Biology subjects

Kang, J. J.

Publications and source records attributed to Kang, J. J..

6 recordsLinked to original sources

bioRxiv2025

Identification of human RNA Polymerase II interactors at early stages of transcription.

RNA Polymerase II (Pol II) transcription is highly regulated at two early steps in the transcription cycle: Pre-Initiation Complex (PIC) assembly with its coupled initiation, and promoter-proximal pausing with its controlled release. Here, we developed an optimized biochemical purification method that captures endogenously tagged chromatin-bound Pol II complexes under native conditions at these rate-limiting steps. We then identified a large set of Pol II interactors by mass spectrometry and determined the footprints of these assemblies on promoters with high resolution. Many well-known and new or understudied factors were identified as associated with the PIC and promoter-proximal paused complexes, indicating that despite decades of efforts, these rate-limiting steps of the transcription cycle are far from being completely understood. The new and understudied factors implicate novel mechanisms of regulation that will need to be characterized to fully understand Pol II regulation.

molecular biology↗

bioRxiv2025

Phosphatidylserine and RhoB connect phosphatidylinositol 4-phosphate and phosphatidic acid metabolism at the plasma membrane

Cells tightly control the homeostatic levels and subcellular localizations of membrane phospholipids through the regulation of the activities of numerous lipid-metabolizing enzymes and lipid transfer proteins. Yet, the mechanisms by which lipid imbalances are sensed and corrected to establish and maintain homeostasis are, in most cases, unknown. Here we present an expanded view of plasma membrane (PM) phosphoinositide metabolism by revealing an unexpected metabolic connection between two key anionic lipids in this membrane, phosphatidylinositol 4-phosphate (PI4P) and phosphatidic acid (PA). PM pools of PI4P are generated by PI 4-kinase Type III (PI4KIII/PI4KA), an essential enzyme whose partial dysfunction leads to numerous hereditary human diseases. We find that depletion of PI4P by pharmacological inhibition of PI4KA increases the activity of phospholipase Ds (PLDs) and the levels of their lipid product, PA, in the PM. Guided by RNA-seq analysis and proximity labeling proteomics, we elucidate how cells connect this PI4P decrease to a compensatory increase in PA levels. Loss of PM PI4P induces a concomitant decrease of phosphatidylserine (PS) levels, and this metabolic rewiring activates a reciprocal relationship between PS synthesis and PLD-mediated PA generation. These metabolic changes also lead to transcriptional and translational upregulation of the small GTPase RhoB, which enhances PLD-mediated PA synthesis and subsequent actin cytoskeletal remodeling. Our study reveals how disease-relevant perturbation of phosphoinositide synthesis induces an integrated response that ultimately boosts levels of PA, a key anionic lipid and metabolic intermediate in phosphoinositide resynthesis.

cell biology↗

bioRxiv2023

3D structural human interactome reveals proteome-wide perturbations by disease mutations

Human genome sequencing studies have identified numerous loci associated with complex diseases. However, translating human genetic and genomic findings to disease pathobiology and therapeutic discovery remains a major challenge at multiscale interactome network levels. Here, we present a deep-learning-based ensemble framework, termed PIONEER (Protein-protein InteractiOn iNtErfacE pRediction), that accurately predicts protein binding partner-specific interfaces for all known protein interactions in humans and seven other common model organisms, generating comprehensive structurally-informed protein interactomes. We demonstrate that PIONEER outperforms existing state-of-the-art methods. We further systematically validated PIONEER predictions experimentally through generating 2,395 mutations and testing their impact on 6,754 mutation-interaction pairs, confirming the high quality and validity of PIONEER predictions. We show that disease-associated mutations are enriched in PIONEER-predicted protein-protein interfaces after mapping mutations from [~]60,000 germline exomes and [~]36,000 somatic genomes. We identify 586 significant protein-protein interactions (PPIs) enriched with PIONEER-predicted interface somatic mutations (termed oncoPPIs) from pan-cancer analysis of [~]11,000 tumor whole-exomes across 33 cancer types. We show that PIONEER-predicted oncoPPIs are significantly associated with patient survival and drug responses from both cancer cell lines and patient-derived xenograft mouse models. We identify a landscape of PPI-perturbing tumor alleles upon ubiquitination by E3 ligases, and we experimentally validate the tumorigenic KEAP1-NRF2 interface mutation p.Thr80Lys in non-small cell lung cancer. We show that PIONEER-predicted PPI-perturbing alleles alter protein abundance and correlates with drug responses and patient survival in colon and uterine cancers as demonstrated by proteogenomic data from the National Cancer Institutes Clinical Proteomic Tumor Analysis Consortium. PIONEER, implemented as both a web server platform and a software package, identifies functional consequences of disease-associated alleles and offers a deep learning tool for precision medicine at multiscale interactome network levels.

