Epigenetic Switch Research Articles

A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells

A long-lived latent reservoir of HIV-1-infected CD4 Tcells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 Tcells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory Tcells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.

Epigenetic switch reshapes epithelial progenitor cell signatures and drives inflammatory pathogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.

EPCO-41. MICRORNA-MEDIATED DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMAS

Abstract Neuron-specific microRNAs miR-9/9* and miR-124 (miR-9/9*-124) are potent neurogenic molecules whose ectopic expression in non-neuronal somatic cells effectively induces direct cell fate conversion into a neuronal fate. Our recent work indicates that the fate transition occurs in distinct steps where miR-9/9*-124 first represses core gene regulatory networks to erase the original non-neuronal cell identity and subsequently activates the neuronal program to confer neuronal fate. Extending these findings, we reason that repression of similar gene regulatory networks by miR-9/9*-124 can also induce fate erasure in cancerous cells using glioblastoma as a paradigm. Glioblastoma multiforme (GBM) is a type of aggressive brain tumor with poor prognosis. Instead of having specific mutation profiles, GBMs present a global genetic network dysregulation which gives rise to a neural stem cell or progenitor cell-like state resembling embryonic neural development. Whereas miR-9/9*-124 plays a key role in inducing mitotic exit and orchestrating neurogenesis during development and neuronal reprogramming, we hypothesize that miR-9/9*-124 will instruct neuronal reprogramming of GBMs. Our results so far demonstrate that miR-9/9*-124 indeed exerts reprogramming activities in patient-derived GBM cell lines. Ectopic expression of miR-9/9*-124 in vitro readily induces GBM cells to exit cell cycle and promotes neuronal identity over time. In GBM intracerebral xenograft mouse model, mice receiving GBM cells overexpressing miR-9/9*-124 were able to survive longer than those with GBMs overexpressing non-specific microRNAs. We are currently carrying out transcriptomic and chromatin accessibility analyses to infer the epigenetic switch as GBM cells are reprogrammed to neurons. Based on these studies, we will further examine whether the GBM cells undergo irreversible fate erasure and are reprogrammed into a stable post-mitotic neuronal state. Our studies will provide insights into the plasticity of GBM fate that can be controlled by microRNAs and the key downstream regulatory nodes underlying fate transition of GBM to permanent post-mitotic state.

PHF6-altered T-ALL Harbor Epigenetic Repressive Switch at Bivalent Promoters and Respond to 5-Azacitidine and Venetoclax.

To assess the impact of PHF6 alterations on clinical outcome and therapeutical actionability in T-cell acute lymphoblastic leukemia (T-ALL). We described PHF6 alterations in an adult cohort of T-ALL from the French trial Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/2005 and retrospectively analyzed clinical outcomes between PHF6-altered (PHF6ALT) and wild-type patients. We also used EPIC and chromatin immunoprecipitation sequencing data of patient samples to analyze the epigenetic landscape of PHF6ALT T-ALLs. We consecutively evaluated 5-azacitidine efficacy, alone or combined with venetoclax, in PHF6ALT T-ALL. We show that PHF6 alterations account for 47% of cases in our cohort and demonstrate that PHF6ALT T-ALL presented significantly better clinical outcomes. Integrative analysis of DNA methylation and histone marks shows that PHF6ALT are characterized by DNA hypermethylation and H3K27me3 loss at promoters physiologically bivalent in thymocytes. Using patient-derived xenografts, we show that PHF6ALT T-ALL respond to the 5-azacytidine alone. Finally, synergism with the BCL2-inhibitor venetoclax was demonstrated in refractory/relapsing (R/R) PHF6ALT T-ALL using fresh samples. Importantly, we report three cases of R/R PHF6ALT patients who were successfully treated with this combination. Overall, our study supports the use of PHF6 alterations as a biomarker of sensitivity to 5-azacytidine and venetoclax combination in R/R T-ALL.

