H1 Linker Research Articles

Linker H1 is an ‘epi’centre of plant defence

The role of linker H1 histones in plant defence has recently been investigated. Sheikh et al. found that Arabidopsis thaliana plants that were lacking all three H1 proteins showed increased disease resistance, but when primed, failed to induce enhanced resistance. Differences in epigenetic patterns could be the cause of defective priming.

Linker histone H1 modulates defense priming and immunity in plants.

Linker H1 histones play an important role in animal and human pathogenesis, but their function in plant immunity is poorly understood. Here, we analyzed mutants of the three canonical variants of Arabidopsis H1 histones, namely H1.1, H1.2 and H1.3. We observed that double h1.1h1.2 and triple h1.1h1.2h1.3 (3h1) mutants were resistant to Pseudomonas syringae and Botrytis cinerea infections. Transcriptome analysis of 3h1 mutant plants showed H1s play a key role in regulating the expression of early and late defense genes upon pathogen challenge. Moreover, 3h1 mutant plants showed enhanced production of reactive oxygen species and activation of mitogen activated protein kinases upon pathogen-associated molecular pattern (PAMP) treatment. However, 3h1 mutant plants were insensitive to priming with flg22, a well-known bacterial PAMP which induces enhanced resistance in WT plants. The defective defense response in 3h1 upon priming was correlated with altered DNA methylation and reduced global H3K56ac levels. Our data place H1 as a molecular gatekeeper in governing dynamic changes in the chromatin landscape of defense genes during plant pathogen interaction.

DDM1-Mediated TE Silencing in Plants.

Epigenetic modifications are indispensable for regulating gene bodies and TE silencing. DECREASE IN DNA METHYLATION 1 (DDM1) is a chromatin remodeller involved in histone modifications and DNA methylation. Apart from maintaining the epigenome, DDM1 also maintains key plant traits such as flowering time and heterosis. The role of DDM1 in epigenetic regulation is best characterised in plants, especially arabidopsis, rice, maize and tomato. The epigenetic changes induced by DDM1 establish the stable inheritance of many plant traits for at least eight generations, yet DDM1 does not methylate protein-coding genes. The DDM1 TE silencing mechanism is distinct and has evolved independently of other silencing pathways. Unlike the RNA-directed DNA Methylation (RdDM) pathway, DDM1 does not depend on siRNAs to enforce the heterochromatic state of TEs. Here, we review DDM1 TE silencing activity in the RdDM and non-RdDM contexts. The DDM1 TE silencing machinery is strongly associated with the histone linker H1 and histone H2A.W. While the linker histone H1 excludes the RdDM factors from methylating the heterochromatin, the histone H2A.W variant prevents TE mobility. The DDM1-H2A.W strategy alone silences nearly all the mobile TEs in the arabidopsis genome. Thus, the DDM1-directed TE silencing essentially preserves heterochromatic features and abolishes mobile threats to genome stability.

Linker histone H1FOO is required for bovine preimplantation development by regulating lineage specification and chromatin structure†.

Linker histone H1 binds to the nucleosome and is implicated in the regulation of the chromatin structure and function. The H1 variant H1FOO is heavily expressed in oocytes and early embryos. However, given the poor homology of H1FOO among mammals, the functional role of H1FOO during preimplantation embryonic development remains largely unknown, especially in domestic animals. Here, we find that H1FOO is not only expressed in oocytes and preimplantation embryos but granulosa cells and spermatids in cattle. We then demonstrate that the interference of H1FOO results in preimplantation embryonic developmental arrest in cattle using either RNA editing or Trim-Away approach. H1FOO depletion leads to a compromised expression of critical lineage-specific genes at the morula stage and affects the establishment of cell polarity. Interestingly, H1FOO depletion causes a significant increase in the expression of genes encoding other linker H1 and core histones. Concurrently, there is an increase of H3K9me3 and H3K27me3, two markers of repressive chromatin and a decrease of H4K16ac, a marker of open chromatin. Importantly, overexpression of bovine H1FOO results in severe embryonic developmental defects. In sum, we propose that H1FOO controls the proper chromatin structure that is crucial for the fidelity of cell polarization and lineage specification during bovine preimplantation development.

Spatial and Temporal Expression of High-Mobility-Group Nucleosome-Binding (HMGN) Genes in Brain Areas Associated with Cognition in Individuals with Down Syndrome.

