06-599 Sigma-AldrichAnti-acetyl-Histone H3 Antibody

CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.

Epigenetic regulation of gene expression is critical for controlling embryonic properties during the embryo-to-seedling phase transition. Here we report that a histone deacetylase19 (HDA19)-associated regulator, scarecrow-like15 (SCL15), is essential for repressing the seed maturation programme in vegetative tissues. SCL15 is expressed in and GFP-tagged SCL15 predominantly localizes to, the vascular bundles particularly in the phloem companion cells and neighbouring specialized cells. Mutation of SCL15 leads to a global shift in gene expression in seedlings to a profile resembling late embryogenesis in seeds. In scl15 seedlings, many genes involved in seed maturation are markedly derepressed with concomitant accumulation of seed 12S globulin; this is correlated with elevated levels of histone acetylation at a subset of seed-specific loci. SCL15 physically interacts with HDA19 and direct targets of HDA19-SCL15 association are identified. These studies reveal that SCL15 acts as an HDA19-associated regulator to repress embryonic traits in seedlings.

The chromatin-modifying enzyme lysine-specific demethylase 1, KDM1A/LSD1 is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells and its overexpression correlated with aggressive disease, poor differentiation and infaust outcome. Here, we show that LSD1 physically binds MYCN both in vitro and in vivo and that such an interaction requires the MYCN BoxIII. We found that LSD1 co-localizes with MYCN on promoter regions of CDKN1A/p21 and Clusterin (CLU) suppressor genes and cooperates with MYCN to repress the expression of these genes. KDM1A needs to engage with MYCN in order to associate with the CDKN1A and CLU promoters. The expression of CLU and CDKN1A can be restored in MYCN-amplified cells by pharmacological inhibition of LSD1 activity or knockdown of its expression. Combined pharmacological inhibition of MYCN and LSD1 through the use of small molecule inhibitors synergistically reduces MYCN-amplified Neuroblastoma cell viability in vitro. These findings demonstrate that LSD1 is a critical co-factor of the MYCN repressive function, and suggest that combination of LSD1 and MYCN inhibitors may have strong therapeutic relevance to counteract MYCN-driven oncogenesis.

Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen.

Therapy resistance is a major clinical problem in cancer medicine and crucial for disease relapse and progression. Therefore, the clinical need to overcome it, particularly for aggressive tumors such as pancreatic cancer, is very high. Aberrant activation of an epithelial-mesenchymal transition (EMT) and an associated cancer stem cell phenotype are considered a major cause of therapy resistance. Particularly, the EMT-activator ZEB1 was shown to confer stemness and resistance. We applied a systematic, stepwise strategy to interfere with ZEB1 function, aiming to overcome drug resistance. This led to the identification of both its target gene miR-203 as a major drug sensitizer and subsequently the class I HDAC inhibitor mocetinostat as epigenetic drug to interfere with ZEB1 function, restore miR-203 expression, repress stemness properties, and induce sensitivity against chemotherapy. Thereby, mocetinostat turned out to be more effective than other HDAC inhibitors, such as SAHA, indicating the relevance of the screening strategy. Our data encourage the application of mechanism-based combinations of selected epigenetic drugs with standard chemotherapy for the rational treatment of aggressive solid tumors, such as pancreatic cancer.

Hydrogen sulfide (H2S) is an endogenous gaseous biological mediator, which regulates, among others, the oxidative balance of cells under normal physiological conditions, as well as in various diseases. Several previous studies have reported that H2S attenuates inflammatory mediator production. In this study, we investigated the role of H2S in chromatin modulation in an in vitro model of lipopolysaccharide (LPS)-induced inflammation and evaluated its effects on inflammatory cytokine production. Tamm-Horsfall protein 1 (THP-1) differentiated macrophages were pre-treated with sodium hydrosulfide (NaHS) (an H2S donor) at 0.01, 0.1, 0.5 or 1 mM for 30 min. To stimulate cytokine production, the cells were challenged with bacterial LPS (1 µg/ml) for 1, 4, 8 or 24 h. Histone H3 acetylation was analyzed by chromatin immunoprecipitation (ChIP), cytokine production was measured by ELISA and histone deacetylase (HDAC) activity was analyzed using a standard biochemical assay. H2S inhibited the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in a concentration-dependent manner; it was most effective at the two highest concentrations used. This effect was associated with a decrease in histone H3 acetylation at the IL-6 and TNF-α promoters in the cells exposed to H2S or H2S + LPS. The findings of the present study suggest that H2S suppresses histone acetylation, which, in turn, inhibits chromatin openness, leading to a decrease in the gene transcription of various pro-inflammatory cytokines. Therefore, this mechanism may contribute to the previously demonstrated anti-inflammatory effects of H2S and various H2S donors.

