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Department of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

	Abstract

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Transcription stimulator S-II relieves RNA polymerase II (RNAPII) from transcription elongation arrest. Mice lacking the S-II gene (S-II KO mice) die at mid-gestation with impaired erythroblast differentiation, and have decreased expression of the Bcl-x gene. To understand a role of S-II in Bcl-x gene expression, we examined the distribution of transcription complex on the Bcl-x gene in S-II KO mice. The amount of RNAPII at intron 2 of the Bcl-x gene was decreased in S-II KO mice, whereas recruitment of transcription initiation factor TFIIB and RNAPII to the promoter was not decreased. Consistently, in vitro transcription analysis revealed the presence of a transcription arrest site in the Bcl-x gene intron 2, and transcription arrest at this site was overcome by S-II. Furthermore, histone acetylation on the coding region of the Bcl-x gene was decreased in S-II KO mice. In the β^major-globin gene, whose expression was also decreased in S-II KO mice, there were no changes in RNAPII distribution or histone acetylation, but the amount of histone H3 occupying the coding region was increased. These results suggest that S-II is involved in transcription of the Bcl-x and β^major-globin gene during erythroblast differentiation, by relieving transcription arrest or affecting histone modification on chromatin template.

	Introduction

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Precise control of gene transcription is essential for the regulation of cell differentiation, proliferation and survival. In early studies of eukaryotic transcription, attention was focused mainly on the proteins regulating transcription initiation, whereas more recent studies suggest that control of the transcription elongation step is also critical for gene expression (Saunders et al. 2006; Li et al. 2007). Many transcription elongation factors, which stimulate transcription by suppressing the pausing of RNA polymerase II (RNAPII) or reactivating stalled RNAPII, have been identified, such as S-II/TFIIS, elongin/SIII, ELL and TFIIF (Sims et al. 2004). The activities and regulatory mechanisms of these transcription elongation factors in vitro are well characterized in transcription systems reconstituted with purified proteins. It is not yet clear, however, if their in vitro activities are the same as that in vivo. Thus, the regulatory mechanisms of transcription elongation in vivo are poorly understood.

To better understand transcription elongation mechanisms in vivo, histone, a major chromatin component, must be taken into consideration. Transcription elongation is disturbed by histones, thus histones must be removed from the chromatin templates when transcription actively occurs (called "histone eviction") (Kristjuhan & Svejstrup 2004; Lee et al. 2004; Zhang et al. 2005). Modification of histones, such as acetylation, also contributes to the regulation of transcription elongation on chromatin template (Kristjuhan & Svejstrup 2004; Schwabish & Struhl 2004; Li et al. 2007).

S-II was originally identified as an RNAPII stimulation factor in vitro (Sekimizu et al. 1979). S-II helps RNAPII to pass through transcriptional blocks on template DNA, such as modified nucleotides and specific sequences that induce transcription arrest (Wind & Reines 2000; Adelman et al. 2005; Charlet-Berguerand et al. 2006). Moreover, recent studies indicate that S-II is necessary to overcome transcriptional barriers imposed by the nucleosomes and to facilitate transcription elongation on the chromatin DNA in vitro (Kireeva et al. 2005; Guermah et al. 2006).

Although S-II is not essential for cell viability in yeast, null mutation renders yeast cells sensitive to oxidative stress and drugs affecting nucleotide metabolism, such as 6-azauracil and mycophenolic acid (Exinger & Lacroute 1992; Koyama et al. 2003). The transcription arrest relief activity of S-II is required for resistance to these drugs (Nakanishi et al. 1995; Koyama et al. 2007). Furthermore, the SSM1 gene, whose expression is decreased in the S-II null mutant, contains transcriptional arrest sites, and S-II helps to relieve transcriptional arrest at this site in vitro (Shimoaraiso et al. 2000). Because the SSM1 gene product functions to induce resistance to 6-azauracil, S-II is suggested to confer resistance to 6-azauracil by stimulating SSM1 gene expression via arrest relief (Shimoaraiso et al. 2000). If S-II is required for this arrest relief in vivo, however, is not known.

