expressed very early during liver development, is sufficient to confer transcriptional activation on a chromatin-repressed AFP template. Transient transfection studies illustrate that HNF3α can activate AFP expression in a non-liver cellular environment, confirming a pivotal role for HNF3α in establishing hepatic-specific gene expression. Restriction enzyme accessibility assays reveal that HNF3α promotes the assembly of an open chromatin structure at the AFP promoter. Combined, these functional and structural data suggest that chromatin assembly establishes a barrier to block inappropriate expression of AFP in non-hepatic tissues and that tissue-specific factors, such as HNF3α, are required to alleviate the chromatin-mediated repression. α-fetoprotein kilobase(s) hepatocyte nuclear factor base pair(s) chloramphenicol acetyltransferase high speed supernatant preinitiation complex During differentiation, patterns of cell-type specific gene expression are established which must be maintained throughout the life of the cell. It is generally assumed that tissue-specific transcription is achieved through selective synthesis and concentration of cell-type restricted transcription factors. These regulatory proteins are usually not as confined in expression as their downstream targets (1Graef I. Crabtree G. Science. 1997; 277: 193-194Crossref PubMed Scopus (11) Google Scholar, 2Henderson A.J. Calame K.L. Crit. Rev. Eukaryotic Gene Expression. 1995; 5: 255-280Crossref PubMed Scopus (12) Google Scholar). Additional levels of control in vivo may rely on chromatin structure that restricts access of ubiquitous or widely expressed regulatory factors, as well as facilitates synergy between transcription activators (3Naar A.M. Beurang P.A. Robinson K.M. Oliner J.D. Avizonis D. Scheek S. Zwicker J. Kadonaga J.T. Tjian R. Genes Dev. 1998; 12: 3020-3031Crossref PubMed Scopus (173) Google Scholar, 4Adams C.C. Workman J.L. Mol. Cell. Biol. 1995; 15: 1405-1421Crossref PubMed Google Scholar). Eukaryotic DNA is highly condensed into chromatin; this compaction results in a general repression of gene expression (see Refs. 5Felsenfeld G. Nature. 1992; 355: 219-224Crossref PubMed Scopus (718) Google Scholar and 6Knezetic J.A. Luse D.S. Cell. 1986; 45: 95-104Abstract Full Text PDF PubMed Scopus (206) Google Scholar, and reviewed in Ref. 7Wolffe A.P. New Biol. 1990; 2: 211-218PubMed Google Scholar). Nuclease sensitivity mapping of a number of genes has indicated a clear pattern of accessible chromatin structure around actively transcribing genes during development (2Henderson A.J. Calame K.L. Crit. Rev. Eukaryotic Gene Expression. 1995; 5: 255-280Crossref PubMed Scopus (12) Google Scholar, 8Aronow B.J. Ebert C.A. Valerius M.T. Potter S.S. Wiginton D.A. Witte D.P. Hutton J.J. Mol. Cell. Biol. 1995; 15: 1123-1135Crossref PubMed Google Scholar, 9Asenbauer H. Klobeck H.G. Eur. J. Immunol. 1996; 26: 142-150Crossref PubMed Scopus (6) Google Scholar, 10Jones B.K. Monks B.R. Liebhaber S.A. Cooke N.E. Mol. Cell. Biol. 1995; 15: 7010-7021Crossref PubMed Scopus (152) Google Scholar, 11McGhee J.D. Wood W.I. Dolan M. Engel J.D. Felsenfeld G. Cell. 1981; 27: 45-55Abstract Full Text PDF PubMed Scopus (258) Google Scholar, 12Nickel B.E. Cattini P.A. Mol. Cell. Endocrinol. 1996; 118: 155-162Crossref PubMed Scopus (11) Google Scholar, 13Stadler J. Larsen A. Engel J.D. Dolan M. Groudine M. 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The liver tumor marker gene, α-fetoprotein (AFP),1 displays strict tissue-specific and developmental regulation in vivo (reviewed in Refs. 15Camper S.A. Godbout R. Tilghman S.M. Prog. Nucleic Acids Res. Mol. Biol. 1989; 36: 131-143Crossref PubMed Scopus (21) Google Scholar and 16Nahon J.-L. Biochimie (Paris). 