Chromatin-mediated Gene Regulation Research Articles

Abstract 577: Deciphering the mechanism of the menin-MLL complex dependency in HCC

Abstract Liver cancer is the third leading cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the most common primary liver cancer (~90%). The 5-year survival rate of patients with advanced HCC is ~18% due to the late prognosis and the moderate clinical benefit of available systemic therapy options. Mutations in chromatin regulators were detected in ~50% of liver cancer. Since many chromatin regulators have been shown a dependency in various cancer types and can modulate therapy response, we focused on identifying new therapeutic targets of HCC among epigenetic modifiers and evaluating them as potential targets for future drug development. We constructed a CRISPR library of 6000 gRNAs targeting 737 genes involved in chromatin-mediated gene regulation. Using the epigenome-focused CRISPR/Cas9 screening in two-dimensional and three-dimensional settings in multiple liver cancer cell lines (HepG2, HLF, PLC/PRF5), we identified a list of epigenetic regulators as potential new targets in HCC. Two of them, MEN1 and ASH2L, are core subunits of the menin-MLL complex mediating H3K4 trimethylation (me3). We validated dependencies of HCC on MEN1 and ASH2L by performing a negative selection CRISPR/Cas9 competitive growth assay in HLF and PLC/PRF5 cell lines. Moreover, treatment of HCC cell lines (HLF, PLC/PRF5, HepG2) with recently developed menin inhibitor SNDX-5613 (revumenib) revealed a dose-dependent reduction in cell proliferation. To determine transcriptional changes associated with disruption of the menin-MLL complex, we performed RNA sequencing of HLF and PLC/PRF5 cells following either inhibition with 5µM SNDX-5613 for 4 days or knockout of the MEN1 or ASH2L gene. We observed the upregulation of KRAS and the oncogenic signature of TGFβ signaling pathways in all menin-MLL complex disrupting conditions. We did not detect global changes in H3K4me3 levels upon the menin inhibition or MEN1 or ASH2L knockouts assessed by immunoblotting. To explore the mechanism of the antiproliferative effect of the menin inhibition on HCC further, we evaluated changes in the binding of menin to chromatin and H3K4me3 levels in HCC cells by performing CUT&RUN following treatment of HLF cells with 5µM SNDX-5613 for 4 days. Menin inhibition led to a drastic removal of menin from the chromatin, while H3K4me3 levels were only mildly decreased. We identified 8765 genes near promoter regions with menin binding and H3K4me3 mark, of which 1952 genes were near regions with simultaneous loss of menin and decreased H3K4me3 levels upon menin inhibition. Integration of our genomics data revealed a list of 30 genes, which are downregulated upon menin inhibition in both HLF and PLC/PRF5 cells (FC≥1.5) and the direct targets of the menin-MLL complex in HLF cells, suggesting their potential role in HCC cell survival. Altogether, we anticipate that menin and ASH2L serve as promising targets and represent an appealing therapeutic strategy for HCC treatment. Citation Format: Margarita M. Dzama, Peyton C. Kuhlers, Jesse R. Raab. Deciphering the mechanism of the menin-MLL complex dependency in HCC [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 577.

Fluorescent Reporter Systems to Investigate Chromatin Effector Proteins in Living Cells.

Epigenetic research faces the challenge of the high complexity and tight regulation in chromatin modification networks. Although many isolated mechanisms of chromatin-mediated gene regulation have been described, solid approaches for the comprehensive analysis of specific processes as parts of the bigger epigenome network are missing. In order to expand the toolbox of methods by a system that will help to capture and describe the complexity of transcriptional regulation, we describe here a robust protocol for the generation of stable reporter systems for transcriptional activity and summarize their applications. The system allows for the induced recruitment of a chromatin regulator to a fluorescent reporter gene, followed by the detection of transcriptional changes using flow cytometry. The reporter gene is integrated into an endogenous chromatin environment, thus enabling the detection of regulatory dependencies of the investigated chromatin regulator on endogenous cofactors. The system allows for an easy and dynamic readout at the single-cell level and the ability to compensate for cell-to-cell variances of transcription. The modular design of the system enables the simple adjustment of the method for the investigation of different chromatin regulators in a broad panel of cell lines. We also summarize applications of this technology to characterize the silencing velocity of different chromatin effectors, removal of activating histone modifications, analysis of stability and reversibility of epigenome modifications, the investigation of the effects of small molecule on chromatin effectors and of functional effector-coregulator relationships. The presented method allows to investigate the complexity of transcriptional regulation by epigenetic effector proteins in living cells.