Previously, we found that S-II gene knockout mice (S-II KO mice) are embryonic lethal (Ito et al. 2006). Mutant embryos have fewer definitive erythrocytes because of an erythroblast differentiation block in the fetal liver, a hematopoietic organ during embryonic development (Ito et al. 2006). In addition, the number of apoptotic cells is dramatically increased in S-II KO mouse fetal liver (Ito et al. 2006), consistent with the reduction in mRNA encoding an anti-apoptotic factor Bcl-xL, a splicing variant derived from the Bcl-x gene. Bcl-x is required for differentiation of definitive erythroblasts, and Bcl-x gene knockout mice are embryonic lethal and have increased erythroblast apoptosis in fetal liver (Motoyama et al. 1995; Dolznig et al. 2002). This observation suggests that increased apoptosis and erythroblast differentiation arrest in S-II KO mice is because of decreased expression of the Bcl-x gene. The functions of S-II in the transcription of the Bcl-x gene, however, remain to be determined. In the present study, we examined the involvement of S-II in the distribution of the transcription complex and the state of histone acetylation on the Bcl-x gene. We also analyzed the contribution of S-II to transcription of other erythroblast-related genes such as the β^major-globin gene.

	Results

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Decreased gene expression during erythroblast differentiation in S-II KO mouse fetal liver

Previously, we reported that Bcl-xL gene expression was decreased in S-II KO mouse fetal liver, which is a hematopoietic organ during embryonic development (Ito et al. 2006). The Bcl-x gene is essential for erythroblast differentiation (Motoyama et al. 1995; Dolznig et al. 2002; Hafid-Medheb et al. 2003), thus the decreased Bcl-x gene expression would be a reason for impaired erythroblast differentiation in S-II KO mice. In the present study, we tried to identify other genes whose expression was decreased in S-II KO mice by microarray analysis, and found that expression of some erythroblast specific genes was decreased in S-II KO mice (data not shown). We confirmed the decrease in expression of these genes in fetal liver by real-time reverse transcription-PCR (RT-PCR). In addition to Bcl-x, the expression of erythroblast-specific genes (Dolznig et al. 2001) such as β^major-globin, glycophorin A (GPA), erythroid specific delta-aminolevulinate synthase (Alas2), carbonic anhydrase 2 (Car2), coproporphyrinogen oxidase (Cpox), hydroxymethylbilane synthase (Hmbs) and transcription factor NF-E2 component p45, were decreased in S-II KO mouse fetal liver (Fig. 1A–H). Expression of another erythropoiesis-related transcription factor GATA-1 was not decreased in S-II KO mice (data not shown). Moreover, expression of the following genes was not decreased in S-II KO mice (Fig. 1I–L); c-myb, which is highly expressed in erythroblast progenitors and is not expressed in erythroblasts (Kastan et al. 1989); albumin (Alb), expressed in hepatocytes (Hoppo et al. 2004); platelet factor 4 (PF4), expressed in megakaryocytes (Pang et al. 2006); and DNase II, expressed in macrophages (Okabe et al. 2005). These results suggest that the transcription of some erythroblast-specific genes is impaired in S-II KO mice.

View larger version (27K): [in this window] [in a new window]   Figure 1  Gene expression analysis during erythroblast differentiation in S-II KO cells. (A–L) RNA prepared from fetal liver of E13.5 mouse embryo was used for analysis. (M–U) Erythroblast progenitors prepared from mouse fetal liver were induced to differentiate and RNA was prepared on differentiation days 0, 1, 2 and 3. Expression levels of each gene were analyzed by real-time RT-PCR and normalized to the Hprt expression level. Relative mRNA expression levels are presented as their ratios to the level of that in wild-type mice (A–L) or wild-type cells before differentiation induction (differentiation at 0 day) (M–U). Values are given as means ± SEM from 5 (A–L) or 3 (M–U) independent experiments. AU; arbitrary unit.

