Biology subjects

Kang, J. J.

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

3 recordsLinked to original sources

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↗