Paul A. Khavari

Insight into population-level gene-environment (GxE) interactions is a major goal in understanding polygenic diseases, such as breast cancer. We integrated massively parallel reporter assays (MPRA), chromatin profiling, and network analysis to identify estrogen-responsive breast cancer variants. Screening 1,604 GWAS-identified breast cancer variants identified 73 estrogen-modulated SNVs (emSNVs). Chromatin accessibility modeling validated allele-specific effects, identifying variants that disrupt pioneer factor binding to chromatin and others that modulate transcription factor (TF) recruitment to pre-accessible enhancers. emSNV targets converged on pathways including mitochondrial metabolism, NF-κB signaling, and chromatin regulation. Aggregating emSNVs into a polygenic risk score (PRSE2) revealed interactions with reproductive risk factors in 13,026 post-menopausal BRCA cases and 108,265 controls, including age at first birth (p=0.0052); a control PRS lacking estrogen-responsive variants showed no interactions. This framework bridges molecular and epidemiological GxE studies to uncover variants whose disease associations depend on environmental context, with implications for understanding polygenic disease risk.

Lineage-determining transcription factors (TFs), such as p63 in epithelial tissues, establish normal tissue identity but can also drive carcinogenesis. The epigenetic basis for these divergent effects, however, is unclear. Here, we show that p63 reorganizes three-dimensional (3D) chromatin topology in squamous cell carcinoma (SCC) through cooperation with the tumor-selective partner TF, FOXK1. Multi-omic profiling across normal and malignant states reveals that p63 enhances connectivity between cis-regulatory elements near oncogenes and promotes a secondary enhancer looping architecture, in which p63 and FOXK1-enriched enhancers indirectly contact target promoters via FOXK1-associated intermediate anchors lacking p63 binding. FOXK1 mediates p63-dependent chromatin looping, as FOXK1 loss selectively weakens p63-dependent loops and reduces transcription of associated target genes. The p63–FOXK1 interaction is observed in human tumor samples but not in normal tissues. Together, our findings define an epigenetic mechanism by which a lineage master TF and tumor-restricted partner TF reshape 3D chromatin structure to drive oncogenic transcription, with implications for tumor cell identity and therapy.

Stratified epithelial differentiation involves transcriptional and proteomic remodeling. Here, multiomic profiling implicated ubiquitin and related posttranslational networks in differentiation dynamics. Systematic perturbation of ubiquitin-like machinery in primary human keratinocytes which uncovered opposite functions of neural-precursor-cell–expressed, developmentally down-regulated 8 (NEDD8) and small ubiquitin-related modifier 2 (SUMO2). Generation of conditional knockout mice established essential roles for NEDD8 in progenitor maintenance, skin regeneration, and inflammation, whereas SUMO2 was required for differentiation. Beyond ubiquitin-proteasome-concordant changes, NEDD8 directed proteomic regulation correlated with RNA abundance. Integration of immunoprecipitation– mass spectrometry with genome-wide suppressor screening revealed context-specific NEDDylation dependencies. Among effectors, heterogeneous nuclear ribonucleoprotein U (HNRNPU) emerged as a posttranscriptional regulator of epithelial cell state whose RNA binding repertoire was modulated by NEDDylation. Thus, NEDD8 and SUMO2 play opposite roles in epithelial homeostasis, regeneration, and inflammation, demonstrating multiple ways ubiquitin-like networks govern tissue homeostasis.

RNA-dependent protein assemblies — including the spliceosome, ribosome and RNA-dependent membraneless organelles — have crucial roles in diverse cellular processes through RNA scaffolding and hierarchical assembly. Various empirical techniques and artificial intelligence algorithms have been developed to help understand the architecture, dynamics and functional implications of RNA–protein complexes, and their further development is underway to comprehensively integrate this information. This Review explores how combining these diverse technologies will enhance our understanding of the biological functions of RNA-dependent protein assemblies. We first explore methodological frontiers, contrasting traditional approaches with new platforms, which enable the identification and tracking of RNA–protein assembly dynamics on the same RNA molecules. We then present avenues for integrating these new experimental techniques with machine-learning methods to improve both predictive models of RNA–protein assembly and functional RNA design. We discuss how the synergy between experimental and digital biology can drive new insights into disease mechanisms and therapeutic strategies, including targeted modulation of pathogenic RNA–protein assemblies. Finally, we examine roadmaps for future research, emphasizing the potential of closed-loop systems that iteratively refine our understanding of RNA–protein assemblies through cycles of hypothesis generation, prediction, experimentation and validation.