Assay for transposase accessible-chromatin with high throughput sequencing (ATAC-seq) analysis the molecular responses of postharvest pear during Penicillium expansum infection

Blue mold decay caused by Penicillium expansum is a global destructive postharvest disease, which has brought huge economic losses to the pear industry. Transcription factors play an important role in pear anti-infection defense, and it has been observed that they have multiple functions. However, the roles of numerous TFs in the defense of pear against P. expansum have not been adequately explored. In this study, ATAC-seq was used to screen the TFs of pear against P. expansum, thereby understanding the chromatin-mediated gene regulation in the defense of pear against infection. The peak of differential expression enriched 9 transcription factors (AGL27, AT5G02460, AT2G28810, AT5G66940, AT3G46070 and OBP1, TCP3, BPC1 and TCP17). The up-regulated genes in our study were implicated in the MAPK signaling pathway-plant, plant hormone signal transduction pathway, plant-pathogen interaction pathway, glutathione metabolism pathway, phenylpropanoid biosynthesis, flavonoid biosynthesis pathway and carotenoid biosynthesis pathway were identified. Especially the transcription factors TGA, gene SAUR and CYCD3 in plant hormone signal transduction pathway and NCED, CYP707A in carotenoid biosynthesis pathway were differentially regulated. RT-qPCR analysis of twelve randomly selected differentially expressed peak-related genes confirmed the accuracy of ATAC-seq analysis of P. expansum infected pear.

Treatment Outcomes of Childhood Picalm:MLLT10+ Acute Leukemias: An International Retrospective Study