DNA replication is a key process in living organisms. DNA polymerase α (Polα) initiates strand synthesis, which is performed by Polε and Polδ in leading and lagging strands, respectively. Whereas loss of DNA polymerase activity is incompatible with life, viable mutants of Polα and Polε were isolated, allowing the identification of their functions beyond DNA replication. In contrast, no viable mutants in the Polδ polymerase-domain were reported in multicellular organisms. Here we identify such a mutant which is also thermosensitive. Mutant plants were unable to complete development at 28°C, looked normal at 18°C, but displayed increased expression of DNA replication-stress marker genes, homologous recombination and lysine 4 histone 3 trimethylation at the SEPALLATA3 (SEP3) locus at 24°C, which correlated with ectopic expression of SEP3. Surprisingly, high expression of SEP3 in vascular tissue promoted FLOWERING LOCUS T (FT) expression, forming a positive feedback loop with SEP3 and leading to early flowering and curly leaves phenotypes. These results strongly suggest that the DNA polymerase δ is required for the proper establishment of transcriptionally active epigenetic marks and that its failure might affect development by affecting the epigenetic control of master genes.

E-cadherin inactivation underpins the progression of invasive lobular breast carcinoma (ILC). In ILC, p120-catenin (p120) translocates to the cytosol where it controls anchorage independence through the Rho-Rock signaling pathway, a key mechanism driving tumor growth and metastasis. We now demonstrate that anchorage-independent ILC cells show an increase in nuclear p120, which results in relief of transcriptional repression by Kaiso. To identify the Kaiso target genes that control anchorage independence we performed genome-wide mRNA profiling on anoikis-resistant mouse ILC cells, and identified 29 candidate target genes, including the established Kaiso target Wnt11. Our data indicate that anchorage-independent upregulation of Wnt11 in ILC cells is controlled by nuclear p120 through inhibition of Kaiso-mediated transcriptional repression. Finally, we show that Wnt11 promotes activation of RhoA, which causes ILC anoikis resistance. Our findings thereby establish a mechanistic link between E-cadherin loss and subsequent control of Rho-driven anoikis resistance through p120- and Kaiso-dependent expression of Wnt11.

GATA1 is a master regulator of erythropoiesis, expression of which is regulated by multiple discrete cis-acting elements. In this study, we examine the activity of a promoter-proximal double GATA (dbGATA) motif, using a Gata1 bacterial artificial chromosome (BAC)-transgenic green fluorescent protein (GFP) reporter (G1BAC-GFP) mouse system. Deletion of the dbGATA motif led to significant reductions in GFP expression in hematopoietic progenitors, while GFP expression was maintained in erythroblasts. Consistently, in mice with a germ line deletion of the dbGATA motif (Gata1(ΔdbGATA) mice), GATA1 expression in progenitors was significantly decreased. The suppressed GATA1 expression was associated with a compensatory increase in GATA2 levels in progenitors. When we crossed Gata1(ΔdbGATA) mice with Gata2 hypomorphic mutant mice (Gata2(fGN/fGN) mice), the Gata1(ΔdbGATA)::Gata2(fGN/fGN) compound mutant mice succumbed to a significant decrease in the progenitor population, whereas both groups of single mutant mice maintained progenitors and survived to adulthood, indicating the functional redundancy between GATA1 and GATA2 in progenitors. Meanwhile, the effects of the dbGATA site deletion on Gata1 expression were subtle in erythroblasts, which showed increased GATA1 binding and enhanced accumulation of active histone marks around the 1st-intron GATA motif of the ΔdbGATA locus. These results thus reveal a novel role of the dbGATA motif in the maintenance of Gata1 expression in hematopoietic progenitors and a functional compensation between the dbGATA site and the 1st-intron GATA motif in erythroblasts.

Microtubule transport of circovirus from the periphery of the cell to the nucleus is essential for viral replication in early infection. How the microtubule is recruited to the viral cargo remains unclear. In this study, we observed that circovirus trafficking is dependent on microtubule polymerization and that incoming circovirus particles colocalize with cytoplasmic dynein and endosomes. However, circovirus binding to dynein was independent of the presence of microtubular α-tubulin and translocation of cytoplasmic dynein into the nucleus. The circovirus capsid (Cap) subunit enhanced microtubular acetylation and directly interacted with intermediate chain 1 (IC1) of dynein. N-terminal residues 42 to 100 of the Cap viral protein were required for efficient binding to the dynein IC1 subunit and for retrograde transport. Knockdown of IC1 decreased virus transport and replication. These results demonstrate that Cap is a direct ligand of the cytoplasmic dynein IC1 subunit and an inducer of microtubule α-tubulin acetylation. Furthermore, Cap recruits the host dynein/microtubule machinery to facilitate transport toward the nucleus by an endosomal mechanism distinct from that used for physiological dynein cargo.Incoming viral particles hijack the intracellular trafficking machinery of the host in order to migrate from the cell surface to the replication sites. Better knowledge of the interaction between viruses and virus proteins and the intracellular trafficking machinery may provide new targets for antiviral therapies. Currently, little is known about the molecular mechanisms of circovirus transport. Here, we report that circovirus particles enter early endosomes and utilize the microtubule-associated molecular motor dynein to travel along microtubules. The circovirus capsid subunit enhances microtubular acetylation, and N-terminal residues 42 to 100 directly interact with the dynein IC1 subunit during retrograde transport. These findings highlight a mechanism whereby circoviruses recruit dynein for transport to the nucleus via the dynein/microtubule machinery.