The Q/R editing site of AMPA receptor GluA2 subunit acts as an epigenetic switch regulating dendritic spines, neurodegeneration and cognitive deficits in Alzheimer’s disease

BackgroundRNA editing at the Q/R site of GluA2 occurs with ~99% efficiency in the healthy brain, so that the majority of AMPARs contain GluA2(R) instead of the exonically encoded GluA2(Q). Reduced Q/R site editing increases AMPA receptor calcium permeability and leads to dendritic spine loss, neurodegeneration, seizures and learning impairments. Furthermore, GluA2 Q/R site editing is impaired in Alzheimer’s disease (AD), raising the possibility that unedited GluA2(Q)-containing AMPARs contribute to synapse loss and neurodegeneration in AD. If true, then inhibiting expression of unedited GluA2(Q), while maintaining expression of GluA2(R), may be a novel strategy of preventing synapse loss and neurodegeneration in AD.MethodsWe engineered mice with the ‘edited’ arginine codon (CGG) in place of the unedited glutamine codon (CAG) at position 607 of the Gria2 gene. We crossbred this line with the J20 mouse model of AD and conducted anatomical, electrophysiological and behavioural assays to determine the impact of eliminating unedited GluA2(Q) expression on AD-related phenotypes.ResultsEliminating unedited GluA2(Q) expression in AD mice prevented dendritic spine loss and hippocampal CA1 neurodegeneration as well as improved working and reference memory in the radial arm maze. These phenotypes were improved independently of Aβ pathology and ongoing seizure susceptibility. Surprisingly, our data also revealed increased spine density in non-AD mice with exonically encoded GluA2(R) as compared to their wild-type littermates, suggesting an unexpected and previously unknown role for unedited GluA2(Q) in regulating dendritic spines.ConclusionThe Q/R editing site of the AMPA receptor subunit GluA2 may act as an epigenetic switch that regulates dendritic spines, neurodegeneration and memory deficits in AD.

Molecular insights into Spindlin1-HBx interplay and its impact on HBV transcription from cccDNA minichromosome

Molecular interplay between host epigenetic factors and viral proteins constitutes an intriguing mechanism for sustaining hepatitis B virus (HBV) life cycle and its chronic infection. HBV encodes a regulatory protein, HBx, which activates transcription and replication of HBV genome organized as covalently closed circular (ccc) DNA minichromosome. Here we illustrate how HBx accomplishes its task by hijacking Spindlin1, an epigenetic reader comprising three consecutive Tudor domains. Our biochemical and structural studies have revealed that the highly conserved N-terminal 2–21 segment of HBx (HBx2–21) associates intimately with Tudor 3 of Spindlin1, enhancing histone H3 “K4me3-K9me3” readout by Tudors 2 and 1. Functionally, Spindlin1-HBx engagement promotes gene expression from the chromatinized cccDNA, accompanied by an epigenetic switch from an H3K9me3-enriched repressive state to an H3K4me3-marked active state, as well as a conformational switch of HBx that may occur in coordination with other HBx-binding factors, such as DDB1. Despite a proposed transrepression activity of HBx2-21, our study reveals a key role of Spindlin1 in derepressing this conserved motif, thereby promoting HBV transcription from its chromatinized genome.

In Vivo Identification of H3K9me2/H3K79me3 as an Epigenetic Barrier to Carcinogenesis.

The highly dynamic nature of chromatin's structure, due to the epigenetic alterations of histones and DNA, controls cellular plasticity and allows the rewiring of the epigenetic landscape required for either cell differentiation or cell (re)programming. To dissect the epigenetic switch enabling the programming of a cancer cell, we carried out wide genome analysis of Histone 3 (H3) modifications during osteogenic differentiation of SH-SY5Y neuroblastoma cells. The most significant modifications concerned H3K27me2/3, H3K9me2, H3K79me1/2, and H3K4me1 that specify the process of healthy adult stem cell differentiation. Next, we translated these findings in vivo, assessing H3K27, H3K9, and H3K79 methylation states in biopsies derived from patients affected by basalioma, head and neck carcinoma, and bladder tumors. Interestingly, we found a drastic decrease in H3K9me2 and H3K79me3 in cancer specimens with respect to their healthy counterparts and also a positive correlation between these two epigenetic flags in all three tumors. Therefore, we suggest that elevated global levels of H3K9me2 and H3K79me3, present in normal differentiated cells but lost in malignancy, may reflect an important epigenetic barrier to tumorigenesis. This suggestion is further corroborated, at least in part, by the deranged expression of the most relevant H3 modifier enzymes, as revealed by bioinformatic analysis. Overall, our study indicates that the simultaneous occurrence of H3K9me2 and H3K79me3 is fundamental to ensure the integrity of differentiated tissues and, thus, their combined evaluation may represent a novel diagnostic marker and potential therapeutic target.

Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis.

Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and β-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.

