Interactions of HP1 Bound to H3K9me3 Dinucleosome by Molecular Simulations and Biochemical Assays

Heterochromatin protein 1 (HP1), associated with heterochromatin formation, recognizes an epigenetically repressive marker, trimethylated lysine 9 in histone H3 (H3K9me3), and generally contributes to long-term silencing. How HP1 induces heterochromatin is not fully understood. Recent experiments suggested that not one, but two nucleosomes provide a platform for this recognition. Integrating previous and new biochemical assays with computational modeling, we provide near-atomic structural models for HP1 binding to the dinucleosomes. We found that the dimeric HP1α tends to bind two H3K9me3s that are in adjacent nucleosomes, thus bridging two nucleosomes. We identified, to our knowledge, a novel DNA binding motif in the hinge region that is specific to HP1α and is essential for recognizing the H3K9me3 sites of two nucleosomes. An HP1 isoform, HP1γ, does not easily bridge two nucleosomes in extended conformations because of the absence of the above binding motif and its shorter hinge region. We propose a molecular mechanism for chromatin structural changes caused by HP1.     

HAMLET binding to α-actinin facilitates tumor cell detachment

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.     

HP1γ links histone methylation marks to meiotic synapsis in mice

During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1γ. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1γ/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1γ/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.     

GBF1 bears a novel phosphatidylinositol-phosphate binding module, BP3K, to link PI3Kγ activity with Arf1 activation involved in GPCR-mediated neutrophil chemotaxis and superoxide production

Most chemoattractants for neutrophils bind to the Gα(i) family of heterotrimeric G protein-coupled receptors (GPCRs) and release Gβγ subunits to activate chemotaxis and superoxide production. GIT2, a GTPase-activating protein for Arf1, forms a complex with Gβγ and is integral for directional sensing and suppression of superoxide production. Here we show that GBF1, a guanine nucleotide exchanging factor for Arf-GTPases, is primarily responsible for Arf1 activation upon GPCR stimulation and is important for neutrophil chemotaxis and superoxide production. We find that GBF1 bears a novel module, namely binding to products of phosphatidyl inositol 3-kinase (PI3K), which binds to products of PI3Kγ. Through this binding, GBF1 is translocated from the Golgi to the leading edge upon GPCR stimulation to activate Arf1 and recruit p22phox and GIT2 to the leading edge. Moreover, GBF1-mediated Arf1 activation is necessary to unify cell polarity during chemotaxis. Our results identify a novel mechanism that links PI3Kγ activity with chemotaxis and superoxide production in GPCR signaling.     

The binding of 14-3-3γ to membranes studied by intrinsic fluorescence spectroscopy

Human 14-3-3 proteins contain two conserved tryptophan residues in each monomer, Trp60 and Trp233 in isoform γ. 14-3-3γ binds to negatively charged membranes and here we show that membrane binding can be monitored by steady-state intrinsic fluorescence spectroscopy. Measurements with W60F and W233F 14-3-3γ mutants revealed that Trp60 is the major contributor to the emission fluorescence, whereas the fluorescence of Trp233, which π-stacks with Tyr184, is quenched. The fluorescence is reduced and red-shifted upon specific binding of a phosphate ligand, and further red-shifted upon binding of 14-3-3γ to the membrane, compatible with solvent exposure of Trp60. Moreover, our results support that membrane binding involves the non-conserved, convex area of 14-3-3γ, and that Trp residues do not intercalate in the bilayer.     

The WD40 protein Morg1 facilitates Par6–aPKC binding to Crb3 for apical identity in epithelial cells

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6–aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6–aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6–aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6–aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.     

沙棘H3K9乙酰化修饰全基因组分析

为了绘制沙棘H3K9乙酰化修饰图谱,确定H3K9乙酰化修饰所调控的基因,该实验通过Western blot验证抗体与组蛋白的结合能力和ChIP-seq验证抗体富集效率,获得全基因组范围内沙棘H3K9乙酰化修饰图谱和调控基因。实验结果表明,H3K9ac抗体与复合物具有较强的结合能力。对富集到的DNA片段进行高通量测序,分别获得2.2×10~7和3.6×10~7条原始序列;唯一比对序列广泛分布于沙棘基因组中,并且在结构基因中的两端具有明显的富集。对富集区进行峰的预测结果显示,共预测出1 011个峰;对峰所处部位基因进行功能预测结果发现,H3K9ac对于沙棘细胞代谢和信号转导基因的表达具有重要调控作用。沙棘片段化DNA的富集以及高通量测序结果证明,抗体能够用于研究沙棘的组蛋白修饰类型,并且绘制了沙棘第一张H3K9乙酰化修饰遗传图谱草图,鉴定出沙棘H3K9乙酰化修饰所调控的基因,为今后研究组蛋白修饰对沙棘基因表达的调控方式奠定了基础。     

Polycomb Repressive Complex 2 and H3K27me3 Cooperate with H3K9 Methylation To Maintain Heterochromatin Protein 1α at Chromatin

Methylation of histone H3 on lysine 9 or 27 is crucial for heterochromatin formation. Previously considered hallmarks of, respectively, constitutive and facultative heterochromatin, recent evidence has accumulated in favor of coexistence of these two marks and their cooperation in gene silencing maintenance. H3K9me2/3 ensures anchorage at chromatin of heterochromatin protein 1α (HP1α), a main component of heterochromatin. HP1α chromoshadow domain, involved in dimerization and interaction with partners, has additional but still unclear roles in HP1α recruitment to chromatin. Because of previously suggested links between polycomb repressive complex 2 (PRC2), which catalyzes H3K27 methylation, and HP1α, we tested whether PRC2 may regulate HP1α abundance at chromatin. We found that the EZH2 and SUZ12 subunits of PRC2 are required for HP1α stability, as knockdown of either protein led to HP1α degradation. Similar results were obtained upon overexpression of H3K27me2/3 demethylases. We further showed that binding of HP1α/β/γ to H3K9me3 peptides is greatly increased in the presence of H3K27me3, and this is dependent on PRC2. These data fit with recent proteomic studies identifying PRC2 as an indirect H3K9me3 binder in mouse tissues and suggest the existence of a cooperative mechanism of HP1α anchorage at chromatin involving H3 methylation on both K9 and K27 residues.     

