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The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions.

Programmed cell death 1 (PD-1) is critical for T regulatory cells (Tregs) to maintain peripheral tolerance to self-antigens. In the tumor microenvironment, interaction between PD-1 and its ligands supports tumor immune evasion. Pembrolizumab blocks interactions of PD-1 with its ligands, enhancing antitumor and clinical responses. We and others have reported that pembrolizumab does not affect function or phenotype of thymic-derived Tregs; however, little is known about its effect on extrathymic differentiation of peripheral Tregs. In this study, we investigated the effect of pembrolizumab on in vitro–induced Tregs (iTregs). Our work showed that PD-1 blockade interferes with iTreg differentiation and has no potential effect on the stability of FOXP3 after differentiation. Additionally, we found that both nontreated and pembrolizumab-treated iTregs were suppressive. However, pembrolizumab-treated iTregs were relatively less suppressive in higher Treg ratios and failed to produce IL-10 compared with their nontreated counterparts. Different methods including transcriptomic analyses confirmed that the downregulation of FOXP3 was mediated by activating mTOR and STAT1 and inhibiting MAPK pathways, shifting the iTreg polarization in favor of Th1 and Th17 subsets. To confirm the role of mTOR activation, we found that rapamycin diminished the effect of pembrolizumab-mediated downregulation of FOXP3. Ingenuity pathway analysis revealed that pembrolizumab-treated iTregs showed upregulation of genes promoting DNA repair and immune cell trafficking, in addition to downregulation of genes supporting cellular assembly and organization. To our knowledge, this is the first study to show that pembrolizumab interferes with differentiation of human FOXP3+ iTregs and to disclose some of the molecular pathways involved.

COVID-19 complications present a huge burden on healthcare systems and warrant a predictive risk model for disease severity of SARS-CoV-2 infection to enable early intervention, prospective decision-making and triaging of patients. We profiled plasma proteins from COVID-19 patients (severe n=50, and mild n=50) and controls (n=50) using function- and pathway-based panels developed with the highly specific proximity extension assays. Several biological pathways were specific for patients with severe complications. Based on these dysregulated profiles, we propose candidate FDA-approved drugs that target multiple upregulated proteins to treat severe complications. In addition, the set of differentially expressed plasma proteins in severe disease contained a robust 46-protein signature, the COVID-19 molecular severity score, which predicts the risk of severe complications. We cross-validated this molecular severity score in an independent cohort and found it useful within three days after hospital admission to predict COVID-19 severity and outcomes. Associated with the molecular severity score, we identified a set of clinical parameters available at admission, that act as a clinical risk score for complications. The molecular and clinical risk scores described in our study may be prognostic tools for severe COVID-19 disease and help alleviate the pressure on healthcare systems during infection peaks.

COVID-19 complications still present a huge burden on healthcare systems and warrant predictive risk models to triage patients and inform early intervention. Here, we profile 893 plasma proteins from 50 severe and 50 mild-moderate COVID-19 patients, and 50 healthy controls, and show that 375 proteins are differentially expressed in the plasma of severe COVID-19 patients. These differentially expressed plasma proteins are implicated in the pathogenesis of COVID-19 and present targets for candidate drugs to prevent or treat severe complications. Based on the plasma proteomics and clinical lab tests, we also report a 12-plasma protein signature and a model of seven routine clinical tests that validate in an independent cohort as early risk predictors of COVID-19 severity and patient survival. The risk predictors and candidate drugs described in our study can be used and developed for personalized management of SARS-CoV-2 infected patients.

