Chromatin-modifying Activities Research Articles

An Emerging Role for Enhancer RNAs in Brain Disorders.

Noncoding DNA undergoes widespread context-dependent transcription to produce noncoding RNAs. In recent decades, tremendous advances in genomics and transcriptomics have revealed important regulatory roles for noncoding DNA elements and the RNAs that they produce. Enhancers are one such element that are well-established drivers of gene expression changes in response to a variety of factors such as external stimuli, cellular responses, developmental cues, and disease states. They are known to act at long distances, interact with multiple target gene loci simultaneously, synergize with other enhancers, and associate with dynamic chromatin architectures to form a complex regulatory network. Recent advances in enhancer biology have revealed that upon activation, enhancers transcribe long noncoding RNAs, known as enhancer RNAs (eRNAs), that have been shown to play important roles in enhancer-mediated gene regulation and chromatin-modifying activities. In the brain, enhancer dysregulation and eRNA transcription has been reported in numerous disorders from acute injuries to chronic neurodegeneration. Because this is an emerging area, a comprehensive understanding of eRNA function has not yet been achieved in brain disorders; however, the findings to date have illuminated a role for eRNAs in activity-driven gene expression and phenotypic outcomes. In this review, we highlight the breadth of the current literature on eRNA biology in brain health and disease and discuss the challenges as well as focus areas and strategies for future in-depth research on eRNAs in brain health and disease.

Orai-mediated calcium entry determines activity of central dopaminergic neurons by regulation of gene expression.

Maturation and fine-tuning of neural circuits frequently require neuromodulatory signals that set the excitability threshold, neuronal connectivity, and synaptic strength. Here, we present a mechanistic study of how neuromodulator-stimulated intracellular Ca2+ signals, through the store-operated Ca2+ channel Orai, regulate intrinsic neuronal properties by control of developmental gene expression in flight-promoting central dopaminergic neurons (fpDANs). The fpDANs receive cholinergic inputs for release of dopamine at a central brain tripartite synapse that sustains flight (Sharma and Hasan, 2020). Cholinergic inputs act on the muscarinic acetylcholine receptor to stimulate intracellular Ca2+ release through the endoplasmic reticulum (ER) localised inositol 1,4,5-trisphosphate receptor followed by ER-store depletion and Orai-mediated store-operated Ca2+ entry (SOCE). Analysis of gene expression in fpDANs followed by genetic, cellular, and molecular studies identified Orai-mediated Ca2+ entry as a key regulator of excitability in fpDANs during circuit maturation. SOCE activates the transcription factor trithorax-like (Trl), which in turn drives expression of a set of genes, including Set2, that encodes a histone 3 lysine 36 methyltransferase (H3K36me3). Set2 function establishes a positive feedback loop, essential for receiving neuromodulatory cholinergic inputs and sustaining SOCE. Chromatin-modifying activity of Set2 changes the epigenetic status of fpDANs and drives expression of key ion channel and signalling genes that determine fpDAN activity. Loss of activity reduces the axonal arborisation of fpDANs within the MB lobe and prevents dopamine release required for the maintenance of long flight.

Orai-mediated calcium entry determines activity of central dopaminergic neurons by regulation of gene expression

Maturation and fine-tuning of neural circuits frequently require neuromodulatory signals that set the excitability threshold, neuronal connectivity, and synaptic strength. Here, we present a mechanistic study of how neuromodulator-stimulated intracellular Ca2+ signals, through the store-operated Ca2+ channel Orai, regulate intrinsic neuronal properties by control of developmental gene expression in flight-promoting central dopaminergic neurons (fpDANs). The fpDANs receive cholinergic inputs for release of dopamine at a central brain tripartite synapse that sustains flight (Sharma and Hasan, 2020). Cholinergic inputs act on the muscarinic acetylcholine receptor to stimulate intracellular Ca2+ release through the endoplasmic reticulum (ER) localised inositol 1,4,5-trisphosphate receptor followed by ER-store depletion and Orai-mediated store-operated Ca2+ entry (SOCE). Analysis of gene expression in fpDANs followed by genetic, cellular, and molecular studies identified Orai-mediated Ca2+ entry as a key regulator of excitability in fpDANs during circuit maturation. SOCE activates the transcription factor trithorax-like (Trl), which in turn drives expression of a set of genes, including Set2, that encodes a histone 3 lysine 36 methyltransferase (H3K36me3). Set2 function establishes a positive feedback loop, essential for receiving neuromodulatory cholinergic inputs and sustaining SOCE. Chromatin-modifying activity of Set2 changes the epigenetic status of fpDANs and drives expression of key ion channel and signalling genes that determine fpDAN activity. Loss of activity reduces the axonal arborisation of fpDANs within the MB lobe and prevents dopamine release required for the maintenance of long flight.

