H3 Lys4 Methylation Research Articles

Structural and Biochemical Insights into MLL1 Core Complex Assembly

Histone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5's β-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analyses of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.

Fine‐tuning the stimulation of MLL1 methyltransferase activity by a histone H3‐based peptide mimetic

The SET1 family of methyltransferases carries out the bulk of histone H3 Lys-4 methylation in vivo. One of the common features of this family is the regulation of their methyltransferase activity by a tripartite complex composed of WDR5, RbBP5, and Ash2L. To selectively probe the role of the SET1 family of methyltransferases, we have developed a library of histone H3 peptide mimetics and report herein the characterization of an Nα acetylated form of histone H3 peptide (NαH3). Binding and inhibition studies reveal that the addition of an acetyl moiety to the N terminus of histone H3 significantly enhances its binding to WDR5 and prevents the stimulation of MLL1 methyltransferase activity by the WDR5-RbBP5-Ash2L complex. The crystal structure of NαH3 in complex with WDR5 reveals that a high-affinity hydrophobic pocket accommodates the binding of the acetyl moiety. These results provide the structural basis to control WDR5-RbBP5-Ash2L-MLL1 activity and a tool to manipulate stem cell differentiation programs.

Lysine-Specific Demethylase 1 Regulates the Embryonic Transcriptome and CoREST Stability

Lysine-specific demethylase 1 (LSD1), which demethylates mono- and dimethylated histone H3-Lys4 as part of a complex including CoREST and histone deacetylases (HDACs), is essential for embryonic development in the mouse beyond embryonic day 6.5 (e6.5). To determine the role of LSD1 during this early period of embryogenesis, we have generated loss-of-function gene trap mice and conditional knockout embryonic stem (ES) cells. Analysis of postimplantation gene trap embryos revealed that LSD1 expression, and therefore function, is restricted to the epiblast. Conditional deletion of LSD1 in mouse ES cells, the in vitro counterpart of the epiblast, revealed a reduction in CoREST protein and associated HDAC activity, resulting in a global increase in histone H3-Lys56 acetylation, but not H3-Lys4 methylation. Despite this biochemical perturbation, ES cells with LSD1 deleted proliferate normally and retain stem cell characteristics. Loss of LSD1 causes the aberrant expression of 588 genes, including those coding for transcription factors with roles in anterior/posterior patterning and limb development, such as brachyury, Hoxb7, Hoxd8, and retinoic acid receptor γ (RARγ). The gene coding for brachyury, a key regulator of mesodermal differentiation, is a direct target gene of LSD1 and is overexpressed in e6.5 Lsd1 gene trap embryos. Thus, LSD1 regulates the expression and appropriate timing of key developmental regulators, as part of the LSD1/CoREST/HDAC complex, during early embryonic development.

Epigenetic enhancement of p66Shc during cellular replicative or premature senescence

The cytoplasmic protein p66Shc is expressed in a wide range of cell types, initially believed to be involved in signaling pathways that regulate cell growth and oxidative stress. Here the epigenetic alterations in the promoter of p66Shc were investigated in replicative senescence and in premature senescence induced by hydrogen peroxide in human embryonic pulmonary fibroblast cells. In both cases p66Shc expression was elevated compared to that seen in growing cultures. However, methylation-specific PCR and bisulfite sequencing revealed that the CpG sites were hypermethylated in all cultures. In addition, quantitative chromatin immunoprecipitation showed increased histone H4 acetylation and histone H3 Lys-4 methylation during replicative senescence, while the increased acetylation of histone H3 and H4, as well as increased H3 Lys-4 methylation, was seen in premature senescence persistence. These findings suggest that histone modifications of p66Shc might be the molecular event in cellular senescence. Taken together, the epigenetic enhancement of p66Shc is associated with the specifically increased histone acetylation and methylation, which may contribute to cellular replicative senescence or premature senescence.

A Modified “Cross-talk” between Histone H2B Lys-120 Ubiquitination and H3 Lys-79 Methylation

Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput. With one set of purification of histones by high pressure liquid chromatography or SDS-PAGE, nearly 20 modification sites including acetylation, propionylation, methylation, and ubiquitination were quantified within 2 h for two samples to be compared. Using this method, the relative levels of H2B ubiquitination and H3 Lys-79 methylation were quantified in the U937 human leukemia cell line, U937 derivative cell lines overexpressing anti-secretory factor 10 (AF10) and mutant AF10 with the deletion of the hDot1 binding domain OM-LZ. We found that H2B ubiquitination is inversely correlated with H3 Lys-79 methylation. Therefore, we propose that a catalytic and inhibitory loop mechanism may better describe the cross-talk relationship between H2B ubiquitination and H3 Lys-79 methylation.