The histone methyltransferase Whsc1 regulates TGFβ-driven macrophage to myofibroblast transition

Abstract In normal wound healing, macrophages regulate early and late inflammation, allowing for resolution of tissue injury and subsequent healing. During normal tissue repair, macrophages undergo transition to myofibroblasts, which promote wound healing. While this critical transition is impaired in diabetic wounds, the underlying mechanisms responsible are unknown. Our group and others have identified that epigenetic regulation of macrophages controls their phenotype in wounds. Since TGFβ drives macrophage to myofibroblast transition, we performed an epigenetic superarray to identify key chromatin modifying enzymes in macrophages (CD11b+CD3− CD19-NK1.1-Ly6G-) from normal diet (ND) and diabetic-induced obese (DIO) murine wounds treated ex vivo with TGFβ. The histone methyltransferase Whsc1, which mediates di- and tri-methylation at H3K36, was significantly increased in DIO macrophages with TGFβ treatment compared to ND macrophages (p<0.05). siRNA knockdown of Whsc1 in BMDMs decreased TGFβ-dependent expression of fibrotic (Acta2, Col1a1, Col3a1) and inflammatory genes (IL-1b, IL-6, and IL-12) (p<0.05). In ChIP experiments, Whsc1 was enriched at Col1a1 and Col3a1 promoters in ND but not DIO BMDMs, and TGFβ inhibited Whsc1 binding as well as H3K36me2 and H3K36me3 at fibrotic gene promoters. Additionally, H3K36me2 and H3K36me3 were decreased in Whsc1-knockdown BMDMs at Col1a1 and Col3a1, validating Whsc1 as an H3K36 methyltransferase that regulates myofibroblast gene expression. These results suggest that TGFβ regulates macrophage to myofibroblast conversion via Whsc1, which we identify as a critical epigenetic switch in tissue repair and potentially an important therapeutic target in diabetic wounds. F32 DK131799-01, 2022 American College of Surgeons Resident Research Scholarship, 2022 VESS/Medtronic Resident Research Award

Epigenetic switching with asymmetric bridging interactions

Gene expression states are often stably sustained in cis despite massively disruptive events like DNA replication. This is achieved by on-going enzymatic activity that maintains parts of the DNA in either heterochromatic (packed) or euchromatic (free) states, each of which is stabilized by both positive feedback and bridging interactions between individual nucleosomes. In contrast to condensed matter, however, the dynamics is not only governed by equilibrium binding interactions but is also mediated by enzymes that recognize and act on specific amino acid tails of the nucleosomes. The mechanical result is that some nucleosomes can bind to one another and form tightly packed polymer configurations, whereas others remain unbound and form free, noncompact polymer configurations. Here, we study the consequences of such an asymmetric interaction pattern on the dynamics of epigenetic switching. We develop a 3D polymer model and show that traits associated with epigenetic switching, such as bistability and epigenetic memory, are permitted by such a model. We find, however, that the experimentally observed burst-like nature of some epigenetic switches is difficult to reproduce by this biologically motivated interaction. Instead, the behavior seen in experiments can be explained by introducing partial confinement, which particularly affects the euchromatic regions of the chromosome.

Glucose depletion enables Candida albicans mating independently of the epigenetic white-opaque switch

The human fungal pathogen Candida albicans can switch stochastically and heritably between a “white” phase and an “opaque” phase. Opaque cells are the mating-competent form of the species, whereas white cells are thought to be essentially “sterile”. Here, we report that glucose depletion, a common nutrient stress, enables C. albicans white cells to undergo efficient sexual mating. The relative expression levels of pheromone-sensing and mating-associated genes (including STE2/3, MFA1, MFα1, FIG1, FUS1, and CEK1/2) are increased under glucose depletion conditions, while expression of mating repressors TEC1 and DIG1 is decreased. Cph1 and Tec1, factors that act downstream of the pheromone MAPK pathway, play opposite roles in regulating white cell mating as TEC1 deletion or CPH1 overexpression promotes white cell mating. Moreover, inactivation of the Cph1 repressor Dig1 increases white cell mating ~4000 fold in glucose-depleted medium relative to that in the presence of glucose. Our findings reveal that the white-to-opaque epigenetic switch may not be a prerequisite for sexual mating in C. albicans in nature.

Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications

This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris–ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.

New frontiers in host-parasite interactions, from cell to organism.

New frontiers in host-parasite interactions, from cell to organism.

