Biology subjects

Lambert, J.-P.

Publications and source records attributed to Lambert, J.-P..

15 recordsLinked to original sources

bioRxiv2026

Interplay of NuA4/TIP60 and PRC2 Complex Activities in Fusion Driven Endometrial Stromal Sarcoma

Low-grade endometrial stromal sarcoma (LGESS) exhibits frequent chromosomal translocations that fuse various subunits of the NuA4/TIP60 co-activator complex to subunits of the Polycomb Repressive Complex 2 (PRC2) complex. LGESS fusion proteins, such as the commonly occurring JAZF1-SUZ12, have been shown to upregulate genes through mislocalization of NuA4/TIP60 activity to Polycomb target genes. In this study, we characterized an interesting recurrent fusion protein in LGESS that fused a NuA4/TIP60 component, MBTD1, to EZHIP. EZHIP is a recently described vertebrate protein that enzymatically inhibits methyltransferase activity of the PRC2 complex and is a potent oncogene. The MBTD1-EZHIP fusion protein forms a chimeric TIP60-PRC2.1 complex and drastically reduces H3K27me3 levels at Polycomb target genes, similar to EZHIP-overexpressing cancers. However, unlike EZHIP overexpression, MBTD1-EZHIP requires mislocalization of NuA4/TIP60 activity through the MBTD1 protein to upregulate oncogenes. Despite differences in the finer molecular mechanisms, MBTD1-EZHIP and the common JAZF1-SUZ12 fusion protein upregulated similar sets of genes in cell lines, showing a convergence of oncogenic gene expression. Surprisingly, unlike in cellular models, JAZF1-SUZ12 translocated patient samples showed not only upregulation of genes driven by increased H4K8ac and decreased H3K27me3 but also downregulation of specific genes due to accumulated H3K27me3, revealing an additional oncogenic mechanism in LGESS.

cancer biology↗

bioRxiv2025

Concomitant acetylation and loading of H2A.Z by NuA4/TIP60 regulate target gene transcription

The human NuA4/TIP60 complex is a multi-subunit, dual enzymatic epigenetic factor and gene regulator. It bears histone acetyltransferase (HAT) activity towards the canonical histones H2A and H4 and the histone variant H2A.Z, a function that has been linked to gene activation. It also acts as a chromatin remodeling enzyme through ATP-dependent exchange of nucleosomal H2A-H2B dimers with H2A.Z-H2B, leading to incorporation of H2A.Z into chromatin at gene regulatory elements. NuA4/TIP60 is unique in merging two enzymatic activities targeting H2A.Z. Both NuA4/TIP60-dependent H2A.Z acetylation and remodeling have been linked to several physiological functions and pathologies, but studies have only focused on either one or the other enzymatic activity, and insights on functional coordination between them are lacking. Here, we leverage our EP400 rapid depletion system to explore and untangle the intricate links between H2A.Z acetylation by Tip60 (the HAT subunit) and loading on chromatin by EP400 (the remodeling subunit) through functional genomic and biochemical analyses. Our data support a mechanism in which H2A.Z is first pre-acetylated to allow for H2A.Zac-H2B dimer association with the complex before incorporation into chromatin, particularly at gene promoters to positively regulate transcription. As both H2A.Z-targeted enzymatic functions of NuA4/TIP60 have been linked to disease, albeit separately, our findings hold important implications for therapeutic intervention, where combinatorial targeting is a promising avenue.

molecular biology↗

bioRxiv2024

Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

Plant homeodomain (PHD) fingers are critical effectors of histone post-translational modifications (PTMs), acting as regulators of gene expression and genome integrity, and frequently presenting in human disease. While most PHD fingers recognize unmodified and methylated states of histone H3 lysine 4 (H3K4), the specific functions of many of the over 100 PHD finger-containing proteins in humans remain poorly understood, despite their significant implications in disease processes. In this study, we undertook a comprehensive analysis of one such poorly characterized PHD finger-containing protein, PHRF1. Using biochemical, molecular, and cellular approaches, we show that PHRF1 robustly binds to histone H3, specifically at its N-terminal region. Through RNA-seq and proteomic analyses, we also find that PHRF1 is intricately involved in transcriptional and RNA splicing regulation and plays a significant role in DNA damage response (DDR). Crucially, mutagenesis of proline 221 to leucine (P221L) in the PHD finger of PHRF1 abolishes histone interaction and fails to rescue defective DDR. These findings underscore the importance of PHRF1-H3 interaction in maintaining genome integrity and provide insight into how PHD fingers contribute to chromatin biology.

biochemistry↗

bioRxiv2024

The combination of optogenetic-induced protein aggregation and proximity biotinylation assays strongly implicates endolysosomal proteins in the early stages of α-synuclein aggregation

