PP1α, PP1β and Wip-1 regulate H4S47 phosphorylation and deposition of histone H3 variant H3.3

Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo, whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo. These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells.     

Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression

The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE.     

Protein H1: a role for chromatin structure in the regulation of bacterial gene expression and virulence?

There has been a recent revival of interest in one of the most abundant Escherichia coli proteins, H1 (also called H-NS). This protein was first identified many years ago as a major component of the bacterial nucleoid, and has been characterized biochemically by several groups. However, no clear function for the protein emerged from these studies. Our thinking has been transformed by recent findings which complement the biochemistry with genetic data. Several mutations, selected over many years by virtue of their diverse effects on gene expression, have turned out to be allelic and to fall within the structural gene for H1. Bringing together the genetics and the biochemistry has demonstrated that the whole is worth more than the sum of the parts! These findings have far-reaching implications for the mechanisms by which gene expression is regulated and also, perhaps, for the control of bacterial virulence.     

Cloning and expression of endo-β-glucanase II gene in Humicola insolens H31-3

Endo-β-glucanase II (EG II) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR. It was cloned into the expression vector pGAPZαA. The resultant recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after being linearized by BspHI digestion. The recombinant Pichia pastoris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation condition and the characteristics of the recombinant EG II were also explored. __________ Translated from Microbiology, 2006, 33(6): 68273 [译自: 微生物学 通报]     

Exploration of protein–protein interaction effects on α-2-macroglobulin in an inhibition of serine protease through gene expression and molecular simulations studies

In Prophenoloxidase (ProPO) cascade, two targets namely serine protease and α-2-macroglobulin are key regulators involved in the defense system of crustaceans. In biological systems, routine role of cell systems requires the understanding in protein–protein interactions through experimental and theoretical concepts, which might yield useful insights into the cellular responses. Response of cells to regulating the immune system is governed by the interactions-involved biomolecular simulations. Unfortunately, studies on the inhibitors (SP and α-2M) that negatively regulate the proPO system or melanization in penaeid shrimp are not yet available. In order to understand how these interactions change the proPO mechanism in Indian white shrimp Fenneropenaeus indicus was determined. In F. indicus, innate immune system is in a sensitive balance of intricate interactions; elucidating these interactions by the integration of in silico and in vitro has great potential. We have determined the expression of both the SP and α-2M enzymes in regulatory mechanism, which are analyzed through qRT-PCR, protein–protein docking, and simulation studies. From this work, we propose a novel approach for studying an organism at the systems level by integrating genome-wide computational analysis and the gene expression data.     

L-type voltage-dependent calcium channels facilitate acetylation of histone H3 through PKCγ phosphorylation in mice with methamphetamine-induced place preference

The present study investigated regulation of histone acetylation by L-type voltage-dependent calcium channels (VDCCs), one of the machineries to provide Ca(2+) signals. Acetylation of histone through the phosphorylation of protein kinase Cγ (PKCγ) in the development of methamphetamine (METH)-induced place preference was demonstrated in the limbic forebrain predominantly but also in the nucleus accumbens of α1C subunit knockout mice. Chronic administration of METH produced a significant place preference in mice, which was dose-dependently inhibited by both chelerythrine (a PKC inhibitor) and nifedipine (an L-type VDCC blocker). Protein levels of acetylated histone H3 and p-PKCγ significantly increased in the limbic forebrain of mice showing METH-induced place preference, and it was also significantly attenuated by pre-treatment with chelerythrine or nifedipine. METH-induced place preference was also significantly attenuated by deletion of half the α1C gene, which is one of the subunits forming Ca(2+) channels. Furthermore, increased acetylation of histone H3 was found in specific gene-promoter regions related to synaptic plasticity, such as Nrxn, Syp, Dlg4, Gria1, Grin2a, Grin2b, Camk2a, Creb, and cyclin-dependent kinase 5, in wild-type mice showing METH-induced place preference, while such enhancement of multiple synaptic plasticity genes was significantly attenuated by a deletion of half the α1C gene. These findings suggest that L-type VDCCs play an important role in the development of METH-induced place preference by facilitating acetylation of histone H3 in association with enhanced expression of synaptic plasticity genes via PKCγ phosphorylation following an increase in the intracellular Ca(2+) concentration.     

