Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Aug;31:83-94.
doi: 10.1016/j.scr.2018.07.009. Epub 2018 Jul 19.

Genomic Functions of Developmental Pluripotency Associated Factor 4 (Dppa4) in Pluripotent Stem Cells and Cancer

Affiliations
Free PMC article

Genomic Functions of Developmental Pluripotency Associated Factor 4 (Dppa4) in Pluripotent Stem Cells and Cancer

Rachel Herndon Klein et al. Stem Cell Res. .
Free PMC article

Abstract

Developmental pluripotency associated factor 4 (Dppa4) is a highly specific marker of pluripotent cells, and is also overexpressed in certain cancers, but its function in either of these contexts is poorly understood. In this study, we use ChIP-Seq to identify Dppa4 binding genome-wide in three distinct cell types: mouse embryonic stem cells (mESC), embryonal carcinoma cells, and 3T3 fibroblasts ectopically expressing Dppa4. We find a core set of Dppa4 binding sites shared across cell types, and also a substantial number of sites unique to each cell type. Across cell types Dppa4 shows a preference for binding to regions with active chromatin signatures, and can influence chromatin modifications at target genes. In 3T3 fibroblasts with enforced Dppa4 expression, Dppa4 represses the cell cycle inhibitor Cdkn2c and activates Ets family transcription factor Etv4, leading to alterations in the cell cycle that likely contribute to the oncogenic phenotype. Dppa4 also directly regulates Etv4 in mESC but represses it in this context, and binds with Oct4 to a set of shared targets that are largely independent of Sox2 and Nanog, indicating that Dppa4 functions independently of the core pluripotency network in stem cells. Together these data provide novel insights into Dppa4 function in both pluripotent and oncogenic contexts.

Keywords: Cell cycle; Chromatin; Dppa4; Histone deacetylase; Oct4; Oncogene; Pluripotent stem cells.

Conflict of interest statement

Disclosure of potential conflicts of interest

The authors have no conflicts of interest to declare.

Figures

Fig. 1.
Fig. 1.
Patterns of Dppa4 binding in E14 mESC, Dppa4 overexpressing 3T3 fibroblasts, and P19 embryonal carcinoma cells. A) Overlap of Dppa4 peaks between 3T3, P19, and E14 cells. B) Heatmap plot of E14, P19, and 3T3 ChIP-Seq reads centered on E14 Dppa4 peaks. C) Comparison of genomic distribution of Dppa4 peaks in E14, P19, and 3T3 cells. D) Dppa4 ChIP-Seq peaks at the Etv4 promoter.
Fig. 2.
Fig. 2.
Dppa4 regulates cell cycle and transcription factor targets in 3T3. A) Overlap of genes downregulated by Dppa4 overexpression in 3T3 and Dppa4 ChIP-Seq peaks in Dppa4 overexpressing 3T3. Hypergeometric distribution was used to calculate probabilities. B) Overlap of genes upregulated by Dppa4 overexpression in 3T3 and Dppa4 ChIP-Seq peaks in Dppa4 overexpressing 3T3. Hypergeometric distribution was used to calculate probabilities. C) Significant gene ontology categories for Dppa4-repressed direct targets. D) Significant gene ontology categories for Dppa4-activated targets. E) qPCR validation of gene expression changes in select Dppa4 targets, n = 4. *p < .05, **p < .01. F) Dppa4 ChIP-qPCR validation of select Dppa4 targets, n = 3. Error bars are the S.E.M.
Fig. 3.
Fig. 3.
Dppa4 influences levels of histone modifications at target genes. A) ChIP-qPCR of H3K4me3 levels at Dppa4 targets in WT and Dppa4 overexpressing 3T3, n = 3. t-test. B) ChIP-qPCR of H3K27ac levels at Dppa4 targets in WT and Dppa4 overexpressing 3T3, n = 3. t-test. *p < .05, **p < .01. Error bars are the S.E.M.
Fig. 4.
Fig. 4.
Characterization of biological impact of downstream targets of Dppa4 in 3T3: Cdkn2c and Etv4. A) Expression of Cdkn2c in WT and Dppa4 expressing 3T3 after transduction and selection for ectopic Cdkn2c expression, compared to WT empty vector (EV) control n = 4. B) Expression of Etv4 in WT and Dppa4 expressing 3T3 after transduction and selection for ectopic Etv4 expression, compared to WT empty vector control n = 4. C) Expression of Etv4 in WT and Dppa4 expressing 3T3 after siRNA knockdown, compared to negative siRNA control in WT 3T3 n = 3. D) Expression of Cdkn2c in WT 3T3 overexpressing Etv4 compared to empty vector control, n = 4. E) Count of number of cells 20 h and 48 h after seeding, n = 3. ANOVA with Post-hoc Tukey HSD test. F) Propidium iodide cell cycle analysis, n = 6. ANOVA with Post-hoc Tukey HSD test. G) Propidium iodide cell cycle analysis, n = 6. ANOVA with Post-hoc Tukey HSD test. *p < .05, **p < .01. Error bars are the S.E.M.
Fig. 5.
Fig. 5.
Dppa4 also regulates cell cycle and transcription factor targets in E14 mESCs. A) Overlap of genes downregulated by Dppa4 disruption in E14 mESC and Dppa4 ChIP-Seq peaks in WT E14 cells. Hypergeometric distribution was used to calculate probabilities. B) Overlap of genes upregulated by Dppa4 knockout in E14 mESC and Dppa4 ChIP-Seq peaks in WT E14 cells. Hypergeometric distribution was used to calculate probabilities. C) Significant gene ontology categories for Dppa4-activated direct targets. D) Significant gene ontology categories for Dppa4-repressed direct targets. E) Dppa4 ChIP-qPCR validation of select Dppa4 targets, n = 2 (independently-generated knockout lines). F) qPCR validation of gene expression changes in select Dppa4 targets, n = 2 (independently-generated knockout lines). t-test. *p < .05, **p < .01. Error bars are the S.E.M.
Fig. 6.
Fig. 6.
Dppa4 influences levels of histone modifications at target genes in E14s. A) ChIP-qPCR of H3K27ac levels at Dppa4 targets in WT and Dppa4−/− E14, n = 2. B) ChIP-qPCR of H3K4me3 levels at Dppa4 targets in WT and Dppa4−/− E14, n = 2. t-test. *p < .05, **p < .01. Error bars are the S.E.M.
Fig. 7.
Fig. 7.
Dppa4 binding overlaps with OCT4 in E14 mESC. A) Plot of OCT4, SOX2, and NANOG ChIP-Seq reads at E14 Dppa4 peaks. B) Overlap of OCT4 and Dppa4 peaks. C) Significantly enriched KEGG Pathways at genes bound by both OCT4 and Dppa4. D) ChIP-qPCR validation of OCT4 binding at OCT4 targets (Oct4, Nanog) and at OCT4-Dppa4 shared targets. E) Endogenous OCT4-DPPA4 Co-immunoprecipitation in NT2 cells. F) Overlap of histone modifications with OCT4-Dppa4 co-bound sites, relative to sites only bound by Dppa4. Error bars are the S.E.M.

Similar articles

See all similar articles

Cited by 1 article

Publication types

MeSH terms

Feedback