Fig. 5

Ino80 localizes to Bmp4 promoter and regulates its expression during embryonic stem cell differentiation. a Quantification of Bmp4, Hex, Lefty1, Cer1, Gata6, and Gata4 expression by RT-qPCR during ESC differentiation in serum containing media without 2i + LIF in monolayer (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). b Cartoon showing the Bmp4 gene, location of upstream enhancers, and immediate downstream convergent gene Gm15217 position of PCR amplicons used in chromatin immunoprecipitation (ChIP) experiments are shown as black bars labeled 1-6. c Localization of Ino80 to regulatory elements of Bmp4 by chromatin immunoprecipitation (ChIP). ChIP was performed with wild-type and Ino80 KO ESC after differentiation as a monolayer for six days in serum containing media lacking 2i + LIF. Position of PCR amplicons are shown in panel a (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). d Cartoon showing the Bmp4 promoter and the location of PCR amplicons used in FAIRE experiments are shown as black bars labeled 1-5. e Changes in chromatin structure ~1.5 Kb upstream of Bmp4 in Ino80 KO ESC. FAIRE was performed with wild-type and Ino80 KO ESC after differentiation in serum containing media lacking 2i + LIF as a monolayer for six days. Position of PCR amplicons is shown in panel d (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). f Increased H3K4me3 occupancy at Bmp4 in Ino80 KO ESC. ChIP was performed with wild-type and Ino80 KO ESC after differentiation as a monolayer for six days in serum containing media lacking 2i + LIF. PCR amplicon 3 from panel b was used for ChIP (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). g Increased SP1 occupancy at Bmp4 in Ino80 KO ESC. ChIP was performed with wild-type and Ino80 KO ESC after differentiation in serum containing media lacking 2i + LIF as a monolayer for six days. PCR amplicon 3 from panel b was used for ChIP (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). h Model for Ino80 function during proximal-distal axis establishment. In wild-type embryos, Ino80 in part functions in the EmE to repress Bmp4 expression. Bmp4 repression in the EmE promotes DVE differentiation. In Ino80 KO embryos, Bmp4 is abnormally expressed in the EmE, which inhibits DVE differentiation. A lack of a DVE prevents the AVE from forming, which subsequently prevents gastrulation. ESC embryonic stem cell, LIF leukemia inhibitory factor, FAIRE formaldehyde assisted isolation of regulatory elements, KO knockout, EmE embryonic ectoderm, DVE distal visceral endoderm, AVE anterior visceral endoderm