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Histone H3 phosphorylation at Ser 10 colocalizes with active transcription sites in MCF-7 breast cancer cells. MCF-7 cells were grown on coverslips in serum- and estrogen-deplete medium ( A and C ), treated with 100 nmol/L TPA for 30 minutes ( B and D ), fixed, and double labeled with anti-H3 pS10 antibodies and anti-BrdUrd antibodies, which detect nascent RNA after in situ incorporation of 5-FU. Two sets of representative cells: untreated and treated. Spatial distribution was visualized by fluorescence microscopy and image deconvolution as described in Materials and Methods. Single optical sections. Yellow in merge signifies colocalization. The boxed area in each merged image is shown enlarged. Bar, 5 A m.
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Histone H3 phosphorylation is a downstream response to activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. This modification is thought to have a role in chromatin remodeling and in the initiation of gene transcription. In MCF-7 breast cancer cells, we observed that phosphorylated histone H3 (phospho-H3) at Ser(10) but not Ser(28...
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... proteins. One downstream event is the phosphorylation of the basic NH 2 -terminal tail of histone H3 at Ser 10 and Ser 28 (H3 pS10 and H3 pS28; ref. 3). Both phospho-modified forms of H3 have been shown to have significant roles in chromosome condensation during mitosis in many organisms, and H3 pS10 has been directly associated with the immediate early gene induction in mouse fibroblasts (4–6). The investigation of H3 phosphorylation in parental murine fibroblasts is well documented, but few studies (7, 8) have been conducted to ascertain its function in human breast cancer cells. Furthermore, the mechanistic involvement of the Ras/MAPK pathway in H3 phosphorylation in breast cancer has not been shown. Trefoil factor 1 (TFF1, formerly pS2) expression is high in estrogen receptor a (ER a )–positive breast cancer and is a useful prognostic marker associated with a favorable response to primary endocrine therapy (9). Although the definitive role of the cysteine- rich secretory TFF1 protein is lacking, it has been reported that TFF1 expression leads to efficient cell mobility that may be important for invasion and metastatic characteristics of aggressive breast cancer (10–12). The upstream regulatory region of the TFF1 gene contains binding sites for different transcription factors that respond to diverse extracellular stimuli, such as growth factors, hormones, and phorbol esters (13). The TFF1 gene responds to both estrogens and phorbol esters but by different mechanisms (14). Although the chromatin remodeling events taking place during estrogen induction of the TFF1 are well characterized (15, 16), little is known about the chromatin modifications of the TFF1 promoter occurring during induction by phorbol esters. Furthermore, although a role for the Ras/MAPK in growth factor– and phorbol ester–induced TFF1 expression is apparent, the consequences of stimulation of the Ras/MAPK pathway on histone modifications bound to the TFF1 promoter have not been investigated. Here, we show that TPA stimulation of the Ras/MAPK pathway results in the recruitment of AP-1, MSK1, but not ER a , and the increased acetylation and S10 phosphorylation of H3 associated with the TFF1 promoter. MCF-7 cells. To determine the stimulatory effect of estrogens and TPA on the Ras/MAPK pathway in epithelial human breast cancer cells, estrogen-dependent MCF-7 cells were cultured under serum- and estrogen-depleted conditions. As EGF, but not TPA, weakly activates JNK/SAPKs and p38, we used TPA to stimulate the Ras/ MAPK in MCF-7 cells. A time course treatment with estradiol at physiologic concentrations displayed no significant increase in phospho-ERK1/2 levels, a hallmark of activated Ras/MAPK signaling (Fig. 1 A ). Furthermore, no change in total MSK1 protein levels was observed, but phospho-p38 levels displayed an increase in the presence of estradiol (6- to 7-fold at 5 minutes; Fig. 1 A ). In contrast, TPA treatment of these cells triggered an immediate and robust elevation in levels of phospho-ERK1/2 (7- to 8-fold at 30 minutes; Fig. 1 B ). Activated phospho-p38 was not detected. To verify that TPA up-regulation of MAPK occurs through the Ras/ Raf/MEK pathway, we used two potent MEK inhibitors PD98059 and UO126 (23). Treatment of cells with either agent before TPA stimulation prevented the TPA-induced increase of phospho-ERK, showing that TPA stimulation occurs through the Ras/MAPK pathway in these cells (Fig. 1 C ). Histone H3 phosphorylation at Ser 10 is increased by TPA. In mouse fibroblasts, H3 pS10 and pS28 is detected in interphase and mitotic cells (3). In indirect immunolocalization studies, we repeated these analyses with MCF-7 cells cultured in complete media. H3 pS10 had a punctate distribution throughout the interphase nuclei of MCF-7 breast cancer cells (Fig. 2 A , arrows, top inset ) and showed a widespread condensed staining in G 2 or scription experiments. sites Under in MCF-7 these cells. conditions, The TPA-induced flow cytometry stimulation analyses of mitotic phase (Fig. 2 A , white arrowheads ) in agreement with H3 revealed pS10 that was 73% also of the examined cells were by in indirect G 0 -G 1 phase immunofluorescence of the cell cycle, previous reports (24, 25). In contrast, H3 pS28 was not detected in with microscopy <9% of and the image cells being deconvolution in the G 2 in -M MCF-7 phase. breast A low cancer level of cells. H3 interphase nuclei, although its staining in G 2 -M cells seemed to be pS10 MCF-7 was cells detected cultured before in estrogen- treatment and (Fig. 2 serum-deplete C ). TPA conditions treatment similar to that of H3 pS10 (Fig. 2 A , bottom inset ). Immunoblot increased displayed a H3 punctate pS10 levels distribution (4.2-fold; of Fig. 2 H3 C pS10. ). This The observation number of shows these analyses of MCF-7 acid-extracted histones isolated from cycling that foci clearly stimulation increased of the upon Ras/MAPK addition signaling of TPA pathways for 30 minutes increases (Fig. 3), the cells confirmed the lack of H3 pS28 in interphase cells (Fig. 2 B ). level consistent of H3 with pS10. the immunoblot analysis (Fig. 2 C ). To determine The intense staining observed for H3 pS10 and pS28 in condensed whether H3 pS10 was positioned next to newly synthesized RNA, regions of chromatin during mitosis was also confirmed by the 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells elevated levels of both phospho-modified forms in colcemid- arrested cells (Fig. 2 B ). These results are in accordance with other studies showing that the staining of nuclei immunochemical stained with antibodies to H3 pS10 or pS28 becomes very intense (3, 24). These observations show that unlike mouse fibroblasts, only H3 pS10 is detected in interphase MCF-7 cells. The effect of TPA treatment of estrogen- and serum-deprived MCF-7 cells on H3 pS10 levels was determined in immunoblot top inset ) and showed a widespread condensed staining in G 2 or scription experiments. sites Under in MCF-7 these cells. conditions, The TPA-induced flow cytometry stimulation analyses of mitotic phase (Fig. 2 A , white arrowheads ) in agreement with H3 revealed pS10 that was 73% also of the examined cells were by in indirect G 0 -G 1 phase immunofluorescence of the cell cycle, previous reports (24, 25). In contrast, H3 pS28 was not detected in with microscopy <9% of and the image cells being deconvolution in the G 2 in -M MCF-7 phase. breast A low cancer level of cells. H3 interphase nuclei, although its staining in G 2 -M cells seemed to be pS10 MCF-7 was cells detected cultured