KDM6A Loss Triggers an Epigenetic Switch That Disrupts Urothelial Differentiation and Drives Cell Proliferation in Bladder Cancer.

A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.

Antagonistic action of GPS2 and KDM1A at enhancers governs alternative macrophage activation by interleukin 4.

The Th2 cytokine interleukin 4 (IL4) promotes macrophage differentiation into alternative subtypes and plays important roles in physiology, in metabolic and inflammatory diseases, in cancer and in tissue regeneration. While the regulatory transcription factor networks governing IL4 signaling are already well-characterized, it is currently less understood which transcriptional coregulators are involved and how they operate mechanistically. In this study, we discover that G protein pathway suppressor 2 (GPS2), a core subunit of the HDAC3 corepressor complex assembled by SMRT and NCOR, represses IL4-dependent enhancer activation in mouse macrophages. Our genome-wide and gene-specific characterization revealed that, instead of directly repressing STAT6, chromatin-bound GPS2 cooperates with SMRT and NCOR to antagonize enhancer activation by lysine demethylase 1A (KDM1A, LSD1). Mechanistically, corepressor depletion increased KDM1A recruitment to enhancers linked to IL4-induced genes, accompanied by demethylation of the repressive histone marks H3K9me2/3 without affecting H3K4me1/2, the classic KDM1A substrates for demethylation in other cellular contexts. This in turn caused enhancer and gene activation already in the absence of IL4/STAT6 and sensitized the STAT6-dependent IL4 responsiveness of macrophages. Thus, our work identified with the antagonistic action of a GPS2-containing corepressor complex and the lysine demethylase KDM1A a hitherto unknown epigenetic corepressor-coactivator switching mechanism that governs alternative macrophage activation.

Effective role of Curcumin on expression regulation of EZH2 histone methyltransferase as a dynamic epigenetic factor in osteogenic differentiation of human mesenchymal stem cells

BackgroundEfficient differentiation of mesenchymal stem cells (MSCs) into a desired cell lineage remains challenging in cell-based therapy and regenerative medicine. Numerous efforts have been made to efficiently promote differentiation of MSCs into osteoblast lineage. Accordingly, epigenetic signatures emerge as a key conductor of cell differentiation. Among them, Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase appears to suppress osteogenesis. Curcumin is an osteoinductive natural polyphenol compound which supposedly modulates epigenetic mechanisms. Hence, the current study aims to address the role of the EZH2 epigenetic factor in osteogenic activity of MSCs after Curcumin treatment. MethodsThe effect of Curcumin on viability and osteogenic differentiation was evaluated at different time points in vitro. The expression level of EZH2 was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) after 14 and 21 days. ResultsMTT results showed no cytotoxic effects at concentrations of 10 and 15 μM of Curcumin and cells survived up to 70 % at all time-points. qRT-PCR results demonstrated that Curcumin significantly enhanced the expression levels of osteogenic markers that included Runx2, Osterix, Collagen type I, Osteopontin and Osteocalcin at day 21. ConclusionsInterestingly, we observed that the expression level of the EZH2 gene was downregulated in the presence of Curcumin compared to the control group during osteogenesis. This study confirmed that Curcumin acts as an epigenetic switch to regulate osteoblast differentiation specifically through the EZH2 suppression.

EPCO-13. IDENTIFYING REGULATORS OF GLIOMA CELL STATE DIVERSITY AND EVOLUTION VIA JOINT SINGLE NUCLEUS RNA AND CHROMATIN ACCESSIBILITY

Abstract Glioma cellular heterogeneity and plasticity represent fundamental obstacles to effective therapies. Understanding the determinants that govern glioma cell diversity and adaptability is critical to overcoming treatment resistance. Recent single cell DNA methylation studies have demonstrated that dynamic epigenetic alterations facilitate glioma cell state shifts and stress response. However, information regarding other modes of epigenetic gene regulation, such as chromatin accessibility, and how therapy shapes cellular heterogeneity remains limited. To determine the critical regulators of glioma cell state plasticity under treatment, we profiled 36 tumors (n = 23 adult patients) with joint single nucleus RNA and ATACseq including 13 longitudinal initial – recurrent pairs that yielded a total of 79,945 cells (37,883 malignant cells). We identified chromatin peaks that were uniquely open in glioma cells (n = 14,462 peaks, FDR < 0.05) compared with tumor microenvironmental cells including neuronal, perivascular, glial, and immune populations. Among malignant cells, differentiated-like cells exhibited more recurrent regions of accessibility than stem-like cells. These populations could be further delineated by differential peak enrichment of transcription factor binding sites in the stem-like (TCF12, ASCL1), differentiated-like (SMARCC1, JUN), and proliferating stem-like (E2F4) malignant cells nominating these transcription factors as master regulators. We confirmed that these cell state-specific open chromatin peaks overlapped enhancer regions via single nucleus multiomics and bulk H3K27ac profiling in 3 patient-derived cell lines. We further demonstrated that there are cell state-specific chromatin changes at recurrence with a trend toward a more open chromatin state that was associated with coordinated transcriptional changes. Finally, we incorporated matched longitudinal whole genome sequencing data to evaluate mutational profiles and differentiate between predominantly epigenetically driven changes and epigenomic co-evolution with the genome. Together, these findings define the key epigenetic switches that shape glioma cell states and facilitate plasticity during tumor progression.