The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.     

Regulated binding of importin-α to protein kinase Cδ in response to apoptotic signals facilitates nuclear import

PKCδ translocates into the nucleus in response to apoptotic agents and functions as a potent cell death signal. Cytoplasmic retention of PKCδ and its transport into the nucleus are essential for cell homeostasis, but how these processes are regulated is poorly understood. We show that PKCδ resides in the cytoplasm in a conformation that precludes binding of importin-α. A structural model of PKCδ in the inactive state suggests that the nuclear localization sequence (NLS) is prevented from binding to importin-α through intramolecular contacts between the C2 and catalytic domains. We have previously shown that PKCδ is phosphorylated on specific tyrosine residues in response to apoptotic agents. Here, we show that phosphorylation of PKCδ at Tyr-64 and Tyr-155 results in a conformational change that allows exposure of the NLS and binding of importin-α. In addition, Hsp90 binds to PKCδ with similar kinetics as importin-α and is required for the interaction of importin-α with the NLS. Finally, we elucidate a role for a conserved PPxxP motif, which overlaps the NLS, in nuclear exclusion of PKCδ. Mutagenesis of the conserved prolines to alanines enhanced importin-α binding to PKCδ and induced its nuclear import in resting cells. Thus, the PPxxP motif is important for maintaining a conformation that facilitates cytosplasmic retention of PKCδ. Taken together, this study establishes a novel mechanism that retains PKCδ in the cytoplasm of resting cells and regulates its nuclear import in response to apoptotic stimuli.     

Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1β with the Nucleosome

Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1β to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1β with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1β-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.     

A pharmacological comparison of [3H]GBR12935 binding to rodent striatal and kidney homogenates: binding to dopamine transporters?

Binding of [3H]GBR12935 to homogenates of mouse and rat striatum and kidney was studied. [3H]GBR12935 bound to both tissue preparations with high affinity (mouse striatum Kd = 2.4 +/- 0.4 nM, n = 4; mouse kidney Kd = 3.8 +/- 0.9 nM, n = 4), in a saturable (striatal Bmax = 1.5 +/- 0.4 pmol/mg protein; kidney Bmax = 4.9 +/- 0.5 pmol/mg protein) and reversible manner. Saturation experiments revealed the presence of a single class of high affinity binding sites in both tissues of both species. Mouse kidney appeared to possess a greater density of [3H]GBR12935 binding sites than the striatum while the reverse situation prevailed for the rat. Although two dopamine uptake inhibitors, namely GBR12909 and benztropine, displaced [3H]GBR12935 binding from striatal and kidney homogenates with a similar affinity in both tissues of these species, unlabelled mazindol, (+/-)cocaine, nomifensine and amfonelic acid were significantly (P < 0.001-0.02) more potent inhibitors of [3H]GBR12935 binding in the striatum than in the kidney. While the pharmacological profile of [3H]GBR12935 binding in the rodent striatum compared well with that of the dopamine transporter reported previously, the pharmacology in the kidney was considerably different to that in the striatum. GBR12909 (1-30 mg/kg, i.p.), a close analog of GBR12935, induced significant antidiuretic and antinatriuretic effects in spontaneously hypertensive rats. These data suggest that while [3H]GBR12935 labels the dopamine uptake sites in the brain, it does not appear to label similar sites in the kidney. The mechanism of action of GBR12909 on sodium and water excretion remains to be determined.     

Dimers and multimers of monoclonal IgG1 exhibit higher in vitro binding affinities to Fcγ receptors

The in vitro binding of monomeric, dimeric and multimeric forms of monoclonal IgG1 molecules, designated mAb1 and mAb2, to the extracellular domains of Fcγ receptors RI, RIIA and RIIIB were investigated using a surface plasmon resonance (SPR) based biosensor technique. Stable noncovalent and covalent dimers of mAb1 and mAb2, respectively, were isolated from CHO cell expressed materials. The dissociation constants of monomeric mAb1 and mAb2 were determined to be 1 nM for the FcγRI-binding and 6–12 μM for the FcγRIIA- and FcγRIIIB-binding. Dimeric mAb1 and mAb2 exhibited increased affinities, by 2-3 fold for FcγRI and 200-800 fold for FcγRIIA and FcγRIIIB. Further increases in binding were observed when the antibodies formed large immune complexes with multivalent antigens, but not in a linear relation with size. The binding properties of monomeric mAb2 were identical with and without a bound monovalent antigen, indicating that antigen-binding alone does not induce measurable change in binding of antibodies to Fcγ receptors. Dimerization is sufficient to show enhancement in the receptor binding. Given the wide distribution of the low-affinity Fcγ receptors on immune effector cells, the increased affinities to aggregated IgG may lead to some biological consequences, depending on the subsequent signal transduction events. The SPR-based in vitro binding assay is useful in evaluating Fcγ receptor binding of various species in antibody-based biotherapeutics.