Approaches to Study Native Chromatin-Modifying Complex Activities and Functions.

The modification of histones—the structural components of chromatin—is a central topic in research efforts to understand the mechanisms regulating genome expression and stability. These modifications frequently occur through associations with multisubunit complexes, which contain active enzymes and additional components that orient their specificity and read the histone modifications that comprise epigenetic signatures. To understand the functions of these modifications it is critical to study the enzymes and substrates involved in their native contexts. Here, we describe experimental approaches to purify native chromatin modifiers complexes from mammalian cells and to produce recombinant nucleosomes that are used as substrates to determine the activity of the complex. In addition, we present a novel approach, similar to the yeast anchor-away system, to study the functions of essential chromatin modifiers by quickly inducing their depletion from the nucleus. The step-by-step protocols included will help standardize these approaches in the research community, enabling convincing conclusions about the specificities and functions of these crucial regulators of the eukaryotic genome.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae.

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Conserved protein Pir2ARS2 mediates gene repression through cryptic introns in lncRNAs

Long non-coding RNAs (lncRNAs) are components of epigenetic control mechanisms that ensure appropriate and timely gene expression. The functions of lncRNAs are often mediated through associated gene regulatory activities, but how lncRNAs are distinguished from other RNAs and recruit effector complexes is unclear. Here, we utilize the fission yeast Schizosaccharomyces pombe to investigate how lncRNAs engage silencing activities to regulate gene expression in cis. We find that invasion of lncRNA transcription into the downstream gene body incorporates a cryptic intron required for repression of that gene. Our analyses show that lncRNAs containing cryptic introns are targeted by the conserved Pir2ARS2 protein in association with splicing factors, which recruit RNA processing and chromatin-modifying activities involved in gene silencing. Pir2 and splicing machinery are broadly required for gene repression. Our finding that human ARS2 also interacts with splicing factors suggests a conserved mechanism mediates gene repression through cryptic introns within lncRNAs.

E2F1 acetylation directs p300/CBP-mediated histone acetylation at DNA double-strand breaks to facilitate repair

E2F1 and retinoblastoma (RB) tumor-suppressor protein not only regulate the periodic expression of genes important for cell proliferation, but also localize to DNA double-strand breaks (DSBs) to promote repair. E2F1 is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here we demonstrate that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases and that this interaction is required for the recruitment of p300 and CBP to DSBs and the induction of histone acetylation at sites of damage. A knock-in mutation that blocks E2F1 acetylation abolishes the recruitment of p300 and CBP to DSBs and also the accumulation of other chromatin modifying activities and repair factors, including Tip60, BRG1 and NBS1, and renders mice hypersensitive to ionizing radiation (IR). These findings reveal an important role for E2F1 acetylation in orchestrating the remodeling of chromatin structure at DSBs to facilitate repair.

Abstract 3026: Rigosertib (RIG) modulates MAPK and hematopoiesis signaling and synergizes Azacitidine (AZA) altering viral mimicry pathway in myelodysplastic syndrome (MDS)