DNA methylation and histone posttranslational modifications are epigenetics processes that contribute to neurophenotype of Down Syndrome (DS). Previous reports present strong evidence that nonhistone high-mobility-group N proteins (HMGN) are epigenetic regulators. They play important functions in various process to maintain homeostasis in the brain. We aimed to analyze the differential expression of five human HMGN genes in some brain structures and age ranks from DS postmortem brain samples. Methodology: We performed a computational analysis of the expression of human HMGN from the data of a DNA microarray experiment (GEO database ID GSE59630). Using the transformed log2 data, we analyzed the differential expression of five HMGN genes in several brain areas associated with cognition in patients with DS. Moreover, using information from different genome databases, we explored the co-expression and protein interactions of HMNGs with the histones of nucleosome core particle and linker H1 histone. Results: We registered that HMGN1 and HMGN5 were significantly overexpressed in the hippocampus and areas of prefrontal cortex including DFC, OFC, and VFC of DS patients. Age-rank comparisons between euploid control and DS individuals showed that HMGN2 and HMGN4 were overexpressed in the DS brain at 16 to 22 gestation weeks. From the BioGRID database, we registered high interaction scores of HMGN2 and HMGN4 with Hist1H1A and Hist1H3A. Conclusions: Overall, our results give strong evidence to propose that DS would be an epigenetics-based aneuploidy. Remodeling brain chromatin by HMGN1 and HMGN5 would be an essential pathway in the modification of brain homeostasis in DS.

Modeling cell-free DNA fragment size densities for non-invasive detection of cancer.

3058 Background: Circulating cell-free DNA (cfDNA) is largely nucleosomal in origin with typical fragment lengths of 167 base-pairs reflecting the length of DNA wrapped around-the histone and H1 linker. Given the nucleosomal origin of cfDNA, we have previously used low coverage whole genome sequencing to evaluate DNA fragmentation profiles to sensitively and specifically detect tumor-derived DNA with altered fragment lengths or coverage. Methods: Here we evaluate the use of Bayesian finite mixtures to model the fragment length distribution and demonstrate how the parameters from these models can be useful to distinguish between individuals with and without cancer. We examined the number of cfDNA fragments by size ranging from 100-220bp and approximated the mixture component location, scale, and weight using Markov Chain Monte Carlo. The performance of the method was determined using a ten-fold, ten repeat cross-validation of Gradient Boosted Machine model using 1) our previously described genome-wide fragmentation profile approach, 2) the parameters from the mixture model and 3) a combination of approaches 1) and 2) as features. Results: In this study of 215 cancer patients and 208 cancer-free individuals, we observed cross-validated AUCs of 1) 0.94, 2) 0.95, and 3) 0.97 among the three approaches. Conclusions: Our findings indicate that parsimonious mixture models may improve detection of cancer in conjunction with fragmentation profile analyses across the genome.

How Human H1 Histone Recognizes DNA.

Linker H1 histone is one of the five main histone proteins (H1, H2A, H2B, H3, and H4), which are components of chromatin in eukaryotic cells. Here we have analyzed the patterns of DNA recognition by free H1 histone using a stepwise increase of the ligand complexity method; the affinity of H1 histone for various single- and double-stranded oligonucleotides (d(pN)n; n = 1–20) was evaluated using their competition with 12-mer [32P]labeled oligonucleotide and protein–oligonucleotide complex delaying on nitrocellulose membrane filters. It was shown that minimal ligands of H1 histone (like other DNA-dependent proteins and enzymes) are different mononucleotides (dNMPs; Kd = (1.30 ± 0.2) × 10−2 M). An increase in the length of single-stranded (ss) homo- and hetero-oligonucleotides (d(pA)n, d(pT)n, d(pC)n, and d(pN)n with different bases) by one nucleotide link regardless of their bases, leads to a monotonic increase in their affinity by a factor of f = 3.0 ± 0.2. This factor f corresponds to the Kd value = 1/f characterizing the affinity of one nucleotide of different ss d(pN)n for H1 at n = 2–6 (which are covered by this protein globule) is approximately 0.33 ± 0.02 M. The affinity of five out of six DNA nucleotide units is approximately 25 times lower than for one of the links. The affinity of duplexes of complementary homo- and hetero-d(pN)20 is only 1.3–3.3-fold higher in comparison with corresponding ss oligonucleotides. H1 histone forms mainly weak additive contacts with internucleoside phosphate groups of ssDNAs and one chain of double-stranded DNAs, but not with the bases.