Homeostasis relies on signaling networks controlled by cell membrane receptors. Although G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors, their specific roles in the epidermis are not fully understood. Dual CRISPR-Flow and single cell Perturb RNA-sequencing knockout screens of all epidermal GPCRs were thus performed, uncovering an essential requirement for adhesion GPCR ADGRL2 (latrophilin 2) in epidermal differentiation. Among potential downstream guanine nucleotide-binding G proteins, ADGRL2 selectively activated Gα13. Follow-up tissue knockouts verified that Gα13 is also required for epidermal differentiation. A cryoelectron microscopy structure in lipid nanodiscs showed that ADGRL2 engages with Gα13 at multiple interfaces, including via an interaction between ADGRL2 intracellular loop 3 and a Gα13-specific QQQ glutamine triplet sequence in its GTPase domain. In situ gene mutation of this interface sequence impaired epidermal differentiation, highlighting an essential new role for an ADGRL2-Gα13 axis in epidermal differentiation.

RNA-binding proteins (RBPs) are essential for post-transcriptional gene regulation, including for RNA modification such as N⁶-methyladenosine (m⁶A), splicing, polyadenylation, localization, translation and decay. Dysregulation of RBPs has been causally linked to a wide array of human diseases, including cancer, neurodegenerative diseases, metabolic disorders and tissue differentiation abnormalities. Although RBPs have traditionally been studied through their RNA, protein and post-translational interactions, growing evidence shows that small biomolecules (SBMs) such as sugars, nucleotides, metabolites such as S-adenosylmethionine (SAM) and NAD(P)H, and drugs can directly bind RBPs and modulate their structure, localization and RNA-binding activity. These context-dependent and concentration-dependent interactions link RBP regulation to cellular metabolism and are a key focus of current research. In this Review, we discuss the expanding landscape of SBM-binding RBPs and the functions of these RBPs in condensate formation, RNA localization, processing and translation. We highlight the molecular principles that underlie these interactions and their functional relevance to human diseases. We also examine recent advances in the identification of SBM–RBP interactions and the innovative methodologies that are driving discoveries in this rapidly advancing field. Together, these insights underscore the potential of SBMs to modulate RBPs and inform novel therapeutic strategies.

Identifying noncoding single nucleotide variants (SNVs) in regulatory DNA linked to polygenic disease risk, the transcription factors (TFs) they bind, and the genes they dysregulate is a goal in polygenic disease research. Here, we use massively parallel reporter analysis of 3451 SNVs linked to risk for polygenic skin diseases with disrupted epidermal homeostasis to identify 355 differentially active SNVs (daSNVs). daSNV target gene analysis, combined with daSNV editing, underscored dysregulated epidermal differentiation as a shared pathomechanism. CRISPR knockout screens of 1772 human TFs revealed 123 TFs essential for epidermal homeostasis, highlighting ZNF217 and CXXC1. 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. High TF-occupancy promoters were “buffered” against ASB. This resource implicates dysregulated binding of specific homeostatic TF families in risk for diverse polygenic skin diseases.

Interactions between genetic variants and environmental factors influence malignancy risk, including for colorectal cancer (CRC). Prevalent CRC susceptibility loci reside predominantly in noncoding regulatory DNA where they may interact with dietary influences to dysregulate expression of specific genes predisposing to neoplasia. The impacts of CRC protective and risk dietary metabolites, butyrate and deoxycholic acid, were thus studied on the transcription-directing activity of 3703 regulatory CRC-associated variants via massively parallel reporter assays (MPRA) in human colonic cells. 1595 variant-dietary metabolite interactions were identified, pointing to dysregulation of MED13L, NKD2, and several modulators of Wnt/β-catenin signaling in potential CRC gene-environment interactions (GxE). Opposing impacts of butyrate and deoxycholic acid were also uncovered, indicating dietary influences may converge on common CRC risk loci and nominating FOSL1 and SP1 as mediators of these opposing responses. Coupling MPRA to relevant environmental factors offers an approach to extend insight into GxE in common human cancers.