Bivalent Histone Modifications Research Articles

Bivalent histone modifications: how phytopathogens evade plant immunity

Bivalent histone modifications regulate gene expression during development, but little is known about their function in plant–microbe interactions. In a recent report, Zhao et al. showed that expression of bivalent chromatin-marked gene 1 (BCG1), containing a pathogen-associated molecular pattern (PAMP) motif, is epigenetically regulated by trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) of histone H3 to evade plant immunity.

Abstract 859: Development of non-smoking lung cancers by indoor carcinogenic aerosols through epigenetic reprogramming of lung stem cells: Bioinformatics and artificial intelligence analysis

Abstract Several scientific studies discovered the toxic effects of indoor aerosols on different organs’ adult stem cells of the human body. The most importantly, the indoor toxic aerosols comprised of numerous carcinogens namely, sodium dodecyl sulfate, phthalic acids from floor cleaners, formaldehyde, acetaldehyde, benzene, toluene, ethyl benzene, xylenes, from air fresheners, Hydramethylnon, Pyrethrins, imiprothrin, cypermethrin, tetramethrin, prallethrin and permethrin from ant and roach Killer. However, there was a lack of analysis revealing the mechanism of toxic effects and epigenetic reprogramming of human lung stem cells and development of non-smoking lung cancers. In this study, we analyzed the toxic effects of these chemicals and the mechanism of epigenetic reprograming on lung epithelial cells using Comparative Toxico-genomics Database (CTD), database of Environmental Health Perspectives (EHP), National Institute of Environmental Health Sciences (NIEHS), Google search browser with generative artificial intelligence (AI) and other biological informatics methods (Reactome, Cytoscape PSICQUIC services, and ChEMBL database). We observed from CTD analysis that the chemical gene interactions as risk factors from indoor aerosols showed transcriptionally highly expressed of 31 common genes, by phthalic acids, sodium dodecyl sulfate, from the total of 1347 genes, 107 common genes by formaldehyde, acetaldehyde from the total of 4193 genes and all other toxic ingredients of indoor aerosols. The epigenetic mechanism of histone modifications (H3K4me3, H3K9me3, H3K27me3, H3K27ac, H3K36me3, H3K4me1, Hsa-mir-1301 of lung epithelial, and miRNAs have been revealed by NIH Roadmap Epigenomics Mapping Consortium and the lung tissue arrays were analyzed with AI and matplotlib program. The heterogenic adult stem cell reprogramming is regulated by epigenetic bivalent histone modifications at transcript starts sequences (TSS) of target genes and our current study revealed that CD44, CD80, ALDH2, ALDH3A1, ALDH3B2 are epigenetically reprogrammed with close interaction of other genes by these toxic ingredients of indoor aerosols. Therefore, our results are significant for the carcinogenic effects of indoor aerosols causing for development of lung cancer epigenetically, which may give a clue for the prevention and treatment of nonsmoking lung cancer. Citation Format: Juan Anderson, Mariah Delgado, Malcolm Lovett, Maya Saunders, Geovannie Lake, Samuel Darko, Rose M. Stiffin, Ayivi Huisso, Marilyn Sherman, Latoya Appleton, Amalya Mihnea, Swarnava Das, Mohammad M. Algahtani, Ravi Vadapalli, Biswarup Basu, Arunima Biswas, Noor Neha, Madhumita Das, Marco A. Ruiz, Mayur Doke, Jayanta K. Das. Development of non-smoking lung cancers by indoor carcinogenic aerosols through epigenetic reprogramming of lung stem cells: Bioinformatics and artificial intelligence analysis [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 859.

Temporally-coordinated bivalent histone modifications of BCG1 enable fungal invasion and immune evasion

Bivalent histone modifications, including functionally opposite H3K4me3 and H3K27me3 marks simultaneously on the same nucleosome, control various cellular processes by fine-tuning the gene expression in eukaryotes. However, the role of bivalent histone modifications in fungal virulence remains elusive. By mapping the genome-wide landscape of H3K4me3 and H3K27me3 dynamic modifications in Fusarium graminearum (Fg) during invasion, we identify the infection-related bivalent chromatin-marked genes (BCGs). BCG1 gene, which encodes a secreted Fusarium-specific xylanase containing a G/Q-rich motif, displays the highest increase of bivalent modification during Fg infection. We report that the G/Q-rich motif of BCG1 is a stimulator of its xylanase activity and is essential for the full virulence of Fg. Intriguingly, this G/Q-rich motif is recognized by pattern-recognition receptors to trigger plant immunity. We discover that Fg employs H3K4me3 modification to induce BCG1 expression required for host cell wall degradation. After breaching the cell wall barrier, this active chromatin state is reset to bivalency by co-modifying with H3K27me3, which enables epigenetic silencing of BCG1 to escape from host immune surveillance. Collectively, our study highlights how fungal pathogens deploy bivalent epigenetic modification to achieve temporally-coordinated activation and suppression of a critical fungal gene, thereby facilitating successful infection and host immune evasion.

