Human SSRP1 Has Spt16-dependent and -independent Roles in Gene Transcription

The facilitating chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16, has been shown to regulate transcription elongation through a chromatin template in vitro and on specific genes in cells. However, its global role in transcription regulation in human cells remains largely elusive. We conducted spotted microarray analyses using arrays harboring 8308 human genes to assess the gene expression profile after knocking down SSRP1 or Spt16 levels in human non-small cell lung carcinoma (H1299) cells. Although the changes of these transcripts were surprisingly subtle, there were ∼170 genes whose transcript levels were either reduced or induced >1.5-fold. Approximately 106 genes with >1.2-fold change at the level of transcripts were the common targets of both SSRP1 and Spt16 (∼1.3%). A subset of genes was regulated by SSRP1 independent of Spt16. Further analyses of some of these genes not only verified this observation but also identified the serum-responsive gene, egr1, as a novel target for both SSRP1 and Spt16. We further showed that SSRP1 and Spt16 are important for the progression of elongation RNA pol II on the egr1 gene. These results suggest that SSRP1 has Spt16-dependent and -independent roles in regulating gene transcription in human cells. The facilitating chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16, has been shown to regulate transcription elongation through a chromatin template in vitro and on specific genes in cells. However, its global role in transcription regulation in human cells remains largely elusive. We conducted spotted microarray analyses using arrays harboring 8308 human genes to assess the gene expression profile after knocking down SSRP1 or Spt16 levels in human non-small cell lung carcinoma (H1299) cells. Although the changes of these transcripts were surprisingly subtle, there were ∼170 genes whose transcript levels were either reduced or induced >1.5-fold. Approximately 106 genes with >1.2-fold change at the level of transcripts were the common targets of both SSRP1 and Spt16 (∼1.3%). A subset of genes was regulated by SSRP1 independent of Spt16. Further analyses of some of these genes not only verified this observation but also identified the serum-responsive gene, egr1, as a novel target for both SSRP1 and Spt16. We further showed that SSRP1 and Spt16 are important for the progression of elongation RNA pol II on the egr1 gene. These results suggest that SSRP1 has Spt16-dependent and -independent roles in regulating gene transcription in human cells. In eukaryotic cells, DNA is packaged with core histones and other chromosomal proteins in the form of chromatin, which limits the accessibility of DNA and inhibits the progression of RNA polymerases as they copy genetic information from the DNA. Thus altering the repressive nature of chromatin is necessary for the cells to implement all of the nuclear activities on chromatin (1Formosa T. Curr. Top Microbiol. Immunol. 2003; 274: 171-201PubMed Google Scholar). There are at least three types of protein complexes for this function (1Formosa T. Curr. Top Microbiol. Immunol. 2003; 274: 171-201PubMed Google Scholar, 2Singer R.A. Johnston G.C. Biochem. Cell Biol. 2004; 82: 419-427Crossref PubMed Scopus (28) Google Scholar). The first type acts by covalently modifying the histones and non-histone chromatin proteins through phosphorylation, acetylation, ubiquitylation, and/or methylation (1Formosa T. Curr. Top Microbiol. Immunol. 