1987; 69: 445-459Crossref PubMed Scopus (56) Google Scholar). AFP is highly expressed during fetal development in endoderm-derived tissues including the yolk sac, liver, and gut. At birth, unlike other liver-specific genes, AFP expression is rapidly repressed (17Tilghman S.M. Oxf. Surv. Eukaryotic Genes. 1985; 2: 160-206PubMed Google Scholar) and is only reactivated in cases of renewed cellular proliferation, including liver regeneration and hepatocellular carcinoma (17Tilghman S.M. Oxf. Surv. Eukaryotic Genes. 1985; 2: 160-206PubMed Google Scholar, 18Nakabayashi H. Hashimoto T. Miyao Y. Tjong K.K. Chan J. Tamaoki T. Mol. Cell. Biol. 1991; 11: 5885-5893Crossref PubMed Google Scholar). Differential AFP expression patterns are accompanied by discrete changes in local chromatin structure as measured by DNase I hypersensitivity (reviewed in Refs. 15Camper S.A. Godbout R. Tilghman S.M. Prog. Nucleic Acids Res. Mol. Biol. 1989; 36: 131-143Crossref PubMed Scopus (21) Google Scholar and 19Godbout R. Ingram R. Tilghman S.M. Mol. Cell. Biol. 1986; 6: 477-487Crossref PubMed Scopus (115) Google Scholar, 20Godbout R. Ingram R.S. Tilghman S.M. Mol. Cell. Biol. 1988; 8: 1169-1178Crossref PubMed Scopus (99) Google Scholar, 21Godbout R. Tilghman S.M. Genes Dev. 1988; 2: 949-956Crossref PubMed Scopus (34) Google Scholar). AFP thus provides an excellent model to assay the potential contribution of chromatin structure to tissue-specific gene regulation. Previous studies using transient transfections and transgenic mice have identified multiple regulatory elements that control AFP expression. These include three distinct enhancers and a promoter/repressor region (19Godbout R. Ingram R. Tilghman S.M. Mol. Cell. Biol. 1986; 6: 477-487Crossref PubMed Scopus (115) Google Scholar, 20Godbout R. Ingram R.S. Tilghman S.M. Mol. Cell. Biol. 1988; 8: 1169-1178Crossref PubMed Scopus (99) Google Scholar, 22Hammer R.E. Krumlauf R. Camper S.A. Brinster R.L. Tilghman S.M. Science. 1987; 235: 53-58Crossref PubMed Scopus (170) Google Scholar, 23Vacher J. Tilghman S.M. Science. 1990; 250: 1732-1735Crossref PubMed Scopus (95) Google Scholar). The enhancers were found to activate a heterologous promoter in non-hepatic cells, while a 1-kilobase fragment containing only the distal and proximal promoter regions exhibited absolute tissue specificity (19Godbout R. Ingram R. Tilghman S.M. Mol. Cell. Biol. 1986; 6: 477-487Crossref PubMed Scopus (115) Google Scholar). The AFP promoter from −1 kb to the start site is therefore a major determinant of liver-specific transcription. Several liver-enriched proteins which direct hepatocyte-specific expression have been shown to bind multiple sites within the AFP promoter (reviewed in Ref. 24Cereghini S. FASEB J. 1996; 10: 267-282Crossref PubMed Scopus (475) Google Scholar), including CAAT/enhancer-binding protein (25Zhang D.-E. Hoyt P.R. Papaconstantinou J. J. Biol. Chem. 1990; 265: 3382-3391Abstract Full Text PDF PubMed Google Scholar), hepatocyte nuclear factor 1 (HNF1; 26), and hepatocyte nuclear factor 3 (HNF3; 27). The HNF3 family is composed of three members, HNF3α, HNF3β, and HNF3γ, which, along with the Drosophila fork head protein, constitute a growing family of winged-helix DNA-binding proteins (28Lai E. Prezioso V.R. Tao W. Chen W.S. Darnell J.E.J. Genes Dev. 1991; 5: 416-427Crossref PubMed Scopus (436) Google Scholar). HNF3/fork head proteins that regulate gene expression in endoderm-derived tissues are required for pattern formation in the embryonic gut (see Ref. 29Dufort D. Schwartz L. Harpal K. Rossant J. Development. 