16-157 Sigma-AldrichProtein A Agarose/Salmon Sperm DNA, 2.5 mL

Histone posttranslational modifications play significant roles in regulating chromatin structure and gene expression. One of the histone modifications, histone citrullination, is catalyzed by an enzyme called peptidylarginine deiminase 4 (PAD4, also called PADI4), which converts both histone arginine (Arg) and mono-methyl arginine residues to citrulline. Recent studies have found that histone citrullination counteracts the effect of histone arginine methylation and functions as a repressive marker to turn off gene expression. Here, we describe assays to study histone citrullination by PAD4 in vitro and in vivo. We also describe approaches to measure histone citrullination levels at gene promoters using chromatin immunoprecipitation assay and analyze the effects of PAD4 inhibitor on cell cycle and apoptosis by flow cytometry. These methods would be useful techniques to study this unique histone modification.

Two related B3 domain transcriptional repressors, HSI2 (HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2)/VAL1 (VP1/ABI3-LIKE1) and HSL1 (HSI2-LIKE1)/VAL2, function redundantly to repress key transcriptional regulators of seed maturation genes in Arabidopsis thaliana seedlings. Using a forward genetic screen designed to isolate trans-acting mutants that affected expression of a transgene containing the glutathione S-transferase F8 promoter::luciferase (GSTF8::LUC) reporter, we identified a novel HSI2 mutant allele, hsi2-4, that exhibits constitutively elevated luciferase expression while expression of the endogenous GSTF8 transcript remains unchanged. The hsi2-4 lesion was found to be a missense mutation that results in the substitution of a conserved cysteine within the plant homeodomain-like (PHD) motif of HSI2. Microarray analysis of hsi2-4 and hsi2-4 hsl1 mutants indicated that the HSI2 PHD-like domain functions non-redundantly to repress a subset of seed maturation genes, including those that encode AGL15 (AGAMOUS-LIKE15), FUSCA3 (FUS3), cruciferins, cupin family proteins, late-embryogenesis abundant protein, oleosins, 2S albumins and other seed-specific proteins in Arabidopsis seedlings. Many genes that are responsive to this mutation in the HSI2 PHD-like domain are enriched in histone H3 trimethylation on lysine 27 residues (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation analysis showed that sequences of the GSTF8::LUC transgene are enriched in H3K27me3 in a HSI2 PHD domain-dependent manner. These results indicate that the transcriptional repression activity of the HSI2 PHD domain could be mediated, at least in part, by its participation in the deposition of H3K27me3 on the chromatin of specific target genes.

A variety of cellular and viral processes are coordinately regulated during adenovirus (Ad) infection to achieve optimal virus production. The Ad late gene product L4-22K has been associated with disparate activities during infection, including the regulation of late gene expression, viral DNA packaging, and infectious virus production. We generated and characterized two L4-22K mutant viruses to further explore L4-22K functions during viral infection. Our results show that L4-22K is indeed important for temporal control of viral gene expression not only because it activates late gene expression but also because it suppresses early gene expression. We also show that the L4-22K protein binds to viral packaging sequences in vivo and is essential to recruit two other packaging proteins, IVa2 and L1-52/55K, to this region. The elimination of L4-22K gave rise to the production of only empty virus capsids and not mature virions, which confirms that the L4-22K protein is required for Ad genome packaging. Finally, L4-22K contributes to adenovirus-induced cell death by regulating the expression of the adenovirus death protein. Thus, the adenovirus L4-22K protein is multifunctional and an integral component of crucial aspects of infection.