Abstract MDS is a heterogeneous stem cell disorder characterized by hyperproliferative bone marrow (BM) and peripheral blood cytopenias involving one or more lineages. AZA, a hypomethylating agent (HMA) is considered 1st line therapy for higher-risk disease. About 50% of MDS patients (pts) respond to AZA, with a median response of 14-24 month (Silverman Cancer Med 9th ed). For pts responding to AZA, most either relapse or progress with worsening BM failure and have a median survival of 4-6 months. Both primary and secondary resistance remains a significant challenge and results in poor survival. We previously reported that RIG, a ras-mimetic identified as a novel anticancer drug, inhibits cell cycle progression, induces apoptosis and acts as a HDAC inhibitor with chromatin modifying activity. In a Phase I/II study the combination of RIG+AZA produced an 85% overall response rate in pts who were HMA naive and 62% in HMA failures (Navada EHA 2017). The ability to reverse the resistance phenotype is a novel observation with clinical implications. In this study, we investigated the effect of AZA and RIG alone or in sequential combination (RIG/AZA; AZA/RIG) (SC) on MDS-L cell line to identify the mechanism of action of the drugs. QPCR array was used to identify the differential gene expression profile. Maximum gene alteration was observed (±2 fold change) on treatment with RIG alone, AZA/RIG and RIG/AZA with differential expression of 20 (14up+6dn), 22 (16up+6dn) and 21 (16up+5dn) genes, respectively. However, AZA alone showed minimal effect with dysregulation, the expression of only 2 genes (1up+1dn). Further, gene expression with 1.5 fold change was used for biological and functional analysis. Interestingly, hematopoiesis cell lineage and JAK-STAT signaling were the pathways most affected in RIG alone, and both the SCs. MAPK signaling is impacted in cells treated with RIG alone and AZA/RIG. This suggests that RIG plays a crucial role in modulating these pathways. However, cytokine-cytokine receptor interactions were impacted only in cells treated with both SCs. RIG-I like receptor signaling was specifically observed only in RIG/AZA. This pathway along with Toll-like receptor (TLR) signaling in RIG/AZA suggests the involvement of viral mimicry. Moreover, TLR signaling dysregulation with AZA also suggests association with viral mimicry and impacts the cell cycle pathway. These results indicate that RIG either alone or in combination acts via the MAPK signaling pathway and impacts hematopoietic signaling. In contrast, AZA had no impact on these signaling pathways. This suggests that RIG in combination with AZA affects important signaling pathways impacting hematopoiesis and may explain the synergy seen in MDS pts. Further study is needed to understand the mechanism of resistance to AZA in MDS patients and identify the potential target for reversal of drug resistance. Citation Format: Stella M. Melana, Richa Rai, Shyamala C. Navada, Rosalie Odchimar-Reissig, Erin P. Demakos, E. Premkumar Reddy, Lewis R. Silverman. Rigosertib (RIG) modulates MAPK and hematopoiesis signaling and synergizes Azacitidine (AZA) altering viral mimicry pathway in myelodysplastic syndrome (MDS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3026.

PS1333 OVEREXPRESSION OF PROTEIN TYROSINE PHOSPHATASE NON-RECEPTOR TYPE1 CAUSES STAT5 DEPHOSPHORYLATION, RESULTING IN SUPPRESSION OF CELL GROWTH SIGNAL IN K562 HUMAN LEUKEMIA CELLS

