cMYC protein interactions and chromatin association in NUT carcinoma

Abstract

The oncogene cMYC (HGNC:7553) is a critical genomic target of the BRD4-NUT (B4N) protein that defines many of the NUTM1-rearrangement cancer subtypes in NUT carcinoma (NC). Previously, we reported that B4N interacts with the EP300 lysine acetyltransferase (KAT3B) and creates hyperacetylated "megadomains" that activate downstream genes such as cMYC. Here we ask how misregulated cMYC in turn interacts with protein partners and target genes in patient-derived NC797 cells, and whether these interactions change in response to B4N inactivation. We used CRISPR-Cas9 mediated knock-in of a BioTAP affinity tag to analyze cMYC protein expressed from its normal chromosomal context. This allowed us to implement a crosslinking purification method termed BioTAP-XL to preserve cMYC integrity and chromatin association for genomic and mass spectrometry-based proteomic analyses. We found that in the NC797 cell line, cMYC interacts primarily with the NuA4 KAT5 lysine acetyltransferase complex, with interactions that are maintained despite a decrease in cMYC levels after JQ1 treatment. We propose that a cascade of aberrant acetyltransferase activities in NC797 cells, first via EP300 recruitment by B4N to mis-regulate cMYC, and then by KAT5 interaction with the resulting overexpressed cMYC protein, drive NC cell proliferation and blockade to differentiation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/489219v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@82ec09org.highwire.dtl.DTLVardef@1566d31org.highwire.dtl.DTLVardef@11ab4dborg.highwire.dtl.DTLVardef@57b741_HPS_FORMAT_FIGEXP M_FIG C_FIG Simple SummaryA longstanding goal in biology is to understand how protein interactions influence and reflect cellular disease states. MYC is a critical regulator of cellular proliferation that is mis-regulated in many cancers, including those with NUTM1-rearrangements featured in this issue. NUT carcinoma cells are dependent on MYC expression, which blocks differentiation. Using a crosslinking approach to identify MYC protein interactions in a NUTM1-rearranged patient cell line, we found that MYC interacts primarily with subunits of the NuA4 lysine acetyltransferase (KAT5), one of two KAT complexes previously discovered as MYC interactors in non-NUTM1 cell lines.