Epigenetic Editing of Ascl1 Gene in Neural Stem Cells by Optogenetics

Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision

Optogenetic regulation of site-specific subtelomeric DNA methylation

Telomere length homeostasis, critical for chromosomal integrity and genome stability, is controlled by intricate molecular regulatory machinery that includes epigenetic modifications. Here, we examine site-specific and spatiotemporal alteration of the subtelomeric methylation of CpG islands using optogenetic tools to understand the epigenetic regulatory mechanisms of telomere length maintenance. Human DNA methyltransferase3A (DNMT3A) were assembled selectively at chromosome ends by fusion to cryptochrome 2 protein (CRY2) and its interacting complement, the basic helix loop helix protein-1 (CIB1). CIB1 was fused to the telomere-associated protein telomere repeat binding factor-1 (TRF1), which localized the protein complex DNMT3A-CRY2 at telomeric regions upon excitation by blue-light monitored by single-molecule fluorescence analyses. Increased methylation was achieved selectively at subtelomeric CpG sites on the six examined chromosome ends specifically after blue-light activation, which resulted in progressive increase in telomere length over three generations of HeLa cell replications. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin modifying enzymes and for loci-specific targeting to regulate the epigenetic pathways at telomeres and other selected genomic regions of interest

A NOTCH3-CXCL12-driven myeloma-tumor niche signaling axis promotes chemoresistance in multiple myeloma

Multiple myeloma (MM) remains incurable due to disease relapse and drug resistance. Notch signals from the tumor microenvironment (TME) confer chemoresistance, but the cellular and molecular mechanisms are not entirely understood. Using clinical and transcriptomic datasets, we found that NOTCH3 is upregulated in CD138+ cells from newly diagnosed MM (NDMM) patients compared to healthy individuals and increased in progression/relapsed MM (PRMM) patients. Further, NDMM patients with high NOTCH3 expression exhibited worse responses to Bortezomib (BOR)-based therapies. Cells of the TME, including osteocytes, upregulated NOTCH3 in MM cells and protected them from apoptosis induced by BOR. NOTCH3 activation (NOTCH3OE) in MM cells decreased BOR anti-MM efficacy and its ability to improve survival in in vivo myeloma models. Molecular analyses revealed that NDMM and PRMM patients with high NOTCH3 exhibit CXCL12 upregulation. TME cells upregulated CXCL12 and activated the CXCR4 pathway in MM cells in a NOTCH3-dependent manner. Moreover, genetic or pharmacologic inhibition of CXCL12 in NOTCH3OE MM cells restored sensitivity to BOR regimes in vitro and in human bones bearing NOTCH3OE MM tumors cultured ex vivo. Our clinical and preclinical data unravel a novel NOTCH3-CXCL12 pro-survival signaling axis in the TME and suggest that osteocytes transmit chemoresistance signals to MM cells

The functional epigenetic landscape of aberrant gene expression in molecular subgroups of newly diagnosed multiple myeloma

Background Multiple Myeloma (MM) is a hematological malignancy with genomic heterogeneity and poor survival outcome. Apart from the central role of genetic lesions, epigenetic anomalies have been identified as drivers in the development of the disease. Methods Alterations in the DNA methylome were mapped in 52 newly diagnosed MM (NDMM) patients of six molecular subgroups and matched with loci-specific chromatin marks to define their impact on gene expression. Differential DNA methylation analysis was performed using DMAP with a ≥10% increase (hypermethylation) or decrease (hypomethylation) in NDMM subgroups, compared to control samples, considered significant for all the subsequent analyses with p<0.05 after adjusting for a false discovery rate. Results We identified differentially methylated regions (DMRs) within the etiological cytogenetic subgroups of myeloma, compared to control plasma cells. Using gene expression data we identified genes that are dysregulated and correlate with DNA methylation levels, indicating a role for DNA methylation in their transcriptional control. We demonstrated that 70% of DMRs in the MM epigenome were hypomethylated and overlapped with repressive H3K27me3. In contrast, differentially expressed genes containing hypermethylated DMRs within the gene body or hypomethylated DMRs at the promoters overlapped with H3K4me1, H3K4me3, or H3K36me3 marks. Additionally, enrichment of BRD4 or MED1 at the H3K27ac enriched DMRs functioned as super-enhancers (SE), controlling the overexpression of genes or gene-cassettes. Conclusions Therefore, this study presents the underlying epigenetic regulatory networks of gene expression dysregulation in NDMM patients and identifies potential targets for future therapies

CRISPR/dCas9-KRAB-Mediated Suppression of S100b Restores p53-Mediated Apoptosis in Melanoma Cells

Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of S100B is poorly correlated (R &lt; 0.3; p &gt; 0.05) to alterations in S100B copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in S100B upregulation in melanoma, we stably suppressed S100b (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Kr&uuml;ppel-associated box (KRAB). Selective combination of S100b-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of S100b in murine B16 melanoma cells without noticeable off-target effects. S100b suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to S100b suppression. S100b-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of S100b therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma

A nuclear WD40 repeat protein PRL1 regulates stability of MYB4 transcription factor in Arabidopsis

MYB4, a member of the R2R3-type subfamily of MYB transcription factor plays a crucial role in regulating the accumulation of UV-B absorbing phenylpropanoids in plants. UV-B exposure for a longer duration down-regulates the expression of MYB4 gene in Arabidopsis. MYB4 protein represses its own expression by binding to its own promoter. However, at present practically nothing is known about the post-translational regulation of MYB4 protein in vivo. Here, we provide evidence that in Arabidopsis MYB4 protein is phosphorylated in vivo and is targeted by the ubiquitin-26S proteasome-dependent pathway. Immunoprecipitation, immunoblotting, and phosphoprotein staining experiments have revealed that both the accumulation pattern and phosphorylation of MYB4 increase in the light condition during the 24 hours time span under long-day conditions. Yeast two-hybrid and bimolecular fluorescence complementation assays have shown that MYB4 directly interacts with a nuclear WD40 repeat protein, PRL1 in vivo. Cell-free protein degradation assay in the absence and presence of proteasome inhibitor indicates that MYB4 is degraded in a ubiquitin proteasome-dependent manner. Furthermore, analyses of MYB4 protein accumulation levels in transgenic atmyb4–1 mutant line expressing 35 S:AtMYB4 (35 S:AtMYB4-atmyb4–1) and atprl1–1 mutant line indicate that PRL1 regulate stability of MYB4 in Arabidopsis. Overall, our results provide important information on the possible mechanism of post-translational modification and regulation of stability of MYB4 protein in Arabidopsis in vivo

Epigenetic Deregulation of Telomere-Related Genes in Newly Diagnosed Multiple Myeloma Patients

High-risk Multiple Myeloma (MM) patients were found to maintain telomere length (TL), below the margin of short critical length, consistent with proactive overexpression of telomerase. Previously, DNA methylation has been shown as a determinant of telomere-related gene (TRG) expression and TL to assess risk in different types of cancer. We mapped genome-wide DNA methylation in a cohort of newly diagnosed MM (NDMM; n = 53) patients of major molecular subgroups, compared to age-matched healthy donors (n = 4). Differential methylation and expression at TRG-loci were analyzed in combination with overlapping chromatin marks and underlying DNA-sequences. We observed a strong correlation (R2 &ge; 0.5) between DNA methylation and expression amongst selective TRGs, such that demethylation at the promoters of DDX1 and TERF1 were associated to their oncogenic upregulation, while demethylation at the bodies of two key tumor suppressors ZNF208 and RAP1A led to downregulation of the genes. We demonstrated that TRG expression may be controlled by DNA methylation alone or in cooperation with chromatin modifications or CCCTC-binding factor at the regulatory regions. Additionally, we showed that hypomethylated DMRs of TRGs in NDMM are stabilized with G-quadruplex forming sequences, suggesting a crucial role of these epigenetically vulnerable loci in MM pathogenesis. We have identified a panel of five TRGs, which are epigenetically deregulated in NDMM patients and may serve as early detection biomarkers or therapeutic targets in the disease

Investigation of antimicrobial physiology of orthorhombic and monoclinic nanoallotropes of sulfur at the interface of transcriptome and metabolome

Nanosized elemental sulfur (ES) is already reported to exert superior antimicrobial efficacy than micron-sized ES, which encourages their use in drugs and therapeutics. The aim of the present study is to explore the possible route and mode of antimicrobial action of orthorhombic (α-SNPs) and monoclinic (β-SNPs) allotropes of sulfur, respectively, at their nano-dimensions. The antimicrobial efficacy of α- and β-SNPs was determined against both the conventionally ES-resistant and ES-susceptible fungi and bacteria. Both the SNPs inhibited the microbial growth, irrespective of their resistance profile to ES and caused significant deformities on the microbial cell surfaces. However, the extent of antimicrobial efficacy was found to be optimum for α-SNPs, which can be attributed to their size, shape, and surface modification. Subsequent transcript profiling, metabolite profiling, and enzymatic analyses revealed that α- and β-SNPs impaired a cluster of mitochondrial enzymes involved in cellular respiration and oxidative phosphorylation. ES and SNPs stress were found to elicit the NADPH-dependent glutathione reductase mediated ES-detoxification response in fungi and caused them to undertake the glyoxylate shunt in favor of energy conservation. A simultaneous study was also undertaken to assess the biocompatible or bio-adverse properties of SNPs in terms of their cytotoxic and genotoxic effects against the human derived lung fibroblast cell line (MRC-5). The present study hence explores the antimicrobial physiology of two novel functional materials and demonstrates their compatibility as a future putative antimicrobial drug