Background: The Myelodysplastic Syndrome (MDS) is a disorder derived from an aberrant pluripotent stem cell, characterized clinically by hyperproliferative bone marrow and peripheral blood cytopenias involving one or more lineages. Azacitidine (AZA), a hypomethylating agent (HMA) is consideredfirst line therapy for patients (pts) with higher-risk disease. AZA is associated with a response rate of 50% and is associated with an increase in overall survival of pts with higher-risk MDS with a median survival of 14–24 months (Silverman et al. JCO 2002; Fenaux et al. Lancet Oncol 2009). Almost all pts treated with AZA develop resistance. Those pts who do not respond initially (primary resistance), and, almost all pts who do respond to AZA relapse (secondary resistance) and develop either bone marrow failure or transform to acute myeloid leukemia. Median survival after HMA failureis only 4–6 months (Prebet et al., 2011). Both primary and secondary resistance remain a significant challenge and result in poor survival. Rigosertib (RIGO) is a Ras-mimetic that interferes with Ras/Raf signaling (Athuluri-Divakar et al, 2016) and also acts as a histone deacetylase inhibitor with chromatin modifying activity (Chaurasia ASCO 2016). The combination of RIGO/AZA had been shown to have a synergistic effect in a sequence dependent manner in vitro(Skiddan et al 2006). A phase I/II study demonstrated that the combination RIGO/AZA can reverse the clinical resistance phenotype in pts failing an HMA with an overall response rate of 90% in HMA naïve pts and 54% in pts failing an HMA (Navada et al, ASH 2018). The ability to reverse the clinical resistance phenotype is a novel observation with clinical implications. Aims: To investigate the effect of AZA and RIGO alone or in sequential combination (RIGO/AZA; AZA/RIGO) (SC) on the MDS-L cell line to identify the mechanism of action of the drugs. Methods: QPCR array was used to identify the differential gene expression profile. Results: Maximum gene alteration was observed (±2 fold change) following treatment with RIGO alone, AZA/RIGO and RIGO/AZA with differential expression of 20 (14up+6dn), 22 (16up+6dn) and 21 (16up+5dn) genes, respectively. AZA alone showed minimal effect on gene expression with altered expression of only 2 genes (1up+1dn). Gene expression with 1.5 fold change was used for the biological and functional analysis. Hematopoietic cell lineage and JAK-STAT signaling pathways were most affected by RIGO alone, and the SCs. MAPK signaling is impacted in cells treated with RIGO alone and by AZA/RIGO suggesting that RIGO plays a crucial role in modulating these pathways. Cytokine-cytokine receptor interactions were impacted only in cells treated by both SCs. The Retinoic acid inducible gene-1 (RIG-1)like receptor signaling was upregulated only by RIGO/AZA sequence. Activation of this pathway along with upregulation of Toll-like receptor (TLR) signaling by RIGO/AZA suggests the involvement of viral mimicry. Moreover, TLR signaling dysregulation with AZA also suggests an association with viral mimicry and AZA also impacts the cell cycle pathway. Summary/Conclusion: These results indicate that RIGO either alone or in combination with AZA may act via the MAPK signaling pathway to impact hematopoietic signaling. RIGO in combination with AZA activates the anti-viral defense pathway and affects TLR and RIG-1 signaling which may explain the synergy seen in MDS pts. Further in-depth study is needed to understand the mechanism of action of resistance to AZA in MDS pts and identify potential targets for the reversal of drug resistance.

PS1332 RIGOSERTIB MODULATES HEMATOPOIESIS GENE SIGNALING PATHWAYS ALONE OR IN SEQUENCED COMBINATION WITH AZACTIDINE ON MDS CELLS IN VITRO

Background:The Myelodysplastic Syndrome (MDS) is a disorder derived from an aberrant pluripotent stem cell, characterized clinically by hyperproliferative bone marrow and peripheral blood cytopenias involving one or more lineages. Azacitidine (AZA), a hypomethylating agent (HMA) is consideredfirst line therapy for patients (pts) with higher‐risk disease. AZA is associated with a response rate of 50% and is associated with an increase in overall survival of pts with higher‐risk MDS with a median survival of 14–24 months (Silverman et al. JCO 2002; Fenaux et al. Lancet Oncol 2009). Almost all pts treated with AZA develop resistance. Those pts who do not respond initially (primary resistance), and, almost all pts who do respond to AZA relapse (secondary resistance) and develop either bone marrow failure or transform to acute myeloid leukemia. Median survival after HMA failureis only 4–6 months (Prebet et al., 2011). Both primary and secondary resistance remain a significant challenge and result in poor survival.Rigosertib (RIGO) is a Ras‐mimetic that interferes with Ras/Raf signaling (Athuluri‐Divakar et al, 2016) and also acts as a histone deacetylase inhibitor with chromatin modifying activity (Chaurasia ASCO 2016). The combination of RIGO/AZA had been shown to have a synergistic effect in a sequence dependent manner in vitro(Skiddan et al 2006). A phase I/II study demonstrated that the combination RIGO/AZA can reverse the clinical resistance phenotype in pts failing an HMA with an overall response rate of 90% in HMA naïve pts and 54% in pts failing an HMA (Navada et al, ASH 2018). The ability to reverse the clinical resistance phenotype is a novel observation with clinical implications.Aims:To investigate the effect of AZA and RIGO alone or in sequential combination (RIGO/AZA; AZA/RIGO) (SC) on the MDS‐L cell line to identify the mechanism of action of the drugs.Methods:QPCR array was used to identify the differential gene expression profile.Results:Maximum gene alteration was observed (±2 fold change) following treatment with RIGO alone, AZA/RIGO and RIGO/AZA with differential expression of 20 (14up+6dn), 22 (16up+6dn) and 21 (16up+5dn) genes, respectively. AZA alone showed minimal effect on gene expression with altered expression of only 2 genes (1up+1dn). Gene expression with 1.5 fold change was used for the biological and functional analysis. Hematopoietic cell lineage and JAK‐STAT signaling pathways were most affected by RIGO alone, and the SCs. MAPK signaling is impacted in cells treated with RIGO alone and by AZA/RIGO suggesting that RIGO plays a crucial role in modulating these pathways. Cytokine‐cytokine receptor interactions were impacted only in cells treated by both SCs. The Retinoic acid inducible gene‐1 (RIG‐1)like receptor signaling was upregulated only by RIGO/AZA sequence. Activation of this pathway along with upregulation of Toll‐like receptor (TLR) signaling by RIGO/AZA suggests the involvement of viral mimicry. Moreover, TLR signaling dysregulation with AZA also suggests an association with viral mimicry and AZA also impacts the cell cycle pathway.Summary/Conclusion:These results indicate that RIGO either alone or in combination with AZA may act via the MAPK signaling pathway to impact hematopoietic signaling. RIGO in combination with AZA activates the anti‐viral defense pathway and affects TLR and RIG‐1 signaling which may explain the synergy seen in MDS pts. Further in‐depth study is needed to understand the mechanism of action of resistance to AZA in MDS pts and identify potential targets for the reversal of drug resistance.

