Ino80 is essential for proximal-distal axis asymmetry in part by regulating Bmp4 expression

Abstract

BackgroundUnderstanding how embryos specify asymmetric axes is a major focus of biology. While much has been done to discover signaling pathways and transcription factors important for axis specification, comparatively little is known about how epigenetic regulators are involved. Epigenetic regulators operate downstream of signaling pathways and transcription factors to promote nuclear processes, most prominently transcription. To discover novel functions for these complexes in axis establishment during early embryonic development, we characterized phenotypes of a mouse knockout (KO) allele of the chromatin remodeling Ino80 ATPase.ResultsIno80 KO embryos implant, but fail to develop beyond the egg cylinder stage. Ino80 KO embryonic stem cells (ESCs) are viable and maintain alkaline phosphatase activity, which is suggestive of pluripotency, but they fail to fully differentiate as either embryoid bodies or teratomas. Gene expression analysis of Ino80 KO early embryos by in situ hybridization and embryoid bodies by RT-PCR shows elevated Bmp4 expression and reduced expression of distal visceral endoderm (DVE) markers Cer1, Hex, and Lefty1. In culture, Bmp4 maintains stem cell pluripotency and when overexpressed is a known negative regulator of DVE differentiation in the early embryo. Consistent with the early embryo, we observed upregulated Bmp4 expression and down-regulated Cer1, Hex, and Lefty1 expression when Ino80 KO ESCs are differentiated in a monolayer. Molecular studies in these same cells demonstrate that Ino80 bound to the Bmp4 promoter regulates its chromatin structure, which correlates with enhanced SP1 binding. These results in combination suggest that Ino80 directly regulates the chromatin structure of the Bmp4 promoter with consequences to gene expression.ConclusionsIn contrast to Ino80 KO differentiated cells, our experiments show that undifferentiated Ino80 KO ESCs are viable, but fail to differentiate in culture and in the early embryo. Ino80 KO ESCs and the early embryo up-regulate Bmp4 expression and down-regulate the expression of DVE markers Cer1, Hex and Lefty1. Based on this data, we propose a model where the Ino80 chromatin remodeling complex represses Bmp4 expression in the early embryo, thus promoting DVE differentiation and successful proximal-distal axis establishment. These results are significant because they show that epigenetic regulators have specific roles in establishing embryonic axes. By further characterizing these complexes, we will deepen our understanding of how the mammalian embryo is patterned by epigenetic regulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0238-5) contains supplementary material, which is available to authorized users.

Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80

“…1b ). Constitutive Ino80 deletion causes embryonic lethality between E8.5 and E10.5 23 , 24 . We therefore leveraged a conditional Ino80 allele 24 to study Ino80 in corticogenesis.…”

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

“…We then asked whether we could observe physiological consequences induced by gene KO using polyclonal populations. The gene encoding the chromatin remodeler INO80 is an essential gene, whose loss leads to embryonic lethality and impairs cell proliferation [ 33 , 34 ]. In agreement, INO80 KO inhibited cell growth and 8 days after Cas9 induction we observed a 7-fold reduction in the number of viable cells compared with the uninduced control ( Fig.…”

Chromatin Remodelling Proteins and Cell Fate Decisions in Mammalian Preimplantation Development

“…Two more chromatin remodelling ATPase proteins that have been implicated in early embryonic development are the INO80 DNA helicase and the SNF2H (SMARCA5) ATPase (Lazzaro and Picketts 2001;Bao and Shen 2007;Gerhold and Gasser 2014), although what their functions might be in preimplantation development are not yet clear. Knockdown of INO80 in zygotes severely impaired blastocyst formation after in vitro culture (Wang et al 2014), but zygotic deletion of INO80 had no detrimental effects until gastrulation, with zygotic INO80-null blastocysts appear morphologically normal and appropriately express markers of epiblast and PrE (Lee et al 2014;Qiu et al 2016). These observations indicate either that maternally-contributed INO80 plays an essential role in early cleavage stages, or that the embryo is able to compensate for a genetic deficiency, but cannot similarly compensate for a knockdown (Rossi et al 2015).…”