Substantial Chromatin Research Articles

ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils.

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and understanding epigenomic regulation. Neutrophils, as a crucial leukocyte type in immune responses, undergo substantial chromatin architectural changes during differentiation and activation, which significantly impact the gene expression necessary for their functions. ATAC-seq has been instrumental in uncovering key transcription factors in neutrophil maturation, revealing pathogen-specific epigenomic signatures, and identifying therapeutic targets for autoimmune diseases. However, neutrophils' sensitivity to the external milieu complicates high-quality ATAC-seq data production. Here, we propose a scalable protocol for preparing ATAC-seq libraries from rodent bone marrow-derived neutrophils, featuring improved immunomagnetic separation to ensure optimal cell viability and high-quality libraries. The vital elements impacting the library quality and optimization principles for methodological extension are discussed in detail. This protocol will support the researchers who are willing to study the chromatin architecture and epigenomic reprogramming of neutrophils, advancing studies in basic and clinical immunology.

Transcription factor network dynamics during the commitment to oncogene-induced senescence

Aberrant oncogenic signaling causes cells to transition into oncogene-induced senescence (OIS) to limit uncontrolled proliferation. Despite being a potent tumor suppressor mechanism, OIS is an unstable cell state susceptible to reprogramming that can promote tumorigenesis. Therefore, elucidating the underlying gene regulatory mechanisms that commit cells to OIS is critical to identifying actionable targets to modulate the senescence state. We previously showed that timely execution of the OIS program is governed by hierarchical transcription factor (TF) networks. However, the gene regulatory mechanisms that prime cells to commit to the OIS fate early upon oncogene hyperactivation are currently not known. Here, we leveraged our time-resolved multi-omic profiling approach to generate TF networks during the first 24 h of oncogenic HRASG12V activation. Using this approach, we demonstrate that the commitment to OIS requires the rearrangement of the TF network on a pre-established epigenomic landscape, priming the cells for the substantial chromatin remodeling that underpins the transition to OIS. Our results provide a detailed map of the chromatin landscape before cells transition to OIS thus offering a platform for manipulation of senescence outcomes of potentially therapeutic value.

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline.

The male mammalian germline is characterized by substantial chromatin remodeling associated with the transition from histones to protamines during spermatogenesis, followed by the reversal to nucleohistones in the male pronucleus preceding the zygotic genome activation. Both transitions are associated with the extensive formation of DNA double-strand breaks (DSBs), requiring an estimated 5 to 10 million transient DSBs per spermatozoa. Additionally, the high transcription rate in early stages of spermatogenesis leads to transcription-coupled damage preceding meiotic homologous recombination, potentially further contributing to the DSB landscape in mature spermatozoa. Once meiosis is completed, spermatozoa remain haploid and therefore cannot rely on error-free homologous recombination, but instead depend on error-prone classical non-homologous end joining (cNHEJ). This DNA damage/repair-scenario is proposed to be one of the main causes of the observed paternal mutation propensity in human evolution. Recent studies have shown that DSBs in the male pronucleus are repaired by maternally provided Polθ in Caenorhabditis elegans through Polθ-mediated end joining (TMEJ). Additionally, population genetic datasets have revealed a preponderance of TMEJ signatures associated with human variation. Since these signatures are the result of the combined effect of TMEJ and DSB formation in spermatozoa and male pronuclei, we used a BLISS-based protocol to analyze recurrent DSBs in mature human sperm heads as a proxy of the male pronucleus before zygotic chromatin remodeling. The DSBs were found to be enriched in (YR)n short tandem repeats and in evolutionarily young SINEs, reminiscent to patterns observed in murine spermatids, indicating evolutionary hotspots of recurrent DSB formation in mammalian spermatozoa. Additionally, we detected a similar DSB pattern in diploid human IMR90 cells when cNHEJ was selectively inhibited, indicating the significant impact of absent cNHEJ on the sperm DSB landscape. Strikingly, regions associated with most retained histones, and therefore less condensed chromatin, were not strongly enriched with recurrent DSBs. In contrast, the fraction of retained H3K27me3 in the mature spermatozoa displayed a strong association with recurrent DSBs. DSBs in H3K27me3 are associated with a preference for TMEJ over cNHEJ during repair. We hypothesize that the retained H3K27me3 may trigger transgenerational DNA repair by priming maternal Polθ to these regions.