Amounts of transcription initiation complex located on the Bcl-x and β^major-globin gene promoters in S-II KO mouse fetal liver are indistinguishable from those in wild-type mice

Previous reports suggested that S-II is involved not only in transcription elongation, but also in recruiting transcription initiation complex to the GAL1 gene promoter in yeast (Prather et al. 2005). This notion was confirmed by using an in vitro transcription system (Kim et al. 2007). We examined if the amounts of initiation complex present at the Bcl-x and β^major-globin gene promoters were decreased in S-II KO erythroblasts. Chromatin immunoprecipitation experiments were carried out using an antibody recognizing the general transcription initiation factor TFIIB against nuclear extract prepared from wild-type or S-II KO mouse fetal livers. The occupancy of TFIIB on the promoter region of Bcl-x or β^major-globin gene was significantly abundant compared to that on the coding region or the promoter region of the MyoD1 gene that is not expressed in E13.5 fetal liver (Sawado et al. 2001) (Fig. 2A). The recruitment of TFIIB to the Bcl-x or β^major-globin gene promoters was not decreased in the S-II KO mouse fetal liver (Fig. 2A). These results suggest that transcription initiation process of the Bcl-x or β^major-globin gene would not be impaired in S-II KO mouse fetal liver.

View larger version (22K): [in this window] [in a new window]   Figure 2  Transcription initiation and elongation status in fetal liver of S-II KO mice. (A) Recruitment of transcription initiation complex to the Bcl-x or βmajor-globin gene promoter. Chromatin immunoprecipitation was carried out using antibody recognizing the general transcription initiation factor TFIIB against nuclear extract prepared from fetal liver of E13.5 mouse embryo. The amount of the indicated genome region was quantified by real-time PCR and the ratio of precipitated DNA to input DNA (% of IP/IN) was determined. In the middle panel (Bcl-x exon 2 and MyoD1 promoter region), only the values of WT are shown. Values are given as means ± SEM (WT, N = 9; S-II KO, N = 8). (B, C) The distribution of RNAPII on the Bcl-x gene in fetal liver of S-II KO mice. Chromatin immunoprecipitation was carried out using RNAPII antibody or control IgG (nIgG) against nuclear extract prepared from fetal liver of E13.5 mouse embryo. Primers corresponding to amplicons named promoter, exon 2, and intron 2 (B) were used for quantification by real-time PCR, and the ratio of precipitated DNA to input DNA (% of IP/IN) was determined. Values are given as means ± SEM (WT, N = 6; S-II KO, N = 3). Asterisk indicates statistically significant difference (Student's t-test, P < 0.05). (D) Fragmentation of chromatin DNA.

We hypothesized that the decreased Bcl-x gene expression in S-II KO mice was because of a defect in transcription elongation. To test this, we analyzed the amount of RNAPII in the coding region of the Bcl-x gene by performing chromatin immunoprecipitation experiments using anti-RNAPII antibody against nuclear extract prepared from fetal liver. Genomic DNA was fragmented to 200–700 base pairs (Fig. 2D). RNAPII association with the Bcl-x gene promoter was not different between the S-II KO and wild-type mice (Fig. 2C), consistent with the results that recruitment of transcription initiation complex was indistinguishable (Fig. 2A). The abundance of RNAPII at exon 2 was also comparable, whereas that at intron 2 was decreased in S-II KO mouse fetal liver compared to wild-type (Fig. 2C). These results suggest that transcription elongation is blocked between exon 2 and intron 2 in S-II KO mouse fetal liver.