ASH2L regulates postnatal neurogenesis through Onecut2-mediated inhibition of TGF-β signaling pathway.

The ability of neural stem/progenitor cells (NSPCs) to proliferate and differentiate is required through different stages of neurogenesis. Disturbance in the regulation of neurogenesis causes many neurological diseases, such as intellectual disability, autism, and schizophrenia. However, the intrinsic mechanisms of this regulation in neurogenesis remain poorly understood. Here, we report that Ash2l (Absent, small or homeotic discs-like 2), one core component of a multimeric histone methyltransferase complex, is essential for NSPC fate determination during postnatal neurogenesis. Deletion of Ash2l in NSPCs impairs their capacity for proliferation and differentiation, leading to simplified dendritic arbors in adult-born hippocampal neurons and deficits in cognitive abilities. RNA sequencing data reveal that Ash2l primarily regulates cell fate specification and neuron commitment. Furthermore, we identified Onecut2, a major downstream target of ASH2L characterized by bivalent histone modifications, and demonstrated that constitutive expression of Onecut2 restores defective proliferation and differentiation of NSPCs in adult Ash2l-deficient mice. Importantly, we identified that Onecut2 modulates TGF-β signaling in NSPCs and that treatment with a TGF-β inhibitor rectifies the phenotype of Ash2l-deficient NSPCs. Collectively, our findings reveal the ASH2L-Onecut2-TGF-β signaling axis that mediates postnatal neurogenesis to maintain proper forebrain function.

ChIP-seq profiling of H3K4me3 and H3K27me3 in an invasive insect, Bactrocera dorsalis.

Introduction: While it has been suggested that histone modifications can facilitate animal responses to rapidly changing environments, few studies have profiled whole-genome histone modification patterns in invasive species, leaving the regulatory landscape of histone modifications in invasive species unclear. Methods: Here, we screen genome-wide patterns of two important histone modifications, trimethylated Histone H3 Lysine 4 (H3K4me3) and trimethylated Histone H3 Lysine 27 (H3K27me3), in adult thorax muscles of a notorious invasive pest, the Oriental fruit fly Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), using Chromatin Immunoprecipitation with high-throughput sequencing (ChIP-seq). Results: We identified promoters featured by the occupancy of H3K4me3, H3K27me3 or bivalent histone modifications that were respectively annotated with unique genes key to muscle development and structure maintenance. In addition, we found H3K27me3 occupied the entire body of genes, where the average enrichment was almost constant. Transcriptomic analysis indicated that H3K4me3 is associated with active gene transcription, and H3K27me3 is mostly associated with transcriptional repression. Importantly, we identified genes and putative motifs modified by distinct histone modification patterns that may possibly regulate flight activity. Discussion: These findings provide the first evidence of histone modification signature in B. dorsalis, and will be useful for future studies of epigenetic signature in other invasive insect species.

Genome-wide analysis of bivalent histone modifications during Drosophila embryogenesis.

In eukaryotes, histone modifications are key epigenetic regulators that are associated with distinct chromatin features. Bivalent histone modifications describe a situation where a subset of promoters have with both activating (H3K4me3) and repressive (H3K27me3) markers in pluripotent cells (e.g., ESCs). However, it remains to be understood whether bivalent histone modifications are stable throughout developmental stages. Here, by systematically analyzing ChIP-seq data of H3K4me3 and H3K27me3, we provided the first panoramic view of bivalent histone modifications in Drosophila from embryonic 0-4 to 20-24 hr. In our study, we found that bivalent histone modifications occur at other locations in the genome in addition to the promoter region. Additionally, the different genomic regions occupied by bivalent histone modifications exhibit spatiotemporal specificity at each stage. Furthermore, gene ontology and motif analysis reflected continuous and gradual changes of target genes during different developmental process. In summary, we suggest that bivalent histone modifications have potential regulatory functions throughout Drosophila embryonic stage.

