Project description:The involvement of G-Protein-Coupled Receptors’ (GPCR) location bias in diverse cellular functions and their misregulation in pathology is an underexplored territory. HCAR1, a GPCR for lactate is linked to cancer progression, mainly due to Warburg effect, but its mechanism of action remains elusive. Here, we show HCAR1 has a nuclear localization, capable of signaling intranuclearly to induce nuclear-ERK and AKT phosphorylation concomitant with higher cancer cell proliferation and survival. We determine its nuclear interactome, proving its involvement in protein-translation and DNA-damage repair. Nuclear HCAR1 (N-HCAR1) directly interacts with chromatin/DNA promoting expression of genes involved in cellular migration. Notably, we show N-HCAR1 particularly regulates a broader transcriptomic signature than its PM counterpart, emphasizing on the facts that functional output of N-HCAR1 is larger than PM localized HCAR1. Our study presents several unprecedented processes by which a GPCR through location-biased activity regulate various cellular functions and how cancer cells exploit these.
Project description:Hepatocyte nuclear factor-4α (HNF4α, NR2A1) is a nuclear receptor which has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4α in the steady state remains to be elucidated. Here we report the native HNF4α isoforms, phosphorylation status and complexes in the steady state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4α, including multiple phosphorylation sites and inter-isoform heterodimerization. The associating complexes identified by label-free semi-quantitative proteomic analysis include the following: the DNA-dependent protein kinase catalytic subunit, histone acetyltransferase complexes, mRNA splicing complex, other nuclear receptor coactivator complexes, the chromatin remodeling complex, and the nucleosome remodeling and histone deacetylation complex. Among the associating proteins, GRB10 interacting GYF protein 2 (GIGYF2, PERQ2) is a new candidate cofactor in metabolic regulation. Moreover, an unexpected heterodimerization of HNF4α and Hepatocyte nuclear factor-4γ was found. A biochemical and genome-wide analysis of transcriptional regulation showed that this heterodimerization activates gene transcription. The genes thus transcribed include the cell death-inducing DEF45-like effector b (CIDEB) gene, which is an important regulator of lipid metabolism in the liver. This suggests that the analysis of the distinctive stoichiometric balance of native HNF4α and its cofactor complexes described here is important for an accurate understanding of transcriptional regulation. Examination of HNF4alpha binding sites with domain-specific antibodies and HNF4gamma binding sites in HepG2 cell.
Project description:Hepatocyte nuclear factor-4α (HNF4α, NR2A1) is a nuclear receptor which has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4α in the steady state remains to be elucidated. Here we report the native HNF4α isoforms, phosphorylation status and complexes in the steady state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4α, including multiple phosphorylation sites and inter-isoform heterodimerization. The associating complexes identified by label-free semi-quantitative proteomic analysis include the following: the DNA-dependent protein kinase catalytic subunit, histone acetyltransferase complexes, mRNA splicing complex, other nuclear receptor coactivator complexes, the chromatin remodeling complex, and the nucleosome remodeling and histone deacetylation complex. Among the associating proteins, GRB10 interacting GYF protein 2 (GIGYF2, PERQ2) is a new candidate cofactor in metabolic regulation. Moreover, an unexpected heterodimerization of HNF4α and Hepatocyte nuclear factor-4γ was found. A biochemical and genome-wide analysis of transcriptional regulation showed that this heterodimerization activates gene transcription. The genes thus transcribed include the cell death-inducing DEF45-like effector b (CIDEB) gene, which is an important regulator of lipid metabolism in the liver. This suggests that the analysis of the distinctive stoichiometric balance of native HNF4α and its cofactor complexes described here is important for an accurate understanding of transcriptional regulation. This SuperSeries is composed of the SubSeries listed below. Refer to individual Series.
Project description:Novel RNA-guided cellular functions are paralleled by an increasing number of RNA binding proteins (RBPs). We present “serial interactome capture” (serIC), a multiple purification procedure of UV-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with maximal specificity. We apply serIC to nuclei of proliferating K562 cells to obtain the first human nuclear interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including fewer factors without known RNA binding domains that are in better agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signaling and double strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs. The nuclear interactome presented here is a stepping-stone towards deciphering of the functional RNA-protein network in the mammalian nucleus.