Abstract 4293: Chromosomal instability causes epigenetic aberrations in cancer

Abstract Chromosomal instability and changes in the epigenome are defining features in most metastatic cancers. Our research links chromosomal missegregation, their containment in micronuclei, and subsequent rupture of the micronuclear envelope to histone post-translational modification aberrations. This relationship was across all tested human and murine cancer cell lines, as well as non-transformed cells. Furthermore, we discovered that the observed changes in histone PTMs were caused by either micronuclear rupture or the persistence of histone PTM status during cell division. Moreover, we revealed substantial chromatin accessibility differences in micronuclei. Notably, there is a distinct bias in chromatin accessibility between promoter regions compared to distal and intergenic regions of the genome, which aligned well with observed changes in histone modifications. The induction of chromosomal instability leads to widespread, heterogeneous, and heritable abnormalities in the epigenetic landscape of cancer cells. Therefore, on top of genomic copy number changes, chromosomal instability causes epigenetic reprogramming, which adds another layer to cancer heterogeneity. Citation Format: Albert S. Agustinus, Duaa Al-Rawi, Bhargavi Dameracharla, Ramya Raviram, Bailey S. Jones, Stephanie Stransky, Lorenzo Scipioni, Jens Luebeck, Melody Di Bona, Danguole Norkunaite, Robert M. Myers, Mercedes Duran, Seongmin Choi, Britta Weigelt, Shira Yomtoubian, Andrew McPherson, Eleonore Toufektchan, Kristina Keuper, Paul S. Mischel, Vivek Mittal, Sohrab P. Shah, John Maciejowski, Zuzana Storchova, Enrico Gratton, Peter Ly, Dan Landau, Mathieu F. Bakhoum, Richard P. Koche, Simone Sidoli, Vineet Bafna, Yael David, Samuel F. Bakhoum. Chromosomal instability causes epigenetic aberrations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4293.

Matrin3 mediates differentiation through stabilizing chromatin loop-domain interactions and YY1 mediated enhancer-promoter interactions

Although emerging evidence indicates that alterations in proteins within nuclear compartments elicit changes in chromosomal architecture and differentiation, the underlying mechanisms are not well understood. Here we investigate the direct role of the abundant nuclear complex protein Matrin3 (Matr3) in chromatin architecture and development in the context of myogenesis. Using an acute targeted protein degradation platform (dTAG-Matr3), we reveal the dynamics of development-related chromatin reorganization. High-throughput chromosome conformation capture (Hi-C) experiments revealed substantial chromatin loop rearrangements soon after Matr3 depletion. Notably, YY1 binding was detected, accompanied by the emergence of novel YY1-mediated enhancer-promoter loops, which occurred concurrently with changes in histone modifications and chromatin-level binding patterns. Changes in chromatin occupancy by Matr3 also correlated with these alterations. Overall, our results suggest that Matr3 mediates differentiation through stabilizing chromatin accessibility and chromatin loop-domain interactions, and highlight a conserved and direct role for Matr3 in maintenance of chromosomal architecture.

Transcriptional and post-transcriptional control of odorant receptor choice in ants

Insects and mammals have independently evolved odorant receptor genes that are arranged in large genomic tandem arrays. In mammals, each olfactory sensory neuron chooses to express a single receptor in a stochastic process that includes substantial chromatin rearrangements. Here, we show that ants, which have the largest odorant receptor repertoires among insects, employ a different mechanism to regulate gene expression from tandem arrays. Using single-nucleus RNA sequencing, we found that ant olfactory sensory neurons choose different transcription start sites along an array but then produce mRNA from many downstream genes. This can result in transcripts from dozens of receptors being present in a single nucleus. Such rampant receptor co-expression at first seems difficult to reconcile with the narrow tuning of the ant olfactory system. However, RNA fluorescence in situ hybridization showed that only mRNA from the most upstream transcribed odorant receptor seems to reach the cytoplasm where it can be translated into protein, whereas mRNA from downstream receptors gets sequestered in the nucleus. This implies that, despite the extensive co-expression of odorant receptor genes, each olfactory sensory neuron ultimately only produces one or very few functional receptors. Evolution has thus found different molecular solutions in insects and mammals to the convergent challenge of selecting small subsets of receptors from large odorant receptor repertoires.

