Histone acetyltransferase p300 regulates the expression of human pituitary tumor transforming gene (hPTTG)

The human pituitary tumor transforming gene (hPTTG) serves as a marker for malignancy grading in several cancers, hPTTG is in volved in multiple cellular pathways including cell transformation, apoptosis, DNA repair, genomic instability, mitotic control and angiogenesis induction. However, the molecular mechanisms underlying hPTTG regulation have not been fully explored. In this study, we found that overexpression of histone acetyltransferase (HAT) p300 upregulated hPTTG at the levels of promoter activity, mRNA and protein expression. Moreover, the HAT activity of p300 was critical for its regulatory function. Chromatin immunoprecipitation (ChIP)analysis revealed that overexpression of p300 elevated the level of histone H3 acetylation on the hPTTG promoter. Additionally, the NF-Y sites at the hPTTG promoter exhibited a synergistic effect on upregulation of hPTTG through interacting with p300. We also found thattreatment of 293T cells with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) increased hPTTG promoter activity. Meanwhile, we provided evidence that HDAC3 decreased hPTTG promoter activity. These data implicate an important role of the histone acetylation modification in the regulation of hPTTG.     

HDAC inhibitors sodium butyrate and sodium valproate do not affect human ncor1 and ncor2 gene expression in HL-60 cells

AIM. This study was designed to examine whether the class I and class IIa histone deacetylase (HDAC) inhibitors, sodium butyrate and sodium valproate alter the expression of human NCOR1 and/or NCOR2 genes coding for N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), respectively. METHODS: Human leukemia HL-60 cells were treated for 24 h with 0.5 and 1 mM sodium butyrate, 1 to 3 mM sodium valproate, 1 mcM all-trans retinoic acid (ATRA) or cotreated with 1 mcM ATRA and 0.5 mM sodium butyrate. The acetylation of histones H3 and H4 was analysed by western blotting. The levels of NCOR1 and NCOR2 mRNA were determined by quantitative real-time PCR. Expression of NCF2 gene coding for the NADPH oxidase subunit p67phox was evaluated as a marker of myeloid differentiation. Results. Both butyrate and valproate increased the acetylation of histone H3 at Lys9 and/or Lys14 as well as histone H4 at Lys12. Both HDAC inhibitors caused a significant increase in NCF2 mRNA levels without affecting NCOR1 or NCOR2 mRNA levels. Similarly, ATRA alone or in combination with butyrate induced NCF2 gene expression without any significant influence on the expression of NCOR1 or NCOR2 genes. CONCLUSION: We conclude that inhibitors of class I and class IIa HDACs do not alter the expression of human NCOR1 or NCOR2 genes and that the onset of myeloid differentiation is not accompanied by induction or repression of these genes in HL-60 cells.     

The MEF2A and MEF2D function as scaffold proteins that interact with HDAC1 or p300 in SOD3 expression in THP-1 cells

Superoxide dismutase 3 (SOD3) is a SOD isozyme and plays a key role in extracellular redox homeostasis. We previously demonstrated that histone acetylation is involved in 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited SOD3 expression in human monocytic THP-1 cells; however, the molecular mechanisms responsible for its expression have not yet been elucidated in detail. The results of the present study demonstrated that the binding of histone deacetylase 1 (HDAC1) to the SOD3 promoter region contributed to SOD3 silencing in basal THP-1 cells. On the other hand, the dissociation of HDAC1 from the SOD3 promoter region and the enrichment of p300, a histone acetyltransferase (HAT), within that region were observed in TPA-induced THP-1 cells. Myocyte enhancer factor 2 (MEF2) functions as a scaffold protein that interacts with histone deacetylases (HDAC) or HAT and regulates gene expression. The present results showed that the MEF2A and MEF2D function as mediators for TPA-elicited SOD3 expression by interacting with HDAC or p300. Additionally, the knockdown of MEF2A or MEF2D in human skin fibroblasts suppressed SOD3 expression at the mRNA and protein levels. Our results provide an insight into epigenetic regulation of redox gene expression, and may ultimately contribute to suppressing the progression of tumours and vascular diseases.     

Differential effects of histone deacetylase inhibitors on interleukin-18 gene expression in myeloid cells

Histone deacetyrase (HDAC) inhibitors induce growth arrest and differentiation of leukemia cell lines and tumor cells derived from a large variety of human tissues. Here we showed that HDAC inhibitors sodium butyrate, TSA, and valproate regulated the expression of Interleukin-18 (IL-18), a cytokine with antitumor and proinflammatory properties, in human acute myeloid leukemia cell lines U937 and HEL. Sodium butyrate increased expression of IL-18 protein and mRNA and activated 1357bp IL-18 gene promoter construct. IL-18 mRNA level was up-regulated by TSA or valproate, which also activated IL-18 full-length promoter. While sodium butyrate or TSA stimulated the 108-bp IL-18 minimal promoter, valproate failed to activate it, indicating that valproate may use a distinct mechanism from sodium butyrate and TSA to activate IL-18 gene expression.     

Histone acetyltransferase p300 regulates the expression of human pituitary rumor transforming gene （hPTTG）

The human pituitary tumor transforming gene （hPTTG） serves as a marker for malignancy grading in several cancers, hPTTG is involved in multiple cellular pathways including cell transformation, apoptosis, DNA repair, genomic instability, mitotic control and angiogenesis induction. However, the molecular mechanisms underlying hPTTG regulation have not been fully explored. In this study, we found that overexpression of histone acetyltransferase （HAT） p300 upregulated hPTTG at the levels of promoter activity, mRNA and protein expression. Moreover, the HAT activity of p300 was critical for its regulatory function. Chromatin immunoprecipitation （CHIP） analysis revealed that overexpression of p300 elevated the level of histone H3 acetylation on the hPTTG promoter. Additionally, the NF-Y sites at the hPTTG promoter exhibited a synergistic effect on upregulation of hPTTG through interacting with p300. We also found that treatment of 293T cells with the histone deacetylase （HDAC） inhibitor Tfichostatin A （TSA） increased hPTTG promoter activity. Meanwhile, we provided evidence that HDAC3 decreased hPTTG promoter activity. These data implicate an important role of the histone acetylafion modification in the regulation of hPTTG.     

