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HDAC1 and Klf4 interplay critically regulates human myeloid leukemia cell proliferation.

Huang Y, Chen J, Lu C, Han J, Wang G, Song C, Zhu S, Wang C, Li G, Kang J, Wang J - Cell Death Dis (2014)

Bottom Line: Here, we found that HDAC1 expression was negatively correlated with that of Krüppel-like factor 4 (Klf4) and that AML patients with lower HDAC1 level had better prognosis.Further, knockdown of HDAC1 in leukemia cells K562, HL-60, and U937 significantly increased Klf4 expression and inhibited cell cycle progression and cell proliferation, similar results were found for HDAC inhibitors (VPA and mocetinostat).We identified HDAC1 as a potential specific target for repressing cell proliferation and inducing cell cycle arrest through interplay and modulation of Klf4 expression, suggests that HDAC1 and Klf4 are potential new molecular markers and targets for clinical diagnosis, prognosis, and treatment of myeloid leukemia.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, PR China.

ABSTRACT
Acute myeloid leukemia (AML) is recognized as a complex disease of hematopoietic stem cell disorders, but its pathogenesis mechanisms, diagnosis, and treatment remain unclear. General histone deacetylase (HDAC) inhibitors have been used in blood cancers including AML, but the lack of gene specificity greatly limits their anti-cancer effects and clinical applications. Here, we found that HDAC1 expression was negatively correlated with that of Krüppel-like factor 4 (Klf4) and that AML patients with lower HDAC1 level had better prognosis. Further, knockdown of HDAC1 in leukemia cells K562, HL-60, and U937 significantly increased Klf4 expression and inhibited cell cycle progression and cell proliferation, similar results were found for HDAC inhibitors (VPA and mocetinostat). Moreover, overexpression or knockdown of Klf4 could markedly block the effects of HDAC1 overexpression or knockdown on leukemia cells in vitro and in vivo, respectively. Mechanistic analyses demonstrated that HDAC1 and Klf4 competitively bound to the promoter region of Klf4 and oppositely regulated Klf4 expression in myeloid leukemia. We identified HDAC1 as a potential specific target for repressing cell proliferation and inducing cell cycle arrest through interplay and modulation of Klf4 expression, suggests that HDAC1 and Klf4 are potential new molecular markers and targets for clinical diagnosis, prognosis, and treatment of myeloid leukemia.

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HDAC1 knockdown-induced Klf4 activation inhibits cell proliferation through upregulation of p21 and p27. (a) QRT-PCR analyses (left panel) and western blotting assays (right panel) showing the effects of HDAC1 knockdown (shHDAC1) on the expression of Klf1, Klf3, Klf4, and Klf7 in K562 cells. ** and *** indicate P<0.01 and P<0.001, respectively. (b) Overexpression and knockdown efficiency of Klf4 after being infected with lentiviruses containing the cDNA (Klf4) and shRNA (shKlf4) for 72 h in K562 cells. GAPDH was used as a loading control. (c) CCK8 assays showing the effects of overexpression and knockdown of Klf4 on cell proliferation in K562, HL-60, and U937 cells at day 1, 2, 3, and 4, respectively. * and ** indicate P<0.05 and P<0.01, respectively. (d) Analyses of the effects of overexpression and knockdown of Klf4 on cell cycle in K562, HL-60, and U937 cells. Cells were collected and fixed using 75% ethanol, stained using PI and measured using FACS assay. * and ** indicate P<0.05 and P<0.01, respectively. (e) QRT-PCR analyses showing the effects of overexpression and knockdown of Klf4 on the expression levels of p21 and p27. GAPDH was used as the internal control. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively. (f) Western blotting assays showing the effects of overexpression and knockdown of Klf4 on the expression levels of p21 and p27. GAPDH was used as the loading control. (g) ChIP-PCR assays of the binding sites of Klf4 at the promoter regions of p21 and p27, according to fold enrichment normalized to normal IgG. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively
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fig3: HDAC1 knockdown-induced Klf4 activation inhibits cell proliferation through upregulation of p21 and p27. (a) QRT-PCR analyses (left panel) and western blotting assays (right panel) showing the effects of HDAC1 knockdown (shHDAC1) on the expression of Klf1, Klf3, Klf4, and Klf7 in K562 cells. ** and *** indicate P<0.01 and P<0.001, respectively. (b) Overexpression and knockdown efficiency of Klf4 after being infected with lentiviruses containing the cDNA (Klf4) and shRNA (shKlf4) for 72 h in K562 cells. GAPDH was used as a loading control. (c) CCK8 assays showing the effects of overexpression and knockdown of Klf4 on cell proliferation in K562, HL-60, and U937 cells at day 1, 2, 3, and 4, respectively. * and ** indicate P<0.05 and P<0.01, respectively. (d) Analyses of the effects of overexpression and knockdown of Klf4 on cell cycle in K562, HL-60, and U937 cells. Cells were collected and fixed using 75% ethanol, stained using PI and measured using FACS assay. * and ** indicate P<0.05 and P<0.01, respectively. (e) QRT-PCR analyses showing the effects of overexpression and knockdown of Klf4 on the expression levels of p21 and p27. GAPDH was used as the internal control. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively. (f) Western blotting assays showing the effects of overexpression and knockdown of Klf4 on the expression levels of p21 and p27. GAPDH was used as the loading control. (g) ChIP-PCR assays of the binding sites of Klf4 at the promoter regions of p21 and p27, according to fold enrichment normalized to normal IgG. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively

Mentions: In consistent with the result of clinical analyses, knockdown of HDAC1 significantly increased the expression of Klfs, particularly Klf4 (Figure 3a). Again, different from HDAC1 knockdown, knockdown of HDAC2, HDAC3, and HDAC8 showed no significant effect on Klf4 expression (Supplementary Figure S3E). Furthermore, using HDACis VPA and mocetinostat, we found that Klf4 is elevated in HDACi-treated cells (Supplementary Figure S1D, S2D, S4A, and S4B). These data demonstrated HDAC1 is negatively correlated with Klf4. Modulation of Klf4 levels by altering shRNA or using an ectopic expression vector can mimic the effects of HDAC1 on cell proliferation, cell cycle, and the expression level of p21 and p27 in human leukemia cell lines K562, HL-60, and U937 (Figures 3b and f). Furthermore, chromatin immunoprecipitation (ChIP)-PCR assays showed that Klf4 can bind to the promoter regions of both p21 and p27 (Figure 3g). Primers used in this assay targets p21 and p27 promoter at six sites (Supplementary Figure S4C). Dual-luciferase reporter gene assay also confirmed that Klf4 could bind at the promoter regions of p27 (Supplementary Figure S4D). All of these results indicate that HDAC1 knockdown inhibits cell proliferation mainly through Klf4 activation, direct binding of Klf4 to p21 and p27 promoter regions, and the induction of cell cycle arrest. Mechanistic studies also indicate that knockdown of HDAC1 increased histone acetylation levels at the Klf4 promoter region and that both HDAC1 and Klf4 can bind to the Klf4 promoter (Figures 4a–c) without direct interaction at the protein level (Supplementary Figure S4E). Cells treated with VPA also showed significantly higher acetylation levels of histone H3 and H4 at the Klf4 promoter region (Supplementary Figure S4F). Furthermore, HDAC1 knockdown also enhanced the binding of Klf4 at its own promoter regions (Figure 4d), which indicates that HDAC1 and Klf4 may compete in binding at the Klf4 promoter region. Primers used in this assay targets Klf4 promoter at four sites (Figure 4e): site 1 (−255, −351), site 2 (−446, −558), site 3 (−610, −745), and site 4 (−1447, −1604). The information of other four sites was included in Supplementary Table S2. Four binding elements of stimulatory protein (Sp1) were indentified on the proximal portion of the Klf4 promoter and were named as Sp1-1, Sp1-2, Sp1-3, and Sp1-4 (Figure 4e). Mutation of the Sp1-1, Sp1-3, or Sp1-4 site significantly killed HDAC1 knockdown-stimulated Klf4 promoter activity whereas Sp1-2 decreased part of the activity of Klf4 promoter (Figure 4f). Data in Supplementary Figure S4G also demonstrated that HDAC1 can mediate the luciferase activities of Klf4 promoter. These studies suggested that transactivation of Klf4 by HDAC1 knockdown or VPA appeared to be mediated through interaction with the Sp1-binding domain on the promoter and is also likely to involve histone acetylation. To investigate the role of Sp1 in Klf4 transcriptional regulation, we performed western blotting analyses to detect the expression and acetylation level of Sp1 upon mocetinostat treatment. Results showed that Sp1 expression was reduced and acetyl-Sp1 level was elevated upon mocetinostat treatment (Supplementary Figure S4H and S4I). Furthermore, mocetinostat repressed the binding of Sp1 at Klf4 promoter (Supplementary Figure S4J). These data indicated that Sp1 may interact with HDAC1 to repress Klf4 transcription, whereas HDACi would repress Sp1-mediated inhibition of Klf4 expression.