1998; 125: 3015-3025Crossref PubMed Google Scholar, reviewed in Ref. 30Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (577) Google Scholar). HNF3α is of particular interest as it has been shown to position nucleosomes within the enhancer of the liver-specific albumin gene (31McPherson C.E. Eun-Yong S. Friedman D.S. Zaret K.S. Cell. 1993; 75: 387-398Abstract Full Text PDF PubMed Scopus (284) Google Scholar,32Shim E.Y. Woodcock C. Zaret K.S. Genes Dev. 1998; 12: 5-10Crossref PubMed Scopus (101) Google Scholar). A role for HNF3 in organizing chromatin is further supported by the three-dimensional structural similarity of the winged helix conformation to the globular domain of linker histones (33Clark K.L. Halay E.D. Lai E. Burley S.K. Nature. 1993; 364: 412-420Crossref PubMed Scopus (1098) Google Scholar, 34Goytisolo F.A. Gerchman S.E., Yu, X. Rees C. Graziano V. Ramakrishnan V. Thomas J.O. EMBO J. 1996; 15: 3421-3429Crossref PubMed Scopus (128) Google Scholar); functional conservation of this domain was confirmed by studies demonstrating the nucleosome-binding properties of HNF3 (35Cirillo L.A. McPherson C.E. Bossard P. Stevens K. Cherian S. Yon Shim E. Clark K.L. Burley S.K. Zaret K.S. EMBO J. 1998; 17: 244-254Crossref PubMed Scopus (303) Google Scholar). However, the mechanism by which HNF3 transactivates hepatic-specific genes remains unclear. We show here that in vitro reconstitution of AFP expression patterns requires both activating and repressive influences. In order to model the tissue-specific expression of the AFP gene, we have used Xenopus laevis egg extracts as a source of histones and nucleosome assembly factors that can reconstitute physiologically spaced nucleosomes in vitro. Our results indicate that the general repressive nature of nucleosomal DNA is necessary to restrict transcription factor access in a non-hepatic environment. Transcription activation, within the context of chromatin, is achieved by binding of hepatic-enriched factors that mediate the restructuring of chromatin into a transcriptionally competent form. Furthermore, we find that HNF3α, in the absence of other hepatic-specific factors, is capable of activating AFP transcription both in vitro and in vivo, demonstrating a critical role for this fork head homolog in programming liver-specific expression patterns. The AFP(1.0)-lacZ vector was constructed by replacing the 177-bp Bam HI-Hin dIII fragment of (pA)3-APΔ44-lacZ (36Spear B.T. Longley T. Moulder S. Wang S.L. Peterson M.L. DNA Cell Biol. 1995; 14: 635-642Crossref PubMed Scopus (21) Google Scholar) with a 1.0-kb Bam HI-Hin dIII fragment containing the AFP promoter/repressor region. The AFP(3.8)-lacZ vector was generated by inserting a 2.8-kb Bam HI fragment containing AFP enhancer element I into the Bam HI site of AFP(1.0)-lacZ. The HNF3α and HNF3β eukaryotic expression vector(s) were a kind gift from Dr. Robert Costa. The HNF3 empty vector was generated by removing the entire HNF3α cDNA insert from the HNF3α vector. The HNF3α bacterial expression vector was kindly provided by Dr. Kenneth Zaret. To obtain immobilized templates, AFP(1.0)-lacZ and AFP(3.8)-lacZ were digested with Eco RI and Cla I. The resulting fragments were Klenow end-filled with biotin 21-dUTP (CLONTECH) and biotin 14-dATP (Life Technologies, Inc.) to generate uniquely biotin-labeled Eco RI sites. Unincorporated nucleotides and small fragments were removed by gel filtration (Chromaspin 1000,CLONTECH). The largest 9.0-kb fragment encompassing the AFP enhancer I and promoter sequences were coupled to streptavidin-coated paramagnetic beads (Dynal) in Kilobase Binding Buffer (Dynal) on a rotating platform at room temperature overnight exactly as described by the manufacturer. Coupled beads were washed 3 times with 2 m NaCl, TE, pH 8.0, and stored in phosphate-buffered saline at 4 °C until use. HeLa and HepG2 cells were transfected with an AFP reporter construct (AFP(1.0)-lacZ) and either an HNF3 empty vector or an HNF3 expression vector along with a CAT control vector using the calcium phosphate protocol as described (36Spear