Lipopolysaccharide increases bitter taste sensitivity via epigenetic changes in Tas2r gene clusters

T2R bitter receptors, encoded by Tas2r genes, are not only critical for bitter taste signal transduction but also important for defense against bacteria and parasites. However, little is known about whether and how Tas2r gene expression are regulated. Here, we show that in an inflammation model mimicking bacterial infection using lipopolysaccharide, the expression of many Tas2rs was significantly upregulated and mice displayed markedly increased neural and behavioral responses to bitter compounds. Using single-cell assays for transposase-accessible chromatin with sequencing (scATAC-seq), we found that the chromatin accessibility of Tas2rs was highly celltype specific and lipopolysaccharide increased the accessibility of many Tas2rs. scATAC-seq also revealed substantial chromatin remodeling in immune response genes in taste tissue stem cells, suggesting potential long-lasting effects. Together, our results suggest an epigenetic mechanism connecting inflammation, Tas2r gene regulation, and altered bitter taste, which mayexplain heightened bitter taste that can occur with infections and cancer treatments.

Intratumoral stem-like CCR4+ regulatory T cells orchestrate the immunosuppressive microenvironment in HCC associated with hepatitis B

Regulatory T cell (Treg) depletion increases antitumor immunity. However, severe autoimmunity can occur following systemic loss of Tregs, which could be avoided by selectively depleting intratumoral Tregs. Herein, we aimed to investigate the role of tumor-infiltrating CCR4+ Tregs in hepatocellular carcinoma (HCC) and to provide a potential target strategy for immunotherapy. CCR4+ Tregs were analyzed by flow cytometry in murine models and clinical samples. The function of tumor-infiltrating and induced CCR4+ Tregs was interrogated by genetic and epigenetic approaches. To block CCR4+ Treg chemotaxis, we developed an N-terminus recombinant protein of CCR4 (N-CCR4-Fc) as a neutralizing pseudo-receptor that effectively bound to its ligand CCL22. The efficacy of CCR4 antagonism as an immunotherapeutic agent was evaluated by tumor weights, growth kinetics and survival curves. CCR4+ Tregs were the predominant type of Tregs recruited to hepatitis B-associated HCC (HBV+ HCC), correlating with sorafenib resistance and HBV load titers. Compared with CCR4- Tregs, CCR4+ Tregs exhibited increased IL-10 and IL-35 expression, and enhanced functionality in suppressing CD8+ T cells. CCR4+ Tregs also displayed PD-1+TCF1+ stem-like properties. ATAC-seq data revealed substantial chromatin remodeling between tumor-infiltrating Tregs (TIL-Tregs) and induced Tregs, suggesting that long-term chromatin reprogramming accounted for the acquisition of enhanced immunosuppressive stem-like specificity by CCR4+ TIL-Tregs. Treatment with a CCR4 antagonist or N-CCR4-Fc blocked intratumoral Treg accumulation, overcame sorafenib resistance, and sensitized tumors to PD-1 checkpoint blockade. Intratumoral stem-like CCR4+ Tregs orchestrated immunosuppressive resource cells in the tumor microenvironment. CCR4 could be targeted to enhance antitumor immunity by specifically blocking infiltration of Tregs into the tumor microenvironment and inhibiting maintenance of the TIL-Treg pool. Targeting regulatory T cells is a promising approach in cancer immunotherapy; however, severe autoimmunity can occur following systemic regulatory T cell loss. This could be avoided by selectively depleting intratumoral regulatory T cells. Herein, targeting intratumoral stem-like CCR4+ regulatory T cells helped to overcome sorafenib resistance and sensitize tumors to immune checkpoint blockade in mouse models of liver cancer. This approach could have wide clinical applicability.

Pre-differentiation exposure to low-dose of atrazine results in persistent phenotypic changes in human neuronal cell lines.

Exposures to organic pesticides, particularly during a developmental window, have been associated with various neurodegenerative diseases later in life. Atrazine (ATZ), one of the most used pesticides in the U.S., is suspected to be associated with increased neurodegeneration later in life but few studies assessed the neurotoxicity of developmental ATZ exposure using human neuronal cells. Here, we exposed human SH-SY5Y cells to 0.3, 3, and 30ppb of ATZ prior to differentiating them into dopaminergic-like neurons in ATZ-free medium to mimic developmental exposure. The differentiated neurons exhibit altered neurite outgrowth and SNCA pathology depending on the ATZ treatment doses. Epigenome changes, such as decreases in 5mC (for 0.3ppb only), H3K9me3, and H3K27me3 were observed immediately after exposure. These alterations persist in a compensatory manner in differentiated neurons. Specifically, we observed significant reductions in 5mC and H3K9me3, as well as, an increase in H3K27me3 in ATZ-exposed cells after differentiation, suggesting substantial chromatin rearrangements after developmental ATZ exposure. Transcriptional changes of relevant epigenetic enzymes were also quantified but found to only partially explain the observed epigenome alteration. Our results thus collectively suggest that exposure to low-dose of ATZ prior to differentiation can result in long-lasting changes in epigenome and increase risks of SNCA-related Parkinson's Disease.

