Biology subjects

Willey, S.

Publications and source records attributed to Willey, S..

5 recordsLinked to original sources

bioRxiv2026

CRISPR-FOIL: A Programmable CRISPR Tool to Engineer and Illuminate Chromatin Folding in Live Human Cells

Chromatin organization plays a critical role in regulating gene expression. Chromatin compaction represses gene expression by physically restricting the access of the transcriptional machinery to DNA, while spatial proximity between enhancers and promoters, often mediated by chromatin loops, is essential for gene activation. To investigate the regulatory mechanisms underlying loop formation and chromatin compaction, as well as their effects on gene expression, we developed CRISPR-FOIL (utilizing CRISPR to FOld and ILluminate chromosomal DNA), a novel programmable platform for engineering chromatin loops and inducing chromatin compaction in live cells. CRISPR-FOIL anchors pairs of genomic loci in proximity by engineered single-guide RNAs (sgRNAs), resulting in an artificial chromatin loop. The fused two CRISPR-Sirius gRNAs enable genomic loci to be visualized through fluorescent RNA coat proteins in various colors. In addition, multiple CRISPR-FOIL complexes can act cooperatively to drive chromatin compaction. These results establish CRISPR-FOIL as a powerful tool for engineering chromatin organization in live cells and highlight its potential as a therapeutic platform for gene regulation and disease control.

cell biology↗

bioRxiv2026

Altered neuronal start codon stringency favors cap-independent repeat-associated non-AUG translation

Intronic GGGGCC repeat expansions in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This expansion supports a non-canonical form of translational initiation known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat proteins that contribute to neurodegeneration. Here, we find that the efficiency of RAN translation and its dependency on the 5' 7-methylguanosine mRNA cap are variable across cell types, with both rodent neurons and human iNeurons favoring cap-independent RAN translation from two distinct repeats (CGG and GGGGCC) across multiple reading frames. Treatment with an eIF4E inhibitor that blocks global cap-dependent translation enhances RAN translation specifically in neurons. Intriguingly, cap-independent RAN translation exhibits less reliance on near-cognate codons for initiation than cap-dependent RAN translation. This finding led us to identify a surprising global increase in start codon stringency in neurons as a contributor to the relatively higher cap-independent RAN translation in this cell type. This effect correlates with a cytoplasmic redistribution of eIF1 in neurons and is reversed with neuronal overexpression of the eukaryotic initiation factor eIF5, which relaxes start codon stringency and selectively enhances cap-dependent RAN translation. Taken together, these findings reveal several neuron-specific features of translational regulation that favor cap-independent RAN translation with implications for nucleotide repeat expansion disorder pathogenesis and neuronal translational regulation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/739042v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@61a6cforg.highwire.dtl.DTLVardef@10e8e64org.highwire.dtl.DTLVardef@a6689aorg.highwire.dtl.DTLVardef@11d683b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Differential Regulation of Large-scale Chromosome Conformations in Osteoblasts and Osteosarcoma

Correct chromosome organization in the cell nucleus is essential for genome function. However, dynamics and regulations of large-scale chromosomal conformations beyond single compartments at larger than ten megabases in vivo in single cells remain largely unknown. Here we use CRISPR-Sirius, a high-resolution and high-sensitivity real-time imaging technique, to directly visualize distinctions in large-scale chromosomal conformations between live osteosarcoma (OS) cells and osteoblasts, suggesting extensive chromatin reorganization during cell transformation. A surprising discovery is that chromosome 19 long arm is primarily extended in osteoblasts and maintained by H3K27me3. Extended chromosome conformation has been reported in fly and mouse but not in human cells yet. However, in OS cells, chromosome 19 primarily folded into collapsed conformations, which reshape in minutes and are regulated by the chromosome architectural proteins CTCF and cohesin in the presence of H3K27ac. Changes in chromosome conformations by knocking down the cohesin subunit RAD21 resulted in altered gene expression, including proto-oncogenes. Transcription inhibition by a small molecule inhibitor did not have detectable effects on large-scale chromosome conformation, suggesting that local transcription events have limited effects on large-scale chromosomal architecture. Our results provide unique insights into the complex regulatory mechanisms of endogenous large-scale chromosome organization in normal and transformed osteogenic tissues. One-sentence summaryRegulations of chromosome territory organization in normal and cancer bone cells visualized by CRISPR-based live-cell imaging.