B.T. Longley T. Moulder S. Wang S.L. Peterson M.L. DNA Cell Biol. 1995; 14: 635-642Crossref PubMed Scopus (21) Google Scholar). Forty-eight hours after transfection, cells were harvested. β-Galactosidase assays were performed as described previously (36Spear B.T. Longley T. Moulder S. Wang S.L. Peterson M.L. DNA Cell Biol. 1995; 14: 635-642Crossref PubMed Scopus (21) Google Scholar) and normalized to CAT activity to control for variations in transfection. Hepatocarcinoma cell extracts were prepared from human HepG2 cells according to the method of Dignam et al. (37Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar) with the following minor modifications. Cells were grown to 70% confluence and harvested by scraping into phosphate-buffered saline. Washed pellets were resuspended in hypotonic buffer (20 mm HEPES, pH 7.9, 10 mm NaCl, 1.5 mm MgCl2, 2 mm dithiothreitol). After swelling 10 min on ice, cells were pelleted and resuspended in hypotonic buffer containing 0.05% Nonidet P-40 prior to Dounce homogenization (Wheaton, type B). Protein extracts were dialyzed against 2 changes of nuclear dialysis buffer (NDB: 20 mmHEPES, pH 7.9, 50 mm KCl, 0.2 mm EDTA, 20% glycerol, 1 mm dithiothreitol, 0.2 mmphenylmethylsulfonyl fluoride) for 2 h. each. Final protein concentration ranged from 7 to 12 μg/μl. HeLa nuclear extract was prepared exactly as described in Current Protocols in Molecular Biology (38Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987)Current Protocols in Molecular Biology (Janssen, K., ed) 1st Ed., Current Protocols, BostonGoogle Scholar). Final protein concentration ranged from 8 to 10 μg/μl.Xenopus egg chromatin assembly extracts were prepared exactly as described previously (39Barton M.C. Emerson B.M. Methods Enzymol. 1996; 274: 299-312Crossref PubMed Scopus (9) Google Scholar). Final protein concentrations ranged from 40 to 60 μg/μl. Recombinant HNF3α protein was expressed in Escherichia coli and purified exactly as described previously (40Zaret K.S. Stevens K. Protein Expression Purif. 1995; 6: 821-825Crossref PubMed Scopus (24) Google Scholar). Final protein concentration was approximately 150 ng/μl. Prior to nucleosome assembly, immobilized AFP templates were preincubated for 20 min at room temperature with the indicated extracts or NDB. Xenopus egg cytoplasmic fraction (HSS) in an amount previously determined to fully repress transcription was added to assemble the bead-DNA into chromatin for 1 h at 22 °C. Prior to transcription, the assembled templates were washed 3 times in NDB (unless otherwise indicated). Washed templates were then in vitro transcribed upon addition of an RNA polymerase II-containing nuclear HeLa extract (37Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar) and an NTP/salts/energy-generating mixture to give final concentrations of 0.6 mm CTP, UTP, GTP, ATP, 5 mm MgCl2, 50 mm KCl, 5 mm creatine phosphate, 10 units/ml of creatine kinase, 0.02% Nonidet P-40 (Sigma), and 12.6 mm HEPES, pH 7.9. After a 60-min incubation at 30 °C, RNA products were purified and analyzed by primer extension and gel electrophoresis (41Waterman M.L. Fischer W.H. Jones K.A. Genes Dev. 1991; 5: 656-669Crossref PubMed Scopus (294) Google Scholar). Nucleosome assembly on the AFP/bead DNA was assessed by micrococcal nuclease (Roche Molecular Biochemicals) digestion after a 2-h incubation at 22 °C with fractionated Xenopus egg extract HSS containing 3 mm ATP and 5 mm MgCl2. Samples were digested and analyzed exactly as described previously (42Barton M.C. Madani N. Emerson B.M. Genes Dev. 1993; 7: 1796-1809Crossref PubMed Scopus (39) Google Scholar). AFP bead/DNA was assembled into chromatin under conditions exactly as described for in vitro transcription analysis and then subjected to restriction enzyme digestion. Chromatin-assembled