Computational de-orphanization of the olive oil biophenol oleacein: Discovery of new metabolic and epigenetic targets

The health promoting effects of extra virgin olive oil (EVOO) relate to its unique repertoire of phenolic compounds. Here, we used a chemoinformatics approach to computationally identify endogenous ligands and assign putative biomolecular targets to oleacein, one of the most abundant secoiridoids in EVOO. Using a structure-based virtual profiling software tool and reference databases containing more than 9000 binding sites protein cavities, we identified 996 putative oleacein targets involving more than 700 proteins. We subsequently identified the high-level functions of oleacein in terms of biomolecular interactions, signaling pathways, and protein-protein interaction (PPI) networks. Delineation of the oleacein target landscape revealed that the most significant modules affected by oleacein were associated with metabolic processes (e.g., glucose and lipid metabolism) and chromatin-modifying enzymatic activities (i.e., histone post-translational modifications). We experimentally confirmed that, in a low-micromolar physiological range (<20 μmol/l), oleacein was capable of inhibiting the catalytic activities of predicted metabolic and epigenetic targets including nicotinamide N-methyltransferase, ATP-citrate lyase, lysine-specific demethylase 6A, and N-methyltransferase 4. Our computational de-orphanization of oleacein provides new mechanisms through which EVOO biophenols might operate as chemical prototypes capable of modulating the biologic machinery of healthy aging.

Sequential Treatment with Rigosertib Followed By Azacitidine Maximizes the Effects on the Interferon Signaling Pathway in Hematopoietic Cells in Myelodysplastic Syndrome (MDS)

