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  • HDAC3 as a molecular chaperone for shuttling phosphorylated TR2 to PML: a novel deacetylase activity-independent function of HDAC3. 19204783

    TR2 is an orphan nuclear receptor specifically expressed in early embryos (Wei and Hsu, 1994), and a transcription factor for transcriptional regulation of important genes in stem cells including the gate keeper Oct4 (Park et al. 2007). TR2 is known to function as an activator (Wei et al. 2000), or a repressor (Chinpaisal et al., 1998, Gupta et al. 2007). Due to the lack of specific ligands, mechanisms triggering its activator or repressor function have remained puzzling for decades. Recently, we found that all-trans retinoic acid (atRA) triggers the activation of extracellular-signal-regulated kinase 2 (ERK2), which phosphorylates TR2 and stimulates its partitioning to promyelocytic leukemia (PML) nuclear bodies, thereby converting the activator function of TR2 into repression (Gupta et al. 2008; Park et al. 2007). Recruitment of TR2 to PML is a crucial step in the conversion of TR2 from an activator to a repressor. However, it is unclear how phosphorylated TR2 is recruited to PML, an essential step in converting TR2 from an activator to a repressor. In the present study, we use both in vitro and in vivo systems to address the problem of recruiting TR2 to PML nuclear bodies. First, we identify histone deacetylase 3 (HDAC3) as an effector molecule. HDAC3 is known to interact with TR2 (Franco et al. 2001) and this interaction is enhanced by the atRA-stimulated phosphorylation of TR2 at Thr-210 (Gupta et al. 2008). Secondly, in this study, we also find that the carrier function of HDAC3 is independent of its deacetylase activity. Thirdly, we find another novel activity of atRA that stimulates nuclear enrichment of HDAC3 to form nuclear complex with PML, which is ERK2 independent. This is the first report identifying a deacetylase-independent function for HDAC3, which serves as a specific carrier molecule that targets a specifically phosphorylated protein to PML NBs. This is also the first study delineating how protein recruitment to PML nuclear bodies occurs, which can be stimulated by atRA in an ERK2-independent manner. These findings could provide new insights into the development of potential therapeutics and in understanding how orphan nuclear receptor activities can be regulated without ligands.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The TGFβ receptor-interacting protein km23-1/DYNLRB1 plays an adaptor role in TGFβ1 autoinduction via its association with Ras. 22637579

    We have previously elucidated the signaling events that are required for TGFβ1 autoinduction (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765-30773). Further, we have reported that the TGFβ receptor (TβR)-interacting protein km23-1 plays an important role in TGFβ signal transduction (Jin, Q., Ding, W., and Mulder, K. M. (2007) J. Biol. Chem. 282, 19122-19132). Here we examined the role of km23-1 in TGFβ1 autoinduction in TGFβ-sensitive epithelial cells. siRNA blockade of km23-1 reduced TGFβ1 mRNA expression, as well as DNA binding and transcriptional activation of the relevant activator protein-1 site in the human TGFβ1 promoter. Further, knockdown of km23-1 inhibited TGFβ-mediated activation of ERK and JNK, phosphorylation of c-Jun, and transactivation of the c-Jun promoter. Sucrose gradient analyses indicate that km23-1 was present in lipid rafts together with Ras and TβRII after TGFβ treatment. Immunoprecipitation/blot analyses revealed the formation of a TGFβ-inducible complex between Ras and km23-1 in vivo within minutes of TGFβ addition. Moreover, we demonstrate for the first time that km23-1 is required for Ras activation by TGFβ. Our results indicate that km23-1 is required for TGFβ1 autoinduction through Smad2-independent Ras/ERK/JNK pathways. More importantly, our findings demonstrate that km23-1 functions as a critical adaptor coupling TβR activation to activation of Ras effector pathways downstream.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Retinoic acid-stimulated sequential phosphorylation, PML recruitment, and SUMOylation of nuclear receptor TR2 to suppress Oct4 expression. 18682553

    We previously reported an intricate mechanism underlying the homeostasis of Oct4 expression in normally proliferating stem cell culture of P19, mediated by SUMOylation of orphan nuclear receptor TR2. In the present study, we identify a signaling pathway initiated from the nongenomic activity of all-trans retinoic acid (atRA) to stimulate complex formation of extracellular signal-regulated kinase 2 (ERK2) with its upstream kinase, mitogen-activated protein kinase kinase (MEK). The activated ERK2 phosphorylates threonine-210 (Thr-210) of TR2, stimulating its subsequent SUMOylation. Dephosphorylated TR2 recruits coactivator PCAF and functions as an activator for its target gene Oct4. Upon phosphorylation at Thr-210, TR2 increasingly associates with promyelocytic leukemia (PML) nuclear bodies, becomes SUMOylated, and recruits corepressor RIP140 to act as a repressor for its target, Oct4. To normally proliferating P19 stem cell culture, exposure to a physiological concentration of atRA triggers a rapid nongenomic signaling cascade to suppress Oct4 gene and regulate cell proliferation.
    Document Type:
    Reference
    Product Catalog Number:
    05-718
    Product Catalog Name:
    Anti-PML Antibody, clone 36.1-104

