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

Zhijun Qiu, Zeinab Elsayed, Veronica Peterkin, Suehyb Alkatib, Dorothy Bennett, Joseph W. Landry

Understanding 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. Ino80 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. In 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.