Abstract 5830: Carcinogenic effects of metal and metalloids through epigenetic reprogramming of human bone marrow stem cells: The bioinformatics database analysis

Abstract Many scientific studies showed that the carcinogenic effects of environmental contamination of several metal and metalloids on bone marrow stem cells of human body. These carcinogens include, Cadmium, Mercury, Chromium, Aluminum, Nickel and Arsenic. However, there was no such analysis for elucidating the mechanism of carcinogenic effects and epigenetic reprogramming of human bone marrow stem cells. In this study, we analyzed the carcinogenic effects of mentioned carcinogens, and the mechanism of epigenetic reprogramming using NIH Toxicology Data Network (TOXNET), Comparative Toxico-genomics Database (CTD), AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database, database of Environmental Health Perspectives (EHP), National Institute of Environmental Health Sciences (NIEHS), and other bioinformatics database. We found from CTD analysis that the chemical gene interactions showed highly expressed of 272 common genes with miR140 by Cadmium, Mercury from total 3704 genes (approximately 7% affected genes). The AACR GENIE database analysis showed CDKN3, is associated with bone cancer (2 clinical data), HDAC7, TP53, MAPK3K1, MAP2K2 (8 clinical data) found among 272 genes regulated by Cadmium and Mercury. The CTD analysis showed the chemical gene interactions of highly expressed of 102 common genes with miR140 by Chromium, Mercury from a total of 1406 genes; 268 common genes with CD84, CD226 by Mercury, Nickel from a total of 5032 genes; 103 common genes with CD226 by Aluminum, Mercury from a total of 2556 genes; 248 common genes by Arsenic, Mercury from total 5508 genes. Among these 5508 genes, 248 common genes with ALDHA1, 247 common genes with CD40, 248 common genes with CD44, 247 common genes with CD423, 247 common genes with miR10A by Arsenic, Mercury from total 2556 genes. The epigenetic mechanisms of histone modifications (H3K9me3, H3K27me3, H3K27ac, H3K36me3, H3K4me1, H3K4me3 of B cells, hemopoietic stem cells, mesenchymal bone marrow, and chondrocyte stem cells and miRNAs) and these carcinogens have been revealed by the database of EHP, NIEHS, the NIH Roadmap Epigenomics Mapping Consortium, the National Center for Biotechnology Information (NCBI), and U.S. National Library of Medicine (NLM) database. Our previous study reported that the regulation of epigenetic bivalent histone modifications at transcript starts sequences (TSS) of target genes for heterogenic adult stem cells (CSCs)reprogramming and our current bioinformatics analysis revealed that TP53 is epigenetically reprogrammed with close interaction of thirteen genes by these carcinogens. Therefore, it is important to find the actual epigenetic mechanism both genomic and epigenomic transcriptional regulation and carcinogenic effects of these carcinogens on bone marrow stem cells, and our current study revealed this important relationship. Citation Format: Benedrica Solomon, Richgena Sturrup, Jada Dixon, Kiyona Jackson, Biancaliz Cruz, Charlissia Ferguson, Taylor Walker, Ralph Elisson, Nathan Harris, Jesennia Bonilla, Rose Mary Stiffin, Marilyn Sherman, Alessandra Manzon, Swarnava Das, Madhumita Das, Jayanta Kumar Das. Carcinogenic effects of metal and metalloids through epigenetic reprogramming of human bone marrow stem cells: The bioinformatics database analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5830.

Cannabinoid CB2 receptors are upregulated via bivalent histone modifications and control primary afferent input to the spinal cord in neuropathic pain

Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root–evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation–qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord.