Transcription arrest in the Bcl-x gene and its relief by S-II

The distribution of RNAPII shown in Fig. 2C suggested that transcription elongation is arrested between exon 2 and intron 2 of the Bcl-x gene in S-II KO mouse fetal liver. An in vitro transcription system can be used to identify transcription arrest sites on template DNA, as reported in histone H3.3 or adenosine deaminase gene (SivaRaman et al. 1990; Kash et al. 1993). Furthermore, these transcription arrests can be relieved by S-II (SivaRaman et al. 1990; Kash et al. 1993). Thus, we searched for transcription arrest sites between Bcl-x exon 2 and intron 2 that could be read-through in an S-II-dependent manner using this experimental technique. Five dC-tailed DNA fragments (Fig. 3A) were used as templates for in vitro transcription. When the transcription reaction was carried out using purified mouse RNAPII, the run-off products were synthesized with all five template DNAs (Fig. 3B, 350–420 bases). When template 3 was used, a transcript with a smaller size was detected (Fig. 3B, filled arrowheads). We suppose that the short transcript was a premature transcript because of transcription arrest at 70 base pairs downstream from the exon 2-intron 2 junction. The addition of 1 µg/mL -amanitin abolished both the run-off and small-size product (Fig. 3C), confirming that they were the transcripts synthesized by RNAPII. Next, we examined if this transcription arrest at intron 2 of the Bcl-x gene could be relieved by S-II. The addition of purified recombinant S-II decreased the amount of the small-size transcript (Fig. 3C, filled arrowheads). These results suggest that intron 2 of the Bcl-x gene contains a transcription arrest site that is read-through in an S-II-dependent manner.

View larger version (29K): [in this window] [in a new window]   Figure 3  Transcription arrest site in the Bcl-x gene intron 2 that can be read-through in an S-II-dependent manner. (A) Templates 1–5 were used as the templates for in vitro transcription. The arrow indicates the position of transcription arrest site. (B) in vitro transcription analysis using purified RNAPII in the absence of S-II. dC-tailed DNA fragments, as shown in (A), were used as a template. Transcripts synthesized in the presence of [-32P] GTP were resolved by electrophoresis on 5% polyacrylamide/8.3 M urea gels, and visualized by autoradiography. (C) In vitro transcription analysis using template 3 in the presence or absence of S-II. indicates that the reaction was carried out in the presence of -amanitin (1 µg/mL).

The transcription elongation complex contains histone acetyl transferase (HAT). HATs acetylate histones on template DNA during transcription elongation (Carey et al. 2006; Govind et al. 2007; Han et al. 2008). Based on the observation that transcription elongation is blocked in S-II KO mouse fetal liver, we assumed that histones around arrest site in the Bcl-x gene (Fig. 3A) are less acetylated in S-II KO mouse fetal liver. We carried out chromatin immunoprecipitation experiments using anti-acetylated histone H3 antibody against nuclear extract prepared from fetal liver, and determined the acetylation levels of histone H3 at the promoter and the coding region of the Bcl-x gene (Fig. 3A). The ratio of acetylated histone H3 to total histone H3 in the Bcl-x gene promoter region in S-II KO mice was indistinguishable from that in wild-type mice (Fig. 4C). In contrast, the acetylation level was decreased at exon 2, just upstream of the arrest site, and at intron 2, 0.9 kb downstream of the arrest site (Figs 3A, 4C). This result suggests that histone acetylation was impaired in fetal liver of S-II KO mice.

View larger version (21K): [in this window] [in a new window]   Figure 4  Decreased histone acetylation on exon 2 and intron 2 of the Bcl-x gene in fetal liver of S-II KO mice. Chromatin immunoprecipitation was carried out using acetylated histone H3 (AcH3) antibody, tri-methylated H3K36 (MeH3) antibody, and total histone H3 antibody against nuclear extract prepared from fetal liver of E13.5 mouse embryo. Primers corresponding to amplicons named promoter, exon 2 and intron 2 (Fig. 2B, 3A) were used for quantification by real-time PCR, and the ratio of precipitated DNA to input DNA (% of IP/IN) was determined. Values are given as means ± SEM (WT, N = 3; S-II KO, N = 4). Asterisk indicates statistically significant difference (Student's t-test, P < 0.05). AU; arbitrary unit. We could not detect statistically significant difference in the levels of total histone H3 between WT and S-II KO mice (P = 0.13) (B, Bcl-x intron 2).