HDAC1 and Klf4 interplay critically regulates human myeloid leukemia cell proliferation.

Huang Y, Chen J, Lu C, Han J, Wang G, Song C, Zhu S, Wang C, Li G, Kang J, Wang J - Cell Death Dis (2014)

HDAC1 knockdown-induced Klf4 activation inhibits cell proliferation through upregulation of p21 and p27. (a) QRT-PCR analyses (left panel) and western blotting assays (right panel) showing the effects of HDAC1 knockdown (shHDAC1) on the expression of Klf1, Klf3, Klf4, and Klf7 in K562 cells. ** and *** indicate P<0.01 and P<0.001, respectively. (b) Overexpression and knockdown efficiency of Klf4 after being infected with lentiviruses containing the cDNA (Klf4) and shRNA (shKlf4) for 72 h in K562 cells. GAPDH was used as a loading control. (c) CCK8 assays showing the effects of overexpression and knockdown of Klf4 on cell proliferation in K562, HL-60, and U937 cells at day 1, 2, 3, and 4, respectively. * and ** indicate P<0.05 and P<0.01, respectively. (d) Analyses of the effects of overexpression and knockdown of Klf4 on cell cycle in K562, HL-60, and U937 cells. Cells were collected and fixed using 75% ethanol, stained using PI and measured using FACS assay. * and ** indicate P<0.05 and P<0.01, respectively. (e) QRT-PCR analyses showing the effects of overexpression and knockdown of Klf4 on the expression levels of p21 and p27. GAPDH was used as the internal control. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively. (f) Western blotting assays showing the effects of overexpression and knockdown of Klf4 on the expression levels of p21 and p27. GAPDH was used as the loading control. (g) ChIP-PCR assays of the binding sites of Klf4 at the promoter regions of p21 and p27, according to fold enrichment normalized to normal IgG. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively
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fig3: HDAC1 knockdown-induced Klf4 activation inhibits cell proliferation through upregulation of p21 and p27. (a) QRT-PCR analyses (left panel) and western blotting assays (right panel) showing the effects of HDAC1 knockdown (shHDAC1) on the expression of Klf1, Klf3, Klf4, and Klf7 in K562 cells. ** and *** indicate P<0.01 and P<0.001, respectively. (b) Overexpression and knockdown efficiency of Klf4 after being infected with lentiviruses containing the cDNA (Klf4) and shRNA (shKlf4) for 72 h in K562 cells. GAPDH was used as a loading control. (c) CCK8 assays showing the effects of overexpression and knockdown of Klf4 on cell proliferation in K562, HL-60, and U937 cells at day 1, 2, 3, and 4, respectively. * and ** indicate P<0.05 and P<0.01, respectively. (d) Analyses of the effects of overexpression and knockdown of Klf4 on cell cycle in K562, HL-60, and U937 cells. Cells were collected and fixed using 75% ethanol, stained using PI and measured using FACS assay. * and ** indicate P<0.05 and P<0.01, respectively. (e) QRT-PCR analyses showing the effects of overexpression and knockdown of Klf4 on the expression levels of p21 and p27. GAPDH was used as the internal control. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively. (f) Western blotting assays showing the effects of overexpression and knockdown of Klf4 on the expression levels of p21 and p27. GAPDH was used as the loading control. (g) ChIP-PCR assays of the binding sites of Klf4 at the promoter regions of p21 and p27, according to fold enrichment normalized to normal IgG. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively
Mentions: In consistent with the result of clinical analyses, knockdown of HDAC1 significantly increased the expression of Klfs, particularly Klf4 (Figure 3a). Again, different from HDAC1 knockdown, knockdown of HDAC2, HDAC3, and HDAC8 showed no significant effect on Klf4 expression (Supplementary Figure S3E). Furthermore, using HDACis VPA and mocetinostat, we found that Klf4 is elevated in HDACi-treated cells (Supplementary Figure S1D, S2D, S4A, and S4B). These data demonstrated HDAC1 is negatively correlated with Klf4. Modulation of Klf4 levels by altering shRNA or using an ectopic expression vector can mimic the effects of HDAC1 on cell proliferation, cell cycle, and the expression level of p21 and p27 in human leukemia cell lines K562, HL-60, and U937 (Figures 3b and f). Furthermore, chromatin immunoprecipitation (ChIP)-PCR assays showed that Klf4 can bind to the promoter regions of both p21 and p27 (Figure 3g). Primers used in this assay targets p21 and p27 promoter at six sites (Supplementary Figure S4C). Dual-luciferase reporter gene assay also confirmed that Klf4 could bind at the promoter regions of p27 (Supplementary Figure S4D). All of these results indicate that HDAC1 knockdown inhibits cell proliferation mainly through Klf4 activation, direct binding of Klf4 to p21 and p27 promoter regions, and the induction of cell cycle arrest. Mechanistic studies also indicate that knockdown of HDAC1 increased histone acetylation levels at the Klf4 promoter region and that both HDAC1 and Klf4 can bind to the Klf4 promoter (Figures 4a–c) without direct interaction at the protein level (Supplementary Figure S4E). Cells treated with VPA also showed significantly higher acetylation levels of histone H3 and H4 at the Klf4 promoter region (Supplementary Figure S4F). Furthermore, HDAC1 knockdown also enhanced the binding of Klf4 at its own promoter regions (Figure 4d), which indicates that HDAC1 and Klf4 may compete in binding at the Klf4 promoter region. Primers used in this assay targets Klf4 promoter at four sites (Figure 4e): site 1 (−255, −351), site 2 (−446, −558), site 3 (−610, −745), and site 4 (−1447, −1604). The information of other four sites was included in Supplementary Table S2. Four binding elements of stimulatory protein (Sp1) were indentified on the proximal portion of the Klf4 promoter and were named as Sp1-1, Sp1-2, Sp1-3, and Sp1-4 (Figure 4e). Mutation of the Sp1-1, Sp1-3, or Sp1-4 site significantly killed HDAC1 knockdown-stimulated Klf4 promoter activity whereas Sp1-2 decreased part of the activity of Klf4 promoter (Figure 4f). Data in Supplementary Figure S4G also demonstrated that HDAC1 can mediate the luciferase activities of Klf4 promoter. These studies suggested that transactivation of Klf4 by HDAC1 knockdown or VPA appeared to be mediated through interaction with the Sp1-binding domain on the promoter and is also likely to involve histone acetylation. To investigate the role of Sp1 in Klf4 transcriptional regulation, we performed western blotting analyses to detect the expression and acetylation level of Sp1 upon mocetinostat treatment. Results showed that Sp1 expression was reduced and acetyl-Sp1 level was elevated upon mocetinostat treatment (Supplementary Figure S4H and S4I). Furthermore, mocetinostat repressed the binding of Sp1 at Klf4 promoter (Supplementary Figure S4J). These data indicated that Sp1 may interact with HDAC1 to repress Klf4 transcription, whereas HDACi would repress Sp1-mediated inhibition of Klf4 expression.