Abstract 2104: 3D spheroid: A rapid drug screening model for epigenetic clinical targets against heterogenic cancer stem cells

Abstract Recent scientific studies showed that transcript start sequences (TSS) are regulated by epigenetic bivalent histone modifications (H3K4me3 and H3K27me3) of target genes for heterogenic cancer stem cells (CSCs) reprogramming. Most of these studies are based on monolayer or 2D culture grew cancer cells. Studies of 3D spheroid models showed a rapid screening of anticancer drugs against CSCs. The 3D-spheroid models closely mimic in vivo tumor microenvironment (TME). Hence, 3D spheroid in vitro model development is a standout amongst other portrayed cancer models regarding 3D cell culture based on its similitude to physiological tumor tissues. Our previous studies of 3D spheroid models showed the effectiveness of small molecules targeted to the bivalent histone modifications against the CSCs in pancreatic cancer. The current study is aimed to explore the epigenetic clinical targets of bivalent histone modifications with the expression of heterogenic CSCs (CD133, CD15 CD44, CD49f, CD24, ALDH1A3, CXCR4) of 3D spheroids in glioblastomas, oral, and colorectal cancers (CRC). The Cancer Stem Cells Therapeutic Target Database Version 2.0 (CSCTTv2.0) reported the highest expression of CSCs in glioblastomas, oral and CRCs that showed resistance to anticancer drugs (such as Bevacizumab, 5-FU, cisplatin, cetuximab, oxaliplatin). The biological informatics methods (String; Cytoscape, BioGrid, Reactome, MatrixDB, Pubchem database) were used in our current study. The NCBI Gene Expression Omnibus (GEO) database showed only 4 studies of 3D spheroids of CSCs which revealed drug-resistant heterogenic CSCs, in 7 samples of glioblastoma spheroids and 4 samples of CSCs in MCF-7 breast cancer spheroids from 6 samples; drug-resistant CSCs of MDA-MB 231 spheroids from 6 samples; CSCs of colorectal cancer (CRC) spheroids from 6 samples, but the clinical the target of bivalent histone modifications with heterogenic CSCs in the glioblastomas, oral and CRCs have not been explored earlier using 3D spheroids for rapid drug screening. The protein-protein interaction (PPI) network of the current study showed a relationship of closely related 17 significant genes. Among these 17 genes, a total of 6 CSCs was closely associated with epigenetic bivalent histone modifications through KDM7A, PHF1and EZH2. We also found from Cytoscape analysis of Reactome network that UniProtKB - P61073 (CXCR4_HUMAN) has significant interaction with other target network genes from 106 network data and it has been mentioned that Plerixafor, an antagonist inhibits CXCR4 activity. Therefore, it is important in designing any small molecules or drugs against heterogenic CSCs of glioblastomas, oral, and CRC with epigenetic target of histone bivalent modifications using a 3D spheroid model which could be rapid screening model for anticancer drug discovery and our current study highlighted this important relationship. Citation Format: Madhumita Das, Maylet Cortes Santana, Stephanie Barraque, Jhoselyn Cardenas, Johnny Amaya Galindo, Misael Cortes, Jose Ramos, Alberto Sanchez Prado, Melissa Tejeda Castillo, Victor Villar, Claudy Vincent, Elizabeth Justo, Mariano Mendez, Indira Mera, Juan Pachon, Kamila Perez, Adara Marin, Nabendu Murmu, Maharaj Biswas, Marco Ruiz, Jayanta Kumar Das. 3D spheroid: A rapid drug screening model for epigenetic clinical targets against heterogenic cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2104.

INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells.

The INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. In the naïve state, INO80 pre-marked gene promoters that would adopt bivalent histone modifications by H3K4me3 and H3K27me3 upon transition into the primed state. In the primed state, in contrast to its known role in H2A.Z exchange, INO80 promoted H2A.Z occupancy at these bivalent promoters and facilitated H3K27me3 installation and maintenance as well as downstream gene repression. Together, our results identified an unexpected function of INO80 in H2A.Z deposition and gene regulation. We showed that INO80-dependent H2A.Z occupancy is a critical licensing step for the bivalent domains, and thereby uncovered an epigenetic mechanism by which chromatin remodeling, histone variant deposition and histone modification coordinately control cell fate.

Conversion of T Follicular Helper Cells to T Follicular Regulatory Cells by Interleukin-2 Through Transcriptional Regulation in Systemic Lupus Erythematosus.