The histone variant H2A.J was previously shown to accumulate in senescent human fibroblasts with persistent DNA damage to promote inflammatory gene expression, but its mechanism of action was unknown. We show that H2A.J accumulation contributes to weakening the association of histone H1 to chromatin and increasing its turnover. Decreased H1 in senescence is correlated with increased expression of some repeated DNA sequences, increased expression of STAT/IRF transcription factors, and transcriptional activation of Interferon-Stimulated Genes (ISGs). The H2A.J-specific Val-11 moderates the transcriptional activity of H2A.J, and H2A.J-specific Ser-123 can be phosphorylated in response to DNA damage with potentiation of its transcriptional activity by the phospho-mimetic S123E mutation. Our work demonstrates the functional importance of H2A.J-specific residues and potential mechanisms for its function in promoting inflammatory gene expression in senescence.

// Waseem Ashraf 1 , Abdulkhaleg Ibrahim 2 , Mahmoud Alhosin 3,4,5 , Liliyana Zaayter 1 , Khalid Ouararhni 2 , Christophe Papin 2 , Tanveer Ahmad 1 , Ali Hamiche 2 , Yves Mely 1 , Christian Bronner 2,* and Marc Mousli 1,* 1 Laboratory of Biophotonics and Pharmacology, Faculty of Pharmacy, University of Strasbourg, Illkirch, France 2 Institute of Genetics and Molecular and Cellular Biology, University of Strasbourg, Illkirch-Graffenstaden, France 3 Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia 4 Cancer Metabolism and Epigenetic Unit, King Abdulaziz University, Jeddah, Saudi Arabia 5 Cancer and Mutagenesis Unit, King Fahd Centre for Medical Research, King Abdulaziz University, Jeddah, Saudi Arabia * These authors are co-last authors Correspondence to: Marc Mousli, email: // Keywords : cancer, biomarkers, epigenetics, UHRF1, DNA methylation Received : February 03, 2017 Accepted : April 02, 2017 Published : April 24, 2017 Abstract Cancer is one of the deadliest diseases in the world causing record number of mortalities in both developed and undeveloped countries. Despite a lot of advances and breakthroughs in the field of oncology still, it is very hard to diagnose and treat the cancers at early stages. Here in this review we analyze the potential of Ubiquitin-like containing PHD and Ring Finger domain 1 (UHRF1) as a universal biomarker for cancers. UHRF1 is an important epigenetic regulator maintaining DNA methylation and histone code in the cell. It is highly expressed in a variety of cancers and is a well-known oncogene that can disrupt the epigenetic code and override the senescence machinery. Many studies have validated UHRF1 as a powerful diagnostic and prognostic tool to differentially diagnose cancer, predict the therapeutic response and assess the risk of tumor progression and recurrence. Highly sensitive, non-invasive and cost effective approaches are therefore needed to assess the level of UHRF1 in patients, which can be deployed in diagnostic laboratories to detect cancer and monitor disease progression.

Programmed cell death 1 (PD-1) is critical for T regulatory cells (Tregs) to maintain peripheral tolerance to self-antigens. In the tumor microenvironment, interaction between PD-1 and its ligands supports tumor immune evasion. Pembrolizumab blocks interactions of PD-1 with its ligands, enhancing antitumor and clinical responses. We and others have reported that pembrolizumab does not affect function or phenotype of thymic-derived Tregs; however, little is known about its effect on extrathymic differentiation of peripheral Tregs. In this study, we investigated the effect of pembrolizumab on in vitro-induced Tregs (iTregs). Our work showed that PD-1 blockade interferes with iTreg differentiation and has no potential effect on the stability of FOXP3 after differentiation. Additionally, we found that both nontreated and pembrolizumab-treated iTregs were suppressive. However, pembrolizumab-treated iTregs were relatively less suppressive in higher Treg ratios and failed to produce IL-10 compared with their nontreated counterparts. Different methods including transcriptomic analyses confirmed that the downregulation of FOXP3 was mediated by activating mTOR and STAT1 and inhibiting MAPK pathways, shifting the iTreg polarization in favor of Th1 and Th17 subsets. To confirm the role of mTOR activation, we found that rapamycin diminished the effect of pembrolizumab-mediated downregulation of FOXP3. Ingenuity pathway analysis revealed that pembrolizumab-treated iTregs showed upregulation of genes promoting DNA repair and immune cell trafficking, in addition to downregulation of genes supporting cellular assembly and organization. To our knowledge, this is the first study to show that pembrolizumab interferes with differentiation of human FOXP3