2003; 274: 171-201PubMed Google Scholar, 2Singer R.A. Johnston G.C. Biochem. Cell Biol. 2004; 82: 419-427Crossref PubMed Scopus (28) Google Scholar). The second type of complex uses ATP hydrolysis to mobilize and/or to alter the structure of nucleosomes, such as the SWI/SNF complex (2Singer R.A. Johnston G.C. Biochem. Cell Biol. 2004; 82: 419-427Crossref PubMed Scopus (28) Google Scholar, 3Vignali M.H.A. Neely K.E. Workman J.L. Mol. Cell. Biol. 2000; 20: 1899-1910Crossref PubMed Scopus (590) Google Scholar, 4Svejstrup J.Q. Curr. Opin. Genet. Dev. 2002; 12: 156-161Crossref PubMed Scopus (34) Google Scholar, 5Akey C.W. Luger K. Curr. Opin. Struct. Biol. 2003; 13: 6-14Crossref PubMed Scopus (163) Google Scholar). The third type of chromatin-modulating complex disrupts and deposits the nucleosomes without utilizing ATP during transcriptional elongation (4Svejstrup J.Q. Curr. Opin. Genet. Dev. 2002; 12: 156-161Crossref PubMed Scopus (34) Google Scholar, 6Belotserkovskaya R. Reinberg D. Curr. Opin. Genet. Dev. 2004; 14: 139-146Crossref PubMed Scopus (105) Google Scholar). One of the latter members is facilitating chromatin transcription (FACT) 2The abbreviations used are: FACT, facilitating chromatin transcription; CHIP, chromatin immunoprecipitation; RT, reverse transcription; HMG, high mobility group; CTD, carboxyl-terminal domain; pol, polymerase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; SRF, serum-response factor; siRNA, short interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PIC, preassembled initiation complex; R, reverse; F, forward; tet, tetracycline. (4Svejstrup J.Q. Curr. Opin. Genet. Dev. 2002; 12: 156-161Crossref PubMed Scopus (34) Google Scholar, 6Belotserkovskaya R. Reinberg D. Curr. Opin. Genet. Dev. 2004; 14: 139-146Crossref PubMed Scopus (105) Google Scholar, 7Orphanides G. LeRoy G. Chang C.H. Luse D.S. Reinberg D. Cell. 1998; 92: 105-116Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). FACT is a heterodimeric complex consisting of Spt16 and SSRP1 (structure-specific recognition protein-1) (8Bruhn S.L. Pil P.M. Essigmann J.M. Housman D.E. Lippard S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2307-2311Crossref PubMed Scopus (235) Google Scholar, 9Orphanides G. Wu W.H. Lane W.S. Hampsey M. Reinberg D. Nature. 1999; 400: 284-288Crossref PubMed Scopus (443) Google Scholar). Both Spt16 and SSRP1 are highly conserved in all eukaryotes. Human Spt16 is a 120-kDa protein and contains a highly acidic and serine-rich carboxyl terminus. It binds to H2A-H2B dimers and to mononucleosomes (10Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). It has 36% identity to its Saccharomyces cerevisiae ortholog Spt16/Cdc68 (10Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). The yeast Spt16/Cdc68 was identified in two independent screens for genes involved in transcription regulation (11Malone E.A. Clark C.D. Chiang A. Winston F. Mol. Cell. Biol. 1991; 11: 5710-5717Crossref PubMed Google Scholar, 12Rowley A. Singer R.A. Johnston G.C. Mol. Cell. Biol. 1991; 11: 5718-5726Crossref PubMed Scopus (100) Google Scholar). Genetic studies in yeast suggest that Spt16/Cdc68 is required for the normal transcription of many loci (11Malone E.A. Clark C.D. Chiang A. Winston F. Mol. Cell. Biol. 1991; 11: 5710-5717Crossref PubMed Google Scholar) and has both positive and negative effects on gene expression (12Rowley A. Singer R.A. Johnston G.C. Mol. Cell. Biol. 1991; 11: 5718-5726Crossref PubMed Scopus (100) Google Scholar). Spt16/Cdc68 is essential for yeast cell growth because spt16 null mutants are nonviable (11Malone E.A. Clark C.D. Chiang A. Winston F. Mol. Cell. Biol. 1991; 11: 5710-5717Crossref PubMed Google Scholar). The mechanism of