Background: MDS is a clonal stem cell disorder characterized by abnormal maturation and differentiation of hematopoietic cells. Azacitidine (AZA), a hypomethylating agent (HMA), was approved by the FDA for patients (pts) with MDS and is the standard of care as 1st line therapy for higher- risk disease. About 50% of MDS pts respond to AZA, with a median duration of response of 14 - 24 month. For those pts responding to an HMA, most either relapse or progress with worsening bone marrow failure (BMF) and have a median survival of 4 to 6 months after treatment failure. Both primary and secondary resistance remains significant clinical problems and result in poor survival. A recent study reveals that AZA activates several immunomodulatory pathways leading to activation of the antiviral defense pathway and upregulation of interferon signaling (IFN) (Chiappinelli et al, 2017). INF has multiple effects on hematopoiesis and appears highly regulated. Interferon can stimulate hematopoietic stem cells (HSC) which may be countered through activation of p38 MAPK; inhibition of the latter may improve hematopoiesis. Short-term effects of IFN stimulate myeloid and erythroid hematopoiesis.Rigosertib (RIG) is a Ras-mimetic that blocks the activation of Ras effector proteins. It has been identified as a novel anti-cancer drug which inhibits cell cycle progression and induces apoptosis of cancer cells (Athuluri-Divakar et al, 2016). It activates apoptosis related pathways by ameliorating several signaling pathways like Akt, p38, MAPK/ JNK, STAT3, β-catenin, GSK3α/β and PLK1 that can be dysregulated in MDS pts (Xu et al, 2014). In vitro, the combination of RIG with AZA (Skidan et al, AACR 2006) showed synergistic effect in inducing cell death in a sequence dependent manner requiring RIG priming. We have reported that it acts as a histone deacetylase inhibitor with chromatin modifying activity (Chaurasia et al, ASCO 2016). In a Phase I/II study the combination of RIG and AZA (NCT 01926587) produced an ORR of 85% in pts who were HMA naïve and importantly 62% in HMA failures (Navada et al, EHA 2017). The ability to reverse the clinical resistance phenotype is a novel observation with important clinical implications and understanding the mechanisms is critical to develop ways of reversing resistance.Methods: We investigated the in vitro effects of RIG combined with AZA on two cell lines one resistant and one sensitive to AZA: BW90, an AZA resistant line and MDS-L, AZA sensitive and in specimens from 2 pts treated on a clinical trial of RIG and AZA in combination, to determine effects on hematopoietic cells and IN signaling.Results: QT-PCR studies demonstrated that individual treatment of MDS-L and BW90 with RIG or combined with AZA in sequential treatment (AZA/RIG or RIG/AZA) altered chromatin remodeler (KDM2a, SET1, JMJD3 and LRWD1) transcript levels in a cell line specific context. Exposure of cells from a bone marrow from a patient prior to treatment with RIG alone or RIG/AZA failed to induce expansion of CD34+ cells and yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSCs) (ANOVA, p=0.006). AZA alone yielded a 3.8 fold expansion of CD34+, with marked decrease in ALDH activity that was inversely proportional to the expansion of CD34+ cells. Expansion of CD34+ cells led to ≥2 fold increase in pluripotent genes (SOX2, OCT4, NANOG and ZIC3) expression levels. QT PCR studies of the effects of AZA or RIG alone or in combination on INF yielded differential effects on INFa and INFb expression as follows compared to control reported as percent fold increase for INFa - AZA 2x; RIG 0.75x, AZA/RIG 1.5x; RIG/AZA 2.5x; INFb AZA 1.3x; RIG 0.6x, AZA/RIG 1.2x; RIG/AZA 0.7x. There was a significant increase in INFa (p=<0.0001) but not INFb, with RIG priming compared to AZA alone or AZA/RIG.Conclusion: Treatments with RIG either alone or combined with AZA results in epigenetic reprogramming of pluripotency genes, and expansion of primitive HSPC. This may serve to regulate HSPC pluripotency and expression of a maturation program. A significant increase in expression of IFNa seen after exposure to RIG/AZA may lead to enhanced hematopoietic function and may explain the differences in the alternative sequence of AZA/RIG. Further studies are underway to define more specific effects on these pathways to explain the impact on the resistance phenotype. DisclosuresNavada:Onconova: Research Funding. Silverman:Mount Sinai School of Medicine: Employment; Onconova Therapeutics Inc.: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding; Johnson and Johnson: Research Funding; Bayer: Research Funding.