This study was undertaken to identify characteristics of follicular regulatory T (Tfr) cells and elucidate the mechanisms by which follicular helper T (Tfh) cells convert to Tfr cells. We probed the phenotype of T helper cells in patients with systemic lupus erythematosus (SLE) and underlying transcriptional regulation using cytokine-induced STAT family factors. Peripheral blood mononuclear cells from 41 patients with SLE and 26 healthy donors were used to sort out the memory Tfh cell subset, and Tfh cells were cultured under various conditions. The phenotype of T helper cells and underlying mechanisms of transcriptional regulation were probed using flow cytometry and quantitative polymerase chain reaction analyses. These analyses evaluated the expression of characteristic markers and phosphorylation of STATs. Chromatin immunoprecipitation was used to evaluate histone modifications. In patients with SLE, the proportion of CD4+CXCR5+FoxP3-PD-1high Tfh cells was increased (P < 0.01), whereas the proportion of CD4+CXCR5+CD45RA-FoxP3high activated Tfr cells was decreased (P < 0.05). Serum interleukin-2 (IL-2) levels were also reduced in patients with SLE. IL-2 induced conversion of memory Tfh cells to functional Tfr cells, which was characterized by CXCR5+Bcl-6+FoxP3high pSTAT3+pSTAT5+ cells. The loci of FOXP3 and BCL6 at STAT binding sites were marked by bivalent histone modifications. Following IL-2 stimulation, STAT3 and STAT5 selectively bound to FOXP3 and BCL6 gene loci accompanied by suppression of H3K27me3. Finally, IL-2 stimulation suppressed the generation of CD38+CD27high plasmablasts in Tfh and B cell coculture assays ex vivo. Impaired function of Tfr cellsmight beattributed to defective IL-2 production. ExogenousIL-2 restores the function of Tfr cells through the conversion of Tfh cells to Tfr cells in patients with SLE. Thus, restoring balance between Tfh and Tfr cells may provide new therapeutic approaches in SLE.

Abstract PO-003: Bivalent histone modifications: Clinical targets against pancreatic cancer stem cell heterogeneity

Abstract Bivalent histone modifications, the pre-transcriptional regulators were known as the chief orchestra underlining the mechanism of development for pancreatic cancer stem cells (PANCSCs) recently. The histone bivalency methylations are related to the transcriptional activation as well as repression of the prominent histone methylations (H3K4me3 and H3K27me3) at the transcript start sequence (TSS). These epigenetic changes are recognized to initiate heterogenic cancer stem cells (CSCs) reprogramming. Recent studies showed that the heterogenic CSCs are more resistant to anti-tumor drug therapy. Our previous study showed the relationship between bivalent histone modifications with the expression of pancreatic CSCs(CD44, CD24, CD133, Ep-CAM, ALDH1A3). Our current study is focused on the importance of designing anti-cancer drugs or epigenetic small molecules against bivalent histone modifications (to inhibit the upregulation of H3K4me3 or down-regulation of H3K27me3) related the expression of heterogenic pancreatic CSCs (CD44, CD24, CD133, Ep-CAM, ALDH1A3). The Cancer Stem Cells Therapeutic Target Database Version 2.0 (CSCTTv2.0) reported the high CD133 as PAN CSCs and high CD133, CD44 showed resistance to anticancer drugs (such as docetaxel, 5-FU, irinotecan, oxaliplatin) in many cancers. The Gemcitabine, a nucleoside analog, and Metformin are only drugs used for pancreatic cancer treatment and showed resistance against heterogenic PANCSCs. The biological informatics analyses (Cytoscape database, HPIDb, Spike, BioGrid, Reactome, MBInfo) were deployed in our study to find the effect of bivalent histone modifications on the TSS of the target genes in heterogenic PANCSCs and we found the effect at TSS of target genes CD44, and EZH2/ENX1. We also found a novel small molecule KY-02111 has an effect to reduce the expression of heterogenic PANCSCs at tumorigenic in vitro 3D spheroids model of PANC-1ORGR (Gemcitabine resistant) cells. Therefore, our preliminary study may give an important clue to find further the role of KY-02111 against the heterogenic pancreatic CSCs by inhibiting the upregulation of H3K4me3 or down-regulation of H3K27me3 as a clinical target for pancreatic cancer therapy. Citation Format: Madhumita Das, Mayur Doke, Edwin Gonzalez, Daima Diaz, Bryan Pineda, Jonathan Celli, Marco Ruiz, Jayanta K. Das. Bivalent histone modifications: Clinical targets against pancreatic cancer stem cell heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-003.

Regulation and Roles of the Nucleolus in Embryonic Stem Cells: From Ribosome Biogenesis to Genome Organization.

