Title: Prolonged glucocorticoid exposure dephosphorylates histone H1 and inactivates the MMTV promoter
Abstract: Article2 March 1998free access Prolonged glucocorticoid exposure dephosphorylates histone H1 and inactivates the MMTV promoter Huay-Leng Lee Huay-Leng Lee Departments of Obstetrics and Gynaecology, Biochemistry and Oncology, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada, N6A 4L6 Search for more papers by this author Trevor K. Archer Corresponding Author Trevor K. Archer Departments of Obstetrics and Gynaecology, Biochemistry and Oncology, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada, N6A 4L6 Search for more papers by this author Huay-Leng Lee Huay-Leng Lee Departments of Obstetrics and Gynaecology, Biochemistry and Oncology, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada, N6A 4L6 Search for more papers by this author Trevor K. Archer Corresponding Author Trevor K. Archer Departments of Obstetrics and Gynaecology, Biochemistry and Oncology, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada, N6A 4L6 Search for more papers by this author Author Information Huay-Leng Lee1 and Trevor K. Archer 1 1Departments of Obstetrics and Gynaecology, Biochemistry and Oncology, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada, N6A 4L6 *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1454-1466https://doi.org/10.1093/emboj/17.5.1454 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Glucocorticoids rapidly induce transcription from the mouse mammary tumour virus (MMTV) promoter via a glucocorticoid receptor (GR)-mediated chromatin disruption event. This remodelling of chromatin is transient such that upon prolonged exposure to hormone the promoter becomes refractory to glucocorticoids. We demonstrate that this refractory state requires the continual presence of hormone and can be reversed by its removal. Our experiments show that the promoter is inactivated via a mechanism whereby histone H1 is dephosphorylated in response to glucocorticoids. Removal of glucocorticoids results in the rephosphorylation of histone H1 and the reacquisition of transcriptional competence by the promoter. This response is specific for the MMTV promoter assembled as chromatin and is not observed for another inducible gene or transiently transfected MMTV DNA. Finally, we demonstrate that H1 on the MMTV promoter is dephosphorylated when the promoter is unresponsive to glucocorticoids. These studies indicate that phosphorylated H1 is intimately linked with the GR-mediated disruption of MMTV chromatin in vivo. Introduction An expanding body of knowledge provides clear mechanistic links between the architecture of DNA as chromatin and the regulation of transcription (Felsenfeld, 1992; Wolffe et al., 1993; Kingston et al., 1996). These investigations include elegant genetic experiments, precise structural characterization of the nucleosome and detailed in vitro biochemical analysis of the transcriptional process (Grunstein, 1990; Arents et al., 1991; Adams and Workman, 1993; Almouzni and Wolffe, 1993). The majority of these studies suggest that the packaging of DNA into nucleosomes acts as a barrier to the initiation of transcription by preventing the access of transcription factors to their recognition sites (Hayes and Wolffe, 1992; Workman and Buchman, 1993). Genetic experiments in Saccharomyces cerevisiae and Tetrahymena thermophila have demonstrated specific roles for the core and linker histones in these processes (Grunstein, 1990; Shen and Gorovsky, 1996). Studies in yeast have defined the contribution of individual histones to the repression of transcription such that the deletion of one or more of the core histones leads to the deregulation of a variety of inducible genes. Specifically, mutation of histone H4 activates transcription (Han and Grunstein, 1988) whereas decreased levels of H2A–H2B dimers de-repress transcription at a number of yeast genes (Norris and Osley, 1987). In Tetrahymena the deletion of H1 leads to specific gene activation without a pronounced effect on global transcription by Pol II or Pol III (Shen and Gorovsky, 1996). This is consistent with the specific repression of the 5S rRNA gene observed with the over-expression of histone H1 in Xenopus laevis (Bouvet et al., 1994). In addition, genetic experiments examining mating type switching in yeast have also identified a number of protein complexes, exemplified by the SWI–SNF complex, which have profound effects on chromatin remodelling and transcription both in vivo and in vitro (Peterson and Herskowitz, 1992; Yoshinaga et al., 1992; Côté et al., 1994; Imbalzano et al., 1994). Beyond these general features attributed to assembling DNA into chromatin, post-translational modifications of core histones have been shown to exert dramatic effects. Perhaps the most well-characterized example of this is the acetylation of lysines in the