Up-Regulation of the μ-Opioid Receptor Gene Is Mediated through Chromatin Remodeling and Transcriptional Factors in Differentiated Neuronal Cells

The effects of morphine are mediated mainly through the mu opioid receptor (MOR). Expression of the MOR is up-regulated during neuronal differentiation in P19 embryonal carcinoma cells and epigenetic changes play an important role in MOR up-regulation. This study investigates the basis for differentiation-dependent alterations of MOR chromatin by studying the recruitment or dissociation of several factors to the remodeled chromatin locus. Chromatin immunoprecipitation assays were used to demonstrate the recruitment of the transcriptional activator Sp1 and the chromatin remodeling factors Brg1 and BAF155 to this promoter, as well as the dissociation of repressors [histone deacetylases, mSin3A, Brm, and methyl-CpG-binding protein 2 (MeCP2)]. Histone modifications (acetylation, induction of histone H3-lys4 methylation, and reduction of H3-lys9 methylation) were consistently detected on this promoter. Overexpression of Sp1 strongly enhanced MOR promoter activity, and the histone deacetylase inhibitor trichostatin A also increased promoter activity. In vitro DNA CpG-methylation of the promoter partially blocked binding of the Sp1 factor but induced MeCP2 binding. Coimmunoprecipitation studies also found novel evidence of an endogenous MeCP2 interaction with Sp3 but a weaker interaction with Sp1. Overall, the results suggest that during neuronal differentiation, MeCP2 and DNA methylation mediate remodeling of the MOR promoter by chromatin remodeling factors (Brg1 and BAF155) from a compacted state to a conformation allowing access for transcriptional factors. Subsequent recruitment of the activating transcription factor Sp1 to the remodeled promoter results in MOR up-regulation.

A Nucleosome Surface Formed by Histone H4, H2A, and H3 Residues Is Needed for Proper Histone H3 Lys36 Methylation, Histone Acetylation, and Repression of Cryptic Transcription

Set2-mediated H3 Lys(36) methylation is a histone modification that has been demonstrated to function in transcriptional elongation by recruiting the Rpd3S histone deacetylase complex to repress intragenic cryptic transcription. Recently, we identified a trans-histone pathway in which the interaction between the N terminus of Set2 and histone H4 Lys(44) is needed to mediate trans-histone H3 Lys(36) di- and trimethylation. In the current study, we demonstrate that mutation of the lysine 44 residue in histone H4 or the Set2 mutant lacking the histone H4 interaction motif leads to intragenic cryptic transcripts, indicating that the Set2 and histone H4 interaction is important to repress intragenic cryptic transcription. We also determine that histone H2A residues (Leu(116) and Leu(117)), which are in close proximity to histone H4 Lys(44), are needed for proper trans-histone H3 Lys(36) methylation. Similar to H4 Lys(44) mutants, histone H2A Leu(116) and Leu(117) mutations exhibited decreased H3 Lys(36) di- and trimethylation, increased histone H4 acetylation, increased resistance to 6-azauracil, and cryptic transcription. Interestingly, the combined histone H4 Lys(44) and H2A mutations have more severe methylation defects and increased H4 acetylation levels. Furthermore, we identify that additional histone H2A and H3 core residues are also needed for H3 Lys(36) di- and trimethylation. Overall, our results show and suggest that multiple H4, H2A, and H3 residues contribute to and form a Set2 docking/recognition site on the nucleosomal surface so that proper Set2-mediated H3 Lys(36) di- and trimethylation, histone acetylation, and transcriptional elongation can occur.

Chemical Approaches for Studying Histone Modifications

Histones form the protein core around which genomic DNA is wrapped in eukaryotic chromatin. Numerous genetic studies have established that the structure and transcriptional state of chromatin are closely related to histone post-translational modifications. Further elucidation of the precise mechanistic roles for individual histone modifications requires the ability to isolate and study homogeneously modified histones. However, the highly heterogeneous nature of histone modifications in vivo poses a significant challenge for such studies. Chemical tools that have enabled biochemical and biophysical studies of site-specifically modified histones are the focus of this minireview.

