Kougnassoukou Tchara, P.-E.

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↗