The anthelmintic activity of chicory (Cichorium intybus L.) herbage has been attributed to sesquiterpene lactones. Chicory leaves contain significant amounts of lactucin (LAC), 8-deoxylactucin (DOL), and lactucopicrin (LPIC), but the proportions of these three sesquiterpene lactones vary among forage chicory cultivars. To determine whether the individual compounds differ in anthelmintic activity, we prepared sesquiterpene lactone-enriched extracts from leaves of two forage chicory cultivars, \'Grasslands Puna\' (Puna) and \'Forage Feast\', and tested their effects on the hatching of a predominantly Haemonchus contortus egg population. The dominant constituents in the Puna and Forage Feast extracts were DOL and LAC, respectively; LPIC concentrations in the two extracts were similar. Extracts from both cultivars inhibited egg hatching at all concentrations tested (P<0.001), but there were significant differences in egg responses to the two extracts (P<0.001). With Puna, egg hatching decreased sharply in a linear fashion when the combined LAC, DOL, and LPIC concentrations ranged from 0 to 5.0mg/ml. A biphasic effect on egg hatching occurred with the Forage Feast extract. The fraction of eggs that hatched decreased gradually to 65% as the sesquiterpene lactone concentrations increased from 0 to 6.7mg/ml. Treatment with higher concentrations resulted in a sharp decline in egg hatchability. Concentrations of sesquiterpene lactones required for 50% lethality were determined by probit dose-effect analysis to be 2.6mg/ml (95% confidence interval: 2.4-2.8mg/ml) for the Puna extract and 6.4mg/ml (95% confidence interval: 5.9-7.2mg/ml) for the Forage Feast extract (P<0.0001). These concentrations provided 1.3 and 1.5mg/ml of DOL and 0.8 and 3.9mg/ml of LAC for Puna and Forage Feast extracts, respectively. Results suggest that LAC has minimal effect on egg hatching and that DOL or other constituent(s) in the extracts is inhibitory. Quantitative analyses of free sesquiterpene lactones in chicory leaf extracts suggest that Puna may be a better cultivar than Forage Feast for use in bioactive pastures for gastrointestinal parasite control in small ruminants.Published by Elsevier B.V.

Malignant pleural mesotheliomas (MPMs) often show CDKN2A and NF2 inactivation, but other highly recurrent mutations have not been described. To identify additional driver genes, we used an integrated genomic analysis of 53 MPM tumor samples to guide a focused sequencing effort that uncovered somatic inactivating mutations in BAP1 in 23% of MPMs. The BAP1 nuclear deubiquitinase is known to target histones (together with ASXL1 as a Polycomb repressor subunit) and the HCF1 transcriptional co-factor, and we show that BAP1 knockdown in MPM cell lines affects E2F and Polycomb target genes. These findings implicate transcriptional deregulation in the pathogenesis of MPM.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue(1). They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers(2) and autoimmune diseases(3). It is well established that transcription is regulated by alterations to the chromatin template(4,5). These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation(6). Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), regulates numerous biological processes, including proliferation, differentiation, and embryonic stem cell self-renewal. Although the DNA sequence to which KLF4 binds is established, the mechanism by which KLF4 controls transcription is not well defined. Small ubiquitin-related modifier (SUMO) is an important regulator of transcription. Here we show that KLF4 is both SUMOylated at a single lysine residue and physically interacts with SUMO-1 in a region that matches an acidic and hydrophobic residue-rich SUMO-interacting motif (SIM) consensus. The SIM in KLF4 is required for transactivation of target promoters in a SUMO-1-dependent manner. Mutation of either the acidic or hydrophobic residues in the SIM significantly impairs the ability of KLF4 to interact with SUMO-1, activate transcription, and inhibit cell proliferation. Our study provides direct evidence that SIM in KLF4 functions as a transcriptional activation domain. A survey of transcription factor sequences reveals that established transactivation domains of many transcription factors contain sequences highly related to SIM. These results, therefore, illustrate a novel mechanism by which SUMO interaction modulates the activity of transcription factors.