Histone eviction does not occur on the chromatin of the β^major-globin gene in S-II KO mouse fetal liver

We next examined the distribution of RNAPII on the β^major-globin gene, whose expression is also decreased in S-II KO mouse fetal liver (Fig. 1). The abundance of RNAPII at the β^major-globin gene promoter or exon 3 did not differ between S-II KO mice and wild-type mice (Fig. 5B). Histone H3 acetylation on the β^major-globin gene was also not decreased in S-II KO mice (Fig. 5C; left panel), whereas the amount of histone H3 on the β^major-globin gene was increased (Fig. 5C; middle panel). These results suggest that histone eviction does not occur on the β^major-globin gene in the absence of S-II in fetal liver.

View larger version (22K): [in this window] [in a new window]   Figure 5  Analysis of the βmajor-globin gene transcription elongation state in fetal liver of S-II KO mice. (A) βmajor-globin gene structure. (B) Chromatin immunoprecipitation was carried out using RNAPII antibody or control IgG (nIgG) against nuclear extract prepared from fetal liver of E13.5 mouse embryo. Primers corresponding to amplicons named promoter and exon 3 (A) were used for quantification by real-time PCR, and the ratio of precipitated DNA to input DNA (% of IP/IN) was determined. Values are given as means ± SEM (WT, N = 4; S-II KO, N = 2). (C) Chromatin immunoprecipitation was carried out using acetylated histone H3 (AcH3) antibody or total histone H3 antibody against nuclear extract prepared from fetal liver of E13.5 mouse embryo. Primers corresponding to amplicons named promoter and exon 3 (A) were used for quantification by real-time PCR, and the ratio of precipitated DNA to input DNA (% of IP/IN) was determined. Values are given as means ± SEM (WT, N = 8; S-II KO, N = 6). Asterisk indicates statistically significant difference (Student's t-test, P < 0.05). AU; arbitrary unit.

	Discussion

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We examined the function of S-II in Bcl-x gene expression. The results of in vitro transcription experiments revealed that transcription is arrested in the Bcl-x intron 2, and that this arrest can be relieved by S-II (Fig. 3). Furthermore, chromatin immunoprecipitation analysis revealed that the amount of RNAPII on the downstream region of this arrest site was decreased in S-II KO mouse fetal liver (Fig. 2C), suggesting that transcription elongation is arrested around this site in vivo in the absence of S-II. These results suggest that Bcl-x gene expression is decreased in S-II KO mouse fetal liver because RNAPII cannot overcome the arrest site in intron 2.

RNAPII processivity is affected by modifications of histone and RNAPII itself such as phosphorylation of Rpb1 C-terminal domain (CTD). In the present study, we found that histone acetylation at exon 2 and intron 2 of the Bcl-x gene, corresponding to the region around the transcription arrest site and downstream, respectively (Fig. 3A), was impaired in S-II KO mouse fetal liver (Fig. 4C). Because acetylation of histone is important for RNAPII processivity (Govind et al. 2007), decreased histone acetylation might be a reason of impaired RNAPII processivity in S-II KO mice. In contrast, methylation of H3K36 is not decreased in S-II KO mice (Fig. 4C). Methylation of H3K36 depends on the CTD phosphorylation of elongating RNAPII (Xiao et al. 2003). Thus, we suppose that the CTD phosphorylation is not significantly decreased in S-II KO mice. In conclusion, it is suggested that S-II is particularly required for histone acetylation. S-II is reported to interact with SAGA (Wery et al. 2004), which has histone acetylation activity and stimulates transcription elongation as well as initiation (Carey et al. 2006; Govind et al. 2007). Furthermore, a recent report suggests that S-II functions synergistically with HAT p300 to stimulate transcription on the chromatin template (Guermah et al. 2006). These observations support the idea that S-II is involved in histone acetylation during transcription elongation. In S-II KO mouse fetal liver, Bcl-x gene expression might be decreased because of both reduced histone acetylation and transcription arrest induced by the DNA sequence.