Cu2+ is required for pyrrolidine dithiocarbamate to inhibit histone acetylation and induce human leukemia cell apoptosis

Pyrrolidine dithiocarbamate (PDTC) has been considered as a potential anticancer drug due to its powerful apoptogenic effect towards cancer cells, where Cu(2+) plays a distinct yet undefined role. Here we report that Cu(2+) is critically needed for PDTC to inhibit histone acetylation in both human leukemia HL-60 cells and human hepatoma Hep3B cells. The inhibition of histone acetylation mainly resulted from the increase of intracellular Cu(2+), but was not due to the inhibition of NF-kappaB activity by PDTC-Cu(2+) since the combinations of Cu(2+) with SN50, MG132 (two known NF-kappaB inhibitors), or bathocuproine disulfonate (BCS, a specific Cu(2+) chelator that does not cross the plasma membrane), did not lead to obvious inhibition of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cells exposed to PDTC-Cu(2+) showed a comparable decrease in histone acetylation levels in HL-60 cells in the absence or presence of the HDAC inhibitors, trichostatin A (TSA) or sodium butyrate (NaBu); the inhibition rates were about 45, 44 and 43%, respectively. PDTC-Cu(2+) had no effect on the activity of HDAC in vitro, but significantly inhibited the HAT activity both in HL-60 cells and in a cell-free in vitro system. PDTC-Cu(2+) also induced HL-60 cell apoptosis, and treating cells with TSA, NaBu or BCS significantly attenuated the apoptosis induced by PDTC-Cu(2+). Collectively, these results showed that inhibition of histone acetylation represents a distinct mechanism for the cytotoxicity of PDTC in the presence of Cu(2+), where HAT is its possible molecular target.     

Role of histone deacetylation in cell-specific expression of endothelial nitric-oxide synthase

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5'-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2'-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.     

Novel protein pp3501 mediates the inhibitory effect of sodium butyrate on SH-SY5Y cell proliferation

Sodium butyrate, a new potential therapeutic drug, improves the efficacy of chemo- and immunotherapy of cancer under unknown mechanisms. A novel gene pp3501 is significantly induced in SH-SY5Y neuroblastoma cells upon sodium butyrate treatment. Therefore, this study has cloned pp3501 cDNA by RT-PCR and generated its recombinant fusion protein and anti-serum subsequently. The pp3501 protein localized mainly in the nucleus, as detected by immunocytochemistry and the expression of pp3501-EGFP fusion protein. pp3501 inhibited the proliferation of SH-SY5Y cells, arrested the cell cycle at G1 phase, and sensitized the SH-SY5Y cells to sodium butyrate treatment. These results provide a new mechanism of sodium butyrate inhibiting cancer cell proliferation as well as a new avenue for the future research on the functions of pp3501.     

Dexamethasone-induced protein degradation in cultured myotubes is p300/HAT dependent

Muscle proteolysis during sepsis and other catabolic conditions is, at least in part, regulated by glucocorticoids. Dexamethasone-treated myotubes are a commonly used in vitro model of muscle wasting. We reported recently that treatment of cultured L6 myotubes with dexamethasone resulted in increased gene and protein expression of the nuclear cofactor p300 but it is not known whether glucocorticoids upregulate p300 histone acetyl transferase (HAT) activity in muscle and whether p300/HAT activity regulates glucocorticoid-induced muscle proteolysis. Here, we found that treatment of cultured L6 myotubes with dexamethasone resulted in increased nuclear p300/HAT activity. Treatment of myotubes with p300 siRNA or transfection of muscle cells with a plasmid expressing p300 that was mutated in its HAT activity domain blocked the dexamethasone-induced increase in protein degradation, supporting a role of p300/HAT in glucocorticoid-induced muscle proteolysis. In addition to increased HAT activity, treatment of the myotubes with dexamethasone resulted in reduced nuclear expression and activity of histone deacetylases (HDACs) 3 and 6. When myotubes were treated with the HDAC inhibitor trichostatin A, protein degradation increased to the same degree as in dexamethasone-treated myotubes. The results suggest that glucocorticoids increase HAT and decrease HDAC activities in muscle, changes that both favor hyperacetylation. The results also provide evidence that dexamethasone-induced protein degradation in cultured myotubes is, at least in part, regulated by p300/HAT activity.     

可逆的组蛋白乙酰化调控p16的表达

p16^INK4a通过抑制CDK4/6的活性而在细胞周期进行中发挥重要的作用,研究发现,组蛋白乙酰转移酶p300能促进p16^INK4a启动子活性,而组蛋白去乙酰化酶HDAC3/4能够逆转由p300介导的p16^INK4a启动子活性的增加,HDAC3/4能够降低p16^INK4a mRNA和蛋白质的水平．染色质免疫沉淀(ChIP)实验结果表明转染p300表达质粒能够逆转由HDAC3/4介导的p16^INK4a启动子组蛋白的低乙酰化状态．此外,免疫荧光实验结果表明HDAC4的核质穿梭起着重要的作用．免疫印迹和染色质免疫沉淀实验证明HDAC的抑制剂丁酸钠盐(NaBu)能通过诱导组蛋白的高乙酰化而促进p16^INK4a的表达．基于这些实验结果,推测出可逆的组蛋白乙酰化参与p16^INK4a基因转录调控的模型．