N-terminal tails of histones H3 and H4 (Roth et al., 1992; Fisher-Adams and Grunstein, 1995). Both biochemical studies using agents that promote hyper-acetylation of the tails and genetic experiments whereby the tails are prevented from being acetylated reveal that hyper-acetylated histones are associated with activated chromatin (Durrin et al., 1991; Lee et al., 1993; Van Lint et al., 1996). In agreement with these studies, analysis of heterochromatic silencing in S.cerevisiae reveals a correlation between hypo-acetylation of histones and a reduction in transcriptional activity (Braunstein et al., 1993). A clear repressive role for chromatin structure has been established by detailed biochemical studies (Hayes and Wolffe, 1992; Adams and Workman, 1993). They reveal that the activities of RNA Pol II and III are significantly inhibited by assembly of target sequences into nucleosomes (Knezetic et al., 1988; Laybourn and Kadonaga, 1991; Hansen and Wolffe, 1994). This nucleosomal structure is also refractory to binding by many, but not all, transcription factors and members of the general transcription machinery (Archer et al., 1991; Workman and Kingston, 1992). Consistent with the observations derived from genetic studies, in vitro nucleosomal reconstitution experiments indicate that the removal of H2A and H2B from nucleosomal arrays results in decreased chromatin compaction and enhanced gene activity (Hansen and Wolffe, 1994). In addition to core histones, the linker histones, by virtue of their participation in chromatin condensation, have been implicated as repressors of transcription (Croston et al., 1991; Shen et al., 1995). In most cases, histone H1 is thought to bind DNA in the nucleosome across the dyad axis and at the linker DNA as it enters and leaves the nucleosome (Ali and Singh, 1987). However, at least for the 5S rRNA gene, recent evidence suggests that a specific and asymmetric binding of the linker histone may be possible (Hayes and Wolffe, 1993; Hayes, 1996; Pruss et al., 1996). The binding of histone H1 in vitro has been shown to stabilize the nucleosome and is proposed to facilitate the folding of nucleosomal arrays into 30 nm chromatin fibres in vivo (Felsenfeld and McGhee, 1986; Kamakaka and Thomas, 1990). Histone H1 can exert a repressive effect on transcription even when transcription factors are in excess (Croston et al., 1991). In vitro studies indicate that the association of histone H1 with nucleosomal cores impairs the binding of transcription factor Gal4-AH and upstream stimulatory factor (USF) to DNA (Juan et al., 1994). This observation is consistent with protein–DNA interaction experiments demonstrating that cross-linking of histone H1 to actively transcribed genes is significantly lower than to transcriptionally silent genes (Dimitrov et al., 1990; Kamakaka and Thomas, 1990; Bresnick et al., 1991; Dedon et al., 1991). The mouse mammary tumour virus (MMTV) represents a well established mammalian system where chromatin structure and transcriptional regulation have been intimately linked (Hager et al., 1993; Truss et al., 1993; Archer et al., 1995). This promoter reproducibly acquires a phased array of six positioned nucleosomes when stably introduced into mammalian cells (Richard-Foy and Hager, 1987). Detailed analysis of the glucocorticoid induced transcription from this promoter demonstrates that the glucocorticoid receptor (GR) initiates a chromatin remodelling event which results in the loading of a pre-initiation complex (PIC) and active transcription from this promoter (Cordingley et al., 1987; Archer et al., 1992). We have confirmed a primary role for chromatin structure in regulating this promoter by the analysis of identical DNA sequences in transient transfection experiments (Archer et al., 1992; Lee and Archer, 1994). Characterization of these transiently transfected molecules reveals that while they are probably associated with nucleosomes they do not display the phased nucleosomal array characteristic of the MMTV promoter in mouse chromatin (Cereghini and Yaniv, 1984; Reeves et al., 1985; Archer et al., 1992; Jeong and Stein, 1993). Consequently these templates are hypersensitive to endonucleases in both the absence and presence of hormone (Archer et al., 1992). As would be predicted by this hypersensitivity, in vivo footprinting studies reveal that transcription factors normally excluded from the endogenous promoter are constitutively bound to the transient template (Archer et al., 1992; Lee and Archer, 1994). However, these transient MMTV templates remain hormone inducible via a mechanism that involves the hormone-dependent recruitment of the TATA binding protein to the MMTV promoter (Lee and Archer, 1994). Previously, we demonstrated that the MMTV promoter was maximally induced 1 h after hormone treatment and became refractory to GR activation after 24 h of continuous hormone