In contrast to the Bcl-x gene, the distribution of RNAPII on the β^major-globin gene was not affected by S-II gene disruption. Thus, it is not clear, if there are any transcription arrest sites in the β^major-globin gene that can be read-through in an S-II-dependent manner. Histone acetylation or recruitment of transcription initiation complex to the β^major-globin gene promoter was not affected by S-II gene deletion. The amount of histone on the β^major-globin gene, however, was increased in S-II KO mouse fetal liver, suggesting that histone eviction caused by progression of the transcription elongation complex is impaired in S-II KO. At this moment, the molecular mechanisms by which S-II induces histone eviction remain to be determined. Elucidation of the function of S-II in chromatin modification, such as histone acetylation of the Bcl-x gene or histone eviction in the β^major-globin gene, would provide further understanding of transcriptional regulation on chromatin templates.

	Experimental procedures

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S-II-deficient mice

S-II-deficient mice generated as previously reported (Ito et al. 2006) were used. All animal experiments were approved by the institutional committee on animal experimentation and carried out in compliance with corresponding animal welfare laws.

Preparation of erythroid progenitor cells and in vitro erythroblast differentiation

Preparation and differentiation of erythroid progenitors from mouse fetal liver were carried out essentially as described previously (Dolznig et al. 2001; Nagata et al. 2007). In brief, fetal livers were dissected from 13.5 days post-coitum (E13.5) mouse embryos and cells were dissociated by gently passing the liver through a 74-µm nylon mesh (Corning, NY). Cells were cultivated in serum-free medium StemPro-34 (GIBCO, Invitrogen Japan KK, Tokyo, Japan) containing 100 ng/mL stem cell factor (PeproTech EC, London, UK), 1 µM dexamethasone (Sigma-Aldrich Japan K.K., Tokyo, Japan), 2 U/mL erythropoietin (a generous gift from Kirin Brewery Co., Ltd) and 40 ng/mL insulin-like growth factor I (Promega KK, Tokyo, Japan). The resulting erythroid progenitors were expanded by daily partial medium changes, maintaining cell density between 1.5 and 4 x 10⁶ cells/mL. After 3 to 5-day culture, growing erythroid progenitor cells were washed twice with phosphate buffered saline and seeded at 2 x 10⁶ cells/mL in differentiation medium; StemPro-34 containing 10 U/mL erythropoietin, 10 ng/mL insulin (Sigma), 3 µM mifepristone (Sigma) and 1 mg/mL holo-transferrin (Sigma). Differentiating cells were maintained at densities of 2 to 6 x 10⁶ cells/mL by daily medium changes.

