DUCOLI, L. | Search bioRxiv

bioRxiv2024

Disease-Linked Regulatory DNA Variants and Homeostatic Transcription Factors in Epidermis

Identifying noncoding single nucleotide variants (SNVs) in regulatory DNA linked to polygenic disease risk, the transcription factors (TFs) they bind, and the target genes they dysregulate is a goal in polygenic disease research. Massively parallel reporter gene analysis (MPRA) of 3,451 SNVs linked to risk for polygenic skin diseases characterized by disrupted epidermal homeostasis identified 355 differentially active SNVs (daSNVs). daSNV target gene analysis, combined with daSNV editing, underscored dysregulated epidermal differentiation as a pathomechanism shared across common polygenic skin diseases. CRISPR knockout screens of 1772 human TFs revealed 108 TFs essential for epidermal progenitor differentiation, uncovering novel roles for ZNF217, CXXC1, FOXJ2, IRX2 and NRF1. Population sampling CUT&RUN of 27 homeostatic TFs identified allele-specific DNA binding (ASB) differences at daSNVs enriched near epidermal homeostasis and monogenic skin disease genes, with notable representation of SP/KLF and AP-1/2 TFs. This resource implicates dysregulated differentiation in risk for diverse polygenic skin diseases.

genetics↗

bioRxiv2024

irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA

RNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation1-3. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease1,4,5, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation6 was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.

molecular biology↗