exposure (Archer et al., 1994a; Lee and Archer, 1994). This ‘silencing’ occurred despite the presence of relevant transcription factors in the nucleus during the refractory phase as evident by the fact that they were able to interact with the transiently introduced MMTV DNA (Lee and Archer, 1994). Consequently, we have suggested that these factors were prevented from binding to their recognition sequences by the reformation of nucleosome B (nuc-B) thus resulting in the inhibition of transcription (Lee and Archer, 1994). This decline in activation following hormone stimulation is characteristic and unique for templates assembled as chromatin as it is not observed when the same sequence is transiently transfected into these cells. This observation suggests that while chromatin structure is vital to the understanding of transcriptional activation, it also plays an important role in the deactivation or cessation of transcription after hormone stimulation. To investigate the mechanisms involved in this loss of activity or transcriptional competence from the promoter we have carried out a series of experiments in which we demonstrate that the maintenance of the refractory state requires the continuous presence of hormone. Prolonged exposure of the cells to hormone induces a refractory or silent state on the promoter that is accompanied by the global dephosphorylation of histone H1. Subsequent experiments demonstrated that the phosphorylation state of histone H1 parallels the activation status of the promoter, and that if H1 phosphorylation is blocked, the MMTV promoter remains refractory to glucocorticoid activation. In contrast, activation of the metallothionein (MT) promoter or the MMTV promoter transiently transfected into the same cells is not influenced by the phosphorylation status of histone H1. A direct role for phosphorylated H1 is suggested by the demonstration that when H1 on the MMTV promoter assembled as chromatin is dephosphorylated, the promoter no longer responds to glucocorticoid. Thus, the phosphorylation of histone H1 plays a pivotal role in transcriptional activation and modulates the ability of the GR to disrupt MMTV chromatin. Results Hormone withdrawal restores transcriptional competency to the MMTV promoter The MMTV promoter has been the focus of numerous studies on the role of chromatin structure in the events leading to the activation of transcription by nuclear hormone receptors (Archer and Mymryk, 1995). The GR induces a rapid transcriptional response from the MMTV promoter by modifying chromatin structure to facilitate the assembly of the transcription pre-initiation complex (Zaret and Yamamoto, 1984; Cordingley et al., 1987; Richard-Foy and Hager, 1987). Prolonged exposure to hormone results in a cessation of transcription such that the promoter returns to near basal levels of activity after 24 h of hormone treatment. This lack of transcription occurs despite the continued presence of hormone and is accompanied by the disruption of transcription complexes and the loss of hypersensitivity as a consequence of chromatin reformation (Lee and Archer, 1994). To explore the mechanism(s) underlying the GR‘s inability to activate transcription we have ascertained if the continued presence of the hormone is required to maintain the refractory phase. The approach we have taken is to examine if the ‘refractory’ promoter could be re-stimulated after hormone withdrawal, growth in the absence of hormone and subsequent hormone re-administration prior to harvesting. A schematic of the experimental approach is presented in Figure 1A. Specifically, cells were either maintained initially in the absence (Figure 1B, lane 1) or presence of hormone for 4 h (Figure 1B, lane 2) and 24 h (Figure 1B, lanes 3–6). Cells grown for 24 h in the presence of hormone were then examined for the effects of continued hormone treatment (Figure 1B, lane 3), hormone removal for 20 h or 1 h prior to the re-addition of hormone (Figure 1B, lanes 5 and 6) and hormone removal without subsequent re-stimulation (Figure 1B, lane 4). Primer extension analysis of total RNA indicated that while 4 h of hormone administration prior to harvesting led to an increase in mRNA accumulation (Figure 1B, compare lanes 1 and 2), 48 h of hormone treatment resulted in a significant reduction in mRNA levels compared with 4 h of treatment (Figure 1B, compare lanes 1, 2 and 3). However, 20 h of dexamethasone withdrawal, prior to 4 h of hormone re-administration, resulted in the reactivation of the promoter and an increase in mRNA accumulation (Figure 1B, compare lanes 3 and 5). In contrast, if hormone was removed for only 1 h during the refractory phase then no increase in mRNA levels was detected if cells were re-treated with 4 h of dexamethasone (Figure 1B, lane 6). These hormone dependent changes were specific for MMTV as no change was observed for actin