Expression and function of Set7/9 in pancreatic islets

Histone tail acetylation and methylation are known to enhance accessibility of islet genes to transcription factors and the basal transcriptional machinery. In this brief report, we follow up on a recent study in which we identified the islet enriched factor Set7/9 as a potentially important histone methyltransferase in β-cells (Deering, et al. Diabetes 2009; 58:185-93). We had suggested that the methylation of H3-Lys4 by Set7/9 enhances accessibility of the insulin gene to the basal transcriptional machinery. Consistent with this hypothesis, we show here that RNA polymerase II occupancy at the insulin and IAPP genes is considerably enhanced in β-cells compared to α cells (or NIH3T3 cells), and that the converse is true for RNA polymerase II occupancy at the glucagon gene. The enrichment of Set7/9 in β-cells appears to be dependent upon Pdx1, as knockdown of Pdx1 in INS-1 β-cells using small hairpin RNAs almost completely abolishes Set7/9 expression. A LacZ expression vector driven by the -6.5 kilobase pair Set7/9 promoter that contains putative Pdx1 binding sites shows β-cell-line-specific expression. Taken together, our data support further the hypothesis that Pdx1-dependent Set7/9 expression may be crucial to enhancing chromatin accessibility and transcription of β-cell genes.

Inhibition of the histone demethylase LSD1 blocks α-herpesvirus lytic replication and reactivation from latency

Reversible methylation of histone tails serve as either positive signals recognized by transcriptional assemblies or negative signals that result in repression 1–4. Invading viral pathogens that depend upon the host cell’s transcriptional apparatus are also subject to the regulatory impact of chromatin assembly and modifications5–8. Here we show that infection by the α-herpesviruses HSV and VZV results in the rapid accumulation of chromatin bearing repressive histone H3-lysine 9 methylation. To enable expression of viral immediate early (IE) genes, both viruses use the cellular transcriptional coactivator HCF-1 to recruit the demethylase LSD1 to the viral immediate early promoters. Depletion of LSD1 or inhibition of its activity with MAO inhibitors results in the accumulation of repressive chromatin and a block to viral gene expression. As HCF-1 is a component of the Set1 and MLL1 histone H3 lysine 4 methyl-transferase complexes 9,10, it thus coordinates modulation of repressive H3-lysine 9 methylation levels with addition of activating H3-lysine 4 trimethylation marks. Strikingly, MAO inhibitors also block the reactivation of HSV from latency in sensory neurons, indicating that the HCF-1 complex is a critical component of the reactivation mechanism. The results support pharmaceutical control of histone modifying enzymes as a strategy for controlling herpesvirus infections.

Gene silencing of HPV16 E6/E7 induced by promoter-targeting siRNA in SiHa cells

Background:Recently, transcriptional gene silencing induced by small interfering RNA (siRNA) was found in mammalian and human cells. However, previous studies focused on endogenous genes.Methods:In this study, we designed siRNA targeting the promoter of human papillomavirus 16 E6/E7 and transfected it into the cervical cancer cell line, SiHa. E6 and E7 mRNA and protein expression were detected in cells treated by promoter-targeting siRNA. Futhermore, cellular growth, proliferation, apoptosis and senescence were detected. Thereafter, we investigated promoter DNA methylation and histone methylation status in cells treated with promoter-targeting siRNA.Results:We found that E6/E7 mRNA and protein were simultaneously reduced, cell growth and proliferation were inhibited and cell death, especially senescence, was remarkably increased. Meanwhile, we also found a significantly increasing histone H3-Lys9 methylation on the promoter when E6/E7 gene expression was inhibited.Interpretation:Our findings suggest that promoter-targeting siRNA effectively and simultaneously knocks down both extraneous HPV 16 E6 and E7 at the transcriptional level, and consequently inhibits proliferation and induces death in HPV 16-infected cells. This transcriptional repression is probably induced by histone modification rather than by alteration of DNA methylation.

Mandatory fortification with folic acid in the United States appears to have adverse effects on histone methylation in women with pre-cancer but not in women free of pre-cancer

To evaluate whether mandatory fortification of grain products with folic acid in the US is associated with changes in histone methylation in cells involved in cervical carcinogenesis. Cervical specimens obtained before (1990 to 1992) and after mandatory folic acid fortification (2000 to 2002) were used to examine the degree of histone methylation (H3 Lys-9) by immunohistochemistry. 91 women (51 before and 40 after fortification) were diagnosed with cervical intraepithelial neoplasia (CIN) grade 3 or carcinoma in situ (CIS) and sections utilized in the study also contained normal, reactive or metaplastic cervical epithelium, CIN 1 or CIN 2. 64 women (34 before and 30 after fortification) were free of CIN and these sections contained only normal or reactive cervical epithelium. Immunohistochemical staining for H3 Lys-9, its assessment in different cell or lesion types and data entry were blinded for fortification status. For each cell type or lesion category we used PROC MIXED in SAS with the specimen identifier as a random effect and the robust variance estimator to estimate age- and race-adjusted intensity score for H3 Lys-9 in the pre- and post-fortification periods. Degree of H3 Lys-9 methylation was significantly higher (P < 0.0001) in ≥CIN 2 lesions (CIN 2, CIN 3 and CIS) than in ≤CIN 1 lesions (CIN 1, normal, reactive and metaplastic), in both pre- and post-fortification CIN 3/CIS specimens. Age- and race-adjusted mean H3 Lys-9 score was significantly higher in all cell or lesion types in CIN 3/CIS specimens obtained in the post-fortification period compared to pre-fortification period (P < 0.05, all comparisons). In contrast, in specimens obtained from women free of CIN, Lys-9 methylation in normal/reactive cervical epithelium was significantly lower in post-fortification specimens than in pre-fortification specimens (P = 0.03). Higher levels of Lys-9 methylation in ≥CIN 2 compared to ≤CIN 1 lesions suggest that higher Lys-9 methylation is associated with progression of lower grade CIN to higher grade CIN. Higher Lys-9 methylation in cervical tissues of women diagnosed with CIN 3 in the post-fortification period than in pre-fortification period suggest that fortification may adversely affect histone methylation in already initiated cells. Lower Lys-9 methylation in normal/reactive cervical cells of women free of CIN in the post-fortification period than pre-fortification on the other hand suggests that fortification is likely to protect against initiation of carcinogenic process in the cervix. These results suggest that mandatory fortification with folic acid in the US seems to have different effects on cancer depending on the stage of carcinogenesis. Because this is the first study to report folic acid fortification-associated differences in histone methylation and because of the limitations inherent to the approach we have taken to demonstrate these differences, validation of the results in other study populations or with other techniques for assessing histone methylation is necessary.

Identification of a minimal cis-element and cognate trans-factor(s) required for induction of Rac2 gene expression during K562 cell differentiation

Rac2 is a Rho family GTPase that is widely expressed in hematopoietic cells and plays a critical role in host defense. This study investigates the mechanisms responsible for increased Rac2 gene expression during myeloid cell differentiation. Treatment of K562 chronic myelogenous leukemia cells with phorbol-12-myristate-13-acetate (PMA) induces megakaryocytic differentiation and Rac2 gene transcription following a lag of 6–12 h. Promoter/luciferase reporter gene assays reveal that a 135 bp cis-element located between − 4223 and − 4008 bp upstream of the Rac2 transcription start site is necessary and sufficient for PMA-induced gene expression. The AP1 transcription factor binds to three cis-elements within the 135 bp Rac2 gene regulatory region both in vitro and in vivo following PMA treatment, and mutagenesis of the AP1 binding sites ablates the PMA responsiveness of the 135 bp Rac2 gene regulatory region. Over-expression of AP1 is sufficient to induce expression of a transiently transfected Rac2 promoter/luciferase plasmid, but not the endogenous Rac2 gene. Induction of AP1 in vitro DNA-binding activity is apparent within 1 h of PMA stimulation. However, AP1 binding to the endogenous Rac2 promoter exhibits a lag of 5–9 h, which correlates with reduced histone H3-Lys9 methylation, increased histone H3 acetylation, and increased nuclease accessibility within the 135 bp Rac2 gene regulatory region. These results demonstrate that PMA induction of Rac2 expression during terminal myeloid differentiation requires the coordinate induction of transcription factors and remodeling of Rac2 gene chromatin structure.

