The global impact of DNA methylation on alternative splicing is largely unknown. Using a genome-wide approach in wild-type and methylation-deficient embryonic stem cells, we found that DNA methylation can either enhance or silence exon recognition and affects the splicing of more than 20% of alternative exons. These exons are characterized by distinct genetic and epigenetic signatures. Alternative splicing regulation of a subset of these exons can be explained by heterochromatin protein 1 (HP1), which silences or enhances exon recognition in a position-dependent manner. We constructed an experimental system using site-specific targeting of a methylated/unmethylated gene and demonstrate a direct causal relationship between DNA methylation and alternative splicing. HP1 regulates this gene's alternative splicing in a methylation-dependent manner by recruiting splicing factors to its methylated form. Our results demonstrate DNA methylation's significant global influence on mRNA splicing and identify a specific mechanism of splicing regulation mediated by HP1.

Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. One probable explanation lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicate that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses reveal that SF3B1 specifically binds nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affects splicing of these exons similarly to SF3B1 knockdown. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice-site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure.

MicroRNA (miRNA) biogenesis initiates co-transcriptionally, but how the Microprocessor machinery pinpoints the locations of short precursor miRNA sequences within long flanking regions of the transcript is not known. Here we show that miRNA biogenesis depends on DNA methylation. When the regions flanking the miRNA coding sequence are highly methylated, the miRNAs are more highly expressed, have greater sequence conservation, and are more likely to drive cancer-related phenotypes than miRNAs encoded by unmethylated loci. We show that the removal of DNA methylation from miRNA loci leads to their downregulation. Further, we found that MeCP2 binding to methylated miRNA loci halts RNA polymerase II elongation, leading to enhanced processing of the primary miRNA by Drosha. Taken together, our data reveal that DNA methylation directly affects miRNA biogenesis.

Chromatin organization and epigenetic markers influence splicing, though the magnitudes of these effects and the mechanisms are largely unknown. Here, we demonstrate that linker histone H1.5 influences mRNA splicing. We observed that linker histone H1.5 binds DNA over splice sites of short exons in human lung fibroblasts (IMR90 cells). We found that association of H1.5 with these splice sites correlated with the level of inclusion of alternatively spliced exons. Exons marked by H1.5 had more RNA polymerase II (RNAP II) stalling near the 3′ splice site than did exons not associated with H1.5. In cells depleted of H1.5, we showed that the inclusion of five exons evaluated decreased and that RNAP II levels over these exons were also reduced. Our findings indicate that H1.5 is involved in regulation of splice site selection and alternative splicing, a function not previously demonstrated for linker histones.

Sexual dimorphisms are responsible for profound metabolic differences in health and behavior. Whether males and females react differently to environmental cues, such as solar ultraviolet (UV) exposure, is unknown. Here we show that solar exposure induces food-seeking behavior, food intake, and food-seeking behavior and food intake in men, but not in women, through epidemiological evidence of approximately 3,000 individuals throughout the year. In mice, UVB exposure leads to increased food-seeking behavior, food intake and weight gain, with a sexual dimorphism towards males. In both mice and human males, increased appetite is correlated with elevated levels of circulating ghrelin. Specifically, UVB irradiation leads to p53 transcriptional activation of ghrelin in skin adipocytes, while a conditional p53-knockout in mice abolishes UVB-induced ghrelin expression and food-seeking behavior. In females, estrogen interferes with the p53–chromatin interaction on the ghrelin promoter, thus blocking ghrelin and food-seeking behavior in response to UVB exposure. These results identify the skin as a major mediator of energy homeostasis and may lead to therapeutic opportunities for sex-based treatments of endocrine-related diseases.

BackgroundT-cell engagers are bispecific molecules directed against the CD3 complex on one end and a tumor specific antigen on the other end, allowing a physical link of T cell to a tumor cell, resulting in tumor killing and immune activation. Bispecific molecules harnessing and redirecting T-cells towards tumor cells are a promising therapeutic agents. Aptamers are single stranded oligonucleotides with binding and recognition propensities similar to those of antibodies. Aptamers have a number of advantages over bispecific antibodies including shorter generation time and low immunogenicity. Thus, aptamers capable of targeting T cells would have great potential for use as anti-cancer therapeuticsMethodsSystematic evolution of ligands by exponential enrichment (SELEX) methodology was employed in order to identify a novel CD3e binding aptamer. CD3 binding aptamer was subsequently linked into a bispecific T cell engager structure with a tumor-targeting aptameric arm. The tumor-targeting aptamer is developed by Aummune's proprietary tailored therapeutic platform.1 based on identifying functional aptamer sequences capable of specifically killing targeted tumor cells and sparing healthy tissue .Exemplary bispecific aptamers were tested for T cell stimulation by flow cytometry. In vivo antitumor activity was investigated in syngeneic and in xenograft tumor models.ResultsWe have successfully identified a novel CD3e –targeting aptamer with a Kd of 31nM. A bispecific T cell engager comprised of this aptamer and a tumor-targeting aptamer induced a potent stimulation of T cells in vitro, resulting in CD69 upregulation and IFNg secretion.Next, the CD3e targeting aptamer was hybridized to tumoricidal aptamers identified by Aummune's platform (VS12) to target either the human colon carcinoma HCT116 cells or (VS32) the murine triple negative breast cancer 4T1 cells. Both bispecific entities (CS6-VS12 and CS6-VS32) effectively lead to inhibition of tumor growth in vivo and increased survival in the corresponding models.ConclusionsOur data above provide a proof-of-concept for Aummune's Bispecific Aptamer efficacy and provide a framework for the clinical development of this novel tailored immune therapeutic agents. Indeed, we are currently in the process of developing a first-in-human clinical study in subjects with solid tumors.ReferenceMamet N, et al, Commun Biol 2020.