mRNA examined from the same cells (Figure 1B, lanes 1–6). This is somewhat surprising, given that glucocorticoids, which activate the promoter though the GR, also appear to be responsible for silencing transcription in cells that received prolonged hormone exposure. Figure 1.RNA accumulation in response to hormone removal and readdition during the refractory phase. (A) Experimental schematic indicating times of hormone exposure, removal and re-exposure prior to harvesting. (B) 1471.1 cells cultured under the conditions in (A); the levels of MMTV and actin mRNAs were analyzed by primer extension from total RNA (20 μg) using gene-specific primers as described in Materials and methods. Cells were cultured without hormone (lane 1), treated with dexamethasone (10−7 M) for 4 h (lane 2) and 48 h (lane 3), or treated with dexamethasone for 24 h (lanes 4 and 5), then hormone was removed for 24 h (lane 4) or 20 h (lane 5) and re-treated with dexamethasone (10−7 M) for 4 h prior to harvest (lane 5). Alternatively, cells were treated with dexamethasone for a period of 43 h, hormone was removed for 1 h and dexamethasone (10−7 M) was added for 4 h prior to harvest (lanes 6). (C) Cells transfected with 10 μg of pLTR LUC, a plasmid which contains the MMTV LTR attached to the firefly Luciferase gene, were cultured under the conditions described in (A). Total RNA (5 μg) was reverse transcribed to cDNA using a primer specific for the Luciferase gene and Superscript reverse transcriptase (Gibco-BRL). The cDNA was analyzed by Taq polymerase amplification with 32P-labeled primers specific for the MMTV LTR. The levels of 18S mRNA were analyzed by primer extension of total RNA (3 μg) as described in Materials and methods. The purified extended products were analyzed on 7% polyacrylamide denaturing gels and exposed to Dupont reflection film. Download figure Download PowerPoint We have established that silencing of the MMTV promoter in response to prolonged exposure to glucocorticoid is observed with stable chromatin templates but not transiently transfected templates (Lee and Archer, 1994). In the next series of experiments, we directly examined the effect of hormone withdrawal and re-addition on the activation of transcription from the transiently transfected MMTV plasmid. In these experiments the transient template displayed continued transactivation upon prolonged exposure to hormone (Figure 1C, compare lanes 1, 2 and 3). This is consistent with our previous studies indicating that transient templates fail to show an inhibition of activity at 24 or 48 h of hormone treatment (Lee and Archer, 1994). Further, in contrast with the chromatin templates, removal of hormone for 1 h is sufficient to allow the re-activation of the promoter upon hormone re-addition (Figure 1B and C, compare lanes 5 and 6). As the establishment of the refractory phase is characterized by a ‘closed’ chromatin structure over nuc-B, we next determined if the changes in mRNA levels seen above correlate with alterations in chromatin structure. As shown in Figure 2, we conducted an in vivo restriction enzyme hypersensitivity assay with cells maintained under a regimen analogous to that described above except that hormone re-addition was for 1 h (Figure 1A). [We have previously established that while mRNA accumulation peaks at 4 h, changes in chromatin structure and transcription factor loading are maximal at 1 h of hormone treatment (Archer et al., 1994a; Lee and Archer, 1994).] Congruent with the RNA analysis, cleavage within nuc-B was elevated 8-fold when cells were re-stimulated with dexamethasone after 23 h of hormone removal (Figure 2A, compare lanes 1, 2 and 5). This is in contrast to cells that were treated with dexamethasone for 48 h, (Figure 2A, lane 3) or had undergone hormone withdrawal without subsequent re-stimulation (Figure 2A, lane 4). In these cells cleavage within nuc-B was similar to that seen in unstimulated cells, 1.3- to 1.6-fold elevation, indicative of a ‘closed’ nucleosomal structure. This suggests that 23 h after the removal of hormone the refractory phase is abrogated and chromatin can once again be remodelled by the GR. Interestingly, cleavage was minimal if hormone was removed from cells for only 1 h prior to hormone re-stimulation, 1.9-fold versus 8-fold (Figure 2A, compare lanes 1, 6 and 5), indicating that short term hormone removal is insufficient to allow reactivation. Thus the recovery of transcriptional competence by the MMTV promoter that has become refractory to the GR requires hormone withdrawal for a period of 20 h. Figure 2.Restoration of restriction enzyme hypersensitivity and the loading of NF1 onto the stable template following hormone withdrawal during the refractory phase. (A) Mouse mammary cells (1471.1) containing the MMTV promoter, mobilized on a BPV-based multicopy plasmid as a stable replicating unit, were treated with