bioinformatics↗

bioRxiv2021

FMRP regulates mRNAs encoding distinct functions in the cell body and dendrites of CA1 pyramidal neurons

Neurons are believed to rely on dendritic localization and translation of mRNAs in order to generate activity-dependent changes in the synaptic plasticity. Here, we develop a strategy combining compartment-specific CLIP and TRAP in conditionally tagged mice to precisely define the ribosome-bound dendritic transcriptome of CA1 pyramidal neurons. This revealed transcripts that have differentially localized alternative 3UTR and splicing isoforms. FMRP targets are overrepresented among dendritic mRNAs, and compartment-specific FMRP-CLIP defined 383 dendritic FMRP targets, and also allowed for segregation of whole-cell FMRP targets into functional modules that are locally regulated by FMRP. In the absence of FMRP, dendritic FMRP targets show increased ribosome association, consistent with reported roles for FMRP in translational repression. Together, the data support a model in which distinct patterns of FMRP localization allow it to differentially regulate the expression of nuclear proteins and synaptic proteins within different compartments of a single neuronal cell type.

neuroscience↗

bioRxiv2020

Phase separation by the Sterile Alpha Motif of Polyhomeotic compartmentalizes Polycomb Group proteins and enhances their activity

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) is important for chromatin compaction and large-scale chromatin organization. Like many SAMs, Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand mechanistically how this occurs, we analyzed the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Condensate formation depends on Ph SAM, and is enhanced by but not strictly dependent on, its polymerization activity. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. Overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, phase separation is an activity of the SAM, which, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.

biochemistry↗

bioRxiv2020

DNA binding reorganizes the intrinsically disordered C-terminal region of PSC in Drosophila PRC1

Polycomb Group (PcG) proteins regulate gene expression by modifying chromatin. A key PcG complex, Polycomb Repressive Complex 1 (PRC1), has two activities: a ubiquitin ligase activity for histone H2A, and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal homology region (HR) of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase for H2A. The intrinsically disordered C-terminal region of PSC (PSC-CTR) compacts chromatin, and inhibits chromatin remodeling and transcription in vitro. Both the PSC-HR and the PSC-CTR are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used cross-linking mass spectrometry (XL-MS) to analyze the conformations of the PSC-CTR in PRC1 and how they change on binding DNA. XL-MS identifies interactions between the PSC-CTR and the core of PRC1, including between the PSC-CTR and PSC-HR. New contacts and overall more compacted PSC-CTR conformations are induced by DNA binding. Protein footprinting of accessible lysine residues in the PSC-CTR reveals an extended, bipartite candidate DNA/chromatin binding surface. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible PSC-CTR. Intramolecular interactions of the PSC-CTR detected by XL-MS can bring the high affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large IDRs that bind nucleic acids. HighlightsO_LIAn intrinsically disordered region (IDR) in Polycomb protein PSC compacts chromatin C_LIO_LICross-linking mass spectrometry (XL-MS) was used to analyze topology of the PSC IDR C_LIO_LIProtein footprinting suggests a bipartite DNA binding surface in the PSC IDR C_LIO_LIA model for the DNA-driven organization of the PSC IDR C_LIO_LICombining XL-MS and protein footprinting is a strategy to understand nucleic acid binding IDRs C_LI O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/130492v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@346c5dorg.highwire.dtl.DTLVardef@1b672b3org.highwire.dtl.DTLVardef@1247bfdorg.highwire.dtl.DTLVardef@48448e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