The nucleolus is the largest compartment of the eukaryotic cell's nucleus. It acts as a ribosome factory, thereby sustaining the translation machinery. The nucleolus is also the subnuclear compartment with the highest transcriptional activity in the cell, where hundreds of ribosomal RNA (rRNA) genes transcribe the overwhelming majority of RNAs. The structure and composition of the nucleolus change according to the developmental state. For instance, in embryonic stem cells (ESCs), rRNA genes display a hyperactive transcriptional state and open chromatin structure compared with differentiated cells. Increasing evidence indicates that the role of the nucleolus and rRNA genes might go beyond the control of ribosome biogenesis. One such role is linked to the genome architecture, since repressive domains are often located close to the nucleolus. This review highlights recent findings describing how the nucleolus is regulated in ESCs and its role in regulating ribosome biogenesis and genome organization for the maintenance of stem cell identity.

Abstract 2431: Identification of the underlining relationship of bivalent histone modifications with pancreatic cancer stem cells by bioinformatic analysis

Abstract Epigenetic alterations are known to involve in cancer stem cells (CSCs) reprogramming. CSCs have been reported to be more resistant to anti-tumor treatments than the non-stem cancer cells, suggesting that surviving CSCs could be responsible for tumor relapse after therapy. The most relevant epigenetic alteration, histone modification has been shown in recent scientific studies to contribute to the regulation of cancer stem cell features in tumor metastasis, progression and response to chemotherapy. The pre-transcriptional regulator, bivalent histone modifications were reported to be involved in the development of CSCs recently. The synchronous presence of functionally opposite histone marks at transcript start sequence (TSS) is known as “Bivalency”. The H3K4me3 as well as H3K27me3, the prominent histone methylations of bivalency, are concerned in transcriptional activation and transcriptional repression respectively. The balance between H3K4me3 and H3K27me3 results in diverse chromatin architectures, ensuing in different transcription states of downstream genes: “poised”, “activated” or “repressed”. The current study is aimed to explore the relationship of bivalent histone modifications, (upregulation or expression of H3K4me3, and down-regulation or repression H3K27me3) the epigenetic regulations with the expression of pancreatic cancer stem cells (CD44, CD24, CD133, Ep-CAM, ALDH1A3) in pancreatic cancer. Protein expression profiles were analyzed using biological informatics methods (string database; Cytoscape database, BioGrid, Reactome, MatrixDB, NCBI Gene and NCBI epigenomics metadata analyses) to identify the relationship of bivalent histone modifications with expressed genes of pancreatic cancer stem cells (CD44, CD24, CD133, Ep-CAM, ALDH1A3). NCBI Biosystems database showed that 197 genes are involved in the Integrated Pancreatic Cancer Pathway model using KEGG database, but the relationship of bivalent histone modifications with the expression of pancreatic cancer stem cells (CD44, CD24, CD133, Ep-CAM, ALDH1A3) in pancreatic cancer has not been showed earlier. Our analysis showed the protein-protein interaction (PPI) network was constructed for such a relationship of closely related 45 significant genes. Among these 45 genes, a total of 7 pancreatic cancer stem cell markers (CSCs) was closely associated with upregulation or expression of H3K4me3, and down-regulation or repression H3K27me3 through EZH2 and PHF19 with a high degree of interaction in the PPI network. We are also able to analyze the role of H3K27ac in regulating target genes in 18 samples of pancreatic cancer cell lines using the ChIP-sequences data set of study SRA 109996 from NCBI NLM Sequence Read Archive from Run Browser Accession number ERR2708462. Therefore, this study identified the significant relationship of bivalent histone modifications associated with pancreatic CSCs which may be the novel biomarkers and target candidates in designing drugs against pancreatic cancer. This work was part of the honors option project (HOP) of Natalia Ortega mentored by Dr. Jayanta K. Das, at MDC north campus. Citation Format: Natalia Ortega, Madhumita Das, Marco A. Ruiz, Jairo Ramos, Jayanta Kumar Das. Identification of the underlining relationship of bivalent histone modifications with pancreatic cancer stem cells by bioinformatic analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2431.

Analysis of Local Chromatin States Reveals Gene Transcription Potential during Mouse Neural Progenitor Cell Differentiation

Chromatin dynamics play a critical role in cell fate determination and maintenance by regulating the expression of genes essential for development and differentiation. In mouse embryonic stem cells (mESCs), maintenance of pluripotency coincides with a poised chromatin state containing active and repressive histone modifications. However, the structural features of poised chromatin are largely uncharacterized. By adopting mild time-course MNase-seq with computational analysis, the low-compact chromatin in mESCs is featured in two groups: one in more open regions, corresponding to an active state, and the other enriched with bivalent histone modifications, considered the poised state. A parameter called the chromatin opening potential index (COPI) is also devised to quantify the transcription potential based on the dynamic changes of MNase-seq signals at promoter regions. Use of COPI provides effective prediction of gene activation potential and, more importantly, reveals a few developmental factors essential for mouse neural progenitor cell (NPC) differentiation.

ADNP Controls Gene Expression Through Local Chromatin Architecture by Association With BRG1 and CHD4.

ADNP (Activity Dependent Neuroprotective Protein) is proposed as a neuroprotective protein whose aberrant expression has been frequently linked to rare neural developmental disorders and cancers, including the recently described neurodevelopmental Helsmoortel-Van der Aa syndrome. Recent studies have suggested that ADNP functions as an important chromatin regulator. However, how ADNP-regulated chromatin mechanisms control gene expression and stem cell fate commitment remains unclear. Here we show that ADNP interacts with two chromatin remodelers, BRG1 and CHD4. ADNP is required for proper establishment of chromatin accessibility, nucleosome configuration, and bivalent histone modifications of developmental genes. Loss of ADNP leads to enhancer over-activation and increased ratio of H3K4me3/H3K27me3 at key primitive endoderm (PrE) gene promoters, resulting in prominent up-regulation of these genes and priming ES cell differentiation toward endodermal cell types. Thus, our work revealed a key role of ADNP in the establishment of local chromatin landscape and structure of developmental genes by association with BRG1 and CHD4. These findings provide further insights into the role of ADNP in the pathology of the Helsmoortel-Van der Aa syndrome.

Long noncoding RNA 91H overexpression contributes to the growth and metastasis of HCC by epigenetically positively regulating IGF2 expression.

Long noncoding RNA 91H is transcribed from the H19/IGF2 locus and contributes to the development of breast and oesophagus cancers by regulating the expression of IGF2, but the regulation mechanism remains poorly characterized. Here, we explored the role of 91H in hepatocellular carcinoma (HCC) and the mechanism of IGF2 expression regulation by 91H. Firstly, the expression of 91H was analysed in HCC by quantitative RT-PCR, the association of 91H with survival was evaluated by the Kaplan-Meier method and the effect of 91H on the growth and invasion of HCC was investigated by the in vitroand in vivo studies. Then, the association of 91H with the expression of IGF2 was evaluated in HCC tissues, and the effect of 91H on the expression of IGF2 was investigated by 91H knockdown. Finally, the binding of RBBP5 to 91H and the binding of RBBP5, activating H3K4me3 mark and repressive H3K27me3 mark to the P3 and P4 promoters of IGF2 gene were studied by RIP and ChIP respectively. The overexpression of 91H was found in HCC and in association with the growth, metastasis and shorter survival time of HCC. The knockdown of 91H down-regulated the IGF2 expression in HCC, and the mechanism was correlated with the decreased enrichment of RBBP5 and H3K4me3 and increased enrichment of H3K27me3 at the bivalent P3 and P4 promoters. The overexpression of 91H promotes tumour growth and metastasis, and is associated with a poor prognosis of HCC at least partially by positively regulating the expression of IGF2 through bivalent histone modification changes characterized by H3K4me3 and H3K27me3 at the P3 and P4 promoters.

EZH2 Supports Osteoclast Differentiation and Bone Resorption Via Epigenetic and Cytoplasmic Targets.