Enrichment of circulating tumor DNA from cell-free DNA of hematopoietic origin.

e13534 Background: In liquid biopsy, circulating tumor DNA (ctDNA) is more fragmented than background cell free DNA, peaking at 147bp (equivalent to a mono-nucleosome) instead of 165bp (nucleosome with an additional 20bp of linker DNA). Isolation of these shorter cell free DNA fragments from longer, extracted cell-free DNA improves detection of ctDNA as demonstrated by enrichment of tumor specific mutations. Nuclease protection of the additional 20bp of linker DNA, conferred by bound linker proteins such as Histone 1, would account for the 165bp peak in host background DNA. We hypothesised that extracting intact nucleosomes with linker DNA using a novel, H1 antigen based, immunocapture approach would enrich the ctDNA fraction in the remaining nucleosomes. Methods: We expressed H1.0 protein in E. coli and following extraction, purification and chemically immobilised it to tosyl-activated magnetic beads. The beads were first used to immunodeplete mono-nucleosomes from HeLa cell digests and the level of immunocaptured nucleosomes was determined by immunoassay targeting intact nucleosomes. The level of nucleosome levels determined before and after depletion was further determined by ELISA targeting H3.1 containing intact nucleosomes. DNA was extracted from the H1 immunocaptured “long” nucleosomes and size profiles compared with the remaining nucleosomes in the supernatant by BioAnalyzer. Then, the method was applied to clinical plasma samples and the size distribution of NGS Libraries (Illumina system) prepared from five colorectal cancer and three healthy samples, their immune depleted supernatants and the immunocaptured nucleosomes were then compared. Enrichment of specific genomic regions was also evaluated. Results: We observed relative enrichment of nucleosomes with short DNA in supernatants following H1 immuno-depletion of the cancer samples as evidenced by a change in size distribution by Bioanalyzer and NGS-sequencing. We also observed potential enrichment of TSS is the H1 immunocaptured nucleosomes consistent with linker DNA positioning of TF binding sites. Conclusions: Histone 1.0 has the highest affinity of H1 mammalian isoforms and successfully immunodepleted plasma samples containing cell free circulating nucleosomes with DNA longer that 147bp. Immobilized H1.0 effectively formed a pseudo-chromatosome by binding to free linker DNA or displacing endogenous H1 and other linker associated proteins. H1 antigen based immuno-depletion offers a simple way to enrich tumour derived nucleosomes and thus cell free DNA.

Amorphous aggregate adducts of linker histone H1 turn highly immunologic in the cancers of oesophagus, stomach, gall bladder and ovary

Hyperglycaemic influence on carcinogenesis and tumour progression is emerging as a link between diabetes and cancer. This work establishes the disturbed structural integrity of nucleosomal linker histone H1 by methyglyoxal (MG) and then correlates the role of modified H1 in the auto-immunopathogenesis of multiple cancers. MG modification caused a loss of free ε-amino groups in H1 and raised its β-sheet structural component with a consequence of non amyloid aggregation. It changed the folding-unfolding denaturation pattern of H1 and attached itself to the lysine residues of the protein eventually making up Nε-(carboxyethyl) lysine. The structural variations act as extra antigenic determinants on H1 that yield aggressive antibody response, when immunised in rabbits. The ELISA tests proved the immunoglobulin response very specific and gel based studies established the preferential binding of antibodies generated against MG-H1 with the modified protein. Cross reaction analysis inferred the multiple specific natures of immunoglobulins with binding tendencies against different inhibitors. The immunoglobulin content in blood sera derived from human subjects with tumours of oesophagus, stomach, gall bladder and ovary confirmed the antibody presence against MG-H1 and competitive ELISA showed their high specificity. This may suggest a link between nucleosomal linker H1, hyperglycaemia, glycoxidation and cancer.

Modulation of chromatin function through linker histone H1 variants.

In this review, the structural aspects of linker H1 histones are presented as a background for characterization of the factors influencing their function in animal and human chromatin. The action of H1 histone variants is largely determined by dynamic alterations of their intrinsically disordered tail domains, posttranslational modifications and allelic diversification. The interdependent effects of these factors can establish dynamic histone H1 states that may affect the organization and function of chromatin regions.

Chromatin remodelling and histone m RNA accumulation in bovine germinal vesicle oocytes.

