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The H2BG53D Oncohistone Directly Upregulates ANXA3 Transcription and Enhances Cell Migration in Pancreatic Ductal Adenocarcinoma

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The H2BG53D Oncohistone Directly Upregulates ANXA3 Transcription and Enhances Cell Migration in Pancreatic Ductal Adenocarcinoma Signal Transduction and Targeted Therapy www.nature.com/sigtrans LETTER OPEN The H2BG53D oncohistone directly upregulates ANXA3 transcription and enhances cell migration in pancreatic ductal adenocarcinoma Signal Transduction and Targeted Therapy (2020) 5:106; https://doi.org/10.1038/s41392-020-00219-2 Dear Editor, when compared with the randomly shuffled peaks with the same Histones are essential proteins in compacting genomic DNA length of G53D peaks (Fig. 1f). A significant number of the and regulating gene expression. Previous studies on histone H3 promoter-bound G53D peaks co-localized with open chromatin oncohistones in pediatric brain cancers1,2 and chondroblastoma3, regions (Fig. 1g) as revealed by ATAC-seq (Fig. 1e), suggesting that documented the transcriptomic reprogramming through the these G53D-H2B enriched regions might impact gene transcrip- alterations of histone modifications. We recently reported the tion. However, the majority of the G53D peaks at intergenic region identification of a novel cancer associated mutation, the H2BG53- and gene bodies are not located at open chromatin regions, to-D in pancreatic ductal adenocarcinoma (PDAC)4. We showed indicating that the G53D mutant H2B might have additional that the H2BG53D mutation weakens the interaction between effects on the chromatin. When correlating the genomic distribu- nucleosomal DNA and histone octamer, subsequently enhances tion of G53D peaks (Fig. 1e–g) to gene expression changes 1234567890();,: transcription in vitro. We further showed that cells expressing the (Fig. 1a–d), we found that only a small proportion of the genes G53D mutant H2B acquired oncogenic phenotypes in our CRISPR- with promoter enrichment of G53D had increased gene expres- Cas9 knock-in model. However, the mechanism by which sion (3 out of 253), suggesting that the promoter-G53D H2BG53D mutation promotes PDAC remains unknown. enrichment alone is insufficient to influence transcription. To To examine the effect of the H2BG53D mutation on gene elucidate whether locus-wide enrichment of G53D-H2B is required expression we conducted RNA-seq using the CRISPR knock-in for altering transcription in vivo, we identified genes showing H2BG53D mutant and isogenic wild-type clones4. We identified differential occupancy of FLAG-tagged histones between wild- 280 upregulated and 262 downregulated genes (Fig. 1a) which are type and H2BG53D mutant cells from 1 kb upstream of TSS to TES. associated with growth factor signaling, cell adhesion, and GESA analysis showed that the 99 genes with elevated gene pathways involved in cancer development (Fig. 1b). Since G53D expression (detected by RNA-seq) and transcription (detected by mutation weakens the DNA–histone interaction, we postulated PRO-seq) (Supplementary Fig. 1) positively correlated with that the increase in gene expression is a consequence of elevated H2BG53D enrichment (Fig. 1h), suggesting that the G53D mutant transcriptional activity at genes containing G53D mutant H2B. We H2B directly promotes transcription of these targets in vivo. More mapped active RNA polymerases by precision nuclear run-on specifically, 31 genes show locus-wide differential enrichment of sequencing (PRO-seq) and identified 559 genes with elevated G53D-H2B, indicating that the locus-wide, rather than local PRO-seq signals and 426 genes with reduced signals (Fig. 1c). On a enrichment of G53D-H2B at promoter, impacts target gene genome-wide scale, we observed significant correlation between transcription (Fig. 1h and Supplementary Table 1). Gene ontology differential gene expression and Pol II occupancy alteration analysis of the RNA-seq data showed that genes involved in between H2BG53D mutant and wild-type cells (Fig. 1d). Gene pathways related to cell migration, such as cell adhesion and focal set enrichment analyses (GSEA) revealed that genes with adhesion, are differentially expressed in the H2BG53D mutant cells significant upregulation have a remarkable increase in Pol II (Fig. 1b), suggesting that the H2BG53D mutation might target cell occupancy in PRO-seq (Supplementary Fig. 1a) and vice versa migration related phenotypes in PDAC. For this reason, among the (Supplementary Fig. 1b). 