Biology subjects

Diehl, M. R.

Publications and source records attributed to Diehl, M. R..

3 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↗

bioRxiv2024

Persistent tailoring of MSC activation through genetic priming

Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a genetic priming method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor IRF1 (interferon response factor 1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-{gamma} (IFN{gamma}). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFN{gamma}-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/578489v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@8be46aorg.highwire.dtl.DTLVardef@40fcdeorg.highwire.dtl.DTLVardef@995a21org.highwire.dtl.DTLVardef@1ec53d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Demultiplexing overlapping signaling scaffold functions to probe lipid messenger coupling to cytoskeletal dynamics.

The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific signaling and regulatory processes. While central to many aspects of cell physiology, this coupling often depends on the function of multi-domain scaffolds that orchestrate transient interactions and dynamic feedback among a spectrum of signaling intermediates and regulatory proteins on organelles. Understanding scaffold protein functions has remained challenging given this complexity. This work employs live-cell imaging and statistical analyses to deconvolve (demultiplex) how the multi-domain scaffold IQGAP1 coordinates phosphoinositide signaling with organelle-specific actin regulation and membrane processing events. Using actin-ensconced endosomes that localize to the basal cortex of polarized epithelial cells as a model system, we demonstrate abilities to dissect how IQGAP1 transitions between different actin and endosomal-membrane tethered states. We provide evidence IQGAP1 functions as a transient inhibitor of actin growth around the endosomes in at least one of these states. While not easily distilled via standard (static) colocalization analyses or traditional pathway perturbations methods, this negative regulation was revealed via a series of dynamic correlation and multiple regression analyses. These methods also uncovered that the negative actin regulation is linked to GTPase-dependent tethering to the endosomal membrane. Moreover, the scaffold transitions underlying this control are shown to depend on the production of PIP3 lipid messengers by the lipid kinase PI3K. Overall, these methods and results provide new insights in to how IQGAP1 act as a signaling hub by orchestrating time-dependent membrane and cytoskeletal protein interactions and provide new routes to dissect scaffold-mediated pathway regulation in a variety of settings.

biophysics↗