A bacterial effector targets host DH‐PH domain RhoGEFs and antagonizes macrophage phagocytosis

Bacterial pathogens often harbour a type III secretion system (TTSS) that injects effector proteins into eukaryotic cells to manipulate host processes and cause diseases. Identification of host targets of bacterial effectors and revealing their mechanism of actions are crucial for understating bacterial virulence. We show that EspH, a type III effector conserved in enteric bacterial pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli and Citrobacter rodentium, markedly disrupts actin cytoskeleton structure and induces cell rounding up when ectopically expressed or delivered into HeLa cells by the bacterial TTSS. EspH inactivates host Rho GTPase signalling pathway at the level of RhoGEF. EspH directly binds the DH‐PH domain in multiple RhoGEFs, which prevents their binding to Rho and thereby inhibits nucleotide exchange‐mediated Rho activation. Consistently, infection of mouse macrophages with EPEC harbouring EspH attenuates phagocytosis of the bacteria as well as FcγR‐mediated phagocytosis. EspH represents the first example of targeting RhoGEFs by bacterial effectors, and our results also reveal an unprecedented mechanism used by enteric pathogens to counteract the host defence system.     

Inhibition of histone deacetylase 2 increases apoptosis and p21Cip1/WAF1 expression, independent of histone deacetylase 1

Histone deacetylases (HDACs) 1 and 2 share a high degree of homology and coexist within the same protein complexes. Despite their close association, each possesses unique functions. We show that the upregulation of HDAC2 in colorectal cancer occurred early at the polyp stage, was more robust and occurred more frequently than HDAC1. Similarly, while the expression of HDACs1 and 2 were increased in cervical dysplasia and invasive carcinoma, HDAC2 expression showed a clear demarcation of high-intensity staining at the transition region of dysplasia compared to HDAC1. Upon HDAC2 knockdown, cells displayed an increased number of cellular extensions reminiscent of cell differentiation. There was also an increase in apoptosis, associated with increased p21Cip1/WAF1 expression that was independent of p53. These results suggest that HDACs, especially HDAC2, are important enzymes involved in the early events of carcinogenesis, making them candidate markers for tumor progression and targets for cancer therapy.     

Altered levels of histone deacetylase OsHDT1 affect differential gene expression patterns in hybrid rice

Hybrids between different inbred varieties display novel patterns of gene expression resulted from parental variation in allelic nucleotide sequences. To study the function of chromatin regulators in hybrid gene expression, the histone deacetylase gene OsHDT1 whose expression displayed a circadian rhythm was over-expressed or inactivated by RNAi in an elite rice parent. Increased OsHDT1 expression did not affect plant growth in the parent but led to early flowering in the hybrid. Nonadditive up-regulation of key flowering time genes was found to be related to flowering time of the hybrid. Over-expression of OsHDT1 repressed the nonadditive expression of the key flowering repressors in the hybrid (i.e. OsGI and Hd1) inducing early flowering. Analysis of histone acetylation suggested that OsHDT1 over-expression might promote deacetylation on OsGI and Hd1 chromatin during the peak expression phase. High throughput differential gene expression analysis revealed that altered OsHDT1 levels affected nonadditive expression of many genes in the hybrid. These data demonstrate that nonadditive gene expression was involved in flowering time control in the hybrid rice and that OsHDT1 level was important for nonadditive or differential expression of many genes including the flowering time genes, suggesting that OsHDT1 may be involved in epigenetic control of parental genome interaction for differential gene expression.     

Modulation of host signaling by a bacterial mimic: structure of the Salmonella effector SptP bound to Rac1

Salmonella spp. utilize a specialized protein secretion system to deliver a battery of effector proteins into host cells. Several of these effectors stimulate Cdc42- and Rac1-dependent cytoskeletal changes that promote bacterial internalization. These potentially cytotoxic alterations are rapidly reversed by the effector SptP, a tyrosine phosphatase and GTPase activating protein (GAP) that targets Cdc42 and Rac1. The 2.3 A resolution crystal structure of an SptP-Rac1 transition state complex reveals an unusual GAP architecture that mimics host functional homologs. The phosphatase domain possesses a conserved active site but distinct surface properties. Binding to Rac1 induces a dramatic stabilization in SptP of a four-helix bundle that makes extensive contacts with the Switch I and Switch II regions of the GTPase.     

UHRF1 recruits the histone acetyltransferase Tip60 and controls its expression and activity

Tat-interactive protein, 60 kDa (Tip60) is a histone acetyltransferase with specificity toward lysine 5 of histone H2A (H2AK5) and plays multiple roles in chromatin remodeling processes. Co-immunoprecipitation experiments performed on Jurkat cells, showed that Tip60 is present in the same macro-molecular complex as UHRF1 (Ubiquitin-like containing PHD and RING domain 1), DNMT1 (DNA methyltransferase 1), and HDAC1 (histone deacetylase 1). Furthermore, immunocytochemistry experiments confirmed that Tip60 co-localizes with the UHRF1/DNMT1 complex. Although down-regulation of UHRF1 by RNA interference enhanced Tip60 expression, a significant decrease of the level of acetylated H2AK5 was observed. Consistently, we have observed that down-regulation of Tip60 and DNMT1 by RNA interference, dramatically reduced the levels of acetylated H2AK5. Altogether, these results suggest that Tip60 is a novel partner of the epigenetic integration platform interplayed by UHRF1, DNMT1 and HDAC1 involved in the epigenetic code replication.     