Live Imaging of Parental Histone Variant Dynamics in UVC-Damaged Chromatin.

In eukaryotic cell nuclei, all DNA transactions, including DNA damage repair, take place on a chromatin substrate, the integrity of which is central to gene expression programs and cell identity. However, substantial chromatin rearrangements accompany the repair response, culminating in the deposition of new histones. How the original epigenetic information conveyed by chromatin may be preserved in this context is a burning question. Elucidating the fate of parental histones, which characterize the pre-damage chromatin state, is a key step forward in deciphering the mechanisms that safeguard epigenome stability. Here, we present an in vivo approach for tracking parental histone H3 variant dynamics in real time after UVC laser-induced damage in human cells.

The DDR at telomeres lacking intact shelterin does not require substantial chromatin decompaction.

Telomeres are protected by shelterin, a six-subunit protein complex that represses the DNA damage response (DDR) at chromosome ends. Extensive data suggest that TRF2 in shelterin remodels telomeres into the t-loop structure, thereby hiding telomere ends from double-stranded break repair and ATM signaling, whereas POT1 represses ATR signaling by excluding RPA. An alternative protection mechanism was suggested recently by which shelterin subunits TRF1, TRF2, and TIN2 mediate telomeric chromatin compaction, which was proposed to minimize access of DDR factors. We performed superresolution imaging of telomeres in mouse cells after conditional deletion of TRF1, TRF2, or both, the latter of which results in the complete loss of shelterin. Upon removal of TRF1 or TRF2, we observed only minor changes in the telomere volume in most of our experiments. Upon codeletion of TRF1 and TRF2, the telomere volume increased by varying amounts, but even those samples exhibiting small changes in telomere volume showed DDR at nearly all telomeres. Upon shelterin removal, telomeres underwent 53BP1-dependent clustering, potentially explaining at least in part the apparent increase in telomere volume. Furthermore, chromatin accessibility, as determined by ATAC-seq (assay for transposase-accessible chromatin [ATAC] with high-throughput sequencing), was not substantially altered by shelterin removal. These results suggest that the DDR induced by shelterin removal does not require substantial telomere decompaction.

Chromatin State Dynamics Underlying CD8 T Cell Differentiation and Dysfunction in Cancer

T cells recognizing tumor-specific antigens are detected in cancer patients but are dysfunctional. Upon antigen encounter, T cells differentiate into discrete phenotypic and functional states. Cellular differentiation is driven by epigenetic remodeling, however, it is not known whether and how epigenetic programming establishes and regulates tumor-specific T cell (TST) dysfunction and determines a T cell's ability to respond to therapeutic interventions such as immune checkpoint blockade (PD-1 and CTLA-4). Here for the first time, we (1) identify chromatin dynamics underlying T cell differentiation to the dysfunctional state in mouse and human tumors and (2) provide insights into the epigenetic and transcriptional regulatory mechanisms determining T cell susceptibility to therapeutic reprogramming.Using a genetic cancer mouse model, we previously showed that CD8 TST become unresponsive early during carcinogenesis at the pre-malignant stage, even before the emergence of a pathologically-defined malignant tumor. While T cell dysfunction was initially reversible, it ultimately became a fixed state that could not be rescued by therapeutic interventions such as PD1 checkpoint blockade.To identify the hierarchical changes in chromatin states resulting in "dysfunction imprinting," we used the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) to map the genome-wide changes in chromatin accessibility in TST cells over the course of cancer development. In parallel, we carried out RNA-Seq to determine the interplay between chromatin remodeling and transcriptional networks. Substantial chromatin remodeling occurred during early T cell activation in the pre-malignant lesion (days 5-7) followed by a second wave of chromatin accessibility changes between days 7 and 14. Strikingly, after the second wave, no further CD8 T cell chromatin remodeling occurred during carcinogenesis, even after progression to an advanced late-stage tumor with an immunosuppressive microenvironment. Interestingly, these 2 distinct chromatin accessibility patterns in TST correlated temporally with the plastic and fixed dysfunctional states and susceptibility to therapeutic reprogramming in vivo. To understand the transition from plastic to fixed dysfunction, we analyzed the differential expression of transcription factors (TF) in conjunction with changes in peak accessibility at TF-binding motifs genome-wide. We identified a network including CD8 T cell regulatory TF such as TCF1, LEF1, BLIMP1, and BACH2 as well as less-well-characterized TF (NR4A2, TOX) potentially controlling differentiation to the dysfunctional state. Moreover, ATAC-Seq analysis of human tumor-infiltrating CD8 T cells revealed similar tumor-associated changes in peak accessibility, and studies are ongoing to assess the associated TF networks.In this study, we have defined discrete chromatin states and associated transcriptional networks underlying plastic and fixed dysfunction in TST, thus providing new insights into the genomic control circuitry of T cell differentiation/dysfunction that may point to new strategies for cellular reprogramming of T cells for cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract PR09: Identifying the epigenetic code of tumor-specific CD8 T cell dysfunction and therapeutic reprogramming