Introduction: Acute leukemia with translocation t(10;11)(p12-13;q14-21) results in generation of the PICALM:MLLT10 fusion gene, and is distinct from t(10;11)(p11-13;q23) which gives rise to KMT2A (formerly MLL) gene rearrangement. Defective PICALM function leads to aberrant hematopoiesis, while defective MLLT10 function impairs chromatin-mediated gene regulation. The PICALM:MLLT10 gene has been reported in patients with Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) predominantly T-cell, Acute Undifferentiated Leukemia (AUL) and Mixed Phenotype Acute Leukemia (MPAL). MLLT10 interacts directly with DOT1L in a complex that regulates the methylation of H3K79. MLLT10 fusions are thought to misdirect DOT1L to the promoters of HOXA genes, leading to hypermethylation of H3K79. This hypermethylation causes constitutive activation of HOXA activity and prevents cell maturation and differentiation. The clinico-biological characteristics, response to conventional treatments, and survival outcomes of childhood PICALM:MLLT10+ leukemias are poorly understood. Methods: This was an international, multicenter, retrospective cohort study. Data were submitted by ten International-BFM (I-BFM) study Group-AML and ALL committees and by the Children`s Oncology Group (COG)-AML Study committee. For study inclusions, patients were required to be ≤ 21 years of age at diagnosis, diagnosed between January 1995 and December 2019 with de novo ALL, AML, AUL or MPAL and PICALM:MLLT10 fusion-positive. Patients with KMT2A-rearranged leukemia or acute promyelocytic leukemia were excluded. Kaplan Meier curves were employed for survival analysis with Cox proportional hazard modelling to compare effect of AML vs ALL therapy, and receipt of hematopoietic stem cell transplant (HSCT) vs no HSCT, on survival. Pearson's Chi Squared was employed as a non-parametric measure to determine the effect of end of induction-1 (EIO-1) MRD positive vs negative status on incidence of relapse. P value cut-off for data to reach statistical significance was <0.05. Results: Ninety-eight children with acute leukemia were included, 60 were males. Median age at diagnosis was 12 years (IQR:9-14.8 years) and median initial white cell count was 40.5 x 109/L (IQR:13.8-168.9). Fifty-five (55.6%) patients were diagnosed with T-ALL, 39 (39.4%) with AML, while precursor-B ALL, AUL and MPAL were each diagnosed in single patients; 1 subtype diagnosis was unknown. Ten (10.1%) patients presented with CNS disease and 13 (13.1%) presented with non-CNS extramedullary manifestations. Of 66 with cytogenetic/molecular data available, 33 (50%) had additional cytogenetic-molecular aberrations; NOTCH1, WT1 and RAS pathway mutations predominated. Most patients received an initial chemotherapy regimen guided by their disease immunophenotype: 58/98 (58.6%) received an ALL-like chemotherapy regimen upfront, 38 (38.4%) received an AML-like regimen, 1 (1.0%) a hybrid regimen, 1 initial chemotherapy regimen was unknown. Five-year event-free survival (EFS) was 50.4% (CI 39.1-64.9). For AML patients, 5-year EFS was 22.5% (CI 9.75-52.0) and for ALL 67.7% (CI 54.2-84.7), p=<0.001. Five-year overall survival (OS) was 56.3% (CI 46.3-68.5). For AML patients, 5-year OS was 26.7% (CI 13.3-53.6) and for ALL 75.7% (CI 64.6-88.8), p=<0.001. Thirty-eight patients died, 22 (57.9%) deaths were disease-related, 4 (10.5%) treatment-related. Patients who received HSCT in CR1, demonstrated a trend towards an inferior 5-year EFS (23.7% vs 56.5%) and OS (42.6% vs 64.5%) than those who did not; however, analysis was limited by small sample size. Four (22.2%) HSCT recipients had ALL but most, 14 (77.8%), had AML, which may account for the unfavourable outcomes associated with HSCT. There was no statistically significant difference in rate of relapse in the EOI-1 MRD-positive compared with MRD-negative patients (p=0.49). Of those who relapsed, 25 (75.8%) had an isolated bone marrow relapse, 3 (9.1%) isolated CNS, 3 (9.1%) other sites and, for 2 patients, site of relapse was unknown. Conclusions: Children with PICALM:MLLT10+ AML have inferior 5-year EFS and OS outcomes compared to their T-ALL counterparts. MRD status at EOI-1 was not predictive of relapse. Targeted therapies with DOT1L inhibitors or DNA demethylating agents should be explored, particularly for patients with AML.

Optimized assay for transposase-accessible chromatin by sequencing (ATAC-seq) library preparation from adult Drosophila melanogaster neurons

Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an untargeted manner. Many studies using ATAC-seq use mammalian or human-derived tissues, and established protocols work well in these systems. However, ATAC-seq is not yet widely used in Drosophila. Vinegar flies present several advantages over mammalian systems that make them an excellent model for ATAC-seq studies, including abundant genetic tools that allow straightforward targeting, transgene expression, and genetic manipulation that are not available in mammalian models. Because current ATAC-seq protocols are not optimized to use flies, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined parameters affecting nuclei isolation, including input size, freezing time, washing, and possible confounds from retinal pigments. Then, we optimized the enzymatic steps of library construction to account for the smaller Drosophila genome size. Finally, we used our optimized protocol to generate ATAC-seq libraries that meet ENCODE quality metrics. Our optimized protocol enables extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.

Temporal signaling, population control, and information processing through chromatin-mediated gene regulation

Chromatin regulation is a key pathway cells use to regulate gene expression in response to temporal stimuli, and is becoming widely used as a platform for synthetic biology applications. Here, we build a mathematical framework for analyzing the response of genetic circuits containing chromatin regulators to temporal signals in mammalian cell populations. Chromatin regulators can silence genes in an all-or-none fashion at the single-cell level, with individual cells stochastically transitioning between active, reversibly silent, and irreversibly silent gene states at constant rates over time. We integrate this mode of regulation with classical gene regulatory motifs, such as autoregulatory and incoherent feedforward loops, to determine the types of responses achievable with duration-dependent signaling. We demonstrate that repressive regulators without long-term epigenetic memory can filter out high frequency noise, and as part of an autoregulatory loop can precisely tune the fraction of cells in a population that expresses a gene of interest. Additionally, we find that repressive regulators with epigenetic memory can sum up and encode the total duration of their recruitment in the fraction of cells irreversibly silenced and, when included in a feed forward loop, enable perfect adaptation. Last, we use an information theoretic approach to show that all-or-none stochastic silencing can be used by populations to transmit information reliably and with high fidelity even in very simple genetic circuits. Altogether, we show that chromatin-mediated gene control enables a repertoire of complex cell population responses to temporal signals and can transmit higher information levels than previously measured in gene regulation.

H4K20 Methylation Is Differently Regulated by Dilution and Demethylation in Proliferating and Cell-Cycle-Arrested Xenopus Embryos.

DNA replication during cell division leads to dilution of histone modifications and can thus affect chromatin-mediated gene regulation, raising the question of how the cell-cycle shapes the histone modification landscape, particularly during embryogenesis. We tackled this problem by manipulating the cell cycle during early Xenopus laevis embryogenesis and analyzing invivo histone H4K20 methylation kinetics. The global distribution of un-, mono-, di-, and tri-methylated histone H4K20 was measured by mass spectrometry in normal and cell-cycle-arrested embryos over time. Using multi-start maximum likelihood optimization and quantitative model selection, we found that three specific biological methylation rate constants were required to explain the measured H4K20 methylation state kinetics. While demethylation is essential for regulating H4K20 methylation kinetics in non-cycling cells, demethylation is very likely dispensable in rapidly dividing cells of early embryos, suggesting that cell-cycle-mediated dilution of H4K20 methylation is an essential regulatory component for shaping its epigenetic landscape during early development. A record of this paper's transparent peer review process is included in the Supplemental Information.

Histone deacetylases as targets in autoimmune and autoinflammatory diseases.

Reversible lysine acetylation of histones is a key epigenetic regulatory process controlling gene expression. Reversible histone acetylation is mediated by two opposing enzyme families: histone acetyltransferases (HATs) and histone deacetylases (HDACs). Moreover, many non-histone targets of HATs and HDACs are known, suggesting a crucial role for lysine acetylation as a posttranslational modification on the cellular proteome and protein function far beyond chromatin-mediated gene regulation. The HDAC family consists of 18 members and pan-HDAC inhibitors (HDACi) are clinically used for the treatment of certain types of cancer. HDACi or individual HDAC member-deficient (cell lineage-specific) mice have also been tested in a large number of preclinical mouse models for several autoimmune and autoinflammatory diseases and in most cases HDACi treatment results in an attenuation of clinical disease severity. A reduction of disease severity has also been observed in mice lacking certain HDAC members. This indicates a high therapeutic potential of isoform-selective HDACi for immune-mediated diseases. Isoform-selective HDACi and thus targeted inactivation of HDAC isoforms might also overcome the adverse effects of current clinically approved pan-HDACi. This review provides a brief overview about the fundamental function of HDACs as epigenetic regulators, highlights the roles of HDACs beyond chromatin-mediated control of gene expression and summarizes the studies showing the impact of HDAC inhibitors and genetic deficiencies of HDAC members for the outcome of autoimmune and autoinflammatory diseases with a focus on rheumatoid arthritis, inflammatory bowel disease and experimental autoimmune encephalomyelitis (EAE) as an animal model of multiple sclerosis.

Accessibility of promoter DNA is not the primary determinant of chromatin-mediated gene regulation.

DNA accessibility is thought to be of major importance in regulating gene expression. We test this hypothesis using a restriction enzyme as a probe of chromatin structure and as a proxy for transcription factors. We measured the digestion rate and the fraction of accessible DNA at almost all genomic AluI sites in budding yeast and mouse liver nuclei. Hepatocyte DNA is more accessible than yeast DNA, consistent with longer linkers between nucleosomes, suggesting that nucleosome spacing is a major determinant of accessibility. DNA accessibility varies from cell to cell, such that essentially no sites are accessible or inaccessible in every cell. AluI sites in inactive mouse promoters are accessible in some cells, implying that transcription factors could bind without activating the gene. Euchromatin and heterochromatin have very similar accessibilities, suggesting that transcription factors can penetrate heterochromatin. Thus, DNA accessibility is not likely to be the primary determinant of gene regulation.