敲除组蛋白去乙酰化酶基因hdac对里氏木霉纤维素酶表达的调控作用

[背景]里氏木霉(Trichoderma reesei)是木霉属中产纤维素酶最具代表性的真菌之一,表观遗传调控是不涉及DNA序列变化的可遗传变化,组蛋白去乙酰化是其中一种。组蛋白去乙酰化酶(histone deacetylase,HDAC)负责脱乙酰化,敲除去乙酰化酶基因可引起菌株孢子、菌丝及纤维素酶活性等的一系列改变。[目的]通过敲除里氏木霉组蛋白去乙酰化酶基因(histone deacetylase,hdac)建立了里氏木霉hdac缺失突变株(T.reesei△hdac),以研究对纤维素酶基因表达的调控作用。[方法]利用Split-Maker技术构建了组蛋白去乙酰化酶基因敲除表达盒,并转化了里氏木霉T.reesei QM9414。经PCR及Southern blotting验证正确后,对突变体T.reesei△hdac连续7 d检测滤纸酶活(filter paper activity,AFP)、羧甲基纤维素钠酶活(carboxymethyl cellulase activity,CMCA),利用RT-qPCR检测纤维素酶及其相关基因cbh1、egl1和xyr1的表达。[结果]突变体T.reesei△hdac两种酶活力均显著高于出发菌株,分别高出8.00、30.00 IU/mL。突变体T.reesei△hdac纤维素酶及其相关基因cbh1、egl1和xyr1的转录水平分别为出发菌株T.reesei QM9414的6.50、6.01和4.51倍。[结论]里氏木霉中纤维素酶的基因表达明显受到组蛋白去乙酰化酶基因(hdac)的调控,这为研究里氏木霉表观遗传调控对纤维素酶的影响提供了新的证据。     

Kinetics and thermodynamics of metal‐binding to histone deacetylase 8

Histone deacetylase 8 (HDAC8) was originally classified as a Zn(II)-dependent deacetylase on the basis of Zn(II)-dependent HDAC8 activity in vitro and illumination of a Zn(II) bound to the active site. However, in vitro measurements demonstrated that HDAC8 has higher activity with a bound Fe(II) than Zn(II), although Fe(II)-HDAC8 rapidly loses activity under aerobic conditions. These data suggest that in the cell HDAC8 could be activated by either Zn(II) or Fe(II). Here we detail the kinetics, thermodynamics, and selectivity of Zn(II) and Fe(II) binding to HDAC8. To this end, we have developed a fluorescence anisotropy assay using fluorescein-labeled suberoylanilide hydroxamic acid (fl-SAHA). fl-SAHA binds specifically to metal-bound HDAC8 with affinities comparable to SAHA. To measure the metal affinity of HDAC, metal binding was coupled to fl-SAHA and assayed from the observed change in anisotropy. The metal K_D values for HDAC8 are significantly different, ranging from picomolar to micromolar for Zn(II) and Fe(II), respectively. Unexpectedly, the Fe(II) and Zn(II) dissociation rate constants from HDAC8 are comparable, k_off ∼0.0006 s⁻¹, suggesting that the apparent association rate constant for Fe(II) is slow (∼3 × 10³ M⁻¹ s⁻¹). Furthermore, monovalent cations (K⁺ or Na⁺) that bind to HDAC8 decrease the dissociation rate constant of Zn(II) by ≥100-fold for K⁺ and ≥10-fold for Na⁺, suggesting a possible mechanism for regulating metal exchange in vivo. The HDAC8 metal affinities are comparable to the readily exchangeable Zn(II) and Fe(II) concentrations in cells, consistent with either or both metal cofactors activating HDAC8.     

Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-beta1 induced murine tissue inhibitor of metalloproteinases-1 gene expression

Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor beta1 (TGF-beta1), although the signalling pathways involved are not clearly defined. Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes. Remarkably, PMA and TGF-beta1 stimulation of Timp-1 show a differential response to TSA or NaB. TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-beta1-induced Timp-1 expression. The repression of TGF-beta1-induced Timp-1 by TSA was maximal at 5 ng.mL(-1), while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is > 500 ng x mL(-1) TSA. A further HDACi, valproic acid, did not block TGF-beta1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1. For either PMA or TGF-beta1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1. The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs. Mutation or deletion of the AP-1 motif (-59/-53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter. In c-Jun-/- cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost. Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif.