Biology subjects

Greenblatt, J.

Publications and source records attributed to Greenblatt, J..

5 recordsLinked to original sources

bioRxiv2021

KRAB Zinc Finger protein Znf684 interacts with Nxf1 to regulate mRNA export

Cys2His2 (C2H2) type zinc finger (ZnF) proteins constitute a large class of proteins that are generally considered to be DNA-binding transcription factors. Using affinity purification followed by mass spectrometry, as well as reciprocal co-immunoprecipitation experiments, we determined that the C2H2-ZnF protein Znf684 interacts physically with several proteins involved in mRNA export, including Nxf1 and Alyref. We utilized individual nucleotide resolution cross-linking immunoprecipitation followed by high throughput sequencing (iCLIP-seq) experiments to show that Znf684 binds directly to specific mRNAs in vivo and has an RNA-binding profile similar to those of Nxf1 and Alyref, suggesting a role in mRNA export regulation. Immunofluorescence microscopy (IF) experiments revealed that Znf684 localizes to both the nucleus and cytoplasm. Using cellular fractionation experiments, we demonstrate that overexpression of Znf684 negatively impacts the export of SMAD3 and other target mRNAs. Taken together, our results suggest that Znf684 regulates the export of a subset of transcripts through physical interactions with Nxf1 and specific target mRNAs.

molecular biology↗

bioRxiv2021

Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2

SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies. IN BRIEFSARS-CoV-2, depends on host cell components for infection and replication. Genome-wide CRISPR screens were performed in multiple human cell lines to elucidate common host dependencies required for SARS-CoV-2 infection. Only ACE2, the cognate SARS-CoV-2 entry receptor, was common amongst cell lines, while all other host genes identified were cell line specific, several of which converged on pathways involved in cell signalling, lipid metabolism, immune pathways, and chromatin modulation. Overall, a network of host factors was identified that will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate productive targeting of host genes and pathways. HIGHLIGHTS- Genome-wide CRISPR screens for SARS-CoV-2 in multiple human cell lines - Identification of wide-ranging cell-type dependent genetic dependencies for SARS-CoV-2 infection - ACE2 is the only common host factor identified across different cell types

systems biology↗

bioRxiv2021

Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization

NSD2 is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36me2), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two PWWP and five PHD domains believed to serve as chromatin reading modules, but their exact function in the regulation of NSD2 activity remains underexplored. Here we report a first-in-class chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 binds potently (Kd of 91 {+/-} 8 nM) to PWWP1, antagonizes its interaction with nucleosomal H3K36me2, and selectively engages endogenous NSD2 in cells. Crystal structures show that UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1 which is juxtaposed to the DNA-binding surface. In cells, UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 as a result of translocations prevalent in multiple myeloma. Mutation of other NSD2 chromatin reader domains also increases NSD2 nucleolar localization, and enhances the effect of UNC6934. Finally we identified two C-terminal nucleolar localization sequences in NSD2 that appear to drive nucleolar accumulation when one or more chromatin reader domains are disabled. These data support a model in which NSD2 chromatin engagement is achieved in a cooperative manner and subcellular localization is controlled by multiple competitive structural determinants. This chemical probe and the accompanying negative control, UNC7145, will be useful tools in defining NSD2 biology.

cell biology↗

bioRxiv2019

Regulation of transcription termination by FUS and TDP-43

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAPII) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. We previously found that symmetrical dimethylation (me2s) of a CTD Arginine residue (R1810 in human) causes recruitment of the Tudor domain of SMN, which interacts with Senataxin. SMN is mutated in spinal muscular atrophy (SMA), and Senataxin is sometimes mutated in Amyotrophic Lateral Sclerosis (ALS). R1810me2s and SMN, like Senataxin, are important for resolving R-loops (DNA:RNA hybrids) at transcription terminators. FUS and TDP-43 (TARDBP) are DNA/RNA binding proteins that are sometimes mutated in ALS and FTD (Frontotemporal dementia). Here we show that TDP-43 and, to some extent, FUS are recruited by the R1810me2s-SMN pathway. Defects in FUS and TDP-43 recruitment influence RNAPII termination and R-loop accumulation, leading to elevated DNA damage at terminators that may contribute to neurodegenerative disorders like ALS and FTD.

molecular biology↗

bioRxiv2018

Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins

Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)--only found in higher eukaryotes--regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p, but enhanced K7ac, indicating RPRD proteins recruit K7 deacetylases, including HDAC1. We also report auto-regulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

molecular biology↗