Epigenetic regulation of histone modifications and Gata6 gene expression induced by maternal diet in mouse embryoid bodies in a model of developmental programming

Abstract

BackgroundDietary interventions during pregnancy alter offspring fitness. We have shown mouse maternal low protein diet fed exclusively for the preimplantation period (Emb-LPD) before return to normal protein diet (NPD) for the rest of gestation, is sufficient to cause adult offspring cardiovascular and metabolic disease. Moreover, Emb-LPD blastocysts sense altered nutrition within the uterus and activate compensatory cellular responses including stimulated endocytosis within extra-embryonic trophectoderm and primitive endoderm (PE) lineages to protect fetal growth rate. However, these responses associate with later disease. Here, we investigate epigenetic mechanisms underlying nutritional programming of PE that may contribute to its altered phenotype, stabilised during subsequent development. We use embryonic stem (ES) cell lines established previously from Emb-LPD and NPD blastocysts that were differentiated into embryoid bodies (EBs) with outer PE-like layer.ResultsEmb-LPD EBs grow to a larger size than NPD EBs and express reduced Gata6 transcription factor (regulator of PE differentiation) at mRNA and protein levels, similar to Emb-LPD PE derivative visceral yolk sac tissue in vivo in later gestation. We analysed histone modifications at the Gata6 promoter in Emb-LPD EBs using chromatin immunoprecipitation assay. We found significant reduction in histone H3 and H4 acetylation and RNA polymerase II binding compared with NPD EBs, all markers of reduced transcription. Other histone modifications, H3K4Me2, H3K9Me3 and H3K27Me3, were unaltered. A similar but generally non-significant histone modification pattern was found on the Gata4 promoter. Consistent with these changes, histone deacetylase Hdac-1, but not Hdac-3, gene expression was upregulated in Emb-LPD EBs.ConclusionsFirst, these data demonstrate ES cells and EBs retain and propagate nutritional programming adaptations in vitro, suitable for molecular analysis of mechanisms, reducing animal use. Second, they reveal maternal diet induces persistent changes in histone modifications to regulate Gata6 expression and PE growth and differentiation that may affect lifetime health.

Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo’s potential, affecting adult health

“…During later gestation, ex vivo LPD or Emb-LPD yolk sac visceral endoderm exhibited increased endocytosis of exogenous radiolabelled tracer, increased numbers of endocytic vesicles at the ultrastructural level and increased megalin protein expression (Watkins et al 2008a). Emb-LPD embryoid bodies grew to a larger size per unit time than NPD embryoid bodies, suggesting that Emb-LPD PE cells may also undergo increased proliferation (Sun et al 2015).…”

Sex and the preimplantation embryo: implications of sexual dimorphism in the preimplantation period for maternal programming of embryonic development

“…Thus, developmental programming during this time is inherent in the embryo and not the result of alteration in maternal function later in pregnancy. Among the changes in the preimplantation embryo that are likely responsible for modifications of postnatal phenotype are alterations in gene expression (Kwong et al 2006; Gad et al 2012; Calle et al 2012), the epigenome (Lucas, 2013; Chen et al 2013; Urrego et al 2014; Sun et al 2015) and patterns of embryonic growth and differentiation (Kwong et al 2000; Eckert et al 2012; Bromfield et al 2014). Such changes may be particularly profound during the preimplantation period because it is at this time that the basic patterns of development are being established, including re-establishment of DNA methylation marks (Rivera and Ross, 2013) and formation of the first differentiated cell lineages from which subsequent organs and tissues are derived.…”

Programmed for sex: Nutrition–reproduction relationships from an inter-generational perspective

“…When these resulting offspring grew up they 365 exhibited comparable dysfunction to offspring that had continued development in the uterus 366 of mothers who had been exposed peri-conceptually to a low protein diet (Watkins et al 367 2008). It appears that one early response to exposure of the mouse embryo to a low protein 368 diet is to cause increased proliferation of the trophectoderm and its outgrowths during 369 implantation (Eckert et al 2012) as well as functional changes to nutrient transport 370 mechanisms (Watkins et al 2008, Fleming et al 2015. Another study has shown that a 371 maternal low protein diet just around the time of conception results, in offspring, in excessive 372 ribosomal DNA (rDNA) transcription and ribosome biogenesis when nutrition after birth is 373 plentiful (Denisenko et al 2016), providing a plausible mechanism to explain increased risk 374 of obesity ('overgrowth') in such individuals.…”

“…Indeed, the authors suggest that rDNA is a 375 plausible candidate for a 'thrifty gene involved in nutrient ulitization control, which is tuned 376 by nutrient availability in utero'. Adaptive changes such as these are considered to sow the 377 seeds for metabolic and other dysfunctions in adulthood (Fleming et al 2015, Xu & Sinclair 378 2015. 379…”

“…The precise epigenetic mechanisms that underlie the various phenotypic changes in offspring 381 affected as a result of maternal peri-conceptual diet remain to be determined, but studies of 382 mouse embryoid bodies derived from cell lines from blastocysts from mothers exposed to 383 normal or low protein diets peri-conceptually, show diet-dependent histone changes that are 384 propagated during cell divisions, which may be involved (Sun et al 2015). Studies focussed 385 on the rDNA gene and how its expression may be altered by peri-conceptual diet of the 386 mother, could be particularly informative as results already point to altered gene methylation 387 13 as being important (Denisenko et al 2016).…”