Bottom Line: Here, we found that HDAC1 expression was negatively correlated with that of Krüppel-like factor 4 (Klf4) and that AML patients with lower HDAC1 level had better prognosis.Further, knockdown of HDAC1 in leukemia cells K562, HL-60, and U937 significantly increased Klf4 expression and inhibited cell cycle progression and cell proliferation, similar results were found for HDAC inhibitors (VPA and mocetinostat).We identified HDAC1 as a potential specific target for repressing cell proliferation and inducing cell cycle arrest through interplay and modulation of Klf4 expression, suggests that HDAC1 and Klf4 are potential new molecular markers and targets for clinical diagnosis, prognosis, and treatment of myeloid leukemia.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, PR China.

ABSTRACT
Acute myeloid leukemia (AML) is recognized as a complex disease of hematopoietic stem cell disorders, but its pathogenesis mechanisms, diagnosis, and treatment remain unclear. General histone deacetylase (HDAC) inhibitors have been used in blood cancers including AML, but the lack of gene specificity greatly limits their anti-cancer effects and clinical applications. Here, we found that HDAC1 expression was negatively correlated with that of Krüppel-like factor 4 (Klf4) and that AML patients with lower HDAC1 level had better prognosis. Further, knockdown of HDAC1 in leukemia cells K562, HL-60, and U937 significantly increased Klf4 expression and inhibited cell cycle progression and cell proliferation, similar results were found for HDAC inhibitors (VPA and mocetinostat). Moreover, overexpression or knockdown of Klf4 could markedly block the effects of HDAC1 overexpression or knockdown on leukemia cells in vitro and in vivo, respectively. Mechanistic analyses demonstrated that HDAC1 and Klf4 competitively bound to the promoter region of Klf4 and oppositely regulated Klf4 expression in myeloid leukemia. We identified HDAC1 as a potential specific target for repressing cell proliferation and inducing cell cycle arrest through interplay and modulation of Klf4 expression, suggests that HDAC1 and Klf4 are potential new molecular markers and targets for clinical diagnosis, prognosis, and treatment of myeloid leukemia.

Show MeSH
Related in: MedlinePlus