before in estrogen- treatment and (Fig. 2 serum-deplete C ). TPA conditions treatment similar to that of H3 pS10 (Fig. 2 A , bottom inset ). Immunoblot increased displayed a H3 punctate pS10 levels distribution (4.2-fold; of Fig. 2 H3 C pS10. ). This The observation number of shows these analyses of MCF-7 acid-extracted histones isolated from cycling that foci clearly stimulation increased of the upon Ras/MAPK addition signaling of TPA pathways for 30 minutes increases (Fig. 3), the cells confirmed the lack of H3 pS28 in interphase cells (Fig. 2 B ). level consistent of H3 with pS10. the immunoblot analysis (Fig. 2 C ). To determine The intense staining observed for H3 pS10 and pS28 in condensed whether H3 pS10 was positioned next to newly synthesized RNA, regions of chromatin during mitosis was also confirmed by the 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells elevated levels of both phospho-modified forms in colcemid- arrested cells (Fig. 2 B ). These results are in accordance with other studies showing that the staining of nuclei immunochemical stained with antibodies to H3 pS10 or pS28 becomes very intense (3, 24). These observations show that unlike mouse fibroblasts, only H3 pS10 is detected in interphase MCF-7 cells. The effect of TPA treatment of estrogen- and serum-deprived MCF-7 cells on H3 pS10 levels was determined in immunoblot scription sites in MCF-7 cells. The TPA-induced stimulation of H3 pS10 was also examined by indirect immunofluorescence microscopy and image deconvolution in MCF-7 breast cancer cells. MCF-7 cells cultured in estrogen- and serum-deplete conditions displayed a punctate distribution of H3 pS10. The number of these foci clearly increased upon addition of TPA for 30 minutes (Fig. 3), consistent with the immunoblot analysis (Fig. 2 C ). To determine whether H3 pS10 was positioned next to newly synthesized RNA, 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells and detected by anti-BrdUrd antibody. Deconvolved images showed that H3 pS10 foci were situated near or overlapped with active transcription sites as noted by the increase in yellow foci upon TPA stimulation (Fig. 3, insets ). Previous studies have shown that histone modification marks associated with transcription (e.g., acetylated K9 and K14 H3) were positioned next but did not coincide with the newly synthesized RNA as detected with 5-FU labeling (26). These results provided evidence that newly phosphorylated S10 H3 was associated with transcriptionally active chromatin. MAPK pathway. The association of TPA-induced H3 pS10 with active transcription sites prompted us to investigate whether MAPK inhibitor UO126 and MSK inhibitor H89 affected estradiol- or TPA-induced TFF1 gene expression. To assess the effects of MAPK inhibitors on TFF1 gene expression, we conducted reverse transcription-PCR (RT-PCR) to determine the levels of TFF1 transcripts relative to those of cyclophilin 33 (17, 27). Treatment of MCF-7 cells cultured under serum- and estradiol-deplete conditions with estradiol or TPA resulted in an immediate and sustained increase in TFF1 gene transcription (3.0-fold for estradiol and for TPA). In the presence of H89, estradiol induction of TFF1 was unaltered (3.0-fold with H89 versus 3.0-fold at 45 minutes; Fig. 4 A and C ). UO126 pretreatment did not prevent the estradiol- induced expression of the ...
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... recruitment of AP-1, MSK1, but not ER a , and the increased acetylation and S10 phosphorylation of H3 associated with the TFF1 promoter. MCF-7 cells. To determine the stimulatory effect of estrogens and TPA on the Ras/MAPK pathway in epithelial human breast cancer cells, estrogen-dependent MCF-7 cells were cultured under serum- and estrogen-depleted conditions. As EGF, but not TPA, weakly activates JNK/SAPKs and p38, we used TPA to stimulate the Ras/ MAPK in MCF-7 cells. A time course treatment with estradiol at physiologic concentrations displayed no significant increase in phospho-ERK1/2 levels, a hallmark of activated Ras/MAPK signaling (Fig. 1 A ). Furthermore, no change in total MSK1 protein levels was observed, but phospho-p38 levels displayed an increase in the presence of estradiol (6- to 7-fold at 5 minutes; Fig. 1 A ). In contrast, TPA treatment of these cells triggered an immediate and robust elevation in levels of phospho-ERK1/2 (7- to 8-fold at 30 minutes; Fig. 1 B ). Activated phospho-p38 was not detected. To verify that TPA up-regulation of MAPK occurs through the Ras/ Raf/MEK pathway, we used two potent MEK inhibitors PD98059 and UO126 (23). Treatment of cells with either agent before TPA stimulation prevented the TPA-induced increase of phospho-ERK, showing that TPA stimulation occurs through the Ras/MAPK pathway in these cells (Fig. 1 C ). Histone H3 phosphorylation at Ser 10 is increased by TPA. In mouse fibroblasts, H3 pS10 and pS28 is detected in interphase and mitotic cells (3). In indirect immunolocalization studies, we repeated these analyses with MCF-7 cells cultured in complete media. H3 pS10 had a punctate distribution throughout the interphase nuclei of MCF-7 breast cancer cells (Fig. 2 A , arrows, top inset ) and showed a widespread condensed staining in G 2 or scription experiments. sites Under in MCF-7 these cells. conditions, The TPA-induced flow cytometry stimulation analyses of mitotic phase (Fig. 2 A , white arrowheads ) in agreement with H3 revealed pS10 that was 73% also of the examined cells were by in indirect G 0 -G 1 phase immunofluorescence of the cell cycle, previous reports (24, 25). In contrast, H3 pS28 was not detected in with microscopy <9% of and the image cells being deconvolution in the G 2 in -M MCF-7 phase. breast A low cancer level of cells. H3 interphase nuclei, although its staining in G 2 -M cells seemed to be pS10 MCF-7 was cells detected cultured before in estrogen- treatment and (Fig. 2 serum-deplete C ). TPA conditions treatment similar to that of H3 pS10 (Fig. 2 A , bottom inset ). Immunoblot increased displayed a H3 punctate pS10 levels distribution (4.2-fold; of Fig. 2 H3 C pS10. ). This The observation number of shows these analyses of MCF-7 acid-extracted histones isolated from cycling that foci clearly stimulation increased of the upon Ras/MAPK addition signaling of TPA pathways for 30 minutes increases (Fig. 3), the cells confirmed the lack of H3 pS28 in interphase cells (Fig. 2 B ). level consistent of H3 with pS10. the immunoblot analysis (Fig. 2 C ). To determine The intense staining observed for H3 pS10 and pS28 in condensed whether H3 pS10 was positioned next to newly synthesized RNA, regions of chromatin during mitosis was also confirmed by the 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells elevated levels of both phospho-modified forms in colcemid- arrested cells (Fig. 2 B ). These results are in accordance with other studies showing that the staining of nuclei immunochemical stained with antibodies to H3 pS10 or pS28 becomes very intense (3, 24). These observations show that unlike mouse fibroblasts, only H3 pS10 is detected in interphase MCF-7 cells. The effect of TPA treatment of estrogen- and serum-deprived MCF-7 cells on H3 pS10 levels was determined in immunoblot top inset ) and showed a widespread condensed staining in G 2 or scription experiments. sites Under in MCF-7 these cells. conditions, The TPA-induced flow cytometry stimulation analyses of mitotic phase (Fig. 2 A , white arrowheads ) in agreement with H3 revealed pS10 that was 73% also of the examined cells were by in indirect G 0 -G 1 phase immunofluorescence of the cell cycle, previous reports (24, 25). In contrast, H3 pS28 was not detected in with microscopy <9% of and the image cells being deconvolution in the G 2 in -M MCF-7 phase. breast