how Spt16/Cdc68 affects transcription is suggested by the placement of Spt16/Cdc68 into a histone group of spt (suppressor of TY) genes that encode histones and also functionally related proteins, including Spt4, Spt5, Spt6, Spt11 and Spt12 (11Malone E.A. Clark C.D. Chiang A. Winston F. Mol. Cell. Biol. 1991; 11: 5710-5717Crossref PubMed Google Scholar). This group of proteins functions by altering chromatin properties and increasing or decreasing their dosage affects on transcription regulation (13Evans D.R. Brewster N.K. Xu Q. Rowley A. Altheim B.A. Johnston G.C. Singer R.A. Genetics. 1998; 150: 1393-1405Crossref PubMed Google Scholar). The partner of Spt16 in the FACT complex, SSRP1, is a high mobility group (HMG) domain containing protein. It binds to cruciform or linear duplex DNA as well as DNA modified by the anticancer drug cisplatin (8Bruhn S.L. Pil P.M. Essigmann J.M. Housman D.E. Lippard S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2307-2311Crossref PubMed Scopus (235) Google Scholar, 14Shirakata M. Huppi K. Usuda S. Okazaki K. Yoshida K. Sakano H. Mol. Cell. Biol. 1991; 11: 4528-4536Crossref PubMed Scopus (87) Google Scholar, 15Hertel L. Foresta P. Barbiero G. Ying G.G. Bonelli G. Baccino F.M. Landolfo S. Gariglio M. Biochimie (Paris). 1997; 79: 717-723Crossref PubMed Scopus (8) Google Scholar, 16Yarnell A.T. Oh S. Reinberg D. Lippard S.J. J. Biol. Chem. 2001; 276: 25736-25741Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The conserved amino terminus of SSRP1 is homologous to the yeast Pob3 protein, whereas the function of the HMG domain is provided by the small HMG-box polypeptide Nhp6 in yeast (17Wittmeyer J. Joss L. Formosa T. Biochemistry. 1999; 38: 8961-8971Crossref PubMed Scopus (106) Google Scholar, 18Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar, 19Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar). The human SSRP1 counterparts of the yeast Pob3 and Nhp6 proteins were detected during apoptosis as the products of caspases 3- and 7-mediated cleavage of SSRP1 (20Landais I. Lee H. Lu H. Cell Death Differ. 2006; 13: 1866-1878Crossref PubMed Scopus (11) Google Scholar), indicating the importance of SSRP1 for cell survival. Indeed, both yeast Pob3 and mammalian SSRP1 are essential for cell (17Wittmeyer J. Joss L. Formosa T. Biochemistry. 1999; 38: 8961-8971Crossref PubMed Scopus (106) Google Scholar, 18Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar, 21Formosa T. Ruone S. Adams M.D. Olsen A.E. Eriksson P. Yu Y. Rhoades A.R. Kaufman P.D. Stillman D.J. Genetics. 2002; 162: 1557-1571Crossref PubMed Google Scholar) and animal (22Cao S. Bendall H. Hicks G.G. Nashabi A. Sakano H. Shinkai Y. Gariglio M. Oltz E.M. Ruley H.E. Mol. Cell. Biol. 2003; 23: 5301-5307Crossref PubMed Scopus (60) Google Scholar) viability. Similar to Spt16, SSRP1 also has an acidic and serine-rich carboxyl terminus that most likely facilitates its binding to histone proteins. Supporting this is the observation that SSRP1 can bind to H3-H4 tetramers (10Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). The current model proposes that FACT disrupts nucleosomes, which allow RNA polymerases to access DNA, and then it reassembles the nucleosomes (10Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar, 21Formosa T. Ruone S. Adams M.D. Olsen A.E. Eriksson P. Yu Y. Rhoades A.R. Kaufman P.D. Stillman D.J. Genetics. 