Atypical protein kinase C (PKC) zeta is an important regulator of inflammation through activation of the nuclear factor-kappaB (NF-kappaB) pathway. Chromatin remodeling on pro-inflammatory genes plays a pivotal role in cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced abnormal lung inflammation. However, the signaling mechanism whereby chromatin remodeling occurs in CS- and LPS-induced lung inflammation is not known. We hypothesized that PKCzeta is an important regulator of chromatin remodeling, and down-regulation of PKCzeta ameliorates lung inflammation by CS and LPS exposures. We determined the role and molecular mechanism of PKCzeta in abnormal lung inflammatory response to CS and LPS exposures in PKCzeta-deficient (PKCzeta(-/-)) and wild-type mice. Lung inflammatory response was decreased in PKCzeta(-/-) mice compared with WT mice exposed to CS and LPS. Moreover, inhibition of PKCzeta by a specific pharmacological PKCzeta inhibitor attenuated CS extract-, reactive aldehydes (present in CS)-, and LPS-mediated pro-inflammatory mediator release from macrophages. The mechanism underlying these findings is associated with decreased RelA/p65 phosphorylation (Ser(311)) and translocation of the RelA/p65 subunit of NF-kappaB into the nucleus. Furthermore, CS/reactive aldehydes and LPS exposures led to activation and translocation of PKCzeta into the nucleus where it forms a complex with CREB-binding protein (CBP) and acetylated RelA/p65 causing histone phosphorylation and acetylation on promoters of pro-inflammatory genes. Taken together, these data suggest that PKCzeta plays an important role in CS/aldehyde- and LPS-induced lung inflammation through acetylation of RelA/p65 and histone modifications via CBP. These data provide new insights into the molecular mechanisms underlying the pathogenesis of chronic inflammatory lung diseases.

Plants fend off potentially damaging ultraviolet (UV)-B radiation by synthesizing and accumulating UV-B-absorbing flavonols that function as sunscreens. Regulation of this biosynthetic pathway is largely transcriptional and controlled by a network of transcription factors, among which the PRODUCTION OF FLAVONOL GLYCOSIDES (PFG) family of R2R3-MYB transcription factors was recently identified with a pivotal function. Here, we describe the response of Arabidopsis seedlings to narrow-band UV-B radiation at the level of phenylpropanoid pathway genes using whole-genome transcriptional profiling and identify the corresponding flavonol glycosides accumulating under UV-B. We further show that the bZIP transcriptional regulator ELONGATED HYPOCOTYL5 (HY5) is required for the transcriptional activation of the PFG1/MYB12 and PFG3/MYB111 genes under UV-B and visible light. A synthetic protein composed of HY5 with the VP16 activation domain is sufficient to activate PFG1/MYB12 expression in planta. However, even though myb11 myb12 myb111 triple mutants have strongly reduced CHS levels in darkness as well as in constant light, neither light- nor UV-B-inducibility seems impaired. Notwithstanding this, absence of the three PFG family transcription factors results in reduced UV-B tolerance, whereas PFG1/MYB12 overexpression leads to an increased tolerance. Thus, our data suggest that HY5-dependent regulation of PFG gene expression contributes to the establishment of UV-B tolerance.

Interaction between acidic activation domains and the activator-binding domains of Swi1 and Snf5 of the yeast SWI/SNF chromatin remodeling complex has previously been characterized in vitro. Although deletion of both activator-binding domains leads to phenotypes that differ from the wild-type, their relative importance for SWI/SNF recruitment to target genes has not been investigated. In the present study, we used chromatin immunoprecipitation assays to investigate the individual and collective importance of the activator-binding domains for SWI/SNF recruitment to genes within the GAL regulon in vivo. We also investigated the consequences of defective SWI/SNF recruitment for target gene activation. We demonstrate that deletion of both activator-binding domains essentially abolishes galactose-induced SWI/SNF recruitment and causes a reduction in transcriptional activation similar in magnitude to that associated with a complete loss of SWI/SNF activity. The activator-binding domains in Swi1 and Snf5 make approximately equal contributions to the recruitment of SWI/SNF to each of the genes studied. The requirement for SWI/SNF recruitment correlates with GAL genes that are highly and rapidly induced by galactose.