bead/DNA was washed once in 1 × React 2 restriction enzyme buffer (50 mm Tris-Cl, pH 8.0, 10 mm MgCl2, 50 mm NaCl; Life Technologies, Inc.) prior to resuspension in the same buffer containing Hin cII (Life Technologies, Inc.) at 25 units/μg of DNA. Following a 30-min incubation at 37 °C, samples were digested with 1 mg/ml proteinase K (Life Technologies, Inc.) in 0.25% SDS, 12.5 mm EDTA, pH 8.0, for 1 h at 37 °C. For the Hin cII fragment release assay (Fig. 5 A), the purified DNA was resuspended in agarose gel loading dye and samples were analyzed by Southern blot as described previously (42Barton M.C. Madani N. Emerson B.M. Genes Dev. 1993; 7: 1796-1809Crossref PubMed Scopus (39) Google Scholar). A 23-bp probe (+5 to +28) was used to detect the 84-bp released fragment (−55 to +29). To measure accessibility at the distal Hin cII site (Fig. 5 B), the purified DNA was resuspended in 1 × React 1 restriction enzyme buffer (50 mm Tris-Cl, pH 8.0, 10 mm MgCl2; Life Technologies, Inc.) and digested to completion with 5 units of Acc I (Life Technologies, Inc.). Digested samples were analyzed by Southern blot using a 24-bp probe (+3333 to +3356) to detect a 4.0-kb Eco/Hin cII fragment. Autoradiograms were scanned and quantified using ImageQuaNT (Molecular Dynamics, version 4.2) software. For the Hin cII fragment release assay, activation over a buffer preincubated control was determined for three independent experiments and the average fold activation (± S.E.) was determined after normalizing to the buffer control. To determine the percent accessibility at the distal Hin cII site, we divided the intensity of the 4.0 kb released fragment by the sum of the 6.4-kb parent band and the released fragment. The fold activation was determined after normalizing to the buffer control. AFP bead/DNA was mock chromatin-assembled by preincubation with protein extracts in the presence or absence of recombinant HNF3α (750 ng) for 20 min prior to a 60-min incubation in egg extract buffer (100 mm KCl, 4 mm MgCl2, 10 mm HEPES, pH 7.2, 100 mm sucrose, 0.1 mm EGTA) ± 3 mm ATP. Assembled bead/DNA was washed 3 times in NDB containing 0.5% Nonidet P-40, and then incubated in an NTP/salts/energy-generating mixture to give final concentrations of 0.6 mm CTP, UTP, GTP, ATP, 5 mm MgCl2, 50 mm KCl, 5 mm creatine phosphate, 10 units/ml of creatine kinase, 0.02% Nonidet P-40 (Sigma) and 12.6 mmHEPES, pH 7.9. After a 60-min incubation at 30 °C, RNA products were purified and analyzed by primer extension and gel electrophoresis (41Waterman M.L. Fischer W.H. Jones K.A. Genes Dev. 1991; 5: 656-669Crossref PubMed Scopus (294) Google Scholar). To explore the basis for tissue-specific AFP regulation, we have employed an in vitro transcription system for AFP. The well characterized human hepatoma cell line HepG2 (43Aden D.P. Fogel A. Plotkin S. Damjanov I. Knowles B.B. Nature. 1979; 282: 615-618Crossref PubMed Scopus (1038) Google Scholar) was used as a source of AFP trans-activating factors. Human cervical carcinoma HeLa cells were chosen as a source of non-liver transcription factors. HepG2 cells actively express both endogenous and transiently introduced AFP, whereas HeLa cells do not (19Godbout R. Ingram R. Tilghman S.M. Mol. Cell. Biol. 1986; 6: 477-487Crossref PubMed Scopus (115) Google Scholar). To establish an in vitro chromatin transcription assay for the AFP gene, we have attached AFP DNA to streptavidin-coated paramagnetic beads. This system, based on a design for transcriptional analysis of the Drosophila hsp70 promoter (44Sandaltzopoulos R. Blank T. Becker P.B. EMBO J. 1994; 13: 373-379Crossref PubMed Scopus (82) Google Scholar), facilitates the rapid purification and concentration of chromatin-assembled templates. A plasmid containing 3.8 kb of mouse AFP regulatory sequence (AFP(3.8)-lacZ), including enhancer I and the distal and proximal promoter elements (36Spear B.T. Longley T. Moulder S. Wang S.L. Peterson M.L. DNA Cell Biol. 1995; 14: 635-642Crossref PubMed Scopus (21) Google Scholar), was restriction enzyme-digested, biotin-end labeled and coupled to streptavidin-coated magnetic beads. The immobilized DNA (AFP/bead DNA) was then transcribed under standard in vitro transcription conditions with either HeLa or HepG2 extract (Fig. 1). Nucleosome-free AFP/bead DNA is efficiently transcribed by both extracts, with peak transcription occurring in the presence of 15 μl of extract, each containing approximately 150 μg of total protein (lanes 3 and 6). These results indicate that restrictions required to direct tissue-specific expression of AFP in vivo cannot be recapitulated on free DNA. To recreate in vitro the physiological constraints conferred by chromatin in vivo, AFP/bead DNA was subjected to nucleosome assembly. Chromatin assembly was achieved by incubation with fractionated Xenopus egg extract. The cytoplasmic fraction or high speed supernatant (HSS) of Xenopus eggs efficiently assembles physiologically spaced nucleosomes (45Rhodes D. Laskey R.A. Methods Enzymol. 1989; 170: 575-585Crossref PubMed Scopus (63) Google Scholar) and has been used in previous chromatin transcription studies (42Barton M.C. Madani N. Emerson B.M. Genes Dev. 1993; 7: 1796-1809Crossref PubMed Scopus (39) Google Scholar, 46Barton M.C. Emerson B.M. Genes Dev. 1994; 8: 2453-2465Crossref PubMed Scopus (51) Google Scholar). Efficiency of chromatin assembly was assessed by micrococcal nuclease digestion. A time course of digestion with micrococcal nuclease indicated the formation of a repetitive array of nucleosomes on the bead/DNA (Fig.2 A). This pattern is identical to that observed on uncoupled AFP DNA incubated in Xenopus HSS (data not shown). We further assessed the extent of chromatin assembly by measuring transcription levels. Assembly of AFP/bead DNA into nucleosomes in the presence of HSS resulted in repression of AFP transcription (Fig. 2 B, compare lanes 1 and 2). This transcriptional repression was maintained during washes in a low salt (50 mm KCl) buffer. However, repression was alleviated by washing the chromatin-assembled bead/DNA in a high salt (3 m KCl) wash buffer (lane 3). As 3 m KCl is a sufficiently high salt concentration to disrupt histone/histone and histone/DNA interactions, these data suggest that the repression of AFP transcription observed in lane 2 is mediated by nucleosome assembly. Together, these results indicate that AFP/bead DNA is efficiently assembled into nucleosomal DNA in the presence of Xenopus egg extract. As shown above (Fig. 1), HepG2 and HeLa extracts transcribe naked AFP DNA templates in vitro with equal efficiencies. To determine whether these transcriptionally competent extracts were capable of establishing active transcription on a chromatin-assembled template, AFP/bead DNA was incubated with increasing amounts of either HeLa or HepG2 extracts or in nuclear extract buffer alone prior to chromatin assembly (Fig.3). This “programming” phase allows transactivators and repressors to bind their respective sites on the nucleosome-free template prior to reconstitution into chromatin. Nucleosome-assembled templates, programmed in this way, were washed in low salt buffer prior to transcription to remove any unassociated proteins and nonspecific transcription repressors; this washing step further ensures that only DNA-associated proteins and protein complexes are present during the in vitro transcription analysis. Chromatin-mediated repression could not be alleviated by providing HeLa factors prior to nucleosome assembly (Fig. 3 A, compare lanes 1 and 2–4). Thus, general transcription factors and non-hepatic transactivators