Mandatory fortification with folic acid in the United States appear to have adverse effects on histone methylation in women with pre‐cancer but not in women free of pre‐cancer

The objective of this study was to evaluate whether mandatory fortification of grain products with folic acid in the USA is associated with changes in histone methylation in cells involved in cervical carcinogenesis. Cervical specimens (free of pre‐cancer or diagnosed with pre‐cancer cervical intraepithelial neoplasia grade 3 [CIN 3]) obtained before (1990‐92) or after mandatory folic acid fortification (2000‐02) were used to examine the degree of histone methylation (H3 Lys‐9) by immunohistochemistry. Age‐ and race‐adjusted mean H3 Lys‐9 score was significantly higher in CIN 3 specimens obtained in the post‐fortification period compared to pre‐fortification period (P &lt; 0.05). In contrast, in specimens obtained from women free of CIN, H3 Lys‐9 scores were significantly lower in post‐fortification specimens compared to pre‐fortification specimens (P &lt; 0.05). Altered histone methylation has emerged as key to DNA methylation‐related gene silencing and this type of gene silencing in cancer is accompanied by an increase in H3 Lys9 methylation. Therefore, higher Lys‐9 methylation in cervical tissues of women diagnosed with CIN 3 in the post‐fortification period compared to pre‐fortification period suggest that fortification may have adverse effects on histone methylation in already initiated cells. Lower Lys‐9 methylation in cervical cells of women free of CIN in the post fortification period compared to pre fortification on the other hand suggest that fortification is likely to protect against the initiation of carcinogenic process in the cervix.Supported by NCI R03 CA 102893

Distinct transcriptional outputs associated with mono- and dimethylated histone H3 arginine 2

Dimethylation of histone H3 Arg2 (H3R2me2) maintains transcriptional silencing by inhibiting Set1 mediated trimethylation of H3K4. Here we demonstrate that Arg2 is also monomethylated (H3R2me1) in yeast but that its functional characteristics are distinct from H3R2me2: (i) H3R2me1 does not inhibit histone H3 Lys4 (H3K4) methylation; (ii) it is present throughout the coding region of genes; and (iii) it correlates with active transcription. Collectively, these results indicate that different H3R2 methylation states have defined roles in gene expression.

Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae

Methylation of specific histone lysine residues regulates gene expression and heterochromatin function, but little is known about its role in DNA repair. To examine how changes in conserved methylated residues of histone H3 affect nucleotide excision repair (NER), viable H3K4R and H3K79R mutants were generated in Saccharomyces cerevisiae. These mutants show decreased UV survival and impaired NER at the transcriptionally silent HML locus, while maintaining normal NER in the constitutively expressed RPB2 gene and transcriptionally repressed, nucleosome loaded GAL10 gene. Moreover, the HML chromatin in these mutants has reduced accessibility to Micrococcal nuclease (MNase). Importantly, chromatin immunoprecipitation analysis demonstrates there is enhanced recruitment of the Sir complex at the HML locus of these mutants, and deletion of the SIR2 or SIR3 genes restores the MNase accessibility and DNA repair efficiency at this locus. Furthermore, following UV irradiation expression of NER genes in these mutants remains at wild type levels, with the exception of RAD16 which decreases by more than 2-fold. These results indicate that impaired NER occurs in the silenced chromatin of H3K79R and H3K4,79R mutants as a result of increased binding of Sir complexes, which may reduce DNA lesion accessibility to repair enzymes.

Age-related difference of site-specific histone modifications in rat liver

Aging is associated with decrease in activities of the transcription, replication and DNA repair that can result in deterioration of cellular and tissue functions. Changes of chromatin structures with age are likely major underling mechanisms for the functional decline. Chromatin consists of DNA and histones as well as non-histone proteins. While age-associated change of DNA methylation is well documented, little information is available on site-specific histone modifications in aging. We studied here age-related change of selected modifications of rat liver histone, i.e., histone H3 Lys9 acetylation (H3K9ac), H3 Lys9 methylation (H3K9me), H3 Ser10 phosphorylation (H3S10ph) and H3 Lys14 acetylation (H3K14ac). H3K9ac was decreased and H3S10ph was increased with age significantly. In view of reports indicating that decrease in acetylation and increase in phosphorylation of H3 histones can suppress gene activity, our findings suggest that a mechanism of decreased chromatin functions with age is due to such epigenetic changes.