31 candidates we focused on the genes that are involved in cell Compared to wild-type cells, a total of 99 genes showed migration and cancer metastasis. We found that ANXA3,an significantly elevated signals in both RNA-seq and PRO-seq in oncogene that has been shown to play important roles in mutant cells (Fig. 1d and Supplementary Table 1). This group of metastasis, is overexpressed in lung, liver5 and ovarian carcino- genes is of interest because their upregulation is likely accoun- mas. However, the functional significance and the clinical table to changes on the transcription level. On the other hand, relevance of ANXA3 in PDAC remain elusive. Hence, we selected increased mRNA level without changes in Pol II occupancy (181 ANXA3 and investigated its function in PDAC in the context of the genes), or vice versa (460 genes), indicate the involvement of H2BG53D mutation. post-transcriptional control (Supplementary Fig. 1c). The G53D-H2B occupancy at ANXA3 was revealed by FLAG To determine the direct effect of the H2BG53D mutation on CUT&RUN (Fig. 1i and Supplementary Fig. 2a) and confirmed by gene regulation, we mapped the genomic localization of the independent CUT&RUN assays (Supplementary Fig. 2b) as well G53D-H2B in CRISPR knock-in cell lines4 by FLAG CUT&RUN assays as MNase FLAG ChIP-qPCR (Supplementary Fig. S2c). The while using H2A CUT&RUN as internal control (Fig. 1e). We elevated expression of ANXA3 (Supplementary Fig. 3a) was identified 928 G53D-specific peaks by differential occupancy confirmed by RT-qPCR and western blot (Supplementary Fig. 3b, analysis compared to wild-type cells, 253 of which reside in the c). To investigate the direct effect of the G53D mutant H2B on promoter region, showing a significant difference (p < 2.2e−16) ANXA3 transcription in vivo, H2BG53D cells were treated with Received: 8 March 2020 Revised: 25 May 2020 Accepted: 13 June 2020 © The Author(s) 2020 Letter 2 a b c d 2 2 1 1 0 0 −1 −1 −2 −2 PRO-seq log2FC G53D_3_R1 WT_7_R2 G53D_3_R2 G53D_32_R1 G53D_32_R2 WT_7_R1 WT_16_R1 WT_16_R2 G53D_32_R1 G53D_32_R2 G53D_3_R1 G53D_3_R2 WT_7_R1 WT_7_R2 WT_16_R1 WT_16_R2 RNA-seq log2FC e p13.3 p13.2 p13.12 p12.3 p12.2 p12.1 p11.2 p11.1 q11.2 q12.1 q12.2 q21 q22.1 q22.2 q23.1 q23.2 q24.1 Chromosome 16 f [0-60] parental p < 2.2e-16 WT_7 [0-60] [0-60] 100 WT_16 [0-60] FLAG G53D_3 [0-60] 80 G53D_32 [0-150] parental 60 [0-150] WT_7 [0-150] 40 WT_16 Downstream H2A [0-150] Gene body G53D_3 [0-150] 20 Intergenic G53D_32 Promoter [0-88] WT_7 0 [0-88] Shuffle WT_16 [0-88] G53D peaks peaks G53D_3 Percentage of H2BG53D specific peaks [0-88] ATAC-seq G53D_32 PHLPP2 AP1G1 ATXN1L ZNF821 IST1 g ATAC−seq overlap h i q21.21 q21.3 q22.2 q23 q24 q25 q26 Non overlap Chromosome 4 15.8% [0-50] parental 11.4% [0-50] WT_7 [0-50] WT_16 85.0% FLAG [0-50] G53D_3 [0-50] Peak Numbers G53D_32 [0-200] parental 16.7% [0-200] WT_7 Downstream Gene body Intergenic Promoter [0-200] 3/18 34/297 57/360 215/253 metric list Ranked score(ES) Enrichment Rank in CUT&RUN log2 fold enrichment WT_16 H2A [0-200] G53D_3 ANXA3 SNAP47 j (58.9 kb) (53.1 kb) [0-200] G53D_32 Ex2-In2 Ex3-In3 Ex7-In7 Ex12-In12 Ex2-In2 In6-Ex7 In6-Ex7 [0-50] WT_7 [0-50] WT_16 1.0 Ex2-In2 1.5 Ex7-In7 0.8 Ex2-In2 1.5 In6-Ex7 * * [0-50] 0.8 WT 16 WT 16 0.6 WT 16 WT 16 G53D_3 G53D 32 1.0 G53D 32 G53D 32 1.0 G53D 32 0.6 ATAC-seq 0.4 [0-50] 0.4 G53D_32 0.5 0.5 ** 0.2 0.2 0 0 0 0 Pre-mRNA expression 0 5 10 20 40 0 5 10 20 40 0 5 10 20 40 Pre-mRNA expression 05102040 ANXA3 Time post-DRB (min) Time post-DRB (min) Time post-DRB (min) Time post-DRB (min) 3 Ex3-In3 ** 1.0 Ex12-In12 ** 2.5 Ex5-In5 k 0.8 2.0 WT 16 WT 16 WT 16 2 G53D 32 0.6 G53D 32 1.5 G53D 32 WT #7 WT #16 G53D #3 G53D #32 * 0.4 1.0 1 r h 0.2 0.5 0 0 0 0.0 0 5 10 20 40 0 5 10 20 40 0 5 10 20 40 NT Pre-mRNA expression Pre-mRNA expression Pre-mRNA expression Pre-mRNA expression Pre-mRNA expression Time post-DRB (min) Time post-DRB (min) Time post-DRB (min) r h 6 m l 20 Gap closure assay 1 * r * h WT 15 0 hr 0 * sh#1 ** 16 hr Pol II r h 10 6 ANXA3 1 H2BG53D 5 r by migrated cells % Area covered h 0 Pol II sh#2 0 2 r h ANXA3 WT H2B 6 ANXA3 G53D–H2B 1 NT WT#7NT WT#16 sh1 WT#7 sh2 WT#7 NT G53D#3 sh1 sh1WT#16 G53D#3 sh2 sh2WT#16 G53D#3 NT G53D#32 sh1 G53D#32 sh2 G53D#3 Signal Transduction and Targeted Therapy (2020) 5:106 Letter 3 Fig. 1 a Heat map showing the differential expression of genes in two wild-type and two H2BG53D mutant clones with two repeats. b Enrichment map of gene sets significantly overrepresented for differentially expressed genes between H2BG53D mutant and wild-type clones (Benjamini–Hochberg adjusted p < 0.05, hypergeometric tests). c Heat map showing the gene loci with differential Pol II occupancy. d Scatter plot illustrating the significant genome-wide correlation between RNA-seq and PRO-seq (Pearson correlation coefficient: 0.37, p < 2.2e−16).
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