Major remodelling of the chromatin enclosed within the germinal vesicle occurs towards the end of oocyte growth in mammals, but the mechanisms involved in this process are not completely understood. In bovine, four distinct stages of chromatin compaction-ranging from a diffused state (GV0) to a fully compacted configuration (GV3)-are linked to the gradual acquisition of developmental potential. To better understand the molecular events and to identify mRNA modulations occurring in the oocyte during the GV0-to-GV3 transition, transcriptomic analysis was performed with the EmbryoGENE microarray platform. The mRNA abundance of several genes decreased as chromatin compaction increased, which correlates with progressive transcriptional silencing that is characteristic of the end of oocyte growth. On the other hand, the abundance of some transcripts increased during the same period, particularly several histone gene transcripts from the H2A, H2B, H3, H4, and linker H1 family. In silico analysis predicted RNA-protein interactions between specific histone transcripts and the bovine stem-loop binding protein 2 (SLBP2), which helps regulate the translation of histone mRNA during oogenesis. These results suggest that some histone-encoding transcripts are actively stored, possibly to sustain the needs of the embryo before genome activation. This dataset offers a unique opportunity to survey which histone mRNAs are needed to complete chromatin compaction during oocyte maturation and which are stockpiled for the first three cell cycles following fertilization.

Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development

Histone variants can replace canonical histones in the nucleosome and modify chromatin structure and gene expression. The histone variant H3.3 preferentially associates with active chromatin and has been implicated in the regulation of a diverse range of developmental processes. However, the mechanisms by which H3.3 may regulate gene activity are unclear and gene duplication has hampered an analysis of H3.3 function in mouse. Here, we report that the specific knockdown of H3.3 in fertilized mouse zygotes leads to developmental arrest at the morula stage. This phenotype can be rescued by exogenous H3.3 but not by canonical H3.1 mRNA. Loss of H3.3 leads to over-condensation and mis-segregation of chromosomes as early as the two-cell stage, with corresponding high levels of aneuploidy, but does not appear to affect zygotic gene activation at the two-cell stage or lineage gene transcription at the morula stage. H3.3-deficient embryos have significantly reduced levels of markers of open chromatin, such as H3K36me2 and H4K16Ac. Importantly, a mutation in H3.3K36 that disrupts H3K36 methylation (H3.3K36R) does not rescue the H3.3 knockdown (KD) phenotype. In addition, H3.3 KD embryos have increased incorporation of linker H1. Knockdown of Mof (Kat8), an acetyltransferase specific for H4K16, similarly leads to excessive H1 incorporation. Remarkably, pan-H1 RNA interference (RNAi) partially rescues the chromosome condensation of H3.3 KD embryos and allows development to the blastocyst stage. These results reveal that H3.3 mediates a balance between open and condensed chromatin that is crucial for the fidelity of chromosome segregation during early mouse development.

Abstract 588: PLK1 inhibitors interact synergistically with HDAC inhibitors in IM-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo.

Abstract Purpose: Previous studies have shown that inhibitors of the serine/threonine kinase PLK1 potently induce cell death in human BCR/ABL+ leukemia cells both sensitive and resistant to imatinib mesylate (IM; Cancer Research 70:1513, 2010). Furthermore, HDACIs are known to potentiate tyrosine kinase lethality in such cells. The goal of the present studies was to determine whether PLK1 inhibitors (e.g. BI2536) and HDACIs interacted synergistically in BCR/ABL+ cells sensitive or resistant to imatinib mesylate in vitro and in vivo and if so, to identify the mechanism(s) underlying this interaction. Experimental Design: K562 and LAMA84 cells, IM-resistant Adult-T315I and BV173/E255K cells, and primary CML cells were exposed to BI2536 with or without vorinostat (Merck) for various intervals. Effects on apoptosis and signaling pathways were determined by flow cytometry, western blotting, and gene transfection. K562 and BV173/E255K cells were used to test the in vivo efficacy of this drug regimen in animal models. Results: Co-treatment with BI2536 and vorinostat synergistically induced cell death in parental or IM-resistant BCR/ABL+ cells and primary CD34+ bone marrow cells but was minimally toxic to normal cells. BI2536/vorinostat co-treatment triggered pronounced mitochondrial dysfunction, caspase activation, PARP cleavage, ROS generation, and DNA damage (manifested by increased expression of γH2A.X, p-ATM, p-ATR), all of which were significantly attenuated by the anti-oxidant TBAP. Combined treatment had little effect on T210 phosphorylation of PLK1, but clearly reduced S137 phospho-PLK1 without changing PLK1 expression. While the agents alone had little effect on BCR/ABL phosphorylation, combined treatment induced modest reductions in phospho-BCR/ABL but not total expression. Exposure of cells to BI2536 alone induced marked G2M arrest, whereas vorinostat co-administration abrogated this effect. PLK1 shRNA knockdown significantly increased HDACI lethality, whereas HDAC 1-3 shRNA knockdown reciprocally increased BI2536-induced apoptosis. Genetic interruption of the DNA damage linker H1.2 partially but significantly reduced PLK1/HDAC inhibitor-mediated cell death, arguing for a functional role for DNA damage in lethality. Finally, BI2536/vorinostat co-treatment dramatically reduced tumor growth in both subcutaneous and systemic BCR/ABL+ leukemia xenograft models (including those resistant to IM), and significantly enhanced animal survival. Notably, the regimen was highly effective in a late stage tumor model which is classically resistant to most therapeutic interventions. Conclusions: These findings suggest that concomitant PLK1 and HDAC inhibition is active against IM-sensitive or refractory CML cells both in vitro and in vivo, and that this strategy warrants further evaluation in the setting of BCR/ABL+ leukemias. Citation Format: Girija Prasad Dasmahapatra, Hiral Patel, Tri Nguyen, Elisa Benson, Steven Grant. PLK1 inhibitors interact synergistically with HDAC inhibitors in IM-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 588. doi:10.1158/1538-7445.AM2013-588

PLK1 Inhibitors Synergistically Potentiate HDAC Inhibitor Lethality in Imatinib Mesylate–Sensitive or –Resistant BCR/ABL+ Leukemia Cells In Vitro and In Vivo

To determine whether Polo-like kinase 1 (PLK1) inhibitors (e.g., BI2536) and histone deacetylase (HDAC) inhibitors (e.g., vorinostat) interact synergistically in the BCR/ABL(+) leukemia cells sensitive or resistant to imatinib mesylate (IM) in vitro and in vivo. K562 and LAMA84 cells sensitive or resistant to imatinib mesylate and primary CML cells were exposed to BI2536 and vorinostat. Effects on cell viability and signaling pathways were determined using flow cytometry, Western blotting, and gene transfection. K562 and BV173/E255K animal models were used to test in vivo efficacy. Cotreatment with BI2536 and vorinostat synergistically induced cell death in parental or imatinib mesylate-resistant BCR/ABL(+) cells and primary CD34(+) bone marrow cells but was minimally toxic to normal cells. BI2536/vorinostat cotreatment triggered pronounced mitochondrial dysfunction, inhibition of p-BCR/ABL, caspase activation, PARP cleavage, reactive oxygen species (ROS) generation, and DNA damage (manifest by increased expression of γH2A.X, p-ATM, p-ATR), events attenuated by the antioxidant TBAP. PLK1 short hairpin RNA (shRNA) knockdown significantly increased HDACI lethality, whereas HDAC1-3 shRNA knockdown reciprocally increased BI2536-induced apoptosis. Genetic interruption of the DNA damage linker H1.2 partially but significantly reduced PLK1/HDAC inhibitor-mediated cell death, suggesting a functional role for DNA damage in lethality. Finally, BI2536/vorinostat cotreatment dramatically reduced tumor growth in both subcutaneous and systemic BCR/ABL(+) leukemia xenograft models and significantly enhanced animal survival. These findings suggest that concomitant PLK1 and HDAC inhibition is active against imatinib mesylate-sensitive or refractory CML and ALL cells both in vitro and in vivo and that this strategy warrants further evaluation in the setting of BCR/ABL(+) leukemias.

Abstract 262: Complex epigenetic alterations induced by MYC during activation of the rDNA locus

Abstract INTRODUCTION: The nucleolus is the site of ribosome biogenesis which is tightly regulated and linked to cellular growth and proliferation. Increased rRNA synthesis and ribosome biogenesis are thought to be important molecular alterations that occur very commonly in cancer cells. The nucleolus is formed around tandem repeats (n=∼400) of the ribosomal RNA genes (18S, 5.8S and 28S) each of which contains an upstream promoter region. Only a limited number of repeat units are activated at any one time, and the inactive silent repeat units (∼50% of all repeats) are characterized by dense methylation of the CpG island of the upstream promoter. Since MYC is overexpressed in most human prostate cancers, can affect nucleolar structure, and can activate rRNA transcription, our goal is to determine precisely how MYC regulates rRNA levels. Despite MYC's ability to activate rRNA transcription, we have previously reported that MYC is unexpectedly required to maintain proper rDNA promoter methylation levels. Interestingly, maintenance of a certain number of these repeats in an inactive form is required for chromosomal stability and proper rRNA accumulation. In this study we further determine the chromatin structure of the rDNA promoter region associated with active and inactive repeats and determine the effects of forced suppression of MYC. METHODS: Chromatin immunoprecipitation (ChIP) was performed for transcription factors for rRNA transcription and several histone modifications. Isolated ChiPed DNA was followed by bisulfite sequencing (ChIP-Bis-Seq) of a region from the rDNA promoter. The functional role of MYC was explored by siRNA mediated knockdown in prostate cancer and lymphoma cell lines. RESULTS: ChIP-Bis-Seq of the rDNA promoter was used to identify two chromatin modifications associated exclusively with methylated CpG DNA of the rDNA promoter (methylated repeats), H3K9me2 and H1 linker, and, 3 transcription factors associated nearly exclusively with unmethylated repeats (MYC, Pol I, and UBTF). Forced suppression of MYC resulted in reductions in the levels of rRNA as expected, and this was accompanied by an induction in the inactive chromatin marks, H3K9me2 and H1 linker. Notably, the induction of these “heterochromatic” marks, does not result in an induction of additional rDNA promoter methylation, which is decreased overall by MYC knockdown. CONCLUSIONS: In addition to MYC's known ability to directly activate Pol I induced transcription, MYC is required to maintain active rDNA promoters by preventing accumulation of inactive chromatin marks such as H1 and H3K9me2. Despite the recruitment of H1 and H3K9me2 to the promoter after MYC suppression, this is not accompanied by an overall increase in rDNA promoter DNA methylation. This is likely the case since MYC is also required to maintain DNA promoter methylation at these sites, and promoter methylation/silencing itself is required for overall production of mature rRNA species. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 262. doi:10.1158/1538-7445.AM2011-262

Promoter activity of the sea urchin ( Paracentrotus lividus ) nucleosomal H3 and H2A and linker H1 α-histone genes is modulated by enhancer and chromatin insulator

Core promoters and chromatin insulators are key regulatory elements that may direct a transcriptional enhancer to prefer a specific promoter in complex genetic loci. Enhancer and insulator flank the sea urchin (Paracentrotus lividus) α-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. This article deals with the specificity of interaction between the H2A enhancer-bound MBF-1 activator and histone gene promoters, and with the mechanism that leads the H1 transcripts to peak at about one-third of the value for nucleosomal H3 and H2A mRNAs. To this end, in vivo competition assays of enhancer and insulator functions were performed. Our evidence suggests that the MBF-1 transcription factor participates also in the expression of the H3 gene and that the sns5 insulator buffers the downstream H1 promoter from the H2A enhancer. Altogether, these results provide a clear demonstration of the enhancer-blocking function of a chromatin insulator in a natural gene context. In addition, they suggest that both the H2A enhancer and the sns5 insulator may account for the diverse accumulation of the linker H1 versus the core nucleosomal histones during early development of the sea urchin embryo.

Histone genes of the razor clamSolen marginatusunveil new aspects of linker histone evolution in protostomes

The association of DNA with histones results in a nucleoprotein complex called chromatin that consists of repetitive nucleosomal subunits. Nucleosomes are joined together in the chromatin fiber by short stretches of linker DNA that interact with a wide diversity of linker H1 histones involved in chromatin compaction and dynamics. Although the long-term evolution of the H1 family has been the subject of different studies during the last 5 years, the lack of molecular data on replication-independent (RI) H1 variants from protostomes has been hampering attempts to complete the evolutionary picture of this histone family in eukaryotes, especially as it pertains to the functional specialization they impart to the chromatin structure in members of this bilaterian lineage. In an attempt to fill this gap, the present work characterizes the histone gene complement from the razor clam Solen marginatus. Molecular evolutionary analyses reveal that the H1 gene from this organism represents one of the few protostome RI H1 genes known to date, a notion which is further supported by its location within the monophyletic group encompassing the RI H1 variants in the overall phylogeny of eukaryotic H1 proteins. Although the detailed characterization of the nucleotide substitution patterns in RI H1 variants agrees with the model of birth-and-death evolution under strong purifying selection, maximum-likelihood approaches unveil the presence of adaptive selection during at least part of the evolutionary differentiation between protostomes and deuterostomes. The presence of increased levels of specialization in RI H1 proteins from deuterostomes as well as the significant differences observed in electrostatic properties between protostome and deuterostome RI H1s represent novel and important preliminary results for future studies of the functional differentiation of this histone H1 lineage across bilaterians.