International audience; Motivation:Single-molecule localization microscopy (SMLM) can play an important role in integrated structural biology approaches to identify, localize and determine the 3D structure of cellular structures. While many tools exist for the 3D analysis and visualization of crystal or cryo-EM structures little exists for 3D SMLM data, which can provide unique insights but are particularly challenging to analyze in three dimensions especially in a dense cellular context.Results:We developed 3DClusterViSu, a method based on 3D Voronoi tessellations that allows local density estimation, segmentation and quantification of 3D SMLM data and visualization of protein clusters within a 3D tool. We show its robust performance on microtubules and histone proteins H2B and CENP-A with distinct spatial distributions. 3DClusterViSu will favor multi-scale and multi-resolution synergies to allow integrating molecular and cellular levels in the analysis of macromolecular complexes.Availability and impementation:3DClusterViSu is available under http://cbi-dev.igbmc.fr/cbi/voronoi3D.Supplementary information:Supplementary figures are available at Bioinformatics online.

// Abdulkhaleg Ibrahim 1, 2, * , Mahmoud Alhosin 3, 4, * , Christophe Papin 1 , Khalid Ouararhni 1 , Ziad Omran 5 , Mazin A. Zamzami 3, 4 , Abdulrahman Labeed Al-Malki 3 , Hani Choudhry 3, 4 , Yves Mely 6 , Ali Hamiche 1 , Marc Mousli 6 and Christian Bronner 1 1 Institut De Genetique Et De Biologie Moleculaire Et Cellulaire (IGBMC), INSERM U1258 CNRS UMR 7104, Universite de Strasbourg, Illkirch, France 2 BioTechnology Research Center (BTRC), Tripoli, Lybia 3 Department of Biochemistry, Cancer Metabolism and Epigenetic Unit, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia 4 Cancer and Mutagenesis Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia 5 College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia 6 CNRS UMR 7021 Laboratoire de Bioimagerie et Pathologies, Universite de Strasbourg, Faculte de Pharmacie, Illkirch, France * These authors contributed equally to this work Correspondence to: Mahmoud Alhosin, email: malhaseen@kau.edu.sa Christian Bronner, email: bronnerc@igbmc.fr Keywords: apoptosis; thymoquinone; tumor suppressor genes; ubiquitination; UHRF1 Received: November 30, 2017 Accepted: May 19, 2018 Published: June 19, 2018 ABSTRACT Down-regulation of UHRF1 (Ubiquitin-like containing PHD and Ring Finger 1) in Jurkat cells, induced by natural anticancer compounds such as thymoquinone, allows re-expression of tumor suppressor genes such as p73 and p16 INK4A . In order to decipher the mechanisms of UHRF1 down-regulation, we investigated the kinetic of expression of HAUSP (herpes virus-associated ubiquitin-specific protease), UHRF1, cleaved caspase-3 and p73 in Jurkat cells treated with thymoquinone. We found that thymoquinone induced degradation of UHRF1, correlated with a sharp decrease in HAUSP and an increase in cleaved caspase-3 and p73. UHRF1 concomitantly underwent a rapid ubiquitination in response to thymoquinone and this effect was not observed in the cells expressing mutant UHRF1 RING domain, suggesting that UHRF1 commits an auto-ubiquitination through its RING domain in response to thymoquinone treatment. Exposure of cells to Z-DEVD, an inhibitor of caspase-3 markedly reduced the thymoquinone-induced down-regulation of UHRF1, while proteosomal inhibitor MG132 had no such effect. The present findings indicate that thymoquinone induces in cancer cells a fast UHRF1 auto-ubiquitination through its RING domain associated with HAUSP down-regulation. They further suggest that thymoquinone-induced UHRF1 auto-ubiquitination followed by its degradation is a key event in inducing apoptosis through a proteasome-independent mechanism.

FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER., Author Summary In the nucleus, DNA is packaged into chromatin. The repeating unit of chromatin, the nucleosome, consists of a histone octamer around which DNA is wrapped into two superhelical turns. The nucleosome is a barrier for various nuclear processes which require access to DNA. To repair lesions on DNA, this barrier has to be overcome by either nucleosome remodeling or by histone eviction. Here we present evidence that FACT, a protein known to be involved in transcription, is also involved in BER, by boosting nucleosome remodeling. Upon in vivo oxidized DNA lesion induction, FACT exhibits a BER-like protein behavior, and it is recruited to the sites of DNA damages. In vitro experiments show that FACT boosts the remodeling activity of the chromatin remodeler RSC and is implicated in DNA repair. The presence of FACT greatly facilitates the removal of uracil from nucleosomal, but not from naked DNA, in a RSC-mediated reaction. Taken collectively, our in vitro and in vivo data reveal a role of FACT in BER by helping the remodeling of chromatin at the sites of lesions.

TRIM24 (TIF1α), TRIM28 (TIF1β) and TRIM33 (TIF1γ) are related cofactors defining a subgroup of the tripartite motif (TRIM) superfamily comprising an N-terminal RING finger E3 ligase and a C-terminal PHD-Bromodomain chromatin interacting module. Increasing evidence highlights the important roles of these proteins as modulators of multiple signaling pathways during normal development and as tumor suppressors. The finding that they interact to form a multiprotein complex suggests new mechanisms to integrate multiple signaling pathways for tumor suppression.

The histone variant H3.3 marks active chromatin by replacing the conventional histone H3.1. In this study, we investigate the detailed mechanism of H3.3 replication-independent deposition. We found that the death domain-associated protein DAXX and the chromatin remodeling factor ATRX (α-thalassemia/mental retardation syndrome protein) are specifically associated with the H3.3 deposition machinery. Bacterially expressed DAXX has a marked binding preference for H3.3 and assists the deposition of (H3.3–H4)2 tetramers on naked DNA, thus showing that DAXX is a H3.3 histone chaperone. In DAXX-depleted cells, a fraction of H3.3 was found associated with the replication-dependent machinery of deposition, suggesting that cells adapt to the depletion. The reintroduced DAXX in these cells colocalizes with H3.3 into the promyelocytic leukemia protein (PML) bodies. Moreover, DAXX associates with pericentric DNA repeats, and modulates the transcription from these repeats through assembly of H3.3 nucleosomes. These findings establish a new link between the PML bodies and the regulation of pericentric DNA repeat chromatin structure. Taken together, our data demonstrate that DAXX functions as a bona fide histone chaperone involved in the replication-independent deposition of H3.3.

CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.

H2A.Z, a widely conserved histone variant, is evicted from chromatin by the histone chaperone ANP32E. However, to date, no deposition chaperone for H2A.Z is known in metazoans. Here, we identify YL1 as a specific H2A.Z-deposition chaperone. The 2.7-A-resolution crystal structure of the human YL1-H2A.Z-H2B complex shows that YL1 binding, similarly to ANP32E binding, triggers an extension of the H2A.Z αC helix. The interaction with YL1 is, however, more extensive and includes both the extended acidic patch and the entire DNA-binding surface of H2A.Z-H2B. Substitution of only four amino acid residues of H2A is sufficient for the formation of an H2A.Z-like interface specifically recognized by YL1. Collectively, our data reveal the molecular basis of H2A.Z-specific recognition by YL1 and shed light on the mechanism of H2A.Z transfer to the nucleosome by the ATP-dependent chromatin-remodeling complexes SRCAP and P400-TIP60.

