Synergistic activity of Nanog, Pou5f3, and Sox19b establishes chromatin accessibility and developmental competence in a context-dependent manner

Abstract

Genome-wide chromatin reprogramming is a fundamental requirement for establishing developmental competence in the newly-formed zygote. In zebrafish, Nanog, Pou5f3 and Sox19b play partially redundant roles in zygotic genome activation, however their interplay in establishing chromatin competency, the context in which they do so and their mechanism of action remain poorly defined. Here, we generated a triple maternal-zygotic nanog-/-;pou5f3-/-;sox19b-/- mutant and assessed the causal relationship between transcription factor (TF) occupancy, chromatin accessibility and genome activation. Analyses of this triple mutant and combinatorial rescues revealed highly synergistic and context-dependent activity of Nanog, Pou5f3, and Sox19b (NPS) in establishing chromatin competency at >50% of active enhancers. Motif analysis revealed a network of TFs that depend on NPS for establishing chromatin accessibility, including the endodermal determinant Eomesa, whose binding we show is regulated by NPS pioneer-like activity. Finally, we demonstrated that NPS play an essential role in establishing H3K27ac and H3K18ac at enhancers and promoters, and that their function in transcriptional activation can be bypassed by targeted recruitment of histone acetyltransferases to individual genes. Altogether, our findings reveal a large network of TFs that function to establish developmental competency, many of which depend on the synergistic and highly context-dependent role of NPS in establishing chromatin accessibility and regulating histone acetylation in order to activate the genome.

The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation

“…To identify the promoter autonomous sequence features that may inform on the regulation of miR-430 gene expression we have remapped the binding data of transcription factors previously described as regulators of the major wave of zebrafish genome activation on our new genome assembly (Lee et al , 2013; Leichsenring et al , 2013; Miao et al , 2020). Publicly available ChIP-seq datasets (Leichsenring et al , 2013; Xu et al , 2012) for these stem cell factors were mapped on the de novo assembled miR-430 contig and signal distribution analysed.…”

“…Previous studies have shown that miR-430 transcription requires H3K27ac deposition, binding of stem cell factors Sox19b/ SoxB1 Nanog and Pou5f1/Pou5f3 (Miao et al , 2020), and it is characterised by CTCF and Rad21 deployment (Chan et al , 2019; Meier et al , 2018; Miao et al , 2020) at a stage when topology associated domains have not yet formed (Kaaij et al , 2018; Wike et al , 2021). However, the question to answer remains: what is the mechanism which triggers miR-430 transcription and that of other minor wave transcribed genes?…”

“…However, the question to answer remains: what is the mechanism which triggers miR-430 transcription and that of other minor wave transcribed genes? While the stem cell factors Pou5f3/Pou5f1, SoxB1/Sox19b and Nanog were previously shown to be required for miR-430 gene activation (Lee et al , 2013; Miao et al , 2020), they are unlikely to selectively regulate minor wave genome activation, as a large number of major wave genes also depend on these factors (Gao et al , 2020; Lee et al , 2013; Miao et al , 2020; Palfy et al , 2020; Veil et al , 2019).…”

“…Additionally, the genomic locus of miR-430 genes is marked by CTCF and Rad21 (factors involved in chromatin looping and higher-order structure) (Meier et al , 2018), suggesting chromosome conformation mechanisms involved in its activation. MiR-430 activation is dependent on BRD4 and competent chromatin marked by histone H3K27 acetylation (ac) (Chan et al , 2019; Miao et al , 2020) and on the pioneer transcription factors (also called stem cell factors): Nanog, Pou5f3, Sox19b (Chan et al , 2019; Leichsenring et al , 2013; Miao et al , 2020). These stem cell factors have been shown to activate main wave of ZGA in fish (Lee et al , 2013; Leichsenring et al , 2013; Palfy et al , 2020; Veil et al , 2019) and homologs Pou5f3 and Sox3 in Xenopus (Gentsch et al , 2019), while their role in the minor wave of genome activation is not yet understood.…”

The DNA-to-cytoplasm ratio broadly activates zygotic gene expression in Xenopus

“…It is now apparent in several fast developing species that hundreds of genes are actively transcribed during cleavage divisions before the MBT, indicating that zygotic genome activation does not necessarily occur at the same time as other MBT events [10][11][12][13][14][15][16][17][18] . Different genes are activated at different times and are regulated by multiple maternally provided transcriptional activators [19][20][21][22] . Moreover, recent work has called into question the extent to which the N/C ratio influences transcription initiation in fish and frogs 21,23 .…”