cell biology↗

bioRxiv2024

TDP43 autoregulation gives rise to shortened isoforms that are tightly controlled by both transcriptional and post-translational mechanisms

The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, highly prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a byproduct of TDP43 autoregulation and cleared by nonsense mediated RNA decay (NMD). The sTDP43-encoding transcripts that escape NMD can lead to toxicity but are rapidly degraded post-translationally. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration in vitro and in vivo via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and provide insight into the consequences of aberrant sTDP43 accumulation in disease.

neuroscience↗

bioRxiv2022

Fluorogenic U-rich internal loop (FLURIL) tagging with bPNA enables intracellular RNA and DNA tracking

We introduce herein a new strategy for intracellular RNA and DNA tracking that is robust, orthogonal and complementary to existing methods: Fluorogenic U-Rich Internal Loop (FLURIL) tagging with cell-permeable fluorophore-labeled bifacial Peptide Nucleic Acids (fbPNAs). Our approach uses an 8-nt (U4xU4) U-rich internal loop (URIL) in the RNA of interest (ROI) as a compact labeling site for fluorogenic triplex hybridization with a bPNA probe (~1 kD). FLURIL tagging thus replaces a 4 bp duplex stem with a labeled 4-base-triple hybrid stem of similar structure and mass. In contrast to existing strategies for RNA tracking, FLURIL tagging can be applied to internal, genetically encoded URIL RNA sites with minimal structural perturbation, co-expression of protein-fusion labels or significant increase in molecular weight and steric bulk. We demonstrate effective FLURIL tagging of intracellular (HEK-293) RNAs, ribonucleoprotein (RNP) complexes and live cell (U2-OS) tracking of genomic loci. FLURIL tracking was internally validated by direct comparison with the most widely used live-cell RNA labeling method, MS2-labeling with MCP-HaloTag and Janelia Fluor dyes. In addition, FLURIL-tagging correctly reported on the endogenous RNP in HEK293 cells formed from TAR DNA binding protein 43 (TDP-43-tdTomato) and UG repeat RNA. The FLURIL strategy was also successfully applied to guide RNA (gRNA) in CRISPR-dCas complexes to enable live cell tracking of a low-copy number genomic locus (IDR3), internally benchmarked against MS2/HaloTag labeling of CRISPR-Sirius gRNA targeted to a proximal locus (IDR2). Notably, FLURIL-tagged IDR2 exhibited similar brightness as loci targeted by CRISPR-Sirius gRNA complexes, which bear 8-MS2 hairpins for protein labeling. Together, these experiments show that FLURIL tagging can simply and reliably track intracellular RNA, RNPs, and DNA, with a streamlined molecular footprint relative to other methods. Importantly, these data also indicate that FLURIL tagging is fully compatible with existing labeling methods without crosstalk and may be used to broaden the scope of intracellular RNA and DNA tracking. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/501035v1_figS1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e6cc24org.highwire.dtl.DTLVardef@1646707org.highwire.dtl.DTLVardef@a80268org.highwire.dtl.DTLVardef@363193_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO FLURIL-tagging of RNAs with bPNA probes. (a) Triplex hybridization of a U-rich internal loop (URIL) with bPNA (blue) via base triple formation between the melamine base (M) and two uracil bases (inset). (b) General schematic of labeling strategy described herein. An RNA of interest is engineered to contain an URIL and expressed within the cell, with a fluorogenic bPNA probe introduced via cell culture media. Successful URIL targeting is reported by an increase in emission (green) and confirmed by a previously established RNA binding protein with a fluorescent protein (red) fusion. C_FIG

cell biology↗