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (− 386/− 374), non-perfect DR1 (− 118/− 106) and HNF4α-specific binding motif (H4-SBM) (− 26/− 14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.     

Glycogen synthase kinase-3β regulates etoposide-induced apoptosis via Bcl-2 mediated caspase-3 activation in C3H10T1/2 cells

Glycogen synthase kinase-3β (GSK3β) controls the survival of osteoblasts during bone development through Wnt canonical signaling. GSK3β is a key factor for osteoblastogenesis, but relatively less is known regarding its role in osteoblast apoptosis. Genotoxic stress induced by etoposide promoted apoptotic signaling by GSK3β activation in C3H10T1/2 cells, a mouse mesenchymal cell line. Etoposide led to the time-dependent activation of GSK3β and caspase-3, which resulted in PARP cleavage. LiCl (a specific inhibitor) and siRNA (gene knock-down) of GSK3β prevented the effects of etoposide on apoptosis. Staurosporine also induced apoptosis in C3H10T1/2 cells, but LiCl could not rescue. Bcl-2 was decreased in the cells by exposure to etoposide. LiCl completely recovered Bcl-2 expression as shown by both the mRNA and the protein expression levels. In conclusion, etoposide-induced apoptosis in C3H10T1/2 cells is mediated by GSK3β, which leads to caspase-3 activation via decrease in Bcl-2 expression. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

socs7, a target gene of microRNA-145, regulates interferon-β induction through STAT3 nuclear translocation in bladder cancer cells

We recently reported that microRNA (miR)-145 is downregulated and induces apoptosis in human bladder cancer cells. Also, it is suggested that the ectopic expression of miR-145 induces apoptosis with the induction of TRAIL expression in several cancer cells. Here, we demonstrated a novel mechanism of apoptosis induction by miR-145 in bladder cancer cells. Exogenous miR-145 in T24 and NKB1 cells markedly increased the expression levels of interferon (IFN)-β, 2′–5′-oligoadenylate synthetase 1, which lies upstream of 2′–5′ oligoadenylates/RNase L system, and TRAIL, and induced apparent caspase-dependent apoptosis that was suppressed by cotreatment with a pan-caspase inhibitor; moreover, these expression levels were reduced by cotreatment with an miR-145 inhibitor. The apoptosis did not depend on Toll-like receptor 3 (TLR3) expression, because TLR3-silencing failed to inhibit IFN-β induction by miR-145. Then, we focused on the suppressor of cytokine signaling 7 (socs7), whose expression level was upregulated in bladder cancer cells compared with its level in normal human urothelial cells, as a putative target gene involved in IFN-β induction by miR-145. Expectedly, exogenous miR-145 decreased the expression level of SOCS7, and socs7-silencing enhanced IFN-β induction by transfection with a TLR3 ligand, polyinosinic acid-polycytidylic acid (PIC). The results of a luciferase reporter assay revealed that miR-145 targeted socs7. In addition, socs7-silencing significantly decreased the level of p-Akt and suppressed the growth of T24 cells. Furthermore, exogenous miR-145 or socs7-silencing promoted nuclear translocation of STAT3. In conclusion, the machinery of IFN-β induction through the regulation of SOCS7 by miR-145 was closely associated with the induction of apoptosis. Moreover, exogenous miR-145 promoted IFN-β induction by targeting socs7, which resulted in the nuclear translocation of STAT3. Additionally, our data indicate that SOCS7 functioned as an oncogene, the finding that revealed a novel mechanism of carcinogenesis in bladder cancer cells.