Dppa2 and Dppa4 directly regulate the Dux-driven zygotic transcriptional program

Abstract

The molecular regulation of zygotic genome activation (ZGA) in mammals remains an exciting area of research. Primed mouse embryonic stem cells contain a rare subset of "2C-like" cells that are epigenetically and transcriptionally similar to the two-cell embryo and thus represent an in vitro approximation for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA in either a Dux-dependent or Dux-independent manner. Here we performed a candidate-based overexpression screen, identifying, among others, developmental pluripotency-associated 2 (Dppa2) and Dppa4 as positive regulators of 2Clike cells and transcription of ZGA genes. In the germline, promoter DNA demethylation coincides with expression of Dppa2 and Dppa4, which remain expressed until embryonic day 7.5 (E7.5), when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during induced pluripotent stem cell (iPSC) reprogramming at the time that 2C-like transcription transiently peaks. Through a combination of overexpression, knockdown, knockout, and rescue experiments together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we teased apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilized. Dppa2 and Dppa4 require Dux to initiate 2C-like transcription, suggesting that they act upstream by directly regulating Dux. Supporting this, ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild-type cells but, in contrast to Dux, can no longer do so in Dppa2/4 double-knockout cells, suggesting that it may act to stabilize rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux up-regulation and activation of the 2C-like transcriptional program, which is subsequently reinforced by Zscan4c.

A distinct metabolic state arises during the emergence of 2‐cell‐like cells

“…Two-cell-like cells emerge from cells that express the transcription factor Zscan4 (Zscan4 + cells) [16], which are yet another subpopulation of ESC cultures constituting approximately 5% of the cell population [17,18]. Early-embryonic-like cells (Zscan4 + and 2-cell-like cells) can be induced in culture through the modulation of specific chromatin pathways, including the chromatin assembly factor 1 (CAF-1) [15] and the non-canonical polycomb repressive complex PRC1.6 [16,19], as well as the transcription factors Dux and Dppa2/4 [12,[20][21][22].…”

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

“…Recent studies have shown that a ZGA-like state can be mimicked in mouse ESCs (Macfarlan et al 2012;Zalzman et al 2010;Bošković et al 2014;Ishiuchi et al 2015;Akiyama et al 2015;Eckersley-Maslin et al 2016;Rodriguez-Terrones et al 2018). Consequently, these cells represent an ideal system for in vitro screening and have been previously used to identify regulators of ZGA (Fu et al 2019;Yan et al 2019;Eckersley-Maslin et al 2019;Rodriguez-Terrones et al 2018). While most of these studies probing ZGA regulators in ESCs have focused on repressors (Fu et al 2019;Rodriguez-Terrones et al 2018), positive inducers of ZGA have thus far not been interrogated in a systematic manner.…”

“…While most of these studies probing ZGA regulators in ESCs have focused on repressors (Fu et al 2019;Rodriguez-Terrones et al 2018), positive inducers of ZGA have thus far not been interrogated in a systematic manner. Such regulators are more relevant given the transcriptionally inactive state prior to ZGA and could be identified in ESCs by assessing the transcriptional changes triggered downstream of their overexpression (Eckersley-Maslin et al 2019).…”

A novel microRNA-based strategy to expand the differentiation potency of stem cells

“…RNA sequencing of these iPSC clones as well as wild-type ESCs was analyzed one month after miR-203 induction ( Fig 1B) and surprisingly revealed that miiPSCs cells were transcriptionally closer to ESCs than Dox-treated wild-type iPSCs both at the genomewide level ( Fig 1C) and when considering a pluripotency-associated signature defined previously 15 (Fig EV1D). In addition, almost every transcript included in a 282-gene signature of 2C blastomeres 16 was induced by miR-203 and sustained during ten days after Dox withdrawal ( Fig 1D and EV1E), including genes such as Zscan4c previously related to the zygotic transcriptional program 17 . An additional comparison of the expression profiles in miiPSCs versus un-induced iPSCs at day 30 after doxycycline withdrawal suggested higher expression of genes related to tissue morphogenesis and embryonic development in those clones in which miR-203 had been transiently induced ( Fig 1B, EV1F and Supplementary Table 1).…”