Hypomethylation of subtelomeric regions in ICF syndrome is associated with abnormally short telomeres and enhanced transcription from telomeric regions

Telomeres and adjacent subtelomeric regions are packaged as heterochromatin in many organisms. The heterochromatic features include DNA methylation, histones H3-Lys9 (Lysine 9) and H4-Lys20 (Lysine 20) methylation and heterochromatin protein1 alpha binding. Subtelomeric DNA is hypomethylated in human sperm and ova, and these regions are subjected to de novo methylation during development. In mice this activity is carried out by DNA methyltransferase 3b (Dnmt3b). Mutations in DNMT3B in humans lead to the autosomal-recessive ICF (immunodeficiency, centromeric region instability, facial anomalies) syndrome. Here we show that, in addition to several satellite and non-satellite repeats, the subtelomeric regions in lymphoblastoid and fibroblast cells of ICF patients are also hypomethylated to similar levels as in sperm. Furthermore, the telomeres are abnormally short in both the telomerase-positive and -negative cells, and many chromosome ends lack detectable telomere fluorescence in situ hybridization signals from either one or both sister-chromatids. In contrast to Dnmt3a/b(-/-) mouse embryonic stem cells, increased telomere sister-chromatid exchange was not observed in ICF cells. Hypomethylation of subtelomeric regions was associated in the ICF cells with advanced telomere replication timing and elevated levels of transcripts emanating from telomeric regions, known as TERRA (telomeric-repeat-containing RNA) or TelRNA. The current findings provide a mechanistic explanation for the abnormal telomeric phenotype observed in ICF syndrome and highlights the link between TERRA/TelRNA and structural telomeric integrity.

Epigenetic Silencing of Tumor Necrosis Factor α during Endotoxin Tolerance

Sustained silencing of potentially autotoxic acute proinflammatory genes like tumor necrosis factor alpha (TNFalpha) occurs in circulating leukocytes following the early phase of severe systemic inflammation. Aspects of this gene reprogramming suggest the involvement of epigenetic processes. We used THP-1 human promonocytes, which mimic gene silencing when rendered endotoxin-tolerant in vitro, to test whether TNFalpha proximal promoter nucleosomes and transcription factors adapt to an activation-specific profile by developing characteristic chromatin-based silencing marks. We found increased TNFalpha mRNA levels in endotoxin-responsive cells that was preceded by dissociation of heterochromatin-binding protein 1alpha, demethylation of nucleosomal histone H3 lysine 9 (H3(Lys(9))), increased phosphorylation of the adjacent serine 10 (H3(Ser(10))), and recruitment of NF-kappaB RelA/p65 to the TNFalpha promoter. In contrast, endotoxin-tolerant cells repressed production of TNFalpha mRNA, retained binding of heterochromatin-binding protein 1alpha, sustained methylation of H3(Lys(9)), reduced phosphorylation of H3(Ser(10)), and showed diminished binding of NF-kappaB RelA/p65 to the TNFalpha promoter. Similar levels of NF-kappaB p50 occurred at the TNFalpha promoter in the basal state, during active transcription, and in the silenced phenotype. RelB, which acts as a repressor of TNFalpha transcription, remained bound to the promoter during silencing. These results support an immunodeficiency paradigm where epigenetic changes at the promoter of acute proinflammatory genes mediate their repression during the late phase of severe systemic inflammation.

Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast

The RNA-induced transcriptional silencing (RITS) complex, containing Ago1, Chp1, Tas3 and centromeric small interfering RNAs (siRNAs), is required for heterochromatic gene silencing at centromeres. Here, we identify a second fission yeast Argonaute complex (Argonaute siRNA chaperone, ARC), which contains, in addition to Ago1, two previously uncharacterized proteins, Arb1 and Arb2, both of which are required for histone H3 Lys9 (H3-K9) methylation, heterochromatin assembly and siRNA generation. Furthermore, whereas siRNAs in the RITS complex are mostly single-stranded, siRNAs associated with ARC are mostly double-stranded, indicating that Arb1 and Arb2 inhibit the release of the siRNA passenger strand from Ago1. Consistent with this observation, purified Arb1 inhibits the slicer activity of Ago1 in vitro, and purified catalytically inactive Ago1 contains only double-stranded siRNA. Finally, we show that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation.