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1.
Mol Oncol ; 17(10): 2109-2125, 2023 10.
Article in English | MEDLINE | ID: mdl-37086156

ABSTRACT

The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models, in vitro human PCa cell lines, and human patient-derived organoids (PDOs) were used to study the expression and activity of IDH enzymes in the normal prostate and PCa. Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography-mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry-based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.


Subject(s)
Citric Acid Cycle , Prostatic Neoplasms , Male , Mice , Animals , Humans , Isocitrate Dehydrogenase/genetics , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Mitochondria/metabolism , Cytosol/metabolism
2.
J Proteome Res ; 22(1): 78-90, 2023 01 06.
Article in English | MEDLINE | ID: mdl-36484504

ABSTRACT

Bromodomains (BRDs) are a family of evolutionarily conserved domains that are the main readers of acetylated lysine (Kac) residues on proteins. Recently, numerous BRD-containing proteins have been proven essential for transcriptional regulation in numerous contexts. This is exemplified by the multi-subunit mSWI/SNF chromatin remodeling complexes, which incorporate up to 10 BRDs within five distinct subunits, allowing for extensive integration of Kac signaling to inform transcriptional regulation. As dysregulated transcription promotes oncogenesis, we sought to characterize how BRD-containing subunits contribute molecularly to mSWI/SNF functions. By combining genome editing, functional proteomics, and cellular biology, we found that loss of any single BRD-containing mSWI/SNF subunit altered but did not fully disrupt the various mSWI/SNF complexes. In addition, we report that the downregulation of BRD7 is common in invasive lobular carcinoma and modulates the interactome of its homologue, BRD9. We show that these alterations exacerbate sensitivities to inhibitors targeting epigenetic regulators─notably, inhibitors targeting the BRDs of non-mSWI/SNF proteins. Our results highlight the interconnections between distinct mSWI/SNF complexes and their far-reaching impacts on transcriptional regulation in human health and disease. The mass spectrometry data generated have been deposited to MassIVE and ProteomeXchange and assigned the identifiers MSV000089357, MSV000089362, and PXD033572.


Subject(s)
Chromosomal Proteins, Non-Histone , Transcription Factors , Humans , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Gene Expression Regulation , Protein Domains
3.
Genes Dev ; 36(11-12): 664-683, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35710139

ABSTRACT

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.


Subject(s)
Endometrial Neoplasms , Sarcoma, Endometrial Stromal , Sarcoma , Chromatin , DNA-Binding Proteins/metabolism , Endometrial Neoplasms/genetics , Endometrial Neoplasms/metabolism , Endometrial Neoplasms/pathology , Female , Histones/metabolism , Humans , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolism , Sarcoma/genetics , Sarcoma, Endometrial Stromal/genetics , Sarcoma, Endometrial Stromal/metabolism , Sarcoma, Endometrial Stromal/pathology , Translocation, Genetic/genetics
4.
Front Mol Biosci ; 9: 831758, 2022.
Article in English | MEDLINE | ID: mdl-35402505

ABSTRACT

Cellular homeostasis requires the orderly expression of thousands of transcripts. Gene expression is regulated by numerous proteins that recognize post-translational modifications-in particular, the acetylation of lysine residues (Kac) on histones. In addition to affecting the general condensation state of the chromatin, acetylated histones act as anchor points for bromodomain (BRD)-containing adapter proteins. BRDs are the primary Kac reader domains in humans, and proteins containing them act as chromatin scaffolds that organize large networks of interactions to regulate transcription. To characterize BRD-dependent interaction networks, we established cell lines in which histone acetylation is dependent on acetate supplementation. To do this, we used genome editing to knock out ATP citrate lyase (ACLY), the enzyme responsible for converting citrate to oxaloacetate and acetyl-CoA in the cytoplasm and nucleus. In our cellular model, removing acetate from the culture medium resulted in the rapid catabolism of acetylated histones to restore the nucleocytoplasmic acetyl-CoA pool. Here we report the use of our new model in functional proteomics studies to characterize BRD-dependent interaction networks on the chromatin.