Abstract Mutant neoantigens are commonly expressed in human solid tumors, and CD8 T cells specific for such antigens are detected in cancer patients. However, we know that these tumor-specific T cells are non-functional because despite their presence, tumors progress and often eventually cause death. Distinct T cell differentiation states are associated with specific epigenetic states that define the T cell's functional and phenotypic properties. It is currently not known what epigenetic changes establish and regulate tumor-specific CD8 T cell dysfunction and whether specific epigenetic modifications in dysfunctional T cells determine the ability to respond to therapeutic interventions such as checkpoint blockade (PD-1 and CTLA-4). Here, for the first time, we (1) characterize the progressive chromatin remodeling underlying T cell differentiation to the dysfunctional state in mouse and human tumors, and (2) provide insights into the epigenetic and transcriptional regulatory mechanisms determining T cell susceptibility to therapeutic reprogramming. Using a genetic cancer mouse model in which tamoxifen treatment induces expression of an oncogenic driver neoantigen, SV40 large T antigen (Tag), we previously showed that tumor-specific CD8 T cells (TCRSV40-I) become unresponsive early during tumorigenesis, even before the emergence of a pathologically-defined malignant tumor (pre-malignant phase). While CD8 T cell dysfunction was initially reversible, it ultimately became a fixed state that could not be rescued by therapeutic interventions such as PD1 checkpoint blockade. To identify the hierarchical changes in chromatin states resulting in “dysfunction imprinting,” we used the “Assay for Transposase-Accessible Chromatin using Sequencing” (ATAC-Seq) to map the genome-wide changes in chromatin accessibility in TCRSV40-I cells over the course of tumor development. In parallel, we carried our RNA-Seq on all samples to determine the interplay between chromatin remodeling and transcriptional networks. Substantial chromatin remodeling occurred during early T cell activation in the pre-malignant lesion (day 5), followed by a second wave of chromatin accessibility changes between day 7 and 14. Strikingly, after the second wave, no further CD8 T cell chromatin remodeling occurred during tumorigenesis, even after progression to an advanced late-stage tumor with an immunosuppressive microenvironment. Interestingly, these 2 distinct chromatin accessibility patterns (states 1 and 2) in dysfunctional TCRSV40-I correlated temporally with the plastic and fixed dysfunctional states and susceptibility to therapeutic reprogramming in vivo. To understand the transition from plastic to imprinted dysfunction, we analyzed the differential expression of transcription factors (TF) in conjunction with changes in peak accessibility at TF-binding motifs genome-wide. We identified a network including CD8 T cell regulatory TF such as TCF1, LEF1, BLIMP1, and BACH2 as well as less-well-characterized TF (NR4A2, TOX) potentially controlling differentiation to the dysfunctional state. Moreover, ATAC-Seq analysis of human tumor-infiltrating CD8 T cells revealed similar tumor-associated changes in peak accessibility, and studies are ongoing to assess the associated TF networks. In this study, we have defined discrete chromatin states and associated transcriptional networks underlying plastic and fixed dysfunction in tumor-specific T cells, thus providing new insights into the genomic control circuitry of T cell differentiation/dysfunction that may point to new strategies for cellular reprogramming of T cells for cancer immunotherapy. Citation Format: Mary Philip, Lauren Fairchild, Liping Sun, Agnes Viale, Steven Camara, Ellen Horste, Taha Merghoub, Jedd D. Wolchok, Christina S. Leslie, Andrea Schietinger. Identifying the epigenetic code of tumor-specific CD8 T cell dysfunction and therapeutic reprogramming [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR09.