A low cancer level of cells. H3 interphase nuclei, although its staining in G 2 -M cells seemed to be pS10 MCF-7 was cells detected cultured before in estrogen- treatment and (Fig. 2 serum-deplete C ). TPA conditions treatment similar to that of H3 pS10 (Fig. 2 A , bottom inset ). Immunoblot increased displayed a H3 punctate pS10 levels distribution (4.2-fold; of Fig. 2 H3 C pS10. ). This The observation number of shows these analyses of MCF-7 acid-extracted histones isolated from cycling that foci clearly stimulation increased of the upon Ras/MAPK addition signaling of TPA pathways for 30 minutes increases (Fig. 3), the cells confirmed the lack of H3 pS28 in interphase cells (Fig. 2 B ). level consistent of H3 with pS10. the immunoblot analysis (Fig. 2 C ). To determine The intense staining observed for H3 pS10 and pS28 in condensed whether H3 pS10 was positioned next to newly synthesized RNA, regions of chromatin during mitosis was also confirmed by the 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells elevated levels of both phospho-modified forms in colcemid- arrested cells (Fig. 2 B ). These results are in accordance with other studies showing that the staining of nuclei immunochemical stained with antibodies to H3 pS10 or pS28 becomes very intense (3, 24). These observations show that unlike mouse fibroblasts, only H3 pS10 is detected in interphase MCF-7 cells. The effect of TPA treatment of estrogen- and serum-deprived MCF-7 cells on H3 pS10 levels was determined in immunoblot scription sites in MCF-7 cells. The TPA-induced stimulation of H3 pS10 was also examined by indirect immunofluorescence microscopy and image deconvolution in MCF-7 breast cancer cells. MCF-7 cells cultured in estrogen- and serum-deplete conditions displayed a punctate distribution of H3 pS10. The number of these foci clearly increased upon addition of TPA for 30 minutes (Fig. 3), consistent with the immunoblot analysis (Fig. 2 C ). To determine whether H3 pS10 was positioned next to newly synthesized RNA, 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells and detected by anti-BrdUrd antibody. Deconvolved images showed that H3 pS10 foci were situated near or overlapped with active transcription sites as noted by the increase in yellow foci upon TPA stimulation (Fig. 3, insets ). Previous studies have shown that histone modification marks associated with transcription (e.g., acetylated K9 and K14 H3) were positioned next but did not coincide with the newly synthesized RNA as detected with 5-FU labeling (26). These results provided evidence that newly phosphorylated S10 H3 was associated with transcriptionally active chromatin. MAPK pathway. The association of TPA-induced H3 pS10 with active transcription sites prompted us to investigate whether MAPK inhibitor UO126 and MSK inhibitor H89 affected estradiol- or TPA-induced TFF1 gene expression. To assess the effects of MAPK inhibitors on TFF1 gene expression, we conducted reverse transcription-PCR (RT-PCR) to determine the levels of TFF1 transcripts relative to those of cyclophilin 33 (17, 27). Treatment of MCF-7 cells cultured under serum- and estradiol-deplete conditions with estradiol or TPA resulted in an immediate and sustained increase in TFF1 gene transcription (3.0-fold for estradiol and for TPA). In the presence of H89, estradiol induction of TFF1 was unaltered (3.0-fold with H89 versus 3.0-fold at 45 minutes; Fig. 4 A and C ). UO126 pretreatment did not prevent the estradiol- induced expression of the TFF1 gene (3.0-fold; Fig. 4 A and C ). However, pretreatment of these cells with H89 reduced the TPA- stimulated TFF1 expression (1.5-fold with H89 as opposed to 3.0- fold; Fig. 4 B and D ). Furthermore, TFF1 transcription was suppressed to basal levels in the presence of UO126 (0.9-fold; Fig. 4 B and D ). These results provided evidence that TPA-induced, but not estradiol-induced, expression of TFF1 occurred through the Ras/MAPK pathway. response of TFF1. The TFF1 proximal promoter contains a specific protein 1 site, an estrogen-responsive element, and an AP-1 site that contribute to conferring estrogen responsiveness (Fig. 5 A ; refs. 17, 22, 28). To ascertain the function of the AP-1 and ERE sites in the TPA-induced activation of the TFF1 promoter, we determined the effect of mutating these elements in the transcription activation of TFF1 promoter. Wild-type and mutated TFF1 promoter/luciferase reporter constructs were transiently transfected into MCF-7. The transfection efficiencies of the reporter constructs were comparable among the three cell populations. TPA treatment stimulated the activity of the wild- type TFF1 promoter 13-fold (Fig. 5 B ). Mutation of the AP-1 site nearly abolished this TPA-mediated response, whereas the ERE mutation had a minor effect (Fig. 5 B ). These results suggest that assay to determine the association of downstream effectors of the the TPA-induced stimulation of the TFF1 gene requires a func- Ras/MAPK pathway on the endogenous TFF1 promoter. From tional AP-1 site. RT-PCR results, we consistently observed that TFF1 expression peaked at 45 minutes of estradiol treatment and at 30 minutes of TPA treatment of MCF-7 cells cultured under serum- and estradiol- We used the chromatin immunoprecipitation deplete conditions. Furthermore, steady-state level of H3 pS10 assay to determine the association of downstream effectors of the Ras/MAPK pathway on the endogenous TFF1 promoter. From RT-PCR results, we consistently observed that TFF1 expression peaked at 45 minutes of estradiol treatment and at 30 minutes of TPA ...
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... showing that TPA stimulation occurs through the Ras/MAPK pathway in these cells (Fig. 1 C ). Histone H3 phosphorylation at Ser 10 is increased by TPA. In mouse fibroblasts, H3 pS10 and pS28 is detected in interphase and mitotic cells (3). In indirect immunolocalization studies, we repeated these analyses with MCF-7 cells cultured in complete media. H3 pS10 had a punctate distribution throughout the interphase nuclei of MCF-7 breast cancer cells (Fig. 2 A , arrows, top inset ) and showed a widespread condensed staining in G 2 or scription experiments. sites Under in MCF-7 these cells. conditions, The TPA-induced flow cytometry stimulation analyses of mitotic phase (Fig. 2 A , white arrowheads ) in agreement with H3 revealed pS10 that was 73% also of the examined cells were by in indirect G 0 -G 1 phase immunofluorescence of the cell cycle, previous reports (24, 25). In contrast, H3 pS28 was not detected in with microscopy <9% of and the image cells being deconvolution in the G 2 in -M MCF-7 phase. breast A low cancer level of cells. H3 interphase nuclei, although its staining in G 2 -M cells seemed to be pS10 MCF-7 was cells detected cultured before in estrogen- treatment and (Fig. 2 serum-deplete C ). TPA conditions treatment similar to that of H3 pS10 (Fig. 2 A , bottom inset ). Immunoblot increased displayed a H3 punctate pS10 levels distribution (4.2-fold; of Fig. 2 H3 C pS10. ). This The observation number of shows these analyses of MCF-7 acid-extracted histones isolated from cycling that foci clearly stimulation increased of the upon Ras/MAPK addition signaling of TPA pathways for 30 minutes increases (Fig. 3), the cells confirmed the lack of H3 pS28 in interphase cells (Fig. 2 B ). level consistent of H3 with pS10. the immunoblot analysis (Fig. 2 C ). To determine The intense staining observed for H3 pS10 and pS28 in condensed whether H3 pS10 was positioned next to newly synthesized RNA, regions of chromatin during mitosis was also confirmed by the 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells elevated levels of both phospho-modified forms in colcemid- arrested cells (Fig. 2 B ). These results are in