Project description:To gain an insight into the PRE DNA-binding protein regulatory network, here, using immunoaffinity purification coupled to the high throughput mass spectrometry, we isolated factors associated with the Combgap, Psq, Zeste and Adf1 PRE DNA-binding proteins. We show that Combgap and Zeste are more tightly associated with the Polycomb repressive complex 1 (PRC1), while Psq interacts strongly with the TrxG proteins, including the BAP SWI/SNF complex. The Adf1 interactome contained Mediator subunits as the top interactors. In addition, Combgap efficiently interacted with AGO2, NELF, and TFIID. Combgap, Psq, and Adf1 have architectural proteins in their networks. We further investigated the existence of direct interactions between different PRE DNA-binding proteins and demonstrated that Combgap-Adf1, Psq-Dsp1, and Pho-Spps can interact in the yeast two-hybrid assay. Overall, our data suggest that Combgap, Psq, Zeste and Adf1 are associated with the protein complexes implicated in different regulatory activities and indicate their potential multifunctional role in the regulation of transcription.
Project description:DNA-protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential for understanding the underlying regulatory mechanisms on a molecular level. Here, we describe a novel co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in cellular chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We show the robustness of CMPP on proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we identify hundreds of putative G4-interacting proteins from various functional classes. Next, we observe high G4 binding affinity and selectivity for several G4 interactors in vitro and confirm direct G4 interactions for one of the top candidates in chromatin. Our studies provide a chemical approach to map protein interactions of specific nucleic acid features in living cells.
Project description:The involvement of G-Protein-Coupled Receptors’ (GPCR) location bias in diverse cellular functions and their misregulation in pathology is an underexplored territory. HCAR1, a GPCR for lactate is linked to cancer progression, mainly due to Warburg effect, but its mechanism of action remains elusive. Here, we show HCAR1 has a nuclear localization, capable of signaling intranuclearly to induce nuclear-ERK and AKT phosphorylation concomitant with higher cancer cell proliferation and survival. We determine its nuclear interactome, proving its involvement in protein-translation and DNA-damage repair. Nuclear HCAR1 (N-HCAR1) directly interacts with chromatin/DNA promoting expression of genes involved in cellular migration. Notably, we show N-HCAR1 particularly regulates a broader transcriptomic signature than its PM counterpart, emphasizing on the facts that functional output of N-HCAR1 is larger than PM localized HCAR1. Our study presents several unprecedented processes by which a GPCR through location-biased activity regulate various cellular functions and how cancer cells exploit these.
Project description:In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge, the necessity to erase and reset genomic methylation. In the male germline RNA-directed DNA methylation silences young active transposable elements (TEs). The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) are proposed to tether MIWI2 to nascent TE transcripts and instruct DNA methylation. The mechanism by which MIWI2 directs de novo TE methylation is poorly understood but central to the immortality of the germline. Here, we define the interactome of MIWI2 in foetal gonocytes that are undergoing de novo genome methylation and identify a novel MIWI2-associated factor, SPOCD1, that is essential for young TE methylation and silencing. The loss of Spocd1 in mice results in male specific infertility and does not impact on piRNA biogenesis nor localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein and its expression is restricted to the period of de novo genome methylation. We found SPOCD1 co-purified in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery as well as constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent TE transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through its association with SPOCD1. In summary, we have identified a novel and essential executor of mammalian piRNA-directed DNA methylation.
Project description:To understand how mutations in Matrin 3 (MATR3) cause amyotrophic lateral sclerosis (ALS) and distal myopathy, we used transcriptome and interactome analysis, coupled with microscopy. Over-expression of wild-type (WT) or F115C mutant MATR3 had little impact on gene expression in neuroglia cells. Only 23 genes, expressed at levels of >100 transcripts showed ≥1.6-fold changes in expression by transfection with WT or mutant MATR3:YFP vectors. We identified ~123 proteins that bound MATR3, with proteins associated with stress granules and RNA processing/splicing being prominent. The interactome of myopathic S85C and ALS-variant F115C MATR3 were virtually identical to WT protein. Deletion of RNA recognition motif (RRM1) or Zn finger motifs (ZnF1 or ZnF2) diminished the binding of a subset of MATR3 interacting proteins. Remarkably, deletion of the RRM2 motif caused enhanced binding of >100 hundred proteins. In live cells, MATR3 lacking RRM2 (ΔRRM2) formed intranuclear spherical structures that fused over time into large structures. Our findings in the cell models used here suggest that MATR3 with disease-causing mutations is not dramatically different from WT protein in modulating gene regulation or in binding to normal interacting partners. The intra-nuclear localization and interaction network of MATR3 is strongly modulated by its RRM2 domain.