Chromatin-Specific Remodeling by HMGB1 and Linker Histone H1 Silences Proinflammatory Genes during Endotoxin Tolerance

Epigenetic silencing of tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) transcription occurs in blood leukocytes of animals and humans after the initiation of severe systemic inflammation (SSI). We previously reported that the epigenetic signature requires induction of NF-kappaB factor RelB, which directs histone H3K9 dimethylation, disrupts assembly of transcription activator NF-kappaB p65, and induces a sustained switch from the euchromatin to heterochromatin. Here, we report the novel findings that intracellular high mobility group box 1 protein (HMGB1) and nucleosome linker histone H1 protein are necessary components of endotoxin-mediated silencing of TNF-alpha in THP-1 human promonocytes. HMGB1 binds the TNF-alpha promoter during transcription silencing and promotes assembly of the repressor RelB. Depletion of HMGB1 by small interfering RNA results in dissociation of RelB from the promoter and partially restores TNF-alpha transcription. Histone H1, which typically displaces HMGB1 from nucleosomal DNA, also binds concomitantly with HMGB1 to the heterochromatin of the silenced TNF-alpha promoter. Combined knockdown of HMGB1 and H1 restores binding of the transcriptionally active NF-kappaB p65 and reestablishes TNF-alpha mRNA levels. Chromatin reimmunoprecipitation experiments demonstrate that HMGB1 and H1 are likely recruited to TNF-alpha sequences independently and that their binding correlates with histone H3K9 dimethylation, as inhibition of histone methylation blocks HMGB1 and H1 binding. Moreover, HMGB1- and H1-mediated chromatin modifications are gene specific during endotoxin silencing in that they also bind and repress acute proinflammatory IL-1beta, while no binding nor repression of antiinflammatory IkappaBalpha is observed. Finally, we find that H1 and HMGB1 bind to the TNF-alpha a promoter in human leukocytes obtained from patients with SSI. We conclude proinflammatory HMGB1 and structural nucleosome linker H1 couple as a component of the epigenetic complex that silences acute proinflammatory TNF-alpha during the assembly of heterochromatin in the SSI phenotype.

Chicken Erythrocyte Histone Octamer Preparation

INTRODUCTIONCore histones can be purified from a variety of cell sources, including Drosophila embryos, HeLa tissue culture cells, calf thymus, or chicken erythrocytes. Chick erythrocytes are an excellent source of cellular histones: Large quantities of source material are readily obtainable, the purified histones have low levels of post-translational modifications, and linker histones can also be purified from the same cell sample. Also, avian histones have an amino acid sequence identical to that of human histones. Histone stocks can be stored successfully for more than a year at 4°C and for several years at -20°C. With this protocol, 200 mL of blood usually yields in excess of 50 mg of purified histone octamers. Additional optional procedures are also presented for the purification of H1 and H5 linker histones, as well as for the preparation of H3/H4 tetramers and H2A/H2B dimers.

Fine Mapping of Posttranslational Modifications of the Linker Histone H1 from Drosophila melanogaster

The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part due to the fact that most of the studies have been performed in organisms that have several H1 variants, which complicates the analyses. We have chosen Drosophila melanogaster, a model organism, which has a single H1 variant, to approach the study of the role of H1 PTMs during embryonic development. Mass spectrometry mapping of the entire sequence of the protein showed phosphorylation only in the ten N-terminal amino acids, mostly at S10. For the first time, changes in the PTMs of a linker H1 during the development of a multicellular organism are reported. The abundance of H1 monophosphorylated at S10 decreases as the embryos age, which suggests that this PTM is related to cell cycle progression and/or cell differentiation. Additionally, we have found a polymorphism in the protein sequence that can be mistaken with lysine methylation if the analysis is not rigorous.