accordance with other studies showing that the staining of nuclei immunochemical stained with antibodies to H3 pS10 or pS28 becomes very intense (3, 24). These observations show that unlike mouse fibroblasts, only H3 pS10 is detected in interphase MCF-7 cells. The effect of TPA treatment of estrogen- and serum-deprived MCF-7 cells on H3 pS10 levels was determined in immunoblot top inset ) and showed a widespread condensed staining in G 2 or scription experiments. sites Under in MCF-7 these cells. conditions, The TPA-induced flow cytometry stimulation analyses of mitotic phase (Fig. 2 A , white arrowheads ) in agreement with H3 revealed pS10 that was 73% also of the examined cells were by in indirect G 0 -G 1 phase immunofluorescence of the cell cycle, previous reports (24, 25). In contrast, H3 pS28 was not detected in with microscopy <9% of and the image cells being deconvolution in the G 2 in -M MCF-7 phase. breast A low cancer level of cells. H3 interphase nuclei, although its staining in G 2 -M cells seemed to be pS10 MCF-7 was cells detected cultured before in estrogen- treatment and (Fig. 2 serum-deplete C ). TPA conditions treatment similar to that of H3 pS10 (Fig. 2 A , bottom inset ). Immunoblot increased displayed a H3 punctate pS10 levels distribution (4.2-fold; of Fig. 2 H3 C pS10. ). This The observation number of shows these analyses of MCF-7 acid-extracted histones isolated from cycling that foci clearly stimulation increased of the upon Ras/MAPK addition signaling of TPA pathways for 30 minutes increases (Fig. 3), the cells confirmed the lack of H3 pS28 in interphase cells (Fig. 2 B ). level consistent of H3 with pS10. the immunoblot analysis (Fig. 2 C ). To determine The intense staining observed for H3 pS10 and pS28 in condensed whether H3 pS10 was positioned next to newly synthesized RNA, regions of chromatin during mitosis was also confirmed by the 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells elevated levels of both phospho-modified forms in colcemid- arrested cells (Fig. 2 B ). These results are in accordance with other studies showing that the staining of nuclei immunochemical stained with antibodies to H3 pS10 or pS28 becomes very intense (3, 24). These observations show that unlike mouse fibroblasts, only H3 pS10 is detected in interphase MCF-7 cells. The effect of TPA treatment of estrogen- and serum-deprived MCF-7 cells on H3 pS10 levels was determined in immunoblot scription sites in MCF-7 cells. The TPA-induced stimulation of H3 pS10 was also examined by indirect immunofluorescence microscopy and image deconvolution in MCF-7 breast cancer cells. MCF-7 cells cultured in estrogen- and serum-deplete conditions displayed a punctate distribution of H3 pS10. The number of these foci clearly increased upon addition of TPA for 30 minutes (Fig. 3), consistent with the immunoblot analysis (Fig. 2 C ). To determine whether H3 pS10 was positioned next to newly synthesized RNA, 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells and detected by anti-BrdUrd antibody. Deconvolved images showed that H3 pS10 foci were situated near or overlapped with active transcription sites as noted by the increase in yellow foci upon TPA stimulation (Fig. 3, insets ). Previous studies have shown that histone modification marks associated with transcription (e.g., acetylated K9 and K14 H3) were positioned next but did not coincide with the newly synthesized RNA as detected with 5-FU labeling (26). These results provided evidence that newly phosphorylated S10 H3 was associated with transcriptionally active chromatin. MAPK pathway. The association of TPA-induced H3 pS10 with active transcription sites prompted us to investigate whether MAPK inhibitor UO126 and MSK inhibitor H89 affected estradiol- or TPA-induced TFF1 gene expression. To assess the effects of MAPK inhibitors on TFF1 gene expression, we conducted reverse transcription-PCR (RT-PCR) to determine the levels of TFF1 transcripts relative to those of cyclophilin 33 (17, 27). Treatment of MCF-7 cells cultured under serum- and estradiol-deplete conditions with estradiol or TPA resulted in an immediate and sustained increase in TFF1 gene transcription (3.0-fold for estradiol and for TPA). In the presence of H89, estradiol induction of TFF1 was unaltered (3.0-fold with H89 versus 3.0-fold at 45 minutes; Fig. 4 A and C ). UO126 pretreatment did not prevent the estradiol- induced expression of the TFF1 gene (3.0-fold; Fig. 4 A and C ). However, pretreatment of these cells with H89 reduced the TPA- stimulated TFF1 expression (1.5-fold with H89 as opposed to 3.0- fold; Fig. 4 B and D ). Furthermore, TFF1 transcription was suppressed to basal levels in the presence of UO126 (0.9-fold; Fig. 4 B and D ). These results provided evidence that TPA-induced, but not estradiol-induced, expression of TFF1 occurred through the Ras/MAPK pathway. response of TFF1. The TFF1 proximal promoter contains a specific protein 1 site, an estrogen-responsive element, and an AP-1 site that contribute to conferring estrogen responsiveness (Fig. 5 A ; refs. 17, 22, 28). To ascertain the function of the AP-1 and ERE sites in the TPA-induced activation of the TFF1 promoter, we determined the effect of mutating these elements in the transcription activation of TFF1 promoter. Wild-type and mutated TFF1 promoter/luciferase reporter constructs were transiently transfected into MCF-7. The transfection efficiencies of the reporter constructs were comparable among the three cell populations. TPA treatment stimulated the activity of the wild- type TFF1 promoter 13-fold (Fig. 5 B ). Mutation of the AP-1 site nearly abolished this TPA-mediated response, whereas the ERE mutation had a minor effect (Fig. 5 B ). These results suggest that assay to determine the association of downstream effectors of the the TPA-induced stimulation of the TFF1 gene requires a func- Ras/MAPK pathway on the endogenous TFF1 promoter. From tional AP-1 site. RT-PCR results, we consistently observed that TFF1 expression peaked at 45 minutes of estradiol treatment and at 30 minutes of TPA treatment of MCF-7 cells cultured under serum- and estradiol- We used the chromatin immunoprecipitation deplete conditions. Furthermore, steady-state level of H3 pS10 assay to determine the association of downstream effectors of the Ras/MAPK pathway on the endogenous TFF1 promoter. From RT-PCR results, we consistently observed that TFF1 expression peaked at 45 minutes of estradiol treatment and at 30 minutes of TPA treatment of MCF-7 cells cultured under serum- and estradiol- deplete conditions. Furthermore, steady-state level of H3 pS10 appeared at its maximum after 30 minutes of TPA induction in our studies (Fig. 2 C ). Thus, we assessed the in situ association of transcription factors and H3 acetylation and phosphorylation at these time points. As a control for the chromatin immunoprecipitation assay, we showed that ER a was not associated with the intron A-exon 2 region of the TFF1 region in cells treated with estradiol (Fig. 6 B ). As further controls for the assay, we confirmed the lack of amplification of the TFF1 promoter fragment in immunoprecipitations using anti-integrin antibodies and when the primary antibody was left out of the assay (Fig. 6 A ). Semiquanti- tative analyses of PCR products from chromatin immunoprecip- itation DNA were normalized against the input, and treated samples (E2 and TPA) were then compared with the untreated to acquire the fold induction. We found that after estradiol stimulation, there was an increase in ER a loading onto the TFF1 promoter (1.7- to 2.7-fold higher than untreated, n = 6), an elevation in H3 acetylation levels (1.1- to 1.8-fold than untreated, n = 5), and a modest increase in the levels of c-Jun (1.1- to 3-fold than ...