H2A.Z is an essential histone variant implicated in the regulation of key nuclear events. However, the metazoan chaperones responsible for H2A.Z deposition and its removal from chromatin remain unknown. Here we report the identification and characterization of the human protein ANP32E as a specific H2A.Z chaperone. We show that ANP32E is a member of the presumed H2A.Z histone-exchange complex p400/TIP60. ANP32E interacts with a short region of the docking domain of H2A.Z through a new motif termed H2A.Z interacting domain (ZID). The 1.48 A resolution crystal structure of the complex formed between the ANP32E-ZID and the H2A.Z/H2B dimer and biochemical data support an underlying molecular mechanism for H2A.Z/H2B eviction from the nucleosome and its stabilization by ANP32E through a specific extension of the H2A.Z carboxy-terminal α-helix. Finally, analysis of H2A.Z localization in ANP32E−/− cells by chromatin immunoprecipitation followed by sequencing shows genome-wide enrichment, redistribution and accumulation of H2A.Z at specific chromatin control regions, in particular at enhancers and insulators. Human protein ANP32E is a histone chaperone that promotes removal of H2A.Z from chromatin. The histone H2A.Z is a variant of histone H2A, one of the canonical histones present in the chromatin of eukaryotic cells. H2A.Z has important functions in transcription and other nuclear processes. Here, Ali Hamiche and colleagues identify the human protein ANP32E as an H2A.Z chaperone capable of promoting the removal of H2A.Z from chromatin. Biochemical and structural data indicate the molecular basis for H2A.Z recognition and eviction by ANP32E, and genome-wide mapping analyses reveal how ANP32E regulates H2A.Z occupancy at important regulatory regions of the genome.

The role of the mitotic phosphorylation of the amino (NH 2 ) terminus of Centromere Protein A (CENP-A), the histone variant epigenetic centromeric marker, remains elusive. Here, we show that the NH 2 terminus of human CENP-A is essential for mitotic progression and that localization of CENP-C, another key centromeric protein, requires only phosphorylation of the CENP-A NH 2 terminus, and is independent of the CENP-A NH 2 terminus length and amino acid sequence. Mitotic CENP-A nucleosomal complexes contain CENP-C and phosphobinding 14-3-3 proteins. In contrast, mitotic nucleosomal complexes carrying nonphosphorylatable CENP-A–S7A contained only low levels of CENP-C and no detectable 14-3-3 proteins. Direct interactions between the phosphorylated form of CENP-A and 14-3-3 proteins as well as between 14-3-3 proteins and CENP-C were demonstrated. Taken together, our results reveal that 14-3-3 proteins could act as specific mitotic “bridges,” linking phosphorylated CENP-A and CENP-C, which are necessary for the platform function of CENP-A centromeric chromatin in the assembly and maintenance of active kinetochores.

Trim24 (Tif1α) and Trim33 (Tif1γ) interact to form a co-repressor complex that suppresses murine hepatocellular carcinoma. Here we show that Trim24 and Trim33 cooperatively repress retinoic acid receptor-dependent activity of VL30-class endogenous retroviruses (ERVs) in liver. In Trim24-knockout hepatocytes, VL30 derepression leads to accumulation of reverse-transcribed VL30 cDNA in the cytoplasm that correlates with activation of the viral-defense interferon responses mimicking the preneoplastic inflammatory state seen in human liver following exogenous viral infection. Furthermore, upon derepression, VL30 long terminal repeats (LTRs) act as promoter and enhancer elements deregulating expression of neighboring genes and generating enhancer RNAs that are required for LTR enhancer activity in hepatocytes in vivo. These data reinforce the role of the TRIM family of proteins in retroviral restriction and antiviral defense and provide an example of an ERV-derived oncogenic regulatory network.

TRIM24 (TIF1α), TRIM28 (TIF1β), and TRIM33 (TIF1γ) are three related cofactors belonging to the tripartite motif superfamily that interact with distinct transcription factors. TRIM24 interacts with the liganded retinoic acid (RA) receptor to repress its transcriptional activity. Germ line inactivation of TRIM24 in mice deregulates RA-signaling in hepatocytes leading to the development of hepatocellular carcinoma (HCC). Here we show that TRIM24 can be purified as at least two macromolecular complexes comprising either TRIM33 or TRIM33 and TRIM28. Somatic hepatocyte-specific inactivation of TRIM24, TRIM28, or TRIM33 all promote HCC in a cell-autonomous manner in mice. Moreover, HCC formation upon TRIM24 inactivation is strongly potentiated by further loss of TRIM33. These results demonstrate that the TIF1-related subfamily of TRIM proteins interact both physically and functionally to modulate HCC formation in mice.

International audience; Retinoic acid (RA), the active derivative of vitamin A, is an important signaling molecule that controls various developmental processes and influence the proliferation and differentiation of a variety of cell types. RA exerts its biological functions primarily through binding to and activating nuclear RA receptors (RARs, which include the RAR alpha, beta and gamma isotypes RARA, RARB and RARC). Aberrant expression or impaired function of these nuclear receptors has been linked to diverse types of cancer. RARs are RA-dependent transcription factors that regulate gene expression through the recruitment of different co-regulators (co-activators and co-repressors). TRIM24 (formerly known as TIF1 alpha) was among the first co-regulators identified as interacting with RARs in a ligand-dependent fashion, and it was recently shown to function in mice as a potent liver-specific tumor suppressor by attenuating Rara-mediated transcription. The fact that Trim24(-/-), but not Trim24(-/-)Rara(+/-), mutant mice are highly predisposed to the development of hepatocellular carcinoma (HCC) has significant implications in cancer research. This result, along with the observation that in response to pharmacological inhibition of the RA signaling, hepatocytes lacking Trim24 loose their ability to proliferate, strongly implicates Rara as a proto-oncogene in hepatocytes and demonstrates that overactivated RA signaling is deleterious to liver homeostasis.

The histone variant mH2A is believed to be involved in transcriptional repression, but how it exerts its function remains elusive. By using chromatin immunoprecipitation and tandem affinity immunopurification of the mH2A1.1 nucleosome complex, we identified numerous genes with promoters containing mH2A1.1 nucleosomes. In particular, the promoters of the inducible Hsp70.1 and Hsp70.2 genes, but not that of the constitutively expressed Hsp70.8, were highly enriched in mH2A1.1. PARP-1 was identified as a part of the mH2A1.1 nucleosome complex and was found to be associated with the Hsp70.1 promoter. A specific interaction between mH2A1.1 and PARP-1 was demonstrated and found to be associated with inactivation of PARP-1 enzymatic activity. Heat shock released both mH2A1.1 and PARP-1 from the Hsp70.1 promoter and activated PARP-1 automodification activity. The data we present point to a novel mechanism for control of Hsp70.1 gene transcription. mH2A1.1 recruits PARP-1 to the promoter, thereby inactivating it. Upon heat shock, the Hsp70.1 promoter-bound PARP-1 is released to activate transcription through ADP-ribosylation of other Hsp70.1 promoter-bound proteins.

Packaging of the DNA in nucleosomes restricts its accessibility to regulatory factors and enzymatic complexes, making a local remodeling of the nucleosome structure a prerequisite to the establishment of protein-DNA interactions. The use of an experimental system in which one nucleosome is reconstituted on a short linear DNA fragment allows gel fractionation of nucleosomes according to their translational positions, whose locations are dependent on the underlying DNA sequence. Nucleosome mobilization by chromatin remodeling factors is easily detected by observing band disappearance in gel, which in turn provides evidence for histone octamer displacement. Here, we provide methods for chromatin assembly that we have been using in our analysis for nucleosome mobilization by chromatin remodeling factors. These methods are straightforward and easy to follow. Thus, they may provide a good starting assay system for analysis of nucleosome movements by other chromatin remodeling machines.