Enhancers compete with a long non-coding RNA for regulation of the Kcnq1 domain.

The imprinted Kcnq1 domain contains a differentially methylated region (KvDMR) in intron 11 of Kcnq1. The Kcnq1ot1 non-coding RNA emerges from the unmethylated paternal KvDMR in antisense direction, resulting in cis-repression of neighboring genes. The KvDMR encompasses the Kcnq1ot1 promoter, CTCF sites and other DNA elements, whose individual contribution to regulation of the endogenous domain is unknown. We find that paternal inheritance of a deletion of the minimal Kcnq1ot1 promoter derepresses the upstream Cdkn1c gene. Surprisingly, Kcnq1ot1 transcripts continue to emerge from alternative sites, evidence that silencing depends, not on the ncRNA, but on the promoter sequence. Detailed analyses of Kcnq1ot during cardiogenesis show substantial chromatin reorganization coinciding with discontinuous RNA production in both wild-type and mutant mice, with loss of imprinting. We show that CTCF binds to both methylated and unmethylated alleles of the KvDMR. Furthermore, we report a multitude of enhancers within the Kcnq1ot1 region, and present conformational dynamics of a novel heart enhancer engaged in Kcnq1 expression. Our results have important implications on tissue-specific imprinting patterns and how transcriptional mechanisms compete to maximize the expression of vital genes, in addition to shifting our perception on the role of the long ncRNA in regulating this imprinted domain.

Nuclear proteome response to cell wall removal in rice (Oryza sativa)

Plant cells are routinely exposed to various pathogens and environmental stresses that cause cell wall perturbations. Little is known of the mechanisms that plant cells use to sense these disturbances and transduce corresponding signals to regulate cellular responses to maintain cell wall integrity. Previous studies in rice have shown that removal of the cell wall leads to substantial chromatin reorganization and histone modification changes concomitant with cell wall re-synthesis. But the genes and proteins that regulate these cellular responses are still largely unknown. Here we present an examination of the nuclear proteome differential expression in response to removal of the cell wall in rice suspension cells using multiple nuclear proteome extraction methods. A total of 382 nuclear proteins were identified with two or more peptides, including 26 transcription factors. Upon removal of the cell wall, 142 nuclear proteins were up regulated and 112 were down regulated. The differentially expressed proteins included transcription factors, histones, histone domain containing proteins, and histone modification enzymes. Gene ontology analysis of the differentially expressed proteins indicates that chromatin & nucleosome assembly, protein-DNA complex assembly, and DNA packaging are tightly associated with cell wall removal. Our results indicate that removal of the cell wall imposes a tremendous challenge to the cells. Consequently, plant cells respond to the removal of the cell wall in the nucleus at every level of the regulatory hierarchy.

Histone H4K20me3 and HP1α are late heterochromatin markers in development, but present in undifferentiated embryonic stem cells

We report here that the formation of heterochromatin in cell nuclei during mouse development is characterised by dynamic changes in the epigenetic modifications of histones. Our observations reveal that heterochromatin in mouse preimplantation embryos is in an immature state that lacks the constitutive heterochromatin markers histone H4 trimethyl Lys20 (H4K20me3) and chromobox homolog 5 (HP1α, also known as CBX5). Remarkably, these somatic heterochromatin hallmarks are not detectable--except in mural trophoblast--until mid-gestation, increasing in level during foetal development. Our results support a developmentally regulated connection between HP1α and H4K20me3. Whereas inner cell mass (ICM) and epiblast stain negative for H4K20me3 and HP1α, embryonic stem (ES) cell lines, by contrast, stain positive for these markers, indicating substantial chromatin divergence. We conclude that H4K20me3 and HP1α are late developmental epigenetic markers, and slow maturation of heterochromatin in tissues that develop from ICM is ectopically induced during ES cell derivation. Our findings suggest that H4K20me3 and HP1α are markers for cell type commitment that can be triggered by developmental or cell context, independently of the differentiation process.