37. Epigenetic reprogramming of brain development pathways during non-small cell lung cancer metastasis to brain

Non-small cell lung cancer (NSCLC) is one of the most common and deadliest cancers, with roughly half of all patients initially presenting with both primary and metastatic disease. While the major events in the metastatic cascade have been identified, a mechanistic understanding of how NSCLC routinely, successfully colonizes the brain is largely unknown. To better understand this process, we profiled a combination of genomic, transcriptomic, and methylomic landscapes of 45 paired NSCLC primary and brain metastasis samples. 75 genes displayed recurrent metastasis enriched variants, largely implicated in focal adhesion and extracellular matrix receptor interactions. Variant allele frequencies over a wide range of epigenetic regulators displayed an increase in metastases, suggesting that epigenetic misregulation may be selected for and possibly contribute to NSCLC metastasis to brain. Consistent with these observations, we observed widespread changes in DNA methylation throughout disease progression, many found within brain-specific active enhancers and correlated with increased nearby gene expression. The greatest recurrent methylation changes during metastatic progression occurred over a subset of DNA methylation valleys (DMVs) enriched for H3K9me3 and bivalent marks H3K27me3 and H3K4me1 in normal lung. Mapping EZH2, the catalytic subunit of polycomb repressive complex 2 (PCR2), binding locations in H1299, a lymph node-derived lung cancer cell line, revealed a pervasive loss of EZH2 binding within DMVs accompanied by an increase in DNA methylation, exemplifying epigenetic switching. The vast majority of these DMR-associated DMVs harbor developmental genes, suggesting that altered epigenetic regulation of developmentally important genes may confer a selective advantage during metastatic progression.

189P LncRNA NKILA as a negative regulator of NFkB in ascites in ovarian cancer

NF-κB-interacting LncRNA (NKILA) plays an inhibitory role in the NF-κB pathway, which is a key regulator of inflammatory cytokines. These mediators, freely floating in ascitic fluid (AF), reflecting the molecular genetic characteristics of tumor cells in ovarian cancer (OC), and associated with epigenetic switches of key pathways of carcinogenesis remain poorly understood. The aim of the work was to study the expression of LncRNA NKILA and marker cytokines of the NF-kB pathway in ascites of patients with OC.

A Clustered Regularly Interspaced Short Palindromic Repeats Knockout Method to Reveal methyl-CpG Binding Domain 4 Function

DNA methylation is an epigenetic mechanism tailored for DNA repression, engineered for regulating genetic expression without direct manipulation of the nucleotide sequence. One component of this process includes methyl-binding proteins (MBD), which have an affinity for methyl groups, and they competitively inhibit transcription factors from binding with genetic promoters. Interestingly, MBD4 is unique because, as opposed to transcriptional repression, it promotes gene repair & demethylation and is associated with various methylation-related diseases, such as Autism. By further studying MBD4, we can identify a potential therapeutic target for MRD and further understand the role of methylation on the epigenome in regards to seasonal plasticity. Therefore, this paper describes a CRISPR Knockout screen to isolate & repress MBD4 from its customary functionality with optimized gRNA targets in Astatotilapia burtoni Cichlid. I expect a morphological change in the Cichlid’s skin color (such change can be identified with computer vision COCO-Style-Dataset-Generator-GUI), which substantiates my belief that MBD4 does, in fact, play a significant role in seasonally-regulated epigenetic switches and can be targeted in methylation treatments. However, the exogenous factors relating to MBD4’s role in methylation remain to be investigated.