Identification of In Planta Protein-Protein Interactions Using IP-MS.

Gene regulation by transcription factors involves complex protein interaction networks, which include chromatin remodeling and modifying proteins as an integral part. Decoding these protein interactions is crucial for our understanding of chromatin-mediated gene regulation. Here, we describe a method for the immunoprecipitation of in planta nuclear protein complexes followed by mass spectrometry (IP-MS) to identify interactions between transcription factors and chromatin remodelers/modifiers in plants. In addition to a step-by-step bench protocol for immunoprecipitation and subsequent mass spectrometry, we provide guidelines and pointers on necessary controls and data analysis approaches.

Epigenome mapping highlights chromatin-mediated gene regulation in the protozoan parasite Trichomonas vaginalis

Trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes trichomoniasis, one of the most common non-viral sexually transmitted diseases. To survive and to maintain infection, T. vaginalis adapts to a hostile host environment by regulating gene expression. However, the mechanisms of transcriptional regulation are poorly understood for this parasite. Histone modification has a marked effect on chromatin structure and directs the recruitment of transcriptional machinery, thereby regulating essential cellular processes. In this study, we aimed to outline modes of chromatin-mediated gene regulation in T. vaginalis. Inhibition of histone deacetylase (HDAC) alters global transcriptional responses and induces hyperacetylation of histones and hypermethylation of H3K4. Analysis of the genome of T. vaginalis revealed that a number of enzymes regulate histone modification, suggesting that epigenetic mechanisms are important to controlling gene expression in this organism. Additionally, we describe the genome-wide localization of two histone H3 modifications (H3K4me3 and H3K27Ac), which we found to be positively associated with active gene expression in both steady and dynamic transcriptional states. These results provide the first direct evidence that histone modifications play an essential role in transcriptional regulation of T. vaginalis, and may help guide future epigenetic research into therapeutic intervention strategies against this parasite.

Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation

Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2B variants as cells undergo arsenic-mediated epithelial to mesenchymal transition. We used electron capture dissociation-based top-down tandem mass spectrometry analysis validated with quantitative reverse transcription real-time polymerase chain reaction to identify changes in the expression levels of H2B variants in inorganic arsenic-mediated epithelial-mesenchymal transition. We identified changes in the expression levels of specific histone H2B variants in two cell types, which are dependent on dose and length of exposure of inorganic arsenic. In particular, we found increases in H2B variants H2B1H/1K/1C/1J/1O and H2B2E/2F, and significant decreases in H2B1N/1D/1B as cells undergo inorganic arsenic-mediated epithelial-mesenchymal transition. The analysis of these histone variants provides a first step toward an understanding of the functional significance of the diversity of histone structures, especially in inorganic arsenic-mediated gene expression and carcinogenesis.

(1)H, (13)C, and (15)N resonance assignments for the tandem PHD finger motifs of human CHD4.

The plant homeodomain (PHD) zinc finger is a structural motif of about 40–60 amino acid residues found in many eukaryotic proteins that are involved in chromatin-mediated gene regulation. The human chromodomain helicase DNA binding protein 4 (CHD4) is a multi-domain protein that harbours, at its N-terminal end, a pair of PHD finger motifs (dPHD) connected by a ~30 amino acid linker. This tandem PHD motif is thought to be involved in targeting CHD4 to chromatin via its interaction with histone tails. Here we report the 1H, 13C and 15N backbone and side-chain resonance assignment of the entire dPHD by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for the determination of the structure, dynamics and histone-binding properties of this tandem domain pair.

Retraction: Sp100 as a Potent Tumor Suppressor: Accelerated Senescence and Rapid Malignant Transformation of Human Fibroblasts through Modulation of an Embryonic Stem Cell Program

Identifying the functions of proteins, which associate with specific subnuclear structures, is critical to understanding eukaryotic nuclear dynamics. Sp100 is a prototypical protein of ND10/PML nuclear bodies, which colocalizes with Daxx and the proto-oncogenic PML. Sp100 isoforms contain SAND, PHD, Bromo, and HMG domains and are highly sumoylated, all characteristics suggestive of a role in chromatin-mediated gene regulation. A role for Sp100 in oncogenesis has not been defined previously. Using selective Sp100 isoform-knockdown approaches, we show that normal human diploid fibroblasts with reduced Sp100 levels rapidly senesce. Subsequently, small rapidly dividing Sp100 minus cells emerge from the senescing fibroblasts and are found to be highly tumorigenic in nude mice. The derivation of these tumorigenic cells from the parental fibroblasts is confirmed by microsatellite analysis. The small rapidly dividing Sp100 minus cells now also lack ND10/PML bodies, and exhibit genomic instability and p53 cytoplasmic sequestration. They have also activated MYC, RAS, and TERT pathways and express mesenchymal to epithelial transdifferentiation (MET) markers. Reintroduction of expression of only the Sp100A isoform is sufficient to maintain senescence and to inhibit emergence of the highly tumorigenic cells. Global transcriptome studies, quantitative PCR, and protein studies, as well as immunolocalization studies during the course of the transformation, reveal that a transient expression of stem cell markers precedes the malignant transformation. These results identify a role for Sp100 as a tumor suppressor in addition to its role in maintaining ND10/PML bodies and in the epigenetic regulation of gene expression.

Genome-wide analysis of chromatin architecture in Oxytricha trifallax: a single-celled eukaryote with 16,000 tiny chromosomes

The pond-dwelling ciliate Oxytricha trifallax is a model system for the study of gene regulatory mechanisms. It possesses a heterochromatin-rich germline micronucleus, and a transcriptionally active somatic macronucleus (MAC). The MAC genome is highly fragmented, consisting of >16,000 unique “nanochromosomes”, with a mean size of 3.2kb. Consequently, only a limited number of nucleosomes can be accommodated in short nanochromosomes. Nanochromosome size does not vary in multiples of ~147bp corresponding to mono-nucleosome-sized DNA, suggesting that nucleosome depleted regions may be especially prevalent. This unusual genome architecture presents novel challenges for the regulation of gene expression. The efficacy and prevalence of chromatin-mediated gene regulation thus remain unclear in Oxytricha. In addition, the paucity of non-coding DNA suggests that promoters in Oxytricha are highly compact, and may be organized differently from other eukaryotes. Our study aims to uncover how the chromatin structure of promoters and coding regions is constrained by chromosome length in the vegetative Oxytricha MAC. To address this, we characterize the chromatin landscape of the MAC through genome-wide mapping of nucleosome positions and DNase I hypersensitive sites. By integrating these studies with existing RNA Polymerase II ChIP-seq and mRNA-seq data, we investigate how chromatin organization varies with nanochromosome length, gene number, and transcriptional output. These data will also be coupled with transcription start site annotations to define promoter architecture in the Oxytricha MAC, including the presence and position of regulatory motifs, the distribution of transcription start sites within a promoter, and local chromatin accessibility. Together, this integrated analysis will test the contribution of chromatin structure to gene expression regulation, and illuminate the variation of eukaryotic gene regulatory mechanisms.

Sp100 as a Potent Tumor Suppressor: Accelerated Senescence and Rapid Malignant Transformation of Human Fibroblasts through Modulation of an Embryonic Stem Cell Program

Identifying the functions of proteins, which associate with specific subnuclear structures, is critical to understanding eukaryotic nuclear dynamics. Sp100 is a prototypical protein of ND10/PML nuclear bodies, which colocalizes with Daxx and the proto-oncogenic PML. Sp100 isoforms contain SAND, PHD, Bromo, and HMG domains and are highly sumoylated, all characteristics suggestive of a role in chromatin-mediated gene regulation. A role for Sp100 in oncogenesis has not been defined previously. Using selective Sp100 isoform-knockdown approaches, we show that normal human diploid fibroblasts with reduced Sp100 levels rapidly senesce. Subsequently, small rapidly dividing Sp100 minus cells emerge from the senescing fibroblasts and are found to be highly tumorigenic in nude mice. The derivation of these tumorigenic cells from the parental fibroblasts is confirmed by microsatellite analysis. The small rapidly dividing Sp100 minus cells now also lack ND10/PML bodies, and exhibit genomic instability and p53 cytoplasmic sequestration. They have also activated MYC, RAS, and TERT pathways and express mesenchymal to epithelial transdifferentiation (MET) markers. Reintroduction of expression of only the Sp100A isoform is sufficient to maintain senescence and to inhibit emergence of the highly tumorigenic cells. Global transcriptome studies, quantitative PCR, and protein studies, as well as immunolocalization studies during the course of the transformation, reveal that a transient expression of stem cell markers precedes the malignant transformation. These results identify a role for Sp100 as a tumor suppressor in addition to its role in maintaining ND10/PML bodies and in the epigenetic regulation of gene expression.

Epigenetic Regulation of the Cardiovascular System

Our genes code for the building blocks of our body, and variations in our genome predispose us to distinct traits, including disease. To add complexity to the interpretation and regulation of our genetic code, the DNA packed into the chromatin forming our chromosomes is modified by factors that alter its expressivity but without changing its sequence. These factors are referred to as epigenetic factors. A recent definition states: “An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.”1 Many factors are involved in establishing epigenetic traits, including DNA methylation, histone variants, chemical modification of histones, position of nucleosomes, and 3D organization of genes in the nucleus, for example. These modifications often act in concert to alter the expression of genes, or to effect allele-specific inheritance. Epigenetic regulation can be established cell-autonomously, through intercellular signaling, and by environmental influences. All cells in the body are subject to epigenetic regulation, and thus understanding this important layer of regulation is key to elucidating mechanisms of cardiovascular development, physiology, and disease (Figure). Figure. Diagrammatic representation of chromatin and chromatin-mediated gene regulation. TF indicates transcription factor. This review series in Circulation Research will explore recent advances in epigenetic research, with an outlook toward the cardiovascular system. The reviews will explore the current state of the art and will focus on the exciting future of epigenetic research as it relates to the cardiovascular system. Epigenetic regulation is commonly considered to include the chemical modification of histone residues that accompany regulated gene expression. Many have noted that the definition of epigenetics quoted above implies heritability of a phenotype through either mitosis or meiosis. At first glance, this might seem …

Chromatin-Mediated Epigenetic Regulation in the Malaria Parasite Plasmodium falciparum

Malaria is a major public health problem in many developing countries, with the malignant tertian parasite Plasmodium falciparum causing the most malaria-associated mortality. Extensive research, especially with the advancement of genomics and transfection tools, has highlighted the fundamental importance of chromatin-mediated gene regulation in the developmental program of this early-branching eukaryote. The Plasmodium parasite genomes reveal the existence of both canonical and variant histones that make up the nucleosomes, as well as a full collection of conserved enzymes for chromatin remodeling and histone posttranslational modifications (PTMs). Recent studies have identified a wide array of both conserved and novel histone PTMs in P. falciparum, indicating the presence of a complex and divergent "histone code." Genome-wide analysis has begun to decipher the nucleosome landscape and histone modifications associated with the dynamic organization of chromatin structures during the parasite's life cycle. Focused studies on malaria-specific phenomena such as antigenic variation and red cell invasion pathways shed further light on the involvement of epigenetic mechanisms in these processes. Here we review our current understanding of chromatin-mediated gene regulation in malaria parasites, with specific reference to exemplar studies on antigenic variation and host cell invasion.