Alpha-synuclein (-syn) aggregation is a defining feature of Parkinsons disease (PD) and related synucleinopathies. Despite significant research efforts focused on understanding -syn aggregation mechanisms, the early stages of this process remain elusive, largely due to limitations in experimental tools that lack the temporal resolution to capture these dynamic events. Here, we introduce UltraID-LIPA, an innovative platform that combines the Light-Inducible Protein Aggregation (LIPA) system with the UltraID proximity-dependent biotinylation assay to identify -syn-interacting proteins and uncover key mechanisms driving its oligomerization. UltraID-LIPA successfully identified 38 -syn-interacting proteins, including both established and novel candidates, highlighting the accuracy and robustness of the approach. Notably, a strong interaction with endolysosomal and membrane-associated proteins was observed, supporting the hypothesis that interactions with membrane-bound organelles are pivotal in the early stages of -syn aggregation. This powerful platform provides new insights into dynamic protein aggregation events, enhancing our understanding of synucleinopathies and other proteinopathies.

neuroscience↗

bioRxiv2024

Coupling proximity biotinylation with genomic targeting to characterize locus-specific changes in chromatin environments

Regulating gene expression involves significant and frequent changes in the chromatin environment at the locus level, especially at regulatory sequences. However, their modulation in response to pharmacological treatments or pathological conditions remain mostly undetermined. Here, we report versatile locus-specific proteomics tools to address this knowledge gap, which combine the targeting ability of the CRISPR/Cas9 system and the protein-labelling capability of the highly reactive biotin ligases TurboID (in CasTurbo) and UltraID (in CasUltra). CasTurbo and CasUltra enabled rapid chromatin protein labelling under mild conditions at repetitive sequences like centromeres and telomeres, as well as non-amplified genes. We applied CasUltra to A375 melanoma cell lines to decipher the protein environment of the MYC promoter and characterize the molecular effects of the bromodomain inhibitor JQ1, which targets bromodomain and extra-terminal (BET) proteins that regulate MYC expression. We quantified the consequences of BET protein displacement from the MYC promoter and found that it was associated with a considerable reorganisation of the chromatin composition. In addition, BET protein retention at the MYC promoter was consistent with a model of increased JQ1 resistance. Thus, through the combination of proximity biotinylation and CRISPR-Cas9-dependent genomic targeting, CasTurbo and CasUltra have successfully demonstrated their utility in profiling the proteome associated with a genomic locus in living cells. In BriefKougnassoukou Tchara et al. report the development and application of CasTurbo and CasUltra, two locus-specific proteomics tools that fuse catalytically dead Cas9 to the engineered biotin ligases TurboID and UltraID. These tools enabled the quantitative mapping of locus-specific chromatin remodelling due to pharmacological inhibition. HighlightsO_LICasTurbo and CasUltra were developed for locus-specific label-free proteomics C_LIO_LICasTurbo mapped the proteins localized to the centromeres and telomeres C_LIO_LIProteins bound to the MYC promoter were quantified in melanoma cells with CasUltra C_LIO_LICasUltra is compatible with investigating pharmacological treatment effects C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/605321v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1f05800org.highwire.dtl.DTLVardef@21754dorg.highwire.dtl.DTLVardef@9c4cc4org.highwire.dtl.DTLVardef@17412a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Systematic profiling of the acetyl lysine machinery reveals a role for MAPKAPK2 in bromodomain inhibitor resistance

Bromodomain (BRD)-containing proteins are chemically tractable multi-domain scaffolding molecules involved in acetyl lysine (Kac) signaling. BRD inhibitors have shown promise in clinical oncology, including melanomas; however, their narrow therapeutic windows and issues with resistance in pre-clinical models highlight the need to better understand the functions of and interconnection between BRD-containing proteins. Here, we use complementary interaction-mapping techniques (affinity purification and proximity-dependent biotinylation) to map the interactions of 39 of the 42 BRD-containing proteins and 110 additional proteins that physically or functionally associate with them. We uncover 3,892 novel interactions and reveal the intricate connectivity of the Kac machinery. Chemical inhibition of multiple BRD classes revealed that inhibiting BETs--but not mSWI/SNF or CREBBP/EP300 proteins--dramatically rewired the interactome. Finally, we identified MAPKAPK2 activity as a critical determinant of BET inhibitor sensitivity in melanoma through its impact on chromatin composition remodeling. In BriefKougnassoukou Tchara et al. generate a static protein interaction map of the human acetyl lysine machinery by coupling two complementary functional proteomics approaches (FLAG affinity purification and proximity-dependent biotinylation) to mass spectrometry. They also investigate network changes upon bromodomain inhibition, and describe a novel resistance mechanism mediated by the p38 stress signaling pathway that causes significant metabolic changes. HighlightsO_LITwo complementary interaction proteomics analyses of the human acetyl lysine machinery were performed. C_LIO_LINovel target- and compound-specific impacts of bromodomain inhibitors were identified. C_LIO_LIMAPKAPK2 was identified as a novel resistance gene to BET bromodomain inhibitors in melanoma. C_LIO_LIBET bromodomain inhibition leads to metabolic adaptation in melanoma. C_LI

cancer biology↗

bioRxiv2023

DNA damage-induced interaction between a lineage addiction oncogenic transcription factor and the MRN complex shapes a tissue-specific DNA Damage Response and cancer predisposition

