Combined gene dosage requirement for SWI/SNF catalytic subunits during early mammalian development

Abstract

Mammalian SWI/SNF complexes utilize either BRG1 or BRM as alternative catalytic subunits with DNA-dependent ATPase activity to remodel chromatin. Although the two proteins are 75 % identical, broadly expressed, and have similar biochemical activities in vitro, BRG1 is essential for mouse embryonic development, while BRM is dispensable. To investigate whether BRG1 and BRM have overlapping functions during mouse embryogenesis, we performed double-heterozygous intercrosses using constitutive null mutations previously created by gene targeting. The progeny of these crosses had a distribution of genotypes that was significantly skewed relative to their combined gene dosage. This was most pronounced at the top and bottom of the gene dosage hierarchy, with a 1.5-fold overrepresentation of Brg1 (+/+) ;Brm (+/+) mice and a corresponding 1.6-fold underrepresentation of Brg1 (+/-) ;Brm (-/-) mice. To account for the underrepresentation of Brg1 (+/-) ;Brm (-/-) mice, timed matings and blastocyst outgrowth assays demonstrated that ~50 % of these embryos failed to develop beyond the peri-implantation stage. These results challenge the idea that BRG1 is the exclusive catalytic subunit of SWI/SNF complexes in ES cells and suggest that BRM also interacts with the pluripotency transcription factors to facilitate self-renewal of the inner cell mass. In contrast to implantation, the Brm genotype did not influence an exencephaly phenotype that arises because of Brg1 haploinsufficiency during neural tube closure and that results in peri-natal lethality. Taken together, these results support the idea that BRG1 and BRM have overlapping functions for certain developmental processes but not others during embryogenesis.

Fig. 1

Runting of Brg1^+/−;Brm^−/− mice. Weight of weaning-age mice from 14 litters of double-heterozygous intercrosses. The weights of Brg1^+/−;Brm^−/− mice are compared to the other 5 genotypes.

Fig. 2

Brg1^+/−;Brm^−/− blastocysts give rise to an increased percentage of abnormal outgrowths. (A, B) Brightfield photographs of normal (A) and abnomal (B) blastocyst outgrowths after 7 days in culture. (A) Normal blastocysts outgrowths hatched from the zona pellucida (ZP), the trophectoderm (TE) attached to and spread out across the bottom of the tissue culture wells, and the inner cell masses (ICM) underwent extensive cell proliferation. (B) Abnormal blastocyst outgrowths failed to hatch from the zona pellucida and died. Pyknotic blastomeres are evident (arrow). (C) Normal and abnormal blastocyst outgrowths for each of the 6 genotypic categories. The percentage value in the abnormal column refers to the percentage of abnormal outgrowths.

Fig. 3

Characterization of J1 antibody binding to BRG1 and BRM. (A)Representative western blot of protein lysates from normal human fibroblasts (NHF1-hTERT) following depletion of BRG1 and BRM, alone and in combination, by RNAi using specific siRNAs as indicated at the top. The first lane is a non-targeted control (NTC) siRNA. Protein lysates were probed with the J1 antibody (top panel), antibodies specific for BRG1 (second panel) or BRM (third panel), and Tubulin (bottom panel) as a loading control. (B) Quantification of BRG1 and BRM levels from western blot above. Protein levels detected by J1, α-BRG1, and α-BRM in siNTC, siBRG1, siBRM, and siBRG1 + siBRM cells are shown. All values were normalized with α-Tubulin and are relative to the siNTC control (set at 100).