Genome-wide nucleosome mapping of Plasmodium falciparum reveals histone-rich coding and histone-poor intergenic regions and chromatin remodeling of core and subtelomeric genes

BackgroundEpigenetic modifications of histones and regulation of chromatin structure have been implicated in regulation of virulence gene families in P. falciparum. To better understand chromatin-mediated gene regulation, we used a high-density oligonucleotide microarray to map the position and enrichment of nucleosomes across the entire genome of P. falciparum at three time points of the intra-erythrocytic developmental cycle (IDC) in vitro. We used an unmodified histone H4 antibody for chromatin immunoprecipitation of nucleosome-bound DNA.ResultsWe observed generally low nucleosomal occupancy of intergenic regions and higher occupancy of protein coding regions. In contract to the overall small fluctuation of nucleosomal occupancy in most coding regions throughout the IDC, subtelomeric genes encoding surface proteins such as var and rif, as well as some core chromosomal genes such as transcription factors, showed large changes in chromatin structure. Telomeres harbored a region with the highest nucleosomal occupancy of the genome and also exhibited large changes with higher nucleosomal occupancy at schizont stages. While many of these subtelomeric genes were previously shown to be modified by H3K9 trimethylation, we also identified some housekeeping genes in core chromosome regions that showed extensive changes in chromatin structure but do not contain this modification. tRNA and basal transcription factor genes showed low nucleosomal occupancy at all times, suggesting of an open chromatin structure that might be permissive for constitutively high levels of expression. Generally, nucleosomal occupancy was not correlated with the steady-state mRNA levels. Several var genes were exceptions: the var gene with the highest expression level showed the lowest nucleosomal occupancy, and selection of parasites for var2CSA expression resulted in lower nucleosomal occupancy at the var2CSA locus. We identified nucleosome-free regions in intergenic regions that may serve as transcription start sites or transcription factor binding sites. Using the nucleosomal occupancy data as the baseline, we further mapped the genome-wide enrichment of H3K9 acetylation and detected general enrichment of this mark in intergenic regions.ConclusionsThese data on nucleosome enrichment changes add to our understanding of the influence of chromatin structure on the regulation of gene expression. Histones are generally enriched in coding regions, and relatively poor in intergenic regions. Histone enrichment patterns allow for identification of new putative gene-coding regions. Most genes do not show correlation between chromatin structure and steady-state mRNA levels, indicating the dominant roles of other regulatory mechanisms. We present a genome-wide nucleosomal occupancy map, which can be used as a reference for future experiments of histone modification mapping.

Characterization of PRMT1 fromPlasmodium falciparum

Arginine methylation is a post-translational modification that affects many cellular processes in eukaryotes. The malaria parasite Plasmodium falciparum encodes three conserved PRMTs (protein arginine N-methyltransferases). We have determined that PfPRMT1 (P. falciparum PRMT1) has authentic type I PRMT activity to form monomethylarginines and asymmetric dimethylarginines. Compared with mammalian PRMT1s, PfPRMT1 possesses a distinctive N-terminal sequence that is approximately 50 amino acids longer and is essential for enzyme activity. Recombinant PfPRMT1 methylated histones H4 and H2A and several conserved substrates involved in RNA metabolism, including fibrillarin, poly(A)-binding protein II, ribosomal protein S2 and a putative splicing factor. Using synthetic peptides and MS, we determined target arginines in several substrates and studied the enzyme kinetics. Whereas the kinetic parameters of recombinant PfPRMT1 on an H4 peptide and S-adenosylmethionine were similar to those of mammalian PRMT1s, PfPRMT1 had much higher substrate-turnover rates. In the histone H4 N-terminus, PfPRMT1 could methylate only Arg3, a mark for transcription activation. Western blotting detected dynamic dimethylation of H4-Arg3 during parasite development, suggesting that histone-arginine methylation may play a conserved role in chromatin-mediated gene regulation. Consistent with the presence of potential substrates in both the cytoplasm and nucleus, green fluorescent protein-tagged PfPRMT1 and untagged PfPRMT1 were localized in both cellular compartments, with the majority in the cytoplasm. in vitro assays showed that PfPRMT1 could be inhibited by several small-molecule inhibitors, with IC50-values in the sub-micromolar range. Most of these compounds also effectively inhibited parasite growth, suggesting that parasite PRMTs are promising targets for developing antiparasitic drugs.