2002; 162: 1557-1571Crossref PubMed Google Scholar). This property gives the FACT complex the ability to regulate transcription initiation (23Biswas D. Yu Y. Prall M. Formosa T. Stillman D.J. Mol. Cell. Biol. 2005; 25: 5812-5822Crossref PubMed Scopus (71) Google Scholar, 24Mason P.B. Struhl K. Mol. Cell. Biol. 2003; 23: 8323-8333Crossref PubMed Scopus (265) Google Scholar), elongation (7Orphanides G. LeRoy G. Chang C.H. Luse D.S. Reinberg D. Cell. 1998; 92: 105-116Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 24Mason P.B. Struhl K. Mol. Cell. Biol. 2003; 23: 8323-8333Crossref PubMed Scopus (265) Google Scholar, 25Saunders A. Werner J. Andrulis E.D. Nakayama T. Hirose S. Reinberg D. Lis J.T. Science. 2003; 301: 1094-1096Crossref PubMed Scopus (232) Google Scholar), and DNA replication (17Wittmeyer J. Joss L. Formosa T. Biochemistry. 1999; 38: 8961-8971Crossref PubMed Scopus (106) Google Scholar, 26Okuhara K. Ohta K. Seo H. Shioda M. Yamada T. Tanaka Y. Dohmae N. Seyama Y. Shibata T. Murofushi H. Curr. Biol. 1999; 9: 341-350Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 27Schlesinger M.B. Formosa T. Genetics. 2000; 155: 1593-1606Crossref PubMed Google Scholar) and also to be involved in DNA damage response (28Keller D.M. Lu H. J. Biol. Chem. 2002; 277: 50206-50213Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 29Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). In addition to its general role, SSRP1 also functions as a co-regulator for several transcription activators, such as serum-response factor (SRF) (30Spencer J.A. Baron M.H. Olson E.N. J. Biol. Chem. 1999; 274: 15686-15693Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and p63 (31Zeng S.X. Dai M.S. Keller D.M. Lu H. EMBO J. 2002; 21: 5487-5497Crossref PubMed Scopus (75) Google Scholar). Despite the current knowledge, it remains obscure if SSRP1 has an Spt16-independent role in gene regulation. Also, it is still unclear if FACT plays a global or gene-specific role in transcriptional regulation in human cells. To address these questions, we generated tet-inducible siRNA cell lines for each of these two proteins using H1299 cells that are p53-deficient (31Zeng S.X. Dai M.S. Keller D.M. Lu H. EMBO J. 2002; 21: 5487-5497Crossref PubMed Scopus (75) Google Scholar). Using these cell lines, we performed spotted microarray analysis and found that the expression of many genes was altered after ablation of the endogenous SSRP1 or Spt16 levels by siRNA. Surprisingly, the effect was moderate. However, there was a subset of genes (∼170) whose expression was either up-regulated or down-regulated after induction of siRNA against SSRP1 or Spt16. We further characterized some of the genes that displayed more apparent changes and found that SSRP1 and Spt16 shared common targets, as well as individually regulated genes. In particular, we identified the serum-responsive gene, egr1 (early growth response 1), as a novel target for both SSRP1 and Spt16. Either SSRP1 or Spt16 was indispensable for the expression of EGR1 in response to serum stimulation. We further elucidated that SSRP1 and Spt16 are important for the progression of RNA pol II on the coding region of the egr1 gene. Hence, our study suggests that SSRP1 and Spt16 indeed work together for the expression of a number of genes, whereas SSRP1 also appears to have an independent role in regulating the expression of a subset of genes in human cells. Plasmids and Antibodies—The pHteto siRNA cloning vector was the generous gift of Mathew Thayer and Dan Stauffer (Oregon Health & Science University, Portland, OR) (32Kuninger D. Stauffer D. Eftekhari S. Wilson E. Thayer M. Rotwein P. Hum. Gene Ther. 2004; 15: 1287-1292Crossref PubMed Scopus (22) Google Scholar). Oligonucleotides ctagGCTCAGGACTGCTCTACCCttcaagagaGGGTAGAGCAGTCCTGAGCtttttggaaa and agcttttccaaaaaGCTCAGGACTGCTCTACCCtctcttgaaGGGTAGAGCAGTCCTGAGC (capital letters indicate the targeting sequences) containing human SSRP1 19-nucleotide targeting sequences or ctagGGAATTAAGACATGGTGTGttcaagagaCACACCATGTCTTAATTCCtttttggaaa and agcttttccaaaaaGGAATTAAGACATGGTGTGtctcttgaaCACACCATGTCTTAATTCC (capital letters indicate the targeting sequences) containing human Spt16 19-nucleotide targeting sequences were annealed and ligated into SpeI and HindIII sites. Oligomers of ssrp1 siRNA, spt16 siRNA, and scrambled siRNA (5′-AAGCGCGCTTTGTAGGATTC-3′) were synthesized (Dharmacon). pcDNA3-FLAG-SSRP1 was described previously (31Zeng S.X. Dai M.S. Keller D.M. Lu H. EMBO J. 2002; 21: 5487-5497Crossref PubMed Scopus (75) Google Scholar). Anti-SSRP1 and anti-Spt16 antibodies were used for Western blot assays, as described previously (31Zeng S.X. Dai M.S. Keller D.M. Lu H. EMBO J. 2002; 21: 5487-5497Crossref PubMed Scopus (75) Google Scholar, 33Li Y. Keller D.M. Scott J.D. Lu H. J. Biol. Chem. 2005; 280: 11869-11875Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Mouse monoclonal SSRP1 antibody 5B10 was generated by Zymed Laboratories Inc. and purified as described previously (20Landais I. Lee H. Lu H. Cell Death Differ. 2006; 13: 1866-1878Crossref PubMed Scopus (11) Google Scholar). The anti-EGR1, anti-SRF, and anti-Spt16 (used in chromatin immunoprecipitation assay) antibodies were purchased from Santa Cruz Biotechnology. The mouse monoclonal RNA polymerase II H5 antibody, which recognizes the RNA pol II Ser-2 phospho-isoform, was purchased from Babco-Covance. The anti-FLAG, anti-α-tubulin, rabbit polyclonal IgG, mouse monoclonal IgG, and mouse monoclonal IgM antibodies were purchased from Sigma. Cell Culture—H1299 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 0.1 mg/ml streptomycin, at 37 °C in 5% CO2. Generation of Inducible Tet-On Cell Line—H1299pcDNA6-TR cells, which stably express the tet repressor, were transfected with pHtetoScramble, pHtetoSSRP1siRNA, or pHtetoSpt16siRNA plasmid and then selected in the medium containing 90 μg/ml hygromycin. Individual colonies were expanded into 12-well plates. To induce the siRNA expression, doxycycline was added to the media at a final concentration of 5 μg/ml. After doxycycline induction, cells were harvested for cell lysate preparation. SSRP1 or Spt16 expression level was checked by Western blot with anti-SSRP1 or anti-Spt16 antibodies. The colonies, which express significantly reduced levels of SSRP1 or Spt16, were maintained and used for further study. Spotted Microarray—One clone for each cell line (clone 19 of H1299pHtetoSSRP1siRNA and clone 20 of H1299pHteto-Spt16siRNA) was used in the spotted microarray experiment. RNA for the Tet-On (doxycycline treatment) samples and the Tet-Off (no doxycycline treatment) samples were prepared from three independent experiments using the Qiagen RNeasy mini kit. RNAs prepared from the SSRP1 and Spt16 siRNA Tet-Off samples were pooled as a control to compare between SSRP1siRNA and Spt16siRNA samples. The samples were sent to the Microarray Core at Oregon Health & Science University. The SMChumC8400A array was used in the experiment. All samples were amplified using linear T7 amplification (Message-Amp, Ambion) and examined for integrity using a BioAnalyzer (Agilent). Reverse transcription was used to synthesize a cDNA containing aminoallyl-modified dUTP (CyScribe Post-labeling; Amersham Biosciences). Using aminoallyl-modified dUTP in both strands eliminates the requirement for dye swap experiments. Aminoallyl-modified cDNA was incubated with Cy-dye esters for a nonenzymatic and covalent attachment of either Cy5 or Cy3 to the cDNA. The