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[Preprint]. 2023 Apr 20:2023.04.18.537367.
doi: 10.1101/2023.04.18.537367.

Cell state-dependent chromatin _targeting in NUT carcinoma

Artyom A Alekseyenko  1   2   3 , Barry M Zee  1   2   4 , Zuzer Dhoondia  1   2 , Hyuckjoon Kang  1   2 , Jessica L Makofske  1   2   5 , Mitzi I Kuroda  1   2
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Cell state-dependent chromatin _targeting in NUT carcinoma

Artyom A Alekseyenko et al. bioRxiv. .

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Abstract

Aberrant transcriptional programming and chromatin dysregulation are common to most cancers. Whether by deranged cell signaling or environmental insult, the resulting oncogenic phenotype is typically manifested in transcriptional changes characteristic of undifferentiated cell growth. Here we analyze _targeting of an oncogenic fusion protein, BRD4-NUT, composed of two normally independent chromatin regulators. The fusion causes the formation of large hyperacetylated genomic regions or megadomains, mis-regulation of c-MYC , and an aggressive carcinoma of squamous cell origin. Our previous work revealed largely distinct megadomain locations in different NUT carcinoma patient cell lines. To assess whether this was due to variations in individual genome sequences or epigenetic cell state, we expressed BRD4-NUT in a human stem cell model and found that megadomains formed in dissimilar patterns when comparing cells in the pluripotent state with the same cell line following induction along a mesodermal lineage. Thus, our work implicates initial cell state as the critical factor in the locations of BRD4-NUT megadomains. These results, together with our analysis of c-MYC protein-protein interactions in a patient cell line, are consistent with a cascade of chromatin misregulation underlying NUT carcinoma.

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Conflict of interest statement

Conflicts of Interest

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. Induction of megadomains in HUES64 human embryonic stem cells.
A. Model for formation of BRD4-NUT megadomains. B. Strategy for comparison of megadomain induction following transfection of HUES64 cells maintained in a pluripotent state or after differentiation along a mesodermal lineage. C. Anti-NUT immunostaining after transient transfection of HUES64 cells with a BRD4-NUT expression plasmid or a pUC19 negative control (400x magnification). Formation of NUT nuclear foci is dependent on expression of the BRD4-NUT transgene.
Figure 2.
Figure 2.. Comparison of BRD4-NUT megadomains by ChIP-seq.
A. Top: Human chromosome 8 IGV browser view of log2 IP/Input BRD4-NUT read counts from ChIP-seq experiments performed in HUES64 (red) and derived mesoderm dME (green). Middle: Boxed regions are enlarged, showing the strong concordance between independent biological replicates. Bottom: Examples of cell type-specific megadomains in hESC and dME are indicated along with their respective genomic sizes. Transcription units are displayed below the ChIP-seq profiles. B. Venn diagrams showing the strong reproducibility of independent biological replicates of the same cell state, and the divergence when comparing the same cell line in different cell states. C. IGV browser view comparing differences between cell states in a 3 Mb region of chromosome 4. D. Metagene profiles and heatmaps of BRD4-NUT megadomains calculated as log2 ChIP/Input. The x-axis shows megadomains scaled to the average size of 180 Kb, flanked by linear 100 Kb segments of upstream and downstream sequences. Top: Each row of the heatmap represents a megadomain sorted in descending order for BRD4-NUT occupancy in HUES64, revealing many HUES64-specific megadomains (left) along with a few that overlap with dME megadomains (right). Bottom: Heat map of dME-specific megadomains sorted in descending order (right) with the corresponding regions of HUES64 (right).
Figure 3.
Figure 3.. Comparison of BRD4-NUT ChIP-seq of the N-MYC, c-MYC and L-MYC loci.
N-MYC, c-MYC, and L-MYC IGV browser views of log2 IP/Input BRD4-NUT read counts from ChIP-seq experiments performed in A. HUES64 and derived mesoderm dME, and B. Ntera2 cells at 0, 4, and 16 hours of retinoic acid induction, which causes differentiation toward a neural lineage. Both N- and c-MYC have large adjacent regulatory regions encompassing non-coding RNA transcripts (such as GACAT3 and PVT1) but lacking additional protein-coding genes, while L-MYC resides in a region with many protein-coding genes. Colored bars below the ChIP profiles indicate megadomains (>96 Kb contiguous occupancy of BRD4-NUT).
Figure 4.
Figure 4.. MYC protein interactions in NC797 cells.
A. Scatterplot depicting enrichment over input of proteins from N-terminally tagged c-MYC using BioTAP-XL affinity purification in NC797 cells. Each gray dot represents an individually identified protein, where its position is a measure of enrichment efficiency based on the number of total peptides identified in pulldown compared to input and normalized to the molecular weight of the protein. The dashed gray lines denote the 99th percentile of enrichment. The right plot is a zoomed in region of the most enriched proteins in the left plot from replicate experiments. B. Comparison of the total peptides mapping to NuA4 and STAGA components in HEK293T and NC797 cells following BioTAP-XL of c-MYC.
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