5.
PLoS Genet ; 16(6): e1008511, 2020 06.
Article in English | MEDLINE | ID: mdl-32598339

ABSTRACT

Ribosomal DNA (rDNA) is the most transcribed genomic region and contains hundreds of tandem repeats. Maintaining these rDNA repeats as well as the level of rDNA transcription is essential for cellular homeostasis. DNA damages generated in rDNA need to be efficiently and accurately repaired and rDNA repeats instability has been reported in cancer, aging and neurological diseases. Here, we describe that the histone demethylase JMJD6 is rapidly recruited to nucleolar DNA damage and is crucial for the relocalisation of rDNA in nucleolar caps. Yet, JMJD6 is dispensable for rDNA transcription inhibition. Mass spectrometry analysis revealed that JMJD6 interacts with the nucleolar protein Treacle and modulates its interaction with NBS1. Moreover, cells deficient for JMJD6 show increased sensitivity to nucleolar DNA damage as well as loss and rearrangements of rDNA repeats upon irradiation. Altogether our data reveal that rDNA transcription inhibition is uncoupled from rDNA relocalisation into nucleolar caps and that JMJD6 is required for rDNA stability through its role in nucleolar caps formation.


Subject(s)
DNA Damage , Jumonji Domain-Containing Histone Demethylases/genetics , RNA, Ribosomal/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , HEK293 Cells , Humans , Jumonji Domain-Containing Histone Demethylases/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , RNA, Ribosomal/metabolism
6.
Genome Res ; 30(1): 107-117, 2020 01.
Article in English | MEDLINE | ID: mdl-31900288

ABSTRACT

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.


Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing , Streptococcus thermophilus/enzymology , Streptococcus thermophilus/genetics , Animals , CRISPR-Associated Protein 9/chemistry , Cell Line , Cells, Cultured , DNA Cleavage , Humans , Mammals , Mice , Mice, Knockout , Structure-Activity Relationship , Substrate Specificity
7.
Nat Commun ; 9(1): 2919, 2018 07 25.
Article in English | MEDLINE | ID: mdl-30046034

ABSTRACT

CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses (bacteriophages, phages) can carry anti-CRISPR (Acr) proteins to evade that immunity. Acrs can also fine-tune the activity of CRISPR-based genome-editing tools. While Acrs are prevalent in phages capable of lying dormant in a CRISPR-carrying host, their orthologs have been observed only infrequently in virulent phages. Here we identify AcrIIA6, an Acr encoded in 33% of virulent Streptococcus thermophilus phage genomes. The X-ray structure of AcrIIA6 displays some features unique to this Acr family. We compare the activity of AcrIIA6 to those of other Acrs, including AcrIIA5 (also from S. thermophilus phages), and characterize their effectiveness against a range of CRISPR-Cas systems. Finally, we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells.


Subject(s)
CRISPR-Associated Protein 9/physiology , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Bacteriophages/genetics , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/physiology , Gene Editing , Humans , Virulence/genetics , Virulence/physiology
8.
Nat Methods ; 14(6): 615-620, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28417998

ABSTRACT

Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.


Subject(s)
CRISPR-Cas Systems/genetics , Cells, Cultured/physiology , DNA Repair/genetics , Gene Editing/methods , Mutagenesis, Site-Directed , Genetic Markers/genetics , Humans
9.
Cell Rep ; 13(3): 621-633, 2015 Oct 20.
Article in English | MEDLINE | ID: mdl-26456817

ABSTRACT

Conventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method used to decipher molecular interaction networks and infer protein function. However, it is sensitive to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and TAL effector nucleases (TALENs) to tag endogenous genes and purified several DNA repair and chromatin-modifying holoenzymes to near homogeneity. We uncovered subunits and interactions among well-characterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small- and large-scale explorations of protein interactions as well as the study of biochemical activities and structure-function relationships.


Subject(s)
CRISPR-Cas Systems , Gene Targeting/methods , Minichromosome Maintenance Proteins/chemistry , Proteomics/methods , Cell Line, Tumor , Genome, Human , Humans , Protein Binding
10.
Expert Rev Respir Med ; 7(1): 43-55, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23362814

ABSTRACT

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease that pathologically increases pulmonary vascular resistance. Ultimately, this leads to right ventricular failure and premature death. Current therapeutic strategies are mainly designed to induce relaxation of the pulmonary arteries, but are not directly aimed to improve vascular remodeling that characterize PAH. Although these treatments modestly improve patient symptoms, pulmonary hemodynamics and survival, none of them are curative and approximately 15% of patients die within 1 year of medical follow-up despite treatment. Within the last 5 years, tremendous advances in our understanding of the PAH pathophysiology have arisen. These advances have a high potential for the development of better patient care by providing novel therapeutic targets. The goal of this report is to review the current PAH treatments, as well as novel therapies that will pave the future in this devastating disease.


Subject(s)
Endothelin Receptor Antagonists , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hypertension, Pulmonary/drug therapy , Protein Kinase Inhibitors/therapeutic use , Familial Primary Pulmonary Hypertension , Humans , Hypertension, Pulmonary/physiopathology , Lung/physiopathology , Pulmonary Artery/physiopathology
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