experimental sample was labeled with Cy5, and the control sample was labeled with Cy3. Following cleanup, selected Cy5 and Cy3 targets were combined and applied to each of two identical slides. Arrays were hybridized using M-series LifterSlips (Erie Scientific) and deep well hybridization chambers (TeleChem). Hybridized arrays were scanned on a ScanArray 4000 XL (PerkinElmer Life Sciences) using ScanArray Express software, and ImaGene (BioDiscovery) was used to extract data from the image. The resulting data file was loaded into GeneSight for normalization using intensity-based local regression (Lowess). The normalized data were used for further segregation and clustering as described in the figure legend. RT-PCR—Total RNA was isolated from cells after different treatments, using the TRIzol (Invitrogen) protocol or Qiagen RNeasy mini kit. Reverse transcription of 5 μg of total RNA was performed in a 20-μl reaction using SuperscriptII reverse transcriptase (Invitrogen) reagent, dNTP, and oligo(dT)15 primer. After reverse transcription, 30 μl of diethyl pyrocarbonate H2O was added to the reaction. 1 μl of reverse transcription reaction was used in the following PCRs with the following primers: egr1 F, 5′-CTGACCGCAGAGTCTTTTCCTG-3′, and R, 5′-TGGGTGCCGCTGAGTAAATG-3′; dusp5 F, 5′-GTGTTGCGTGGATGTAAAACCC-3′, and R, 5′-GCTCCTCCTCTGCTTGATGTAATC-3′; plau F, 5′-CACACACTGCTTCATTGATTACCC-3′, and R, 5′-TTTTGGTGGTGACTTCAGAGCC-3′; id2 F, 5′-CGTGAGGTCCGTTAGGAAAAACAG-3′, and R, 5′-CTGACAATAGTGGGATGCGAGTC-3′; gapdh F, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′, and R, 5′-CCACCCATGGCAAATTCCATGGCA-3′; ssrp1 F, 5′-GAGCGATGACTCAGGAGAAG-3′, and R, 5′-TTACTCATCGGATCCTG-3′; spt16 F, 5′-AGATATGTGACGTGTATAACG-3′, and R, 5′-CTTCAGCTTCTCGAGTTTTAT-3′; and β-actin F, 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′, and R, 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′. Real Time PCR—1 μl of RT reaction was used in the real time PCR. A 15-μl reaction was performed using the SYBR Green PCR Master Mix (Applied Biosystems), according to the manufacturer's protocol, and amplified on the ABI7900HT. Threshold cycles (Ct) for three replicate reactions were determined using SDS2. The relative transcript abundance was calculated following normalization with the GAPDH amplicon. The data for p21 and EGR1 were collected at 80 °C, and the data for other target genes were collected at 83 °C. The following primers were used: egr1 F, 5′-CCTCCCTCTCTACTGGAGTGGAA-3′, and R, 5′-GAAGAACTTGGACATGGCTGTTTC-3′; dusp5 F, 5′-CTCAGGGTAGGTTCTCGGGACT-3′, and R, 5′-GGCGAACTCTGAGGTGCAAG-3′; plau F, 5′-ATTCCTGCCAGGGAGACTCAG-3′, and R, 5′-TTGTCCTTCAGGGCACATCC-3′; id2 F, 5′-CAGTCCCGTGAGGTCCGTTA-3′, and R, 5′-CACCAGCTCCTTGAGCTTGG-3′; p21 F, 5′-CTGGACTGTTTTCTCTCGGCTC-3′, and R, 5′-TGTATATTCAGCATTGTGGGAGGA-3′; gapdh (83 °C) F, 5′-TGGAGTCCACTGGCGTCTTC-3′, and R, 5′-TTCACACCCATGACGAACATG-3′; and gapdh (80 °C) F, 5′-GATTCCACCCATGGCAAATTC-3′, and R, 5′-AGCATCGCCCCACTTGATT-3′. Transient Transfection—H1299 cells (60% confluence in 60-mm plates) were transfected with 3 μg of pcDNA3 or pcDNA3-FLAG-SSRP1 using TransFectin (Bio-Rad). Total RNA was extracted after 48 h of transfection. siRNA Transient Transfection and Serum Stimulation Assays—H1299 cells (60% confluence in 60-mm plates) were transfected with 30 nm of the scramble, SSRP1siRNA, or Spt16siRNA using SiLentFect (Bio-Rad). At the same time, DMEM containing 10% FBS was changed to DMEM containing 0.25% FBS for serum starvation. After 48 h, cells were cultured in DMEM with 20% FBS for serum stimulation. The cells were harvested at different time points for Western