Reverse transcription and real-time quantitative PCR analysis

Total RNA was prepared using the RNeasy Mini Kit (Qiagen KK, Tokyo, JAPAN), and 500 ng of RNA was reverse-transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Real-time PCR was carried out with an ABI Prism 7700 Sequence Detection System using TaqMan Universal PCR Master Mix (Applied Biosystems), SYBR Premix ExTaq (Takara Bio, Shiga, Japan) or SYBR Green PCR Master Mix (Applied Biosystems). TaqMan Gene Expression Assays (Applied Biosystems) were used to quantify the following mRNA: Alb (Mm00619261), PF4 (Mm00451315), DNaseII (Mm00438463) and hypoxanthine guanine phosphoribosyl transferase (Hprt) (Mm0044696). Expression of other genes was analyzed using the SYBR Green system. The sequences of primer sets used in the SYBR Green system were as follows (sense primer/antisense primer); Bcl-xL, 5'-ACTGTGCGTGGAAAGCGTAGA-3'/5'-GATCCACAAAA GTGTCCCAGC-3'; β^major-globin, 5'-GGTGAACGCCGAT GAAGTTG-3'/5'-TTACCCATGATAGCAGAGGCAGAG-3'; GPA, 5'-CCCAGTATGACCGAGAGCACA-3'/5'-TCTTCAT TAGGAGTCTGCTCA-3'; Alas2, 5'-CCCGAAGCCATTCAT TTCCT-3'/5'-ATAACCGAAAGCCTGGCTTCC-3'; Car2, 5'-TTGAAGATTGGACCTGCCTCA-3'/5'-GGATCGAAGTTA GCAAAGGCC-3'; Cpox, 5'-TGGTCCCTTCCTATGTTCCCA-3'/5'-AAACTCCACATACCGCCCTCTC-3'; Hmbs, 5'-ATTGG AGCCATCTGCAAACG-3'/5'-TGGTTCCCACGGCACTTTT-3'; NF-E2 p45, 5'-GGGAGCTTGGAGAGATGGAAC-3'/5'-GGGTGCTTGAGGCTCAAAAG-3'; c-myb, 5'-ATTATCTGC CCAACCGGACAG-3'/5'-ATAACAGACCAACGCTTCGGA-3'. Specific amplifications of each gene were confirmed by measuring the dissociation curves for the amplified products and by agarose gel electrophoresis (data not shown).

Chromatin immunoprecipitation assays

Chromatin immunoprecipitation assays were carried out essentially as described previously (Johnson et al. 2001). In brief, fetal liver cells from E13.5 mouse embryos were prepared as described, and protein-DNA cross-linking was carried out by treating cells with formaldehyde at a final concentration of 0.4% for 10 min at room temperature with gentle agitation. Glycine (0.125 M) was added to quench the reaction. Cells were then collected by centrifugation and washed in phosphate buffered saline. Nuclei were isolated by incubation in cell lysis buffer (10 mM Tris, 10 mM NaCl, 0.2% Nonidet P-40 [pH 8]) containing protease inhibitor cocktail (Sigma) for 10 min on ice followed by centrifugation. Nuclei were lysed in nuclei lysis buffer (50 mM Tris, 10 mMM EDTA, 1% sodium dodecyl sulfate (SDS) [pH 8]) for 1 h on ice. The lysate was sonicated (Shimadzu SUS-103, 28 kHz, 30 s x 40 cycle) to generate chromatin fragments with an average size of approximately 500 bp (Fig. 2D). Soluble chromatin was diluted fivefold with IP dilution buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.01% SDS, 1% Triton X-100, protease inhinbitor cocktail [pH 8]) and precleared by Protein A-Sepharose. Precleared samples were incubated with the following antibodies or normal IgG for 3 h at 4 °C; anti-TFIIB (sc-274, Santa Cruz), anti-RNAPII (CTD4H8, 05-623, Upstate) (Kristjuhan et al. 2002; Kristjuhan & Svejstrup 2004), anti-acetylated histone H3 (06-599, Upstate) (Lee et al. 2004; Schwabish & Struhl 2004; Govind et al. 2007; Han et al. 2008), anti-tri-methyl H3K36 antibody (ab9050, Abcam), anti-histone H3 (ab1791, Abcam) (Kristjuhan & Svejstrup 2004; Zhang et al. 2005; Govind et al. 2007). Immune complexes were collected by incubation with Protein A-Sepharose for 2 h at 4 °C. Protein A-Sepharose pellets were washed twice with IP wash buffer 1 (20 mM Tris, 50 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100 [pH 8]), once with IP wash buffer 2 (10 mM Tris, 0.25 M LiCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxycholate [pH 8]), and twice with TE (10 mM Tris, 1 mM EDTA [pH 8]). Immune complexes were eluted with IP elution buffer (0.1 M NaHCO₃, 1% SDS), followed by incubation with RNaseA (0.003 mg/mL) for 5 h at 65 °C. Samples were digested with proteinase K (0.24 mg/mL) for 12 h at 45 °C. DNA was purified by extraction with phenol : chloroform followed by ethanol precipitation. Input and immunoprecipitated DNA were quantified by real-time PCR using SYBR Green system as described. The sequences of primer sets were as follows (sense primer/anti-sense primer); Bcl-x promoter region, 5'-ACACTAA ACCCATACCTCCG-3'/5'-GTCGCCGGTAACTCAGCAAA-3'; Bcl-x exon 2 region, 5'-TTCGGGATGGAGTAAACTGGG-3'/5'-CCGACTCACCAATACCTGCA-3'; Bcl-x intron 2 region, 5'TTGCGGCTTCTGAGACGTATT-3'/5'-GCCCCTTCCAC CTCACTTC-3'; β^major-globin promoter region, 5'-GACAAACA TTATTCAGAGGGAGTA-3'/5'-AAGCAAATGTGAGGAGC AACTGAT-3'; β^major-globin exon 3 region, 5'-GCCCTGGCTC ACAAGTACCA-3'/5'-TTCACAGGCAAGAGCAGGAA-3'. Occupancy values were calculated by determining the apparent immunoprecipitation efficiency (% of immunoprecipitated (IP) vs. input (IN) sample).