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA Damage Response (DDR) programs. However, some cells, in skin for example, are normally exposed to high levels of DNA damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Here we show, using melanoma as a model, that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a non-transcriptional role in shaping the DDR. On exposure to DNA damaging agents, MITF is phosphorylated by ATM/DNA-PKcs, and unexpectedly its interactome is dramatically remodelled; most transcription (co)factors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks, and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement, high MITF levels are associated with increased SNV burden in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of ATM/DNA-PKcs-phosphorylated MITF. Our data suggest that a non-transcriptional function of a lineage-restricted transcription factor contributes to a tissue-specialised modulation of the DDR that can impact cancer initiation.

molecular biology↗

bioRxiv2022

eIf2α-regulated translation modulates the early adhesion of mesenchymal-like cells

Cellular invasion is a complex process that requires several interdependent biological mechanisms, which are initiated by changes in adhesion that establish a morphology favorable for migration. Hence, the regulation of adhesion potential is a rate-limiting step in metastasis. Our previous work revealed that de novo translation is necessary to regulate the adhesion of mesenchymal-like cells; however, the underlying translational regulatory mechanism and the identity of newly synthesized proteins needed for the adhesion process remain unidentified. Here, we identify a mechanotransduction pathway linking force-mediated protein unfolding to translational reprogramming via the activation of the integrated stress response (ISR). Specifically, we demonstrate that phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2) during early adhesion events leads to selective translation of mRNAs encoding proteins critical for adhesion complex formation, mechanosensing, and contractility. These findings uncover a mechanosensitive translational control axis that links intracellular force generation to cell adhesion and stress adaptation, with implications for understanding mesenchymal cell adhesion and morphological behavior

cell biology↗

bioRxiv2021

Recurrent chromosomal translocations in sarcomas create a mega-complex that mislocalizes NuA4/TIP60 to Polycomb target loci

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 (ESS) and Ossifying FibroMyxoid Tumors (OFMT) leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a co-activator 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 mega-complex 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 ESS, JAZF1-SUZ12, is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating similar outcomes as 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 employ 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.

molecular biology↗

bioRxiv2021

Misregulation of translation drives prostate cancer drug resistance

Emerging evidence associates translation factors and regulators to tumorigenesis. Recent advances in our ability to perform global translatome analyses indicate that our understanding of translational changes in cancer resistance is still limited. Here, we generated an enzalutamide-resistant prostate cancer (PCa) model, which recapitulated key features of clinical enzalutamide-resistant PCa. Using this model and polysome profiling, we investigated global translation changes that occur during the acquisition of PCa resistance. We found that enzalutamide-resistant cells exhibit a discordance in biological pathways affected in their translatome relative to their transcriptome, a deregulation of proteins involved in translation, and an overall decrease in translational efficiency. We also show that genomic alterations in proteins with high translational efficiency in enzalutamide-resistant cells are good predictors of poor patient prognosis. Additionally, long non-coding RNAs in enzalutamide-resistant cells show increased association with ribosomes, higher translation efficiency, and an even stronger correlation with poor patient prognosis. Taken together, this suggests that aberrant translation of coding and non-coding genes are strong indicators of PCa enzalutamide-resistance. Our findings thus point towards novel therapeutic avenues that may target enzalutamide resistant PCa.

cancer biology↗

bioRxiv2020

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links associated with Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion decreased cellular viability and altered the expression of selected targets. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

molecular biology↗

bioRxiv2020

Proximity proteomics reveal EPH tyrosine kinase receptors' regulation of cell polarity and identify Par3 scaffold protein as a novel effector of EPH signaling