blotting or RNA extraction. For Western blotting, the cell lysates were prepared as described (31Zeng S.X. Dai M.S. Keller D.M. Lu H. EMBO J. 2002; 21: 5487-5497Crossref PubMed Scopus (75) Google Scholar), and 30 μg of cell lysates were used. Cell Growth Assays—H1299pHScramble, H1299pHtetoSSRP1siRNA, and H1299pHtetoSpt16siRNA inducible cell lines were seeded at 4 × 105 cells/60-mm plate and induced for siRNA expression by adding 5 μg/ml doxycycline to the media. After 4 days of induction, the cell number was counted, and viable cells were compared among the scrambled siRNA-, SSRP1siRNA-, and Spt16siRNA-expressing cells. Chromatin Immunoprecipitation (ChIP)-Real Time PCR—H1299 cells were cultured in DMEM containing 0.25% FBS for serum starvation. After 48 h, cells were switched to media containing 20% FBS and harvested at 0, 5, and 30 min post-serum stimulation. ChIP assays were carried out as described previously (31Zeng S.X. Dai M.S. Keller D.M. Lu H. EMBO J. 2002; 21: 5487-5497Crossref PubMed Scopus (75) Google Scholar, 34Gomes N.P. Bjerke G. Llorente B. Szostek S.A. Emerson B.M. Espinosa J.M. Genes Dev. 2006; 20: 601-612Crossref PubMed Scopus (213) Google Scholar) with the indicated antibodies. After reverse of cross-linking, DNA was purified by miniprep kit (Qiagen) and eluted in 50 μl of elution buffer. 1 μl of ChIP DNA or input DNA was used as templates in real time PCRs. A 20-μl reaction was performed using the SYBR Green PCR Master Mix (Applied Biosystems), according to the manufacturer's protocol, and amplified on the ABI7300. Threshold cycles (Ct) for three replicate reactions were determined using the 7300 system SDS software. The relative fold change among the ChIP DNA samples was calculated following normalization with the input DNA. The following primers were used in the real time PCR: primers for negative control, egr1 upstream 5990F, 5′-CACGGCCTGAACAGTGCAC-3′, and egr1 upstream 5839R, 5′-AGAAAGCCAGTGGAACCATCC-3′; primers for promoter region, egr1 upstream 440F, 5′-CCCGGAAATGCCATATAAGGAGC-3′, and egr1 upstream 291R, 5′-AGTTCCCGCGTTGCCCCT-3′; egr1 upstream192F, 5′-GGGTGCAGGATGGAGGTGC-3′, and egr1 upstream 37R, 5′-TTGAAGGGTCTGGAACGGCA-3′; primers for coding region, egr1 downstream 1292F, 5′-AACGAGAAGGTGCTGGTGGA-3′, and egr1 downstream 1408R, 5′-CCACAAGGTGTTGCCACTGTT-3′; egr1 downstream 2705F, 5′-TCAGAGCCAAGTCCTCCCTCT-3′, and egr1 downstream 2836R, 5′-GAAGAACTTGGACATGGCTGTTTC-3′; c-myc downstream 4192F, CAGGCTCCTGGCAAAAGGT, and c-myc downstream 4266R, CAGTGGGCTGTGAGGAGGTT. The Establishment of siRNA-inducible Cell Lines—To determine whether SSRP1 and Spt16 are required for global or gene-specific transcription in human cells, we established inducible SSRP1 or Spt16 siRNA tet H1299 cell lines. In the presence of doxycycline, the expression of siRNA can be induced to down-regulate its target mRNA. Indeed, as shown in Fig. 1, both the protein and the mRNA levels of either SSRP1 or Spt16 were markedly knocked down when the cells were cultured with doxycycline. As a control, the level of neither α-tubulin nor β-actin was changed in either of the SSRP1- or Spt16-knock-down cells. Also, the mRNA level of Spt16 was not changed in the SSRP1-knockdown cells and vice versa for the Spt16-knockdown cells. These results indicate that we have established tet-inducible siRNA cell lines for SSRP1 and Spt16, respectively. Reduction of SSRP1 or Spt16 Level by siRNA Alters the Transcription of a Common Set of Genes—To compare the gene expression profiles obtained from SSRP1- and Spt16-knock-down cells, we used a common reference (a pool of RNA samples from both of the siRNA cell