In vitro transcription assays

In vitro transcription assays were carried out essentially as described previously (Shimoaraiso et al. 2000). In brief, plasmid pGEM-Bclx contained sequences of the murine Bcl-x gene fragment inserted into pGEM-3Zf(+). The 3'-deoxycytidine-extended templates were prepared as described (Shilatifard et al. 1996) by using linearized pGEM-Bclx, and adding oligodeoxycytidylate residues with terminal deoxynucleotidyl transferase, followed by digestion with EcoRI and PstI. The fragments of Bcl-x gene used were +633 to +983 (fragment 1), +948 to +1317 (fragment 2), +1269 to 1689 (fragment 3), +1691 to +2051 (fragment 4) and +2390 to +2738 (fragment 5), where +1 indicates the transcription start site. Transcription reactions with purified RNAPII and dC-tailed DNA contained 60 mM Tris–acetate (pH 8.0), 5 mM Mg-acetate, 5 % glycerol, 100 mM ammonium sulfate and 6 mM spermidine. Reactions contained 0.6 µg DNA and three units of mouse RNAPII. RNAPII and tailed DNA were first incubated 5 min at 30 °C without nucleoside triphosphates in a total volume of 25 µL. ATP, CTP, UTP (0.6 mM each), GTP (20 µM) and 30 µCi of [-³²P] GTP (3000 Ci/mmol, GE Healthcare) were added, followed 1 min later by 100 µg/mL of heparin and 0.8 mM of NTPs. In reaction with S-II, purified recombinant mouse S-II (100 ng) was added and incubated for 30 min at 30 °C. Reactions were stopped by adding 0.8 mL of a stop mixture comprised of 2 mM EDTA, 0.5 M ammonium acetate, and 1 µg of Escherichia coli tRNA. Nucleic acids were collected by ethanol precipitation, resolved by electrophoresis on 5% (w/v) polyacrylamide / 8.3 M urea gels, and visualized by autoradiography.

	Acknowledgements

 
We thank Kirin Brewery Co., Ltd for the recombinant human erythropoietin. This work was supported by a research grant from Japan Society for the Promotion of Science (JSPS) to T.I. M.N. and H.K. are the recipients of JSPS Postdoctoral Research Fellowship for Young Scientist.

	Footnotes

 
Communicated by: Hiroshi Handa

^* Correspondence: sekimizu{at}mol.f.u-tokyo.ac.jp

	References

Top Abstract Introduction Results Discussion Experimental procedures References

 
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Received: 22 July 2008
Accepted: 2 December 2008

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