dexamethasone (10−7 M) for 24 h followed by hormone withdrawal for 23 h (lane 5) or 24 h (lane 4) and were either not given any hormone (lane 4) or were treated with dexamethasone (10−7 M) for 1 h prior to harvest (lane 5). Alternatively, cells were treated with dexamethasone for a period of 46 h, hormone was removed for 1 h and dexamethasone (10−7 M) was added for 1 h prior to harvest (lane 6). Cells that did not receive any hormone or were treated with dexamethasone for 1 or 48 h are shown in lanes 1, 2 and 3, respectively. The nuclei were isolated and digested with DpnII in vivo as previously described. Following purification, all the samples were digested to completion with HaeIII in vitro as an internal standard for reiterative primer extension analysis using a 32P-labeled primer. The purified extended products were separated on a 5% polyacrylamide denaturing gel before autoradiography at −80°C. The arrows indicate HaeIII and DpnII cleavage products. (B) 1471.1 cells were treated as described in Figure 2A and nuclei were isolated and digested with HaeIII and Exonuclease III. The purified DNA were recut with HaeIII in vitro, treated with Mung bean nuclease and then analyzed by primer extension with Taq polymerase using primers that were specific for the MMTV promoter. Download figure Download PowerPoint Coincident with the GR-induced remodelling of MMTV chromatin is the hormone dependent formation of a PIC at the promoter. As shown in Figure 2B, this complex can be monitored by examining the hormone dependent loading of transcription factor NF1 using in vivo footprinting analysis (Figure 2B, lanes 1 and 2). Consequently, it was important to examine the assembly of the PIC when the promoter was re-activated during the refractory phase upon hormone withdrawal and re-addition. NF1 binding was observed in cells that were treated with hormone for 24 h and subsequently had hormone removed for 23 h prior to re-stimulation, but not in cells that were maintained in media containing hormone for 48 h (Figure 2B, compare lanes 3 and 5). Consistent with the restriction enzyme hypersensitivity assay, NF1 was not detected when cells were re-stimulated with dexamethasone after only 1 h of hormone removal (Figure 2B, lane 6). These experiments confirm that chronic exposure to glucocorticoid results in a non-inducible promoter. Further, they demonstrate that hormone removal for a protracted period restores the promoter to a ‘conformation’ that can be re-activated upon a second exposure to hormone. Phosphorylation status of histone H1 and transcriptional competency of the MMTV promoter Our experiments suggest that it is the distinct arrangement of chromatin during the refractory phase that prevents the GR-mediated activation of MMTV transcription (Figures 1 and 2). This new arrangement of chromatin that results from prolonged exposure to hormone is itself reversible upon removal of the hormone (Figure 2). Consequently, we turned our attention to reversible aspects of chromatin structure, such as post-translational modification of histones, which might contribute to the loss and recovery of transcriptional competence at this promoter. In addition to the disruption of core histones, the GR-mediated activation of MMTV transcription has been correlated with the displacement of histone H1 (Bresnick et al., 1991). This linker histone is known to undergo a number of post-translational modifications including phosphorylation (Bradbury, 1992; Roth and Allis, 1992). The phosphorylation of histone H1 influences its interaction with DNA and has been proposed to result in an ‘opening’ of chromatin to increase transcription factor access to DNA (Roth and Allis, 1992). To pursue this possibility, we have examined the level of phosphorylated H1 in cells exposed to dexamethasone under a similar time course that results in the loss of transcriptional competence at the promoter (Figure 3). Western blot analysis indicated that phosphorylated H1 was abundant in untreated cells (Figure 3, lane 1) and in cells that were treated with dexamethasone for 1 h (Figure 3, lane 2). However, histone H1 was progressively dephosphorylated in the continued presence of dexamethasone. After 7 h of hormone treatment the level of phosphorylated H1 present was decreased (Figure 3, lane 3) and became barely detectable after 24 h of dexamethasone treatment (Figure 3, lane 4). Under these conditions no changes in total H1 levels were evident (Figure 3, lanes 1–4). Hence, the ‘opening’ and ‘closing’ of nuc-B, the ability of transcription factors to bind chromatin and the level of transcription following hormone treatment correlate tightly with the phosphorylation and the dephosphorylation of histone H1. Figure 3.Histone H1 is progressively dephosphorylated in the continued presence of dexamethasone. Mouse 1471.1 cells were either not given any hormone (lane 1) or were treated with