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Tumor suppressor functions of DAXX through histone H3.3 deposition pathway in pancreatic neuroendocrine tumor

ABSTRACT: Purpose: Pancreatic neuroendocrine tumors (PanNETs) have considerable malignant potential. Frequent somatic mutations and loss of DAXX protein expression have been frequently found in PanNETs. DAXX is known as a transcriptional repressor, however, molecular functions underlying loss of DAXX remain unclear in PanNETs. Experimental Design: We evaluated DAXX-knockdown (KD) and -knockout (KO) PanNET cells were analyzed for in vitro and vivo. The target genes were screened by microarray and chromatin immunoprecipitation (ChIP) assays for DAXX, histone H3.3 and H3K9me3 complex. Results: Microarray and ChIP analyses of DAXX-KD/KO identified 13 genes as the direct targets of DAXX transcriptional repressor. Among them, 5 genes were suppressed by DAXX/H3.3/H3K9me3 pathway. DAXX-KD/KO increased sphere forming activity, but it was suppressed by knockdown of the target gene. In xenograft models, tumorigenicity was significantly increased in DAXX-KO cells with high expression of the target. Clinically, higher recurrence was recognized in PanNETs with low expression of DAXX and high expression of the target than others. Conclusions: DAXX plays as a tumor suppressor and DAXX/H3.3 complex suppresses target genes by promoting H3K9me3 in PanNETs. DAXX and its target molecule might be effective biomarkers and therapeutic candidates.

ORGANISM(S): Homo sapiens

PROVIDER: GSE93050 | GEO | 2018/06/28

REPOSITORIES: GEO

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Project description:Endogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1 mediated H3K9me3 and DNA methylation. However, the protein network which regulates the deposition of these chromatin modifications is still incompletely understood. Here, we performed a genome-wide sgRNA screen for genes involved in ERV silencing and identified the GHKL ATPase protein Morc3 as a top-scoring hit. Morc3 knock-out cells display de-repression, reduced H3K9me3, and increased chromatin accessibility of distinct ERV families. We found that the Morc3 ATPase cycle and Morc3 SUMOylation are important for ERV chromatin regulation. Proteomic analysis revealed that Morc3 mutant proteins fail to interact with the histone H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation and Daxx SUMO binding. Notably, in Morc3 ko cells, we observed strongly reduced histone H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as a critical regulator of Daxx-mediated histone H3.3 incorporation to ERV regions. This dataset comprises several experiments addressing different questions: 1. ChIP-MS experiment to determine the protein interaction context of Morc3 using a Morc3-3xFLAG knock-in ES cell line compared to wild type ES cells (Experiment 20200408). 2. ChIP-MS experiments to investigate changes in the protein interaction context of the Morc3 mutant rescue cell lines. Comparison of Morc3 knock-out cell lines with re-expression of Morc3-CW-3xFLAG mutant (Ref. #3111), Morc3-ATP-binding-3xFLAG and Morc3-SUMOylation-3xFLAG mutants (Ref. #3635), and Morc3-deltaN-3xFLAG mutant (Ref. #5174) compared to wt Morc3-3XFLAG rescue. 3. ChIP-MS experiment to determine if the interaction between Morc3 and Daxx is mediated through this C-terminal SIM, comparing Daxx knock-out cell lines with re-expression of wild type 3xFLAG-Daxx protein or 3xFLAG-Daxx ∆SIM, which lacks the C-terminal SIM domain. (Ref. #3301)

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Tumor suppressor functions of DAXX through histone H3.3 deposition pathway in pancreatic neuroendocrine tumor

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