Abstract LB-342: Targeted gene control with bifunctional molecules to harness endogenous chromatin-modifying activity

Abstract Epigenetic pathways are commonly found disrupted in many human diseases including cancer, yet much remains unknown about the mechanisms by which dysregulation of chromatin regulatory pathways leads to disease pathology. To investigate and control these complex interactions in live cells, we developed a novel system that will unveil the underlying mechanisms of proteins responsible for modulating chromatin states using a gene-specific manipulation strategy. To explore how dynamic changes in acetylation levels impact chromatin structure we generated an endogenous reporter system at the murine Oct4 locus with a Gal4 DNA binding array upstream of an EGFP reporter allele. In our system, a FKBP protein fused to a Gal4 DNA-binding-domain serves as a protein anchor to the DNA binding array. Next, we synthesized novel cell-permeable, bifunctional molecules that include FK506 (which binds to FKBP) linked to a component that selectively binds to a histone tail chromatin-modifying enzyme. We characterized the effects of the Chemical Epigenetic Modifiers (CEM)s using flow cytometry, high-content microscopy and chromatin immunoprecipitation. We show that our lead repressive CEM can repress GFP expression by 50%. Next, we have made our system more modular by creating a deactivated Cas9 (dCas9)-FKBP fusion protein. The FKBP portion allows us to recruit our CEMs to any region of the genome to which we design a guide RNA. To study the addition of histone acetylation we utilized our new activating, HAT-binding CEMs and designed our dCas9-FKBP to target low-expression reporters. Our lead activating CEM increases expression over 20-fold. We are now using our activating CEM system to target tumor suppressor genes in hard to treat cancers. To show proof-of-principle, we have targeted the tumor suppressor p53 in a colon cancer cell line. We hypothesize that our activating CEM platform will activate the repressed P53 allele leading to slower tumor cell proliferation and apoptosis. This work represents a novel therapeutic strategy that is capable of modulating gene expression in a reversible and dose-dependent manner. Citation Format: Anna M. Chiarella, Kyle V. Butler, Jian Jin, Nathaniel A. Hathaway. Targeted gene control with bifunctional molecules to harness endogenous chromatin-modifying activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-342.

Stable Heritable Germline Silencing Directs Somatic Silencing at an Endogenous Locus

The importance of transgenerationally inherited epigenetic states to organismal fitness remains unknown as well-documented examples are often not amenable to mechanistic analysis or rely on artificial reporter loci. Here we describe an induced silenced state at an endogenous locus that persists, at 100% transmission without selection, for up to 13 generations. This unusually persistent silencing enables a detailed molecular genetic analysis of an inherited epigenetic state. We find that silencing is dependent on germline nuclear RNAi factors and post-transcriptional mechanisms. Consistent with these later observations, inheritance does not require the silenced locus, and we provide genetic evidence that small RNAs embody the inherited silencing signal. Notably, heritable germline silencing directs somatic epigenetic silencing. Somatic silencing does not require somatic nuclear RNAi but instead requires both maternal germline nuclear RNAi and chromatin-modifying activity. Coupling inherited germline silencing to somatic silencing may enable selection for physiologically important traits.

The Complex Role of the ZNF224 Transcription Factor in Cancer.

ZNF224 is a member of the Kruppel-associated box zinc finger proteins (KRAB-ZFPs) family. It was originally identified as a transcriptional repressor involved in gene-specific silencing through the recruitment of the corepressor KAP1, chromatin-modifying activities, and the arginine methyltransferase PRMT5 on the promoter of its target genes. Recent findings indicate that ZNF224 can behave both as a tumor suppressor or an oncogene in different human cancers. The transcriptional regulatory properties of ZNF224 in these systems appear to be complex and influenced by specific sets of interactors. ZNF224 can also act as a transcription cofactor for other DNA-binding proteins. A role for ZNF224 in transcriptional activation has also emerged. Here, we review the state of the literature supporting both roles of ZNF224 in cancer. We also examine the functional activity of ZNF224 as a transcription factor and the influence of protein partners on its dual behavior. Increasing information on the mechanism through which ZNF224 can operate could lead to the identification of agents capable of modulating ZNF224 function, thus potentially paving the way to new therapeutic strategies for treatment of cancer.

Emerging Roles of Epigenetic Regulator Sin3 in Cancer.

Revolutionizing treatment strategies is an urgent clinical need in the fight against cancer. Recently the scientific community has recognized chromatin-associated proteins as promising therapeutic candidates. However, there is a need to develop more targeted epigenetic inhibitors with less toxicity. Sin3 family is one such target which consists of evolutionary conserved proteins with two paralogues Sin3A and Sin3B. Sin3A/B are global transcription regulators that provide a versatile platform for diverse chromatin-modifying activities. Sin3 proteins regulate key cellular functions that include cell cycle, proliferation, and differentiation, and have recently been implicated in cancer pathogenesis. In this chapter, we summarize the key concepts of Sin3 biology and elaborate the recent advancements in the role of Sin3 proteins in cancer with specific examples in multiple endocrine neoplasia type 2, pancreatic ductal adenocarcinoma, and triple negative breast cancer. Finally, a program to create an integrative approach for screening antitumor agents that target chromatin-associated factors like Sin3 is presented.