P53-paralog DNp73 oncogene is repressed by IFNα/STAT2 through the recruitment of the Ezh2 polycomb group transcriptional repressor.

The DNp73 proteins act as trans-repressors of p53 and p73-dependent transcription and exert both anti-apoptotic activity and pro-proliferative activity. DNp73s are frequently up-regulated in a variety of human cancers, including human hepatocellular carcinomas (HCCs). Increased levels of DNp73 proteins confer to HCC cells resistance to apoptosis and, irrespective to p53 status, a chemoresistant phenotype. Here, we show that interferon (IFN)α down-regulates DNp73 expression in primary human hepatocytes (PHHs) and HCC cell lines. IFNα has been used as pro-apoptotic agent in the treatment of malignancies and there is increasing evidence of IFNα effectiveness in HCC treatment and prevention of recurrence. The precise mechanisms by which class I IFNs exert their anti-proliferative and anti-tumor activity remain unclear. IFNα binding to its receptor activates multiple intracellular signaling cascades regulating the transcription of numerous direct target genes through the recruitment of a complex comprising of STAT1, STAT2 and IFN regulatory factor (IRF)9 to their promoters. We found that, in response to IFNα, the P2p73 promoter undergoes substantial chromatin remodeling. Histone deacetylases (HDACs) replace histone acetyl transferases. STAT2 is recruited onto the endogenous P2p73 promoter together with the polycomb group protein Ezh2, leading to increased H3K27 methylation and transcriptional repression. The reduction of DNp73 levels by IFNα is paralleled by an increased susceptibility to IFNα-triggered apoptosis of Huh7 hepatoma cells. Our results show, for the first time, that IFN-stimulated gene factor 3 recruitment may serve both in activating and repressing gene expression and identify the down-regulation of DNp73 as an additional mechanism to counteract the chemoresistance of liver cancer cells.

Differential Histone Modification and Protein Expression Associated with Cell Wall Removal and Regeneration in Rice (Oryza sativa)

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

Epigenetic Instability of Cytokine and Transcription Factor Gene Loci Underlies Plasticity of the T Helper 17 Cell Lineage

Phenotypic plasticity of T helper 17 (Th17) cells suggests instability of chromatin structure of key genes of this lineage. We identified epigenetic modifications across the clustered Il17a and Il17f and the Ifng loci before and after differential IL-12 or TGF-beta cytokine signaling, which induce divergent fates of Th17 cell precursors. We found that Th17 cell precursors had substantial remodeling of the Ifng locus, but underwent critical additional modifications to enable high expression when stimulated by IL-12. Permissive modifications across the Il17a-Il17f locus were amplified by TGF-beta signaling in Th17 cells, but were rapidly reversed downstream of IL-12-induced silencing of the Rorc gene by the transcription factors STAT4 and T-bet. These findings reveal substantial chromatin instability of key transcription factor and cytokine genes of Th17 cells and support a model of Th17 cell lineage plasticity in which cell-extrinsic factors modulate Th17 cell fates through differential effects on the epigenetic status of Th17 cell lineage factors.

Development and analysis of a germline BAC resource for the sea lamprey, a vertebrate that undergoes substantial chromatin diminution

Over the last several years, the sea lamprey (Petromyzon marinus) has grown substantially as a model for understanding the evolutionary fundaments and capacity of vertebrate developmental and genome biology. Recent work on the lamprey genome has resulted in a preliminary assembly of the lamprey genome and led to the realization that nearly all somatic cell lineages undergo extensive programmed rearrangements. Here we describe the development of a bacterial artificial chromosome (BAC) resource for lamprey germline DNA and use sequence information from this resource to probe the subchromosomal structure of the lamprey genome. The arrayed germline BAC library represents approximately 10x coverage of the lamprey genome. Analyses of BAC-end sequences reveal that the lamprey genome possesses a high content of repetitive sequences (relative to human), which show strong clustering at the subchromosomal level. This pattern is not unexpected given that the sea lamprey genome is dispersed across a large number of chromosomes (n approximately 99) and suggests a low-copy DNA targeting strategy for efficiently generating informative paired-BAC-end linkages from highly repetitive genomes. This library therefore represents a new and biologically informed resource for understanding the structure of the lamprey genome and the biology of programmed genome rearrangement.