  • Lin28 modulates cell growth and associates with a subset of cell cycle regulator mRNAs in mouse embryonic stem cells. 19147696

    Lin28 is highly expressed in human and mouse embryonic stem (ES) cells. Here, we show that in mouse ES cells, specific repression of Lin28 results in decreased cell proliferation, while overexpression of Lin28 accelerates cell proliferation. Further, Lin28 associates specifically with ribonucleoprotein particles containing mRNAs for cyclins A and B and cdk4. Importantly, changes in Lin28 levels lead to corresponding changes in the levels of these proteins, and sequences from the 3' untranslated regions of cyclin B and cdk4 mRNAs exhibit stimulatory effects on translation of reporter genes in a Lin28-dependent fashion. Thus, we postulate that Lin28 may play a role in the regulation of translation of genes important for the growth and maintenance of pluripotent cells.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Nucleosome stability mediated by histone variants H3.3 and H2A.Z. 17575053

    Nucleosomes containing the histone variant H3.3 tend to be clustered in vivo in the neighborhood of transcriptionally active genes and over regulatory elements. It has not been clear, however, whether H3.3-containing nucleosomes possess unique properties that would affect transcription. We report here that H3.3 nucleosomes isolated from vertebrates, regardless of whether they are partnered with H2A or H2A.Z, are unusually sensitive to salt-dependent disruption, losing H2A/H2B or H2A.Z/H2B dimers. Immunoprecipitation studies of nucleosome core particles (NCPs) show that NCPs that contain both H3.3 and H2A.Z are even less stable than NCPs containing H3.3 and H2A. Intriguingly, NCPs containing H3 and H2A.Z are at least as stable as H3/H2A NCPs. These results establish an hierarchy of stabilities for native nucleosomes carrying different complements of variants, and suggest how H2A.Z could play different roles depending on its partners within the NCP. They also are consistent with the idea that H3.3 plays an active role in maintaining accessible chromatin structures in enhancer regions and transcribed regions. Consistent with this idea, promoters and enhancers at transcriptionally active genes and coding regions at highly expressed genes have nucleosomes that simultaneously carry both H3.3 and H2A.Z, and should therefore be extremely sensitive to disruption.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The histone demethylase PHF8 is essential for cytoskeleton dynamics. 22850744

    PHF8 is a histone demethylase associated with X-linked mental retardation. It has been described as a transcriptional co-activator involved in cell cycle progression, but its physiological role is still poorly understood. Here we show that PHF8 controls the expression of genes involved in cell adhesion and cytoskeleton organization such as RhoA, Rac1 and GSK3β. A lack of PHF8 not only results in a cell cycle delay but also in a disorganized actin cytoskeleton and impaired cell adhesion. Our data demonstrate that PHF8 directly regulates the expression of these genes by demethylating H4K20me1 at promoters. Moreover, c-Myc transcription factor cooperates with PHF8 to regulate the analysed promoters. Further analysis in neurons shows that depletion of PHF8 results in down-regulation of cytoskeleton genes and leads to a deficient neurite outgrowth. Overall, our results suggest that the mental retardation phenotype associated with loss of function of PHF8 could be due to abnormal neuronal connections as a result of alterations in cytoskeleton function.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3408
    Product Catalog Name:
    Anti-Tubulin Antibody, beta, clone KMX-1

  • Wnt proteins regulate acetylcholine receptor clustering in muscle cells. 22309736

    The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish.In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4.These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4.
    Document Type:
    Reference
    Product Catalog Number:
    05-1050
    Product Catalog Name:
    4G10® Platinum, Anti-Phosphotyrosine Antibody (mouse monoclonal cocktail IgG2b)

  • Direct interplay among histones, histone chaperones, and a chromatin boundary protein in the control of histone gene expression. 22907759

    In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription.
    Document Type:
    Reference
    Product Catalog Number:
    07-677

  • Role of SUMO modification of human PCNA at stalled replication fork. 22457066

    DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • Coxsackievirus and adenovirus receptor (CAR) mediates trafficking of acid sensing ion channel 3 (ASIC3) via PSD-95. 22809504

    We have previously shown that the Coxsackievirus and adenovirus receptor (CAR) can interact with post-synaptic density 95 (PSD-95) and localize PSD-95 to cell-cell junctions. We have also shown that activity of the acid sensing ion channel (ASIC3), a H(+)-gated cation channel that plays a role in mechanosensation and pain signaling, is negatively modulated by PSD-95 through a PDZ-based interaction. We asked whether CAR and ASIC3 simultaneously interact with PSD-95, and if so, whether co-expression of these proteins alters their cellular distribution and localization. Results indicate that CAR and ASIC3 co-immunoprecipitate only when co-expressed with PSD-95. CAR also brings both PSD-95 and ASIC3 to the junctions of heterologous cells. Moreover, CAR rescues PSD-95-mediated inhibition of ASIC3 currents. These data suggest that, in addition to activity as a viral receptor and adhesion molecule, CAR can play a role in trafficking proteins, including ion channels, in a PDZ-based scaffolding complex.
    Document Type:
    Reference
    Product Catalog Number:
    AB5927
    Product Catalog Name:
    Anti-Acid Sensitive Ion Channel 3 Antibody, 285-304 of extracellular domain of rat ASIC3