The adenovirus E1A oncoprotein N-terminal transcriptional repression domain enhances p300 autoacetylation and inhibits histone H3 Lys18 acetylation.

Expression of the adenovirus E1A N-terminal transcription repression domain alone (E1A 1-80) represses transcription from specific promoters such as HER2 [1] and from reconstituted chromatin [2]. Significantly, E1A 1-80 can induce the death of human breast cancer cells over-expressing the HER2 oncogene [1] as well as other cancer cells. Here, we report that E1A 1-80 alone is sufficient to inhibit H3K18 acetylation in vivo and p300-mediated H3K18 acetylation in reconstituted chromatin. Of interest, hypoacetylation of H3K18 has been correlated with the survival of tumor cells and the poor prognosis of many cancers [3, 4]. E1A 1-80 enhances p300 autoacetylation and concurrently inhibits H3K18 acetylation in chromatin in a dose-dependent manner. Pre-acetylation of p300 by incubation with acetyl-CoA alone reduces p300's ability to acetylate H3K18 in chromatin. Additional acetylation of p300 in the presence of E1A 1-80 produces stronger inhibition of H3K18 acetylation. These findings indicate that autoacetylation of p300 greatly reduces its ability to acetylate H3K18. The results reported here combined with our previous findings suggest that E1A can repress transcription by multiple strategies, including altering the chromatin modifying activity of p300 and dissociating TFIID from the TATA box thus disrupting formation of the transcription pre-initiation complex [5, 6].

The role of long non-coding RNAs in neurodevelopment, brain function and neurological disease.

Long non-coding RNAs (lncRNAs) are transcripts with low protein-coding potential that represent a large proportion of the transcriptional output of the cell. Many lncRNAs exhibit features indicative of functionality including tissue-restricted expression, localization to distinct subcellular structures, regulated expression and evolutionary conservation. Some lncRNAs have been shown to associate with chromatin-modifying activities and transcription factors, suggesting that a common mode of action may be to guide protein complexes to target genomic loci. However, the functions (if any) of the vast majority of lncRNA transcripts are currently unknown, and the subject of investigation. Here, we consider the putative role(s) of lncRNAs in neurodevelopment and brain function with an emphasis on the epigenetic regulation of gene expression. Associations of lncRNAs with neurodevelopmental/neuropsychiatric disorders, neurodegeneration and brain cancers are also discussed.

The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription

The SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription.

The many faces of estrogen signaling.

Estrogens have long been known as important regulators of the female reproductive functions; however, our understanding of the role estrogens play in the human body has changed significantly over the past years. It is now commonly accepted that estrogens and androgens have important functions in both female and male physiology and pathology. This is in part due to the local synthesis and action of estrogens that broadens the role of estrogen signaling beyond that of the endocrine system. Furthermore, there are several different mechanisms through which the three estrogen receptors (ERs), ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER1) are able to regulate target gene transcription. ERα and ERβ are mostly associated with the direct and indirect genomic signaling pathways that result in target gene expression. Membrane-bound GPER1 is on the other hand responsible for the rapid non-genomic actions of estrogens that activate various protein-kinase cascades. Estrogen signaling is also tightly connected with another important regulatory entity, i.e. epigenetic mechanisms. Posttranslational histone modifications, microRNAs (miRNAs) and DNA methylation have been shown to influence gene expression of ERs as well as being regulated by estrogen signaling. Moreover, several coregulators of estrogen signaling also exhibit chromatin-modifying activities further underlining the importance of epigenetic mechanisms in estrogen signaling. This review wishes to highlight the newer aspects of estrogen signaling that exceed its classical endocrine regulatory role, especially emphasizing its tight intertwinement with epigenetic mechanisms.