Biology subjects

Freeman, W.

Publications and source records attributed to Freeman, W..

4 recordsLinked to original sources

bioRxiv2025

snoCLASH Reveals Extensive snoRNA-mRNA Interaction Networks

Small nucleolar RNAs (snoRNAs) are classically defined as guides for modification of ribosomal RNA (rRNA) and small nuclear RNA (snRNA), yet increasing evidence suggests that box C/D snoRNAs also interact with non-canonical transcripts. Systematic discovery of such interactions has been hindered by overwhelming rRNA abundance and technical limitations in RNA-RNA capture. Here, we present snoCLASH, an optimized snoRNA:RNA binding protein (RBP)-based crosslinking, ligation, and sequencing framework that integrates phenol-toluol extraction, polyA enrichment, nuclear fractionation, rRNA depletion, and dual-reference chimeric read analysis to enable transcriptome-scale identification of snoRNA-non-rRNA interactions. Applying this approach reveals thousands of snoRNA-associated mRNA regions spanning coding and regulatory elements and enriched for RBPs linked to epitranscriptomic regulation. Using this framework, we identify high-confidence snoRNA-mRNA interactions and functionally validate OGA mRNA as a snoCLASH-discovered target undergoes snoRNA-dependent 2-O-methylation with downstream effects on protein expression. Together, this work establishes snoCLASH as a platform for discovering and validating non-canonical snoRNA targets beyond rRNA. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/693487v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@13d9a4corg.highwire.dtl.DTLVardef@15d12fdorg.highwire.dtl.DTLVardef@189ac06org.highwire.dtl.DTLVardef@17ceeea_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Blocking necroptosis reduces inflammation and tumor incidence in a mouse model of diet-induced hepatocellular carcinoma

Background & AimsNonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. As necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis- induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet- induced HCC. MethodsMale and female wild-type (WT) mice or mouse models where necroptosis is blocked (Ripk3-/- or Mlkl-/- mice) were fed a control diet or choline-deficient low fat diet (CD-LFD) or CD-high fat diet (CD-HFD) for 6 months. Changes in inflammation, immune cell infiltration, activation of oncogenic pathways, and tumor incidence were assessed by gene expression analysis, western blotting, and flow cytometry. RNA sequencing (RNA-seq) was performed to assess the changes in liver transcriptome. ResultsBlocking necroptosis by deleting either Ripk3 or Mlkl reduced markers of inflammation [proinflammatory cytokines (TNF, IL-6, and IL-1{beta}), F4/80+ve macrophages, CCR2+ve infiltrating monocytes], inflammation associated oncogenic pathways (JNK, PD-L1/PD-1, {beta}-catenin), and HCC in male mice. In female mice, blocking necroptosis reduced HCC independent of inflammation. Blocking necroptosis reduced cell senescence markers in males and females, suggesting a novel cross-talk between necroptosis and cell senescence. ConclusionsOur data show that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. In female mice necroptosis contributes to HCC independent of inflammation. Thus, our study suggests that necroptosis is a valid target for NAFLD-mediated HCC. SynopsisNecroptosis is a cell death pathway that mediate inflammation. Blocking necroptosis attenuated chronic inflammation by reducing recruitment and activation of liver macrophages, which in turn reduced activation of oncogenic pathways and progression of NAFLD to HCC in mice. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/502666v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1bf82d7org.highwire.dtl.DTLVardef@26fc68org.highwire.dtl.DTLVardef@18538f4org.highwire.dtl.DTLVardef@15e338b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2017

Caloric-restriction induced alterations in CG and non-CG methylation attenuate age-associated changes in the old brain.

Brain aging is marked by cognitive decline and susceptibility to neurodegeneration. Caloric-restriction (CR) increases neurogenesis, improves memory function, and protects from age-associated neurological disorders. Epigenetic mechanisms, including DNA methylation, are vital to normal CNS cellular and memory functions, and are dysregulated with aging. The beneficial effects of CR have been proposed to work through epigenetic processes, but this is largely unexplored. We therefore tested whether life-long CR prevents age-related DNA methylation changes in the brain. Hippocampal DNA from young (3 months) and old (24 months) male mice fed ad libitum and 24 month old mice fed a 40% calorierestricted diet from 3 months of age were examined by genome-wide bisulfite sequencing to measure methylation with base-specificity. Over 27 million CG and CH (non-CG) sites were examined. Of the ~40,000 differentially methylated CGs (dmCGs) and ~80,000 CHs (dmCHs) with aging, >1/3 were prevented by CR and were found across genomic regulatory regions and gene pathways. CR also caused alterations to CG and CH methylation at sites not differentially methylated with aging, and these CR-specific changes demonstrated a different pattern of regulatory element and gene pathway enrichment than those affected by aging. CR-specific DNMT1 and TET3 promoter hypermethylation corresponded to reduced gene expression. These findings demonstrate that CR attenuates age-related CG and CH hippocampal methylation changes, in combination with CR-specific methylation that may also contribute to the neuroprotective effects of CR. The prevention of age-related methylation alterations is also consistent with the pro-longevity effects of CR working through an epigenetic mechanism.

neuroscience↗

bioRxiv2017

Sexually divergent DNA methylation programs with hippocampal aging

DNA methylation is a central regulator of genome function and altered methylation patterns are indicative of biological aging and mortality. Age-related cellular, biochemical, and molecular changes in the hippocampus lead to cognitive impairments and greater vulnerability to neurodegenerative disease that varies between the sexes. The role of hippocampal epigenomic changes with aging in these processes is unknown as no genome-wide analyses of age-related methylation changes have considered the factor of sex in a controlled animal model. High-depth, genome-wide bisulfite sequencing of young (3 month) and old (24 month) male and female mouse hippocampus revealed that while total genomic methylation amounts did not change with aging, specific sites in CG and non-CG (CH) contexts demonstrated age-related increases or decreases in methylation that were predominantly sexually divergent. Differential methylation with age for both CG and CH sites was enriched in intergenic, and intronic regions and under-represented in promoters, CG islands and specific enhancer regions in both sexes suggesting that certain genomic elements are especially labile with aging, even if the exact genomic loci altered are predominantly sex-specific. Life-long sex differences in autosomal methylation at CG and CH sites were also observed. The lack of genome-wide hypomethylation, sexually divergent aging response, and autosomal sex differences at CG sites were confirmed in human data. These data reveal sex as a previously unappreciated central factor of hippocampal epigenomic changes with aging. In total, these data demonstrate an intricate regulation of DNA methylation with aging by sex, cytosine context, genomic location, and methylation level.

neuroscience↗