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... nuclei of MCF-7 breast cancer cells (Fig. 2 A , arrows, top inset ) and showed a widespread condensed staining in G 2 or scription experiments. sites Under in MCF-7 these cells. conditions, The TPA-induced flow cytometry stimulation analyses of mitotic phase (Fig. 2 A , white arrowheads ) in agreement with H3 revealed pS10 that was 73% also of the examined cells were by in indirect G 0 -G 1 phase immunofluorescence of the cell cycle, previous reports (24, 25). In contrast, H3 pS28 was not detected in with microscopy <9% of and the image cells being deconvolution in the G 2 in -M MCF-7 phase. breast A low cancer level of cells. H3 interphase nuclei, although its staining in G 2 -M cells seemed to be pS10 MCF-7 was cells detected cultured before in estrogen- treatment and (Fig. 2 serum-deplete C ). TPA conditions treatment similar to that of H3 pS10 (Fig. 2 A , bottom inset ). Immunoblot increased displayed a H3 punctate pS10 levels distribution (4.2-fold; of Fig. 2 H3 C pS10. ). This The observation number of shows these analyses of MCF-7 acid-extracted histones isolated from cycling that foci clearly stimulation increased of the upon Ras/MAPK addition signaling of TPA pathways for 30 minutes increases (Fig. 3), the cells confirmed the lack of H3 pS28 in interphase cells (Fig. 2 B ). level consistent of H3 with pS10. the immunoblot analysis (Fig. 2 C ). To determine The intense staining observed for H3 pS10 and pS28 in condensed whether H3 pS10 was positioned next to newly synthesized RNA, regions of chromatin during mitosis was also confirmed by the 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells elevated levels of both phospho-modified forms in colcemid- arrested cells (Fig. 2 B ). These results are in accordance with other studies showing that the staining of nuclei immunochemical stained with antibodies to H3 pS10 or pS28 becomes very intense (3, 24). These observations show that unlike mouse fibroblasts, only H3 pS10 is detected in interphase MCF-7 cells. The effect of TPA treatment of estrogen- and serum-deprived MCF-7 cells on H3 pS10 levels was determined in immunoblot top inset ) and showed a widespread condensed staining in G 2 or scription experiments. sites Under in MCF-7 these cells. conditions, The TPA-induced flow cytometry stimulation analyses of mitotic phase (Fig. 2 A , white arrowheads ) in agreement with H3 revealed pS10 that was 73% also of the examined cells were by in indirect G 0 -G 1 phase immunofluorescence of the cell cycle, previous reports (24, 25). In contrast, H3 pS28 was not detected in with microscopy <9% of and the image cells being deconvolution in the G 2 in -M MCF-7 phase. breast A low cancer level of cells. H3 interphase nuclei, although its staining in G 2 -M cells seemed to be pS10 MCF-7 was cells detected cultured before in estrogen- treatment and (Fig. 2 serum-deplete C ). TPA conditions treatment similar to that of H3 pS10 (Fig. 2 A , bottom inset ). Immunoblot increased displayed a H3 punctate pS10 levels distribution (4.2-fold; of Fig. 2 H3 C pS10. ). This The observation number of shows these analyses of MCF-7 acid-extracted histones isolated from cycling that foci clearly stimulation increased of the upon Ras/MAPK addition signaling of TPA pathways for 30 minutes increases (Fig. 3), the cells confirmed the lack of H3 pS28 in interphase cells (Fig. 2 B ). level consistent of H3 with pS10. the immunoblot analysis (Fig. 2 C ). To determine The intense staining observed for H3 pS10 and pS28 in condensed whether H3 pS10 was positioned next to newly synthesized RNA, regions of chromatin during mitosis was also confirmed by the 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells elevated levels of both phospho-modified forms in colcemid- arrested cells (Fig. 2 B ). These results are in accordance with other studies showing that the staining of nuclei immunochemical stained with antibodies to H3 pS10 or pS28 becomes very intense (3, 24). These observations show that unlike mouse fibroblasts, only H3 pS10 is detected in interphase MCF-7 cells. The effect of TPA treatment of estrogen- and serum-deprived MCF-7 cells on H3 pS10 levels was determined in immunoblot scription sites in MCF-7 cells. The TPA-induced stimulation of H3 pS10 was also examined by indirect immunofluorescence microscopy and image deconvolution in MCF-7 breast cancer cells. MCF-7 cells cultured in estrogen- and serum-deplete conditions displayed a punctate distribution of H3 pS10. The number of these foci clearly increased upon addition of TPA for 30 minutes (Fig. 3), consistent with the immunoblot analysis (Fig. 2 C ). To determine whether H3 pS10 was positioned next to newly synthesized RNA, 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells and detected by anti-BrdUrd antibody. Deconvolved images showed that H3 pS10 foci were situated near or overlapped with active transcription sites as noted by the increase in yellow foci upon TPA stimulation (Fig. 3, insets ). Previous studies have shown that histone modification marks associated with transcription (e.g., acetylated K9 and K14 H3) were positioned next but did not coincide with the newly synthesized RNA as detected with 5-FU labeling (26). These results provided evidence that newly phosphorylated S10 H3 was associated with transcriptionally active chromatin. MAPK pathway. The association of TPA-induced H3 pS10 with active transcription sites prompted us to investigate whether MAPK inhibitor UO126 and MSK inhibitor H89 affected estradiol- or TPA-induced TFF1 gene expression. To assess the effects of MAPK inhibitors on TFF1 gene expression, we conducted reverse transcription-PCR (RT-PCR) to determine the levels of TFF1 transcripts relative to those of cyclophilin 33 (17, 27). Treatment of MCF-7 cells cultured under serum- and estradiol-deplete conditions with estradiol or TPA resulted in an immediate and sustained increase in TFF1 gene transcription (3.0-fold for estradiol and for TPA). In the presence of H89, estradiol induction of TFF1 was unaltered (3.0-fold with H89 versus 3.0-fold at 45 minutes; Fig. 4 A and C ). UO126 pretreatment did not prevent the estradiol- induced expression of the TFF1 gene (3.0-fold; Fig. 4 A and C ). However, pretreatment of these cells with H89 reduced the TPA- stimulated TFF1 expression (1.5-fold with H89 as opposed to 3.0- fold; Fig. 4 B and D ). Furthermore, TFF1 transcription was suppressed to basal levels in the presence of UO126 (0.9-fold; Fig. 4 B and D ). These results provided evidence that TPA-induced, but not estradiol-induced, expression of TFF1 occurred through the Ras/MAPK pathway. response of TFF1. The TFF1 proximal promoter contains a specific protein 1 site, an estrogen-responsive element, and an AP-1 site that contribute to conferring estrogen responsiveness (Fig. 5 A ; refs. 17, 22, 28). To ascertain the function of the AP-1 and ERE sites in the TPA-induced activation of the TFF1 promoter, we determined the effect of mutating these elements in the transcription activation of TFF1 promoter. Wild-type and mutated TFF1 promoter/luciferase reporter constructs were transiently transfected into MCF-7. The transfection efficiencies of the reporter constructs were comparable among the three cell populations. TPA treatment stimulated the activity of the wild- type TFF1 promoter 13-fold (Fig. 5 B ). Mutation of the AP-1 site nearly abolished this TPA-mediated response, whereas the ERE mutation had a minor effect (Fig. 5 B ). These results suggest that assay to determine the association of downstream effectors of the the TPA-induced stimulation of the TFF1 gene requires a func- Ras/MAPK pathway on the endogenous TFF1 promoter. From tional AP-1 site. RT-PCR results, we consistently observed that TFF1 expression peaked at 45 minutes of estradiol treatment and at 30 minutes of TPA treatment of MCF-7 cells cultured under serum- and estradiol- We used the chromatin immunoprecipitation deplete conditions. Furthermore, steady-state level of H3 pS10 assay to determine the association of downstream effectors of the Ras/MAPK pathway on the endogenous TFF1 promoter. From RT-PCR results, we consistently observed that TFF1 expression peaked at 45 minutes of estradiol treatment and at 30 minutes of TPA treatment of MCF-7 cells cultured under serum- and estradiol- deplete conditions. Furthermore, steady-state level of H3 pS10 appeared at its maximum after 30 minutes of TPA induction in our studies (Fig. 2 C ). Thus, we assessed the in situ association of transcription factors and H3 acetylation and phosphorylation at these time points. As a control for the chromatin immunoprecipitation assay, we showed that ER a was not associated with the intron A-exon 2 region of the TFF1 region in cells treated with estradiol (Fig. 6 B ). As further controls for the assay, we confirmed the lack of amplification of the TFF1 promoter fragment in immunoprecipitations using anti-integrin antibodies and when the primary antibody was left out of the assay (Fig. 6 A ). Semiquanti- tative analyses of PCR products from chromatin immunoprecip- itation DNA were normalized against the input, and treated samples (E2 and TPA) were then compared with the untreated to acquire the fold induction. We found that after estradiol stimulation, there was an increase in ER a loading onto the TFF1 promoter (1.7- to 2.7-fold higher than untreated, n = 6), an elevation in H3 acetylation levels (1.1- to 1.8-fold than untreated, n = 5), and a modest increase in the levels of c-Jun (1.1- to 3-fold than untreated, n = 5) and MSK1 (1.1- to 3-fold higher than untreated, n = 6), which coincided with a modest increase in H3 pS10 (1.2- to 4-fold than untreated, n = 8; Fig. 6 A ). Upon TPA stimulation, there was a prominent recruitment of c-Jun and MSK1 (3.3- to 6-fold, n = 5 and 1.4- to 6-fold higher than untreated, n = 6, respectively) to the TFF1 promoter and increased levels of H3 pS10 (1.5- to 5.7-fold higher ...
Context 5
... cells. The TPA-induced stimulation of H3 pS10 was also examined by indirect immunofluorescence microscopy and image deconvolution in MCF-7 breast cancer cells. MCF-7 cells cultured in estrogen-and serum-deplete conditions displayed a punctate distribution of H3 pS10. The number of these foci clearly increased upon addition of TPA for 30 minutes (Fig. 3), consistent with the immunoblot analysis (Fig. 2C). To determine whether H3 pS10 was positioned next to newly synthesized RNA, 5-FU was incorporated into nascent RNA transcripts of MCF-7 cells Figure 2. Histone H3 phosphorylation at Ser 10 is abundant in MCF-7 cells and increases after TPA treatment. A, cycling MCF-7 cells maintained ...
Context 6
... Res 2006; 66: (9). May 1, 2006 and detected by anti-BrdUrd antibody. Deconvolved images showed that H3 pS10 foci were situated near or overlapped with active transcription sites as noted by the increase in yellow foci upon TPA stimulation (Fig. 3, insets). Previous studies have shown that histone modification marks associated with transcription (e.g., acetylated K9 and K14 H3) were positioned next but did not coincide with the newly synthesized RNA as detected with 5-FU labeling (26). These results provided evidence that newly phosphorylated S10 H3 was associated with transcriptionally ...
Citations
... H3S10ph levels are also increased in breast cancer cells and, similarly to other epithelial cancers, increase after TPA treatment via the activity of RAS/MAPK pathway. Promoter region of a breast cancer marker gene and an inferior prognostic factor, trefoil factor 1 (TFF1) is occupied by MSK1 and decorated with H3S10ph [103]. The MSK-mediated phosphorylation of H3S10 leads to the recruitment of 14-3-3 adaptor and BRG1 to the enhancer and upstream promoter regions of TFF1 [104]. ...
Background
With the discovery that more than half of human cancers harbor mutations in chromatin proteins, deregulation of epigenetic mechanisms has been recognized a hallmark of malignant transformation. Post-translational modifications (PTMs) of histone proteins, as main components of epigenetic regulatory machinery, are also broadly accepted as therapeutic target. Current “epigenetic” therapies target predominantly writers, erasers and readers of histone acetylation and (to a lesser extent) methylation, leaving other types of PTMs largely unexplored. One of them is the phosphorylation of serine 10 on histone H3 (H3S10ph).
Main body
H3S10ph is emerging as an important player in the initiation and propagation of cancer, as it facilitates cellular malignant transformation and participates in fundamental cellular functions. In normal cells this histone mark dictates the hierarchy of additional histone modifications involved in the formation of protein binding scaffolds, transcriptional regulation, blocking repressive epigenetic information and shielding gene regions from heterochromatin spreading. During cell division, this mark is essential for chromosome condensation and segregation. It is also involved in the function of specific DNA–RNA hybrids, called R-loops, which modulate transcription and facilitate chromosomal instability. Increase in H3S10ph is observed in numerous cancer types and its abundance has been associated with inferior prognosis. Many H3S10-kinases, including MSK1/2, PIM1, CDK8 and AURORA kinases, have been long considered targets in cancer therapy. However, since these proteins also participate in other critical processes, including signal transduction, apoptotic signaling, metabolic fitness and transcription, their chromatin functions are often neglected.
Conclusions
H3S10ph and enzymes responsible for deposition of this histone modification are important for chromatin activity and oncogenesis. Epigenetic-drugs targeting this axis of modifications, potentially in combination with conventional or targeted therapy, provide a promising angle in search for knowledge-driven therapeutic strategies in oncology.
... In the past few years, an increasing number of studies have reported that the dysregulation of PTMs is implicated in the development and progression of several diseases, including cancer (19)(20)(21)(22). Song et al. identified that parkin may inhibit cell growth by decreasing ribosomal protein SA (RPSA) expression and inducing phosphorylation of cytokeratin 8/18 (23). ...
The dbPTM (http://dbPTM.mbc.nctu.edu.tw/) has been maintained for over 10 years with the aim to provide functional and structural analyses for post-translational modifications (PTMs). In this update, dbPTM not only integrates more experimentally validated PTMs from available databases and through manual curation of literature but also provides PTM-disease associations based on non-synonymous single nucleotide polymorphisms (nsSNPs). The high-throughput deep sequencing technology has led to a surge in the data generated through analysis of association between SNPs and diseases, both in terms of growth amount and scope. This update thus integrated disease-associated nsSNPs from dbSNP based on genome-wide association studies. The PTM substrate sites located at a specified distance in terms of the amino acids encoded from nsSNPs were deemed to have an association with the involved diseases. In recent years, increasing evidence for crosstalk between PTMs has been reported. Although mass spectrometry-based proteomics has substantially improved our knowledge about substrate site specificity of single PTMs, the fact that the crosstalk of combinatorial PTMs may act in concert with the regulation of protein function and activity is neglected. Because of the relatively limited information about concurrent frequency and functional relevance of PTM crosstalk, in this update, the PTM sites neighboring other PTM sites in a specified window length were subjected to motif discovery and functional enrichment analysis. This update highlights the current challenges in PTM crosstalk investigation and breaks the bottleneck of how proteomics may contribute to understanding PTM codes, revealing the next level of data complexity and proteomic limitation in prospective PTM research.
... For MSK1 to elicit an appropriate biological response, it is crucial that it be properly recruited to the promoter region of target genes. Multiple studies have revealed that many transcriptional activators, such as Elk1, activator protein-1, NF-κB, and RARα, have a role in recruiting MSK1 to specific promoter regions for localized modification of chromatin 8,10,41,42 . However, no reports have clearly demonstrated p53-dependent recruitment of kinases to chromatin to modulate transcription. ...
New drugs to fight tumors could be developed, thanks to research revealing how a protein called MSK1 interacts with and assists another protein, “p53,” which has well-established tumor-suppressing activity. Researchers led by Jeong Hyeon Park at Massey University in New Zealand and Jaehoon Kim at the Korea Advanced Institute of Science and Technology in South Korea studied the function of the MSK1 protein in cultured human cells. They uncovered details of a previously unknown direct interaction between the MSK1 and p53 proteins. This allows the MSK1-p53 combination to increase the activity of a gene that codes for another protein, p21. The p21 protein then participates in further molecular processes that can restrict cell growth and multiplication. Drugs that control MSK1 activity might offer new opportunities to combat the uncontrolled multiplication of cancer cells.
... As H3K9acS10ph and H3K27acS28ph are typically mutually exclusive on a given allele, it is likely that MSK1/2 complexes with PCAF or CBP/p300 are recruited separately to different alleles. Specific transcription factors (for example, NF-κB, ELK-1, AP-1) recruit MSK1/2 complexes to the regulatory regions of IE genes (Espino et al., 2006;Joo and Jetten, 2008;Zhang et al., 2008). Once recruited, depending on the acetylation state of K27, MSK phosphorylates H3 at S10 or S28 of nucleosomes located at these regulatory regions. ...
Stimulation of the MAPK pathway results in mitogen- and stress-activated protein kinase 1/2 (MSK1/2)-catalyzed phosphorylation of histone H3 at serine 10 or 28 and expression of immediate-early (IE) genes. In 10T1/2 mouse fibroblasts, phosphorylation of H3S10 and H3S28 occurs on different H3 molecules and in different nuclear regions. Likewise, we show that mitogen-induced H3S10 and H3S28 phosphorylation occurs in separate pools in human primary fibroblasts. High-resolution imaging studies on both cell types have revealed that H3S10 and H3S28 phosphorylation events can be induced in a single cell but on different alleles, giving rise to H3S10ph and H3S28ph epi-alleles. Co-immunoprecipitation and inhibition studies demonstrate that CBP/p300-mediated H3K27 acetylation is required for MSK1/2 to phosphorylate S28. Although the K9ac and S10ph marks coexist on H3, S10 phosphorylation is not dependent on K9 acetylation by PCAF. We propose that random targeting of H3S10 or H3S28 results from the stochastic acetylation of H3 by CBP/p300 or PCAF, a process comparable to transcriptional bursting causing temporary allelic imbalance. In 10T1/2 cells expressing Jun, at least two out of three alleles per cell were induced, a sign of high expression level. H3S10ph and H3S28ph redundant role may enable a rapid and efficient IE gene induction.
... Phosphorylation of H3 at S10 and S28 (leading to H3S10ph and H3S28ph, respectively) is crucial during chromosome condensation in mitosis and during transcriptional activation of immediate early genes. Interestingly, growth factors that induce Ras/MAPK and increase H3S10ph at transcriptionally active loci are implicated in aberrant gene expression, as observed in breast cancer progression [146]. Phosphorylation of histone H3 at specific residues is recognized by the 14-3-3 chaperones, which comprise seven isoforms in mammals. ...
To guarantee appropriate management of genetic information, chromatin is posttranslationally modified in histones as well as in DNA. Posttranslational modifications (PTMs) of histones include acetylation, methylation, phosphorylation, ubiquitination, ADP-ribosylation, and SUMOylation, among others. The main role of these PTMs is to regulate chromatin compaction, DNA accessibility, and to serve as binding sites for effector molecules; hence, they represent an overall modulation of any biological process involving DNA transactions. In the past 20 years, a considerable effort has been made to understand the specific role of these modifications in cell physiology and their impact on human health. In this chapter, we summarize the current knowledge on the implications of histone PTMs in diverse human pathologies. We focus on the identified changes in histone modifications and associated enzymes in human diseases, as well as on their potential role in clinical diagnosis.
... MSK1 is downstream of MAPK [17], and activation of Histone H3 can occur as a result of MSK1 phosphorylation. Hyper-phosphorylation of histone H3 on serine 10 site may cause cell chromatin structural changes to open transcriptional factor promoter regions leading to enhanced gene transcription, the outcome of which is cell and stimulus dependent, and can range from cellular differentiation and cell proliferation to cell transformation and neoplasia [18][19][20]. ...
... In the case of MAPK signaling in ht-UtLM cells, genistein increased phospho-MAPK p44/42 levels resulting in a high predilection for MSK1 and histone H3 phosphorylation at serine10 site that induced epigenetic changes that led to enrichment of promoter regions of immediate early response genes known to correspond to cell proliferation and tumorigenesis [25]. It was reported that MSK1 is recruited to promoters of target genes by transcription factors such as Elk1 and the progesterone receptor [19,20,28]. In the cells there is a change in transcription factors composition and activity, which may alter the dynamics of MSK1 recruitment to target promoters to activate histone H3 leading to chromatin structure alterations [18] and in turn increase target promoter expression levels during tumorigenesis. ...
Background
The phytoestrogen, genistein at low doses nongenomically activates mitogen-activated protein kinase p44/42 (MAPKp44/42) via estrogen receptor alpha (ERα) leading to proliferation of human uterine leiomyoma cells. In this study, we evaluated if MAPKp44/42 could activate downstream effectors such as mitogen- and stress-activated protein kinase 1 (MSK1), which could then epigenetically modify histone H3 by phosphorylation following a low dose (1 μg/ml) of genistein. ResultsUsing hormone-responsive immortalized human uterine leiomyoma (ht-UtLM) cells, we found that genistein activated MAPKp44/42 and MSK1, and also increased phosphorylation of histone H3 at serine10 (H3S10ph) in ht-UtLM cells. Colocalization of phosphorylated MSK1 and H3S10ph was evident by confocal microscopy in ht-UtLM cells (r = 0.8533). Phosphorylation of both MSK1and H3S10ph was abrogated by PD98059 (PD), a MEK1 kinase inhibitor, thereby supporting genistein’s activation of MSK1 and Histone H3 was downstream of MAPKp44/42. In proliferative (estrogenic) phase human uterine fibroid tissues, phosphorylated MSK1 and H3S10ph showed increased immunoexpression compared to normal myometrial tissues, similar to results observed in in vitro studies following low-dose genistein administration. Real-time RT-PCR arrays showed induction of growth-related transcription factor genes, EGR1, Elk1, ID1, and MYB (cMyb) with confirmation by western blot, downstream of MAPK in response to low-dose genistein in ht-UtLM cells. Additionally, genistein induced associations of promoter regions of the above transcription factors with H3S10ph as evidenced by Chromatin Immunoprecipitation (ChIP) assays, which were inhibited by PD. Therefore, genistein epigenetically modified histone H3 by phosphorylation of serine 10, which was regulated by MSK1 and MAPK activation. Conclusion
Histone H3 phosphorylation possibly represents a mechanism whereby increased transcriptional activation occurs following low-dose genistein exposure.
... The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification (4). Contemporary research has implicated the dysregulation of PTMs in severe pathological events, including cancer, disease and drug resistance, motivating a thorough investigation of protein modification dynamics (5)(6)(7)(8)(9)(10). Mass spectrometry (MS)-based experiments provide a practical means of the site-specific identification of PTMs in proteomics (11). ...
... Many proteins undergo PTMs that involve physical or chemical changes to their side chains, causing cancer or other diseases; other PTMs may be used diagnostically (5)(6)(7)(8)(9)(10). Accordingly, the disease annotations in the KEGG Disease Database (59), the Online Mendelian Inheritance in Man database (OMIM) (60) and Human Protein Reference Database (HPRD) (61) were integrated to identify associations between diseases and PTM-associated proteins. ...
Owing to the importance of the post-translational modifications (PTMs) of proteins in regulating biological processes, the dbPTM (http://dbPTM.mbc.nctu.edu.tw/) was developed as a comprehensive database of experimentally verified PTMs from several databases with annotations of potential PTMs for all UniProtKB protein entries. For this 10th anniversary of dbPTM, the updated resource provides not only a comprehensive dataset of experimentally verified PTMs, supported by the literature, but also an integrative interface for accessing all available databases and tools that are associated with PTM analysis. As well as collecting experimental PTM data from 14 public databases, this update manually curates over 12 000 modified peptides, including the emerging S-nitrosylation, S-glutathionylation and succinylation, from approximately 500 research articles, which were retrieved by text mining. As the number of available PTM prediction methods increases, this work compiles a non-homologous benchmark dataset to evaluate the predictive power of online PTM prediction tools. An increasing interest in the structural investigation of PTM substrate sites motivated the mapping of all experimental PTM peptides to protein entries of Protein Data Bank (PDB) based on database identifier and sequence identity, which enables users to examine spatially neighboring amino acids, solvent-accessible surface area and side-chain orientations for PTM substrate sites on tertiary structures. Since drug binding in PDB is annotated, this update identified over 1100 PTM sites that are associated with drug binding. The update also integrates metabolic pathways and protein-protein interactions to support the PTM network analysis for a group of proteins. Finally, the web interface is redesigned and enhanced to facilitate access to this resource.