provided by the HeLa extract are insufficient to mediate assembly of a chromatin template which supports transcription. In contrast, incubating AFP/bead DNA with HepG2 extract during the programming stage, prior to nucleosome assembly, resulted in transcriptionally active templates (lanes 5–7). Comparison with a nucleosome-free AFP template control transcription (data not shown) revealed that the HepG2 preincubation rescued 90% (±3.9% S.E.) of free DNA transcription levels, indicating almost complete derepression of the chromatin template. An equivalent level of derepression was obtained when hepatoma factors were introduced concomitant with egg extract addition (Fig. 3 B, compare lanes 2 and 4, 7 and 9). Hepatoma factors can, therefore, successfully compete with histones to bind their respective sites during on-going chromatin assembly and maturation. Addition of a low salt wash step immediately after pre-binding of the hepatoma extract did not significantly alter hepatoma-mediated assembly into an active chromatin template, indicating the stability of the protein/DNA interactions (data not shown). To confirm that preincubation with cellular extract under transcription conditions did not disrupt nucleosome assembly, chromatin templates preincubated with buffer, HepG2, or HeLa extract were subjected to a limit digest with micrococcal nuclease (Fig.3 C). Southern analysis of the digested DNA was performed using either a transcriptional start site oligo (+5 to +28) or the full-length AFP(3.8)-lacZ plasmid as a probe. Equivalent amounts of mononucleosomes were observed under all three transcription conditions, indicating that HepG2-mediated transcription activation was not due to gross inhibition of nucleosome assembly. Nucleosome-free AFP templates which lack the enhancer element but retain 1.0 kb of upstream regulatory sequence (AFP(1.0)-lacZ), are transcribed as efficiently as the full-length enhancer-containing construct (AFP(3.8)-lacZ) in in vitro transcription assays with nuclear HeLa extract (data not shown). Transcription analysis of chromatin-assembled templates indicates that the enhancer is not required to establish hepatoma-activated AFP expression in vitro (Fig. 3 B, lanes 6–9). These results indicate that 1.0 kb of AFP upstream regulatory sequence is sufficient to confer liver-specific expression, consistent with previous transfection studies by Tilghman and colleagues (19Godbout R. Ingram R. Tilghman S.M. Mol. Cell. Biol. 1986; 6: 477-487Crossref PubMed Scopus (115) Google Scholar). These data demonstrate that HepG2 extract contains factors which are capable of stably associating with AFP regulatory sequences and directing the assembly of an accessible chromatin structure. HeLa extract is unable to program transcriptionally competent nucleosome-assembled DNA, indicating either a lack of essential activators or the presence of repressors which only function within the context of chromatin. Thus, our in vitro chromatin transcription assay system successfully recapitulates the restricted AFP expression pattern observed in vivo, indicating the importance of chromatin structure for tissue-specific regulation of AFP. These results along with the robust transcription of nucleosome-free AFP DNA observed in HeLa extract (Fig. 1), suggest that hepatic-specific factors may enhance the formation of functional preinitiation complexes on the nucleosome-assembled AFP template. One likely candidate for such an hepatic-enriched factor is the winged helix protein, HNF3, which regulates the transcription of numerous liver-specific genes including albumin, transthyretin, and phosphoenolpyruvate carboxykinase (reviewed in Refs. 30Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (577) Google Scholar an
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