lines without doxycycline treatment) in spotted microarray. The alterations of 8308 genes were compared between the SSRP1 and Spt16 siRNA samples (three samples per cell line). The genes with more than a 1.2-fold change in both SSRP1 siRNA and Spt16 siRNA samples were displayed by unsupervised hierarchical clustering. This analysis revealed that ∼118 genes were either up-regulated or down-regulated with >1.2-fold change in both of the SSRP1 and Spt16 siRNA samples (supplemental Fig. S1A). Most of the genes (106 genes) appeared to be the common targets for both SSRP1 and Spt16, suggesting that the regulation of these genes may be executed by the FACT complex. There were more down-regulated genes (73 or 75 genes) than up-regulated genes (45 or 43 genes) in the SSRP1 or Spt16 siRNA samples. These results suggest that SSRP1 and Spt16 work together to enhance the expression of most of their target genes, although they may also act to repress the expression of a subset of genes in human cells. This result is consistent with previous results in yeast (12Rowley A. Singer R.A. Johnston G.C. Mol. Cell. Biol. 1991; 11: 5718-5726Crossref PubMed Scopus (100) Google Scholar). However, the 118 genes should not be the final number of SSRP1 and Spt16 targets in human cells because the cDNA array used for our study only contained 8308 genes. For example, p21, a previously identified target for SSRP1 (31Zeng S.X. Dai M.S. Keller D.M. Lu H. EMBO J. 2002; 21: 5487-5497Crossref PubMed Scopus (75) Google Scholar), was not in this array. Even with this limited number of genes in the array, our gene expression profile data suggest that FACT may not play a global role in gene transcription, as only ∼1.3% of the tested genes (106 of 8308 genes) displayed similar changes at their transcript levels in both SSRP1 and Spt16 knockdown samples (supplemental Fig. S1A). To further analyze the 118 affected genes, we classified them into 10 different groups based on their functions in biological processes. As shown in supplemental Fig. S1B, they are involved in a broad spectrum of functions, including cell growth/maintenance (23 or 27 of 118), nucleic acid metabolism (19 or 21 of 118), signal transduction (8 or 9 of 118), protein metabolism (11 of 118), biosynthesis (3 of 118), cell adhesion (3 of 118), etc. Most of the SSRP1 and Spt16 target genes (73 or 81 of 118) encode either novel proteins with unknown functions or proteins with unclassified functions or hypothetical proteins, and thus are put into the unclassified group (supplemental Fig. S1B). These results indicate that SSRP1 and Spt16 have a relatively broad role in various cellular activities. However, a majority of them are involved in cell growth and/or maintenance and metabolism, which are essential for cell growth. These data support the notion that SSRP1 and Spt16 are essential for cell viability (17Wittmeyer J. Joss L. Formosa T. Biochemistry. 1999; 38: 8961-8971Crossref PubMed Scopus (106) Google Scholar, 18Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar, 21Formosa T. Ruone S. Adams M.D. Olsen A.E. Eriksson P. Yu Y. Rhoades A.R. Kaufman P.D. Stillman D.J. Genetics. 2002; 162: 1557-1571Crossref PubMed Google Scholar). Indeed, ablation of either SSRP1 or Spt16 by inducible siRNA in H1299 cells severely reduced the number of viable cells (supplemental Fig. S1D). This result was also repeated in 293 cells (data not shown). It was surprising that the changes of gene expression were no more than 4-fold after knockdown of SSRP1 or Spt16 (supplemental Fig. S1, A and C; Tables 1, 2, 3). These moderate changes could be due to two possibilities