The EPH family is the largest among receptor tyrosine kinases (RTKs) in humans. In contrast to other RTKs, EPH receptors (EPHRs) cognate ligands, ephrins, are tethered to the cell surface. This results in EPHR-ephrin signaling being mainly involved in short-range cell-cell communication events that regulate cell adhesion, migration and tissue boundary formation. Although EPHRs functions have been broadly studied, the molecular mechanisms by which they control these processes are far from being understood. To address this, we sought to identify new effector proteins acting downstream of EPHRs and determine their role in EPHR-regulated functions. To unravel EPHR-associated signaling complexes under native conditions, we applied a mass spectrometry-based approach, namely BioID proximity labeling. We obtained a composite proximity network from EPHA4, -B2, -B3 and -B4 receptors that comprises 395 proteins, most of which were not previously linked to EPH signaling. A gene ontology and pathway term analysis of the most common candidates highlighted cell polarity as a novel function associated with EPHR activity. We found that EPHA1 and EPHB4 expression is restricted to the basal and lateral membrane domains in polarized Caco-2 3D spheroidal cell cultures. We further discovered that their depletion impairs the compartmentalized distribution of polarity proteins as well as overall spheroid morphogenesis. Moreover, we examined the contribution of a number of candidates, selected from EPHR proximity networks, via loss-of-function in an EPHR-dependent cell segregation assay. We found that depletion of the signaling scaffold PAR-3 blocks cell sorting. We also delineated a signalling complex involving the C-terminal SRC kinase (CSK), whose recruitment to PAR-3 complexes is dependent on EPHR signals. Our work sheds a new light on EPHR signaling networks and describes conceptually novel the mechanisms by which EPHRs signal at the membrane to contribute to the regulation of cellular phenotypes.

molecular biology↗

bioRxiv2020

The specificity of SRC Homology 3 (SH3) domain interactions is modulated by their protein context in vivo

Protein-protein interactions (PPIs) between modular binding domains and their target peptide motifs are thought to largely depend on the intrinsic binding specificities of the domains. By combining deletion, mutation, swapping and shuffling of SRC Homology 3 (SH3) domains and measuring their impact on protein interactions, we find that most SH3s do not autonomously dictate PPI specificity in vivo. The identity of the host protein and the position of the SH3 domains within their host are both critical for PPI specificity, for cellular functions and for key biophysical processes such as phase separation. Our work demonstrates the importance of the interplay between a modular PPI domain such as SH3 and its host protein in establishing specificity to wire PPI networks.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2019

JMJD6 participates in the maintenance of ribosomal DNA integrity in response to DNA damage

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 as rDNA repeats instability has been reported in cancer, aging and neurological diseases. Here, we describe that the histone demethylase JMJD6 is rapidly recruited at nucleolar DNA damage and is crucial for the relocalisation of rDNA in nucleolar caps. Yet, JMJD6 is dispensable for rDNA transcription inhibition. Mass spectrometry study 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 reveals that rDNA transcription inhibition is uncoupled from rDNA relocalisation into nucleolar caps and that JMJD6 is required for rDNA stability upon the rDNA damage response through its role in nucleolar caps formation.\n\nAuthor summaryRibosomal DNA is the most transcribed genomic region composed of repeated sequences. Transcribed rDNA is essential for cellular homeostasis and cell proliferation. Numerous pathologies such as cancer and neurological disorders are described to present defective rDNA repeats maintenance. The mechanisms involved in the control of rDNA integrity involve major DNA repair pathways such as NonHomologous End-Joining and Homologous Recombination. However, how they are controlled and orchestrated is poorly understood. Here, we identified JMJD6 as a new member of the maintenance of rDNA integrity. We observed that JMJD6 controls the recruitment of NBS1 in the nucleolus in order to lead to the proper response to DNA damage at rDNA repeats.\n\nAuthor contributionsYC and DT wrote the manuscript with input from their coauthors. JF, CC, JH, S-KM, JL, J-PL and YC performed the experiments. JC supervised JF and JH during purification and mass spectrometry analysis. YC and DT conceived the project, designed research and coordinated the studies.

cell biology↗

bioRxiv2019

A proximity biotinylation map of a human cell

Compartmentalization is a defining characteristic of eukaryotic cells, partitioning cellular processes into discrete subcellular locations. High throughput microscopy1 and biochemical fractionation coupled with mass spectrometry2-6 have helped to define the proteomes of a variety of organelles and macromolecular structures. However, many other intracellular compartments have remained refractory to such approaches, due for example to difficulty in purifying non-membrane bound structures. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach for defining the composition of cellular compartments in living cells7-10. Here we present a BioID-based map of a human cell based on 192 markers from 32 different subcellular compartments, comprising 35,902 high confidence proximity interactions, and defining the intracellular locations of 4,145 unique proteins in HEK 293 cells. Our localization predictions meet or exceed previous-approaches, with higher specificity, and enabled the discovery of proteins at the mitochondrial outer membrane-endoplasmic reticulum (ER) interface that are critical for mitochondrial homeostasis. Based on this dataset, we have established humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to better understand BioID datasets.

molecular biology↗