... Overactive Ras-MAPK pathway and elevated MSK1 activity were observed in various cancerous tissues and cell lines [14,15]. MSK1 is responsible for histone H3 phosphorylation of estrogen-responsive Trefoil Factor-1 (TFF-1) promoter in breast cancer MCF-7 cells [16]. MSK1 also regulates transcriptional activation of NFκ B-dependent pro-inflammatory genes in response to cigarette smoke [17]. ...
Mitogen- and Stress-Activated Kinase 1 (MSK1) is a nuclear kinase that serves as active link between extracellular signals and the primary response of gene expression. However, the involvement of MSK1 in malignant transformation and cancer development is not well understood. In this study, we aimed to explore the role of MSK1 in Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1)-promoted carcinogenesis of nasopharyngeal carcinoma (NPC).
The level of MSK1 phosphorylation at Thr581 was detected by the immunohistochemical analysis in NPC tissues and normal nasopharynx tissues, and its correlation with LMP1 was analyzed in NPC tissues and cell lines. Using MSK1 inhibitor H89 or small interfering RNA (siRNA)-MSK1, the effects of MSK1 on LMP1-promoted CNE1 cell proliferation and transformation were evaluated by CCK-8 assay, flow cytometry and focus-forming assay respectively. Furthermore, the regulatory role of MSK1-mediated histone H3 phosphorylation at Ser10 on the promoter activity and expression of Fra-1 or c-Jun was determined by reporter gene assay and western blotting analysis.
Immunohistochemical analysis revealed that the level of MSK1 phosphorylation at Thr581 was significantly higher in the poorly differentiated NPC tissues than that in normal nasopharynx tissues (P < 0.001). Moreover, high level of phosphorylated MSK1 was positively correlated with the expression of LMP1 in NPC tissues (r = 0.393, P = 0.002) and cell lines. MSK1 inhibitor H89 or knockdown of MSK1 by siRNA dramatically suppressed LMP1-promoted CNE1 cell proliferation, which was associated with the induction of cell cycle arrest at G0/G1 phase. In addition, the anchorage-independent growth promoted by LMP1 was blocked in MSK1 knockdown cells. When the activity or expression of MSK1 was inhibited, LMP1-induced promoter activities of Fra-1 and c-Jun as well as their protein levels were greatly reduced. It was found that only H3 WT, but not mutant H3 S10A, dramatically increased LMP1 induction of Fra-1 and c-Jun genes compared with mock cells.
Increasing MSK1 activity is critically important for LMP1-promoted cell proliferation and transformation in NPC, which may be correlated with its induction of Fra-1 and c-Jun through phosphorylation of histone H3 at Ser10.
... These data are in concordance with previous observations showing that nucleosomal organization of c-Ha-Ras oncogene transformed fibroblasts is more decondensed than that of normal parental fibroblasts (27). Several groups have also reported a global increase in phosphorylation of histone H3 at serine 10 in transformed cells (29,46,47) that may also be related to a more open chromatin conformation (46) and it is indispensable for cell transformation (29). More recently, Liu and colleagues have shown that Ras-PI3K signaling downregulates H3K56ac, which is associated to alteration on proliferation and cell migration of tumor cells (8); interestingly, they showed that Ras-PI3K-AKT pathway specifically targets H3K56ac via CBP/p300 degradation mediated by MDM2 ubiquitin ligase (8). ...
Cell transformation is clearly linked to epigenetic changes. However, the role of the histone-modifying enzymes in this process
is still poorly understood. In this study, we investigated the contribution of the histone acetyltransferase (HAT) enzymes
to Ras-mediated transformation. Our results demonstrated that lysine acetyltransferase 5, also known as Tip60, facilitates
histone acetylation of bulk chromatin in Ras-transformed cells. As a consequence, global H4 acetylation (H4K8ac and H4K12ac)
increases in Ras-transformed cells, rendering a more decompacted chromatin than in parental cells. Furthermore, low levels
of CREB-binding protein (CBP) lead to hypoacetylation of retinoblastoma 1 (Rb1) and cyclin-dependent kinase inhibitor 1B (Cdkn1b or p27Kip1) tumour suppressor gene promoters to facilitate Ras-mediated transformation. In agreement with these data, overexpression
of Cbp counteracts Ras transforming capability in a HAT-dependent manner. Altogether our results indicate that CBP and Tip60 coordinate
histone acetylation at both local and global levels to facilitate Ras-induced transformation.
... Hormones (estradiol [11]), growth factors such as insulin-like growth factor I (IGF-I), IGF-II, epidermal growth factor (EGF) or transforming growth factor (TGF) [11][12][13], phorbol esters [11,14], plasminogen activator and Fos and Jun oncoproteins [11] have been reported to regulate the expression of the TFF1 through the activation of the gene promoter containing an estrogen-response element (ERE) and a TPA-response element (TRE) [15]. The peptide was found to be overexpressed in approximately 50 % of primary breast carcinomas [16], mainly estrogen receptor alpha-positive (ERα+) ones. ...
... Nevertheless, these low estrogen levels in postmenopausal women are still able to induce estrogenic response in a way to stimulate TFF1 expression in breast cancer cells. In addition, TFF1 expression in breast cancer may also be induced by growth factors, such as IGF-I [15,25], via signaling pathway that are related or not to ER, thus making it estrogen-independent. On the basis of our results, we assumed that TFF1 expression is predominantly regulated by growth factors in ER-negative tumors of postmenopausal patients while additionally regulated through indirect estrogen-dependent ER signaling pathways in ER-positive tumors of these patients [51]. ...
Background. A role of an estrogen-regulated, autocrine motogenic factor was assumed to be a major biological role of trefoil factor 1 (TFF1) in breast cancer. TFF1 is regarded as a predictive factor for positive response to endocrine therapy in breast cancer patients. The aim of our study was to examine TFF1 level distribution in breast carcinomas in order to distinguish estrogen-independent from estrogen-dependent TFF1 expression and to evaluate clinical usefulness of TFF1 status in early breast cancer during the first 3 years of follow-up.
Methods. The study included 226 patients with primary operable invasive early breast carcinomas for whom an equal, a 3-year follow-up was conducted. TFF1 levels as well as estrogen receptor (ER) and progesterone receptor (PR) levels were measured in cytosolic extracts of tumor samples by immunoradiometric assay or by use of classical biochemical method, respectively. Non-parametric statistical tests were applied for data analyses.
Results. Statistical analysis revealed that TFF1 levels were significantly higher in premenopausal patients (p=0.02), or in tumors with: lower histological grade (p<0.001), positive ER or PR status (p<0.001, in both cases). On the basis of TFF1 level distribution between ER-negative and ER-positive postmenopausal patients with tumors of different histological grade, 14 ng/mg was set as the cut-off value to distinguish estrogen-independent from estrogen-dependent TFF1 expression in breast cancer. Depending on menopausal and PR status, positive TFF1 status identified patients at opposite risk for relapse among ER-positive patients with grade II tumors. Among ER- and PR-positive premenopausal patients with grade II tumors, TFF1 status alone identified patients at opposite risk for relapse.
Conclusions. Determination of TFF1 status might identify patients at different risk for relapse and help in making decision on administering adjuvant therapy for early breast cancer patients during the first 3 years of follow-up.