dexamethasone (10−7 M) for a period of 1, 7 or 24 h (lanes 2–4 respectively). Total histones were prepared from nuclei by acid extraction as described in Materials and methods. Histones (40 μg) were separated on a 16% acrylamide acid–urea gel, transferred to a nitrocellulose membrane and Western blot analysis was performed with a polyclonal antibody against phosphorylated H1. As a loading control the blot was then stained with amido black to reveal total histone H1 present. Download figure Download PowerPoint If the ‘closing’ of nuc-B after 24 h of dexamethasone treatment results from the dephosphorylation of histone H1 this would imply that histone H1 is rephosphorylated under conditions that reactivate transcription (Figure 1B). Indeed, in sharp contrast to cells that were grown in media containing dexamethasone for a period of 48 h (Figure 4, lane 4), histone H1 was highly phosphorylated after hormone was removed from cells for 23 or 24 h (Figure 4, lanes 5 and 6). Furthermore, histone H1 remained dephosphorylated when dexamethasone was removed for only 1 h from cells exposed to hormone for 46 h (Figure 4, lane 7). This is consistent with the restriction enzyme hypersensitivity and transcription factor loading assay, indicating that 1 h of hormone removal was insufficient to obtain activation during the refractory phase (Figures 1 and 2). Finally, while the phosphorylation status of H1 was altered by hormone treatment, the levels of total histone H1 were unchanged by hormone administration (Figure 3 and data not shown). These observations strengthen the correlation between H1 phosphorylation and induction of transcription from the MMTV promoter, linking changes in histone H1 phosphorylation with precise alterations in the hormone responsiveness of the MMTV promoter assembled as chromatin. Figure 4.Histone H1 is rephosphorylated after prolonged hormone withdrawal. Cells were treated with dexamethasone (10−7 M) for 24 h, hormone was removed for 23 h (lane 6) or 24 h (lane 5) and cells were either not given any hormone (lane 5) or were treated with dexamethasone (10−7 M) for 1 h prior to harvest (lane 6). Alternatively, cells were treated with dexamethasone for a period of 46 h, hormone was removed for 1 h and dexamethasone (10−7 M) was added for 1 h prior to harvest (lane 7). Cells that did not receive any hormone or were treated with dexamethasone for 1 and 48 h are shown in lanes 2, 3 and 4 respectively. Purified histone H1 (10 μg) (lane 1) and total histones (40 μg) (lanes 2–7) were separated on a 16% acrylamide acid–urea gel, transferred to a nitrocellulose membrane and Western blot analysis was performed using a polyclonal antibody against phosphorylated H1. Download figure Download PowerPoint These results imply that the phosphorylation of histone H1 plays an important role in the recovery from the refractory phase, including the ability of the GR to modify MMTV chromatin and activate transcription. Thus we would predict that blocking H1 rephosphorylation when hormone is removed would prevent the reactivation of the promoter. For these experiments, we used the kinase inhibitor staurosporine to prevent the rephosphorylation of H1 following hormone removal. Western blot analysis of the phosphorylation state of histone H1 indicated that staurosporine inhibited histone H1 rephosphorylation after hormone removal (Figure 5A, lanes 6 and 7) to a level that was not detectable by the chemiluminescent detection procedure. In fact, the level of phosphorylated H1 was lower in cells treated with staurosporine (Figure 5A, lanes 5, 6 and 7) than in cells treated with dexamethasone for 48 h (Figure 5A, lane 2). In contrast, cells not treated with staurosporine displayed high levels of phosphorylated H1 following dexamethasone removal (Figure 5A, lanes 3 and 4). Figure 5.Effects of the kinase inhibitor staurosporine on MMTV activation. (A) Staurosporine inhibits the rephosphorylation of H1. Dexamethasone (10−7 M) was added to cells for 24 h and hormone was removed for 23 h (lanes 4 and 7) or 24 h (lanes 3 and 6), either in the absence (lanes 2–4) or the presence (lanes 5–7) of staurosporine (100 ng/ml). After hormone removal, cells were either not given any hormone (lanes 3 and 6) or were treated with dexamethasone (10−7 M) for 1 h prior to harvest (lanes 4 and 7). Cells that were treated with dexamethasone (10−7 M) for 48 h are shown in lanes 2 and 5, respectively. Purified histone H1 (10 μg) (lane 1) and total histones (35 μg) (lanes 2–7) were separated on a 16% acrylamide acid–urea gel, transferred to a nitrocellulose membrane, and Western blot analysis was performed using a polyclonal antibody against phosphorylated H1. (B) Staurosporine inhibits MMTV transcription from chromatin. Levels of MMTV and 18S rRNA transcripts in 1471.1 cells were analyzed by primer extension from total RNA (20 μg) using primers specific for either