Biology subjects

Hilton, I.

Publications and source records attributed to Hilton, I..

5 recordsLinked to original sources

bioRxiv2026

Induction of hysteretic chromatin states by CRISPRa reveals locus-specific encoding of transcriptional memory

Transcriptional memory is a phenomenon that allows cells to 'remember' prior stimulatory history, which can alter subsequent gene expression patterns and shape development, immunity, and disease. Although various epigenetic adaptations have been implicated, it remains unclear whether the mechanics of transcription alone are sufficient to install durable memory independently of complex activation and global phenotypic changes induced by inflammatory and environmental stimuli. Here, we employ CRISPR activation (CRISPRa) to uncouple gene activation from global signaling and test whether memory can be established selectively by a local transcriptional pulse in primary mesenchymal stromal cells (MSCs). Key stimulus-responsive genes (IDO1, CXCL11) remain sensitized for weeks after either IFN{gamma} exposure or locus-specific CRISPRa perturbations. Histone PTM profiling links this memory to epigenetic hysteresis involving persistent loss of promoter H3K27me3. Single-molecule chromatin mapping by DAF-seq showed that this adaptation does not coincide with gains in promoter accessibility, but suggests it supports increased transcription factor reacquisition in rechallenged cells. Finally, while H3K27me3 loss appears to be a key hallmark of strongly induced memory genes, multi-omic analyses showed that persistent H3K27me3 hysteresis also occurs at a small subset of non-memory genes. Coordination of H3K27me3 hysteresis with additional cis factors therefore likely shapes transcriptional memory in a locus-specific fashion, underscoring the utility of targeted CRISPRa perturbations for dissecting these responses.

molecular biology↗

bioRxiv2026

Loss of Syndecan-3 Drives a Pro-inflammatory, EndMT-prone Phenotype in Aortic Valve Endothelial Cells

The earliest events in calcific aortic valve disease (CAVD) occur at the valve endothelium, where the cells lining the leaflet undergo NF-kappaB (Nuclear Factor kappa B)-driven inflammatory activation and endothelial-to-mesenchymal transition (EndMT). The endothelial glycocalyx (EG) sets the threshold at which endothelial cells (ECs) respond to such cues, yet its composition and function in the aortic valve remain almost entirely uncharacterized. Here we define the cell-surface proteoglycan (CPG) landscape of human aortic valve endothelial cells (HAVECs) under inflammatory stimulation and identify syndecan-3 (SDC3) as a regulator of valve endothelial activation. Among the proteoglycans examined, SDC3 was highly expressed and inflammation-responsive. Silencing SDC3 in unstimulated HAVECs was sufficient to upregulate NF-kappaB pathway components and the leukocyte adhesion molecules vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-selectin, and it amplified their induction upon TNF-alpha challenge. Pharmacological inhibition of IKKbeta; abolished this difference, placing SDC3 upstream of NF-kappaB, while SDC3 overexpression attenuated tumor necrosis factor-alpha (TNF-alpha)-induced VCAM1, the inverse of the knockdown effect. Loss of SDC3 further primed HAVECs toward a mesenchymal, EndMT-prone phenotype under transforming growth factor-beta 2 (TGF-beta2) stimulation, marked by increased smooth muscle protein 22-alpha (SM22alpha) and reduced platelet endothelial cell adhesion molecule (PECAM1/CD31). Together, these findings identify SDC3 as an endogenous restraint on the inflammatory and mesenchymal programs that characterize the endothelial phase of CAVD and establish the valve endothelial glycocalyx as a previously unappreciated yet potent regulator of aortic valve endothelial dysfunction. Preserving or augmenting SDC3 may warrant investigation as a means of holding valve endothelial cells in a quiescent state.

pathology↗

bioRxiv2023

Predicting the effect of CRISPR-Cas9-based epigenome editing

Epigenetic regulation orchestrates mammalian transcription, but functional links between them remain elusive. To tackle this problem, we use epigenomic and transcriptomic data from 13 ENCODE cell types to train machine learning models to predict gene expression from histone post-translational modifications (PTMs), achieving transcriptome-wide correlations of [~] 0.70 - 0.79 for most cell types. Our models recapitulate known associations between histone PTMs and expression patterns, including predicting that acetylation of histone subunit H3 lysine residue 27 (H3K27ac) near the transcription start site (TSS) significantly increases expression levels. To validate this prediction experimentally and investigate how natural vs. engineered deposition of H3K27ac might differentially affect expression, we apply the synthetic dCas9-p300 histone acetyltransferase system to 8 genes in the HEK293T cell line and to 5 genes in the K562 cell line. Further, to facilitate model building, we perform MNase-seq to map genome-wide nucleosome occupancy levels in HEK293T. We observe that our models perform well in accurately ranking relative fold-changes among genes in response to the dCas9-p300 system; however, their ability to rank fold-changes within individual genes is noticeably diminished compared to predicting expression across cell types from their native epigenetic signatures. Our findings highlight the need for more comprehensive genome-scale epigenome editing datasets, better understanding of the actual modifications made by epigenome editing tools, and improved causal models that transfer better from endogenous cellular measurements to perturbation experiments. Together these improvements would facilitate the ability to understand and predictably control the dynamic human epigenome with consequences for human health.

bioinformatics↗

bioRxiv2022

Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription

To infer potential causal relationships between 3D chromatin structure, enhancers, and gene transcription, we mapped each feature in a genome-wide fashion across eight narrowly-spaced timepoints of macrophage activation. Enhancers and genes connected by loops exhibited stronger correlations between histone H3K27 acetylation and expression than can be explained by genomic distance or physical proximity alone. Changes in acetylation at looped distal enhancers preceded changes in gene expression. Changes in gene expression exhibit a directional bias at differential loop anchors; gained loops are associated with increased expression of genes oriented away from the center of the loop, while lost loops were often accompanied by high levels of transcription with the loop boundaries themselves. Taken together, these results are consistent with a reciprocal relationship in which loops can facilitate increased transcription by connecting promoters to distal enhancers while high levels of transcription can impede loop formation. HIGHLIGHTSO_LILPS + IFN{gamma} triggers genome-wide changes in chromatin looping, enhancer acetylation, and gene expression C_LIO_LILooped enhancer-promoter pairs exhibit ordered and correlated changes in acetylation and expression C_LIO_LIChanges in gene expression exhibit a directional bias at differential loop anchors C_LIO_LILost loops are associated with high levels of transcription within loop boundaries C_LI

genomics↗

bioRxiv2021

Transgenic mice for in vivo epigenome editing with CRISPR-based systems

The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

genomics↗