Key osteoclast (OCL) regulatory gene promoters in bone marrow-derived monocytes harbor bivalent histone modifications that combine activating Histone 3 lysine 4 tri-methyl (H3K4me3) and repressive H3K27me3 marks, which upon RANKL stimulation resolve into repressive or activating architecture. Enhancer of zeste homologue 2 (EZH2) is the histone methyltransferase component of the polycomb repressive complex 2, which catalyzes H3K27me3 modifications. Immunofluorescence microscopy reveals that EZH2 localization during murine osteoclastogenesis is dynamically regulated. Using EZH2 knockdown and small molecule EZH2 inhibitor GSK126, we show that EZH2 plays a critical epigenetic role in OCL precursors (OCLp) during the first 24 hours of RANKL activation. RANKL triggers EZH2 translocation into the nucleus where it represses OCL-negative regulators MafB, Irf8, and Arg1. Consistent with its cytoplasmic localization in OCLp, EZH2 methyltransferase activity is required during early RANKL signaling for phosphorylation of AKT, resulting in downstream activation of the mTOR complex, which is essential for induction of OCL differentiation. Inhibition of RANKL-induced pmTOR-pS6RP signaling by GSK126 altered the translation ratio of the C/EBPβ-LAP and C/EBPβ-LIP isoforms and reduced nuclear translocation of the inhibitory C/EBPβ-LIP, which is necessary for transcriptional repression of the OCL negative-regulatory transcription factor MafB. EZH2 in multinucleated OCL is primarily cytoplasmic and mature OCL cultured on bone segments in the presence of GSK126 exhibit defective cytoskeletal architecture and reduced resorptive activity. Here we present new evidence that EZH2 plays epigenetic and cytoplasmic roles during OCL differentiation by suppressing MafB transcription and regulating early phases of PI3K-AKT-mTOR-mediated RANKL signaling, respectively. Consistent with its cytoplasmic localization, EZH2 is required for cytoskeletal dynamics during resorption by mature OCL. Thus, EZH2 exhibits complex roles in supporting osteoclast differentiation and function. © 2019 American Society for Bone and Mineral Research.

Cold stress induces enhanced chromatin accessibility and bivalent histone modifications H3K4me3 and H3K27me3 of active genes in potato

BackgroundCold stress can greatly affect plant growth and development. Plants have developed special systems to respond to and tolerate cold stress. While plant scientists have discovered numerous genes involved in responses to cold stress, few studies have been dedicated to investigation of genome-wide chromatin dynamics induced by cold or other abiotic stresses.ResultsGenomic regions containing active cis-regulatory DNA elements can be identified as DNase I hypersensitive sites (DHSs). We develop high-resolution DHS maps in potato (Solanum tuberosum) using chromatin isolated from tubers stored under room (22 °C) and cold (4 °C) conditions. We find that cold stress induces a large number of DHSs enriched in genic regions which are frequently associated with differential gene expression in response to temperature variation. Surprisingly, active genes show enhanced chromatin accessibility upon cold stress. A large number of active genes in cold-stored tubers are associated with the bivalent H3K4me3-H3K27me3 mark in gene body regions. Interestingly, upregulated genes associated with the bivalent mark are involved in stress response, whereas downregulated genes with the bivalent mark are involved in developmental processes. In addition, we observe that the bivalent mark-associated genes are more accessible than others upon cold stress.ConclusionsCollectively, our results suggest that cold stress induces enhanced chromatin accessibility and bivalent histone modifications of active genes. We hypothesize that in cold-stored tubers, the bivalent H3K4me3-H3K27me3 mark represents a distinct chromatin environment with greater accessibility, which may facilitate the access of regulatory proteins required for gene upregulation or downregulation in response to cold stress.

Genome-Wide Mapping of Bivalent Histone Modifications in Hepatic Stem/Progenitor Cells.

The “bivalent domain,” a distinctive histone modification signature, is characterized by repressive trimethylation of histone H3 at lysine 27 (H3K27me3) and active trimethylation of histone H3 at lysine 4 (H3K4me3) marks. Maintenance and dynamic resolution of these histone marks play important roles in regulating differentiation processes in various stem cell systems. However, little is known regarding their roles in hepatic stem/progenitor cells. In the present study, we conducted the chromatin immunoprecipitation (ChIP) assay followed by high-throughput DNA sequencing (ChIP-seq) analyses in purified delta-like 1 protein (Dlk+) hepatic stem/progenitor cells and successfully identified 562 genes exhibiting bivalent domains within 2 kb of the transcription start site. Gene ontology analysis revealed that these genes were enriched in developmental functions and differentiation processes. Microarray analyses indicated that many of these genes exhibited derepression after differentiation toward hepatocyte and cholangiocyte lineages. Among these, 72 genes, including Cdkn2a and Sox4, were significantly upregulated after differentiation toward hepatocyte or cholangiocyte lineages. Knockdown of Sox4 in Dlk+ cells suppressed colony propagation and resulted in increased numbers of albumin+/cytokeratin 7+ progenitor cells in colonies. These findings implicate that derepression of Sox4 expression is required to induce normal differentiation processes. In conclusion, combined ChIP-seq and microarray analyses successfully identified bivalent genes. Functional analyses of these genes will help elucidate the epigenetic machinery underlying the terminal differentiation of hepatic stem/progenitor cells.