β-catenin, Tcf7l1, and Esrrb mediate seeding density-dependent gene regulation during mouse embryonic stem cell differentiation. β-catenin, Tcf7l1, and Esrrb mediate seeding density-dependent gene regulation during mouse embryonic stem cell differentiation

Cell density affects numerous biological processes, including gene expression and cell fate specification. However, mechanistic understanding of how changes in cell density alter the transcriptome is lacking. Here, we reveal that the expression of thousands of genes in mouse embryonic stem cells is affected by cell density. Furthermore, we find that low cell density enhances the efficiency of differentiation. Mechanistically, β-catenin is localized primarily to adherens junctions during conditions of high cell-cell contact. At low seeding density during differentiation, we observe that β-catenin translocates to the nucleus and co-activates target genes in concert with Tcf7l1, leading to the induction of lineage markers. Meanwhile, Esrrb sustains the expression of high density-specific, pluripotency-associated genes, but low seeding density differentiation reduces its occupancy on its target loci. Our demonstration of factors that transcriptionally regulate genes responsive to cell density contributes to our understanding of gene regulation in stem cells and has implications for reproducibility, as density can vary between substantially labs and experimental protocols. Overall design: Expression and transcription factor occupancy in mouse embryonic stem cells at high and low cell seeding density.

细胞密度会影响诸多生物学过程，包括基因表达与细胞命运特化。然而，目前学界对细胞密度改变如何调控转录组的机制仍缺乏深入认知。本研究发现，小鼠胚胎干细胞中数千个基因的表达水平均受细胞密度调控。此外，本研究还观察到低细胞密度可提升干细胞分化效率。从机制层面来看，当细胞间接触紧密时，β-连环蛋白（β-catenin）主要定位于黏着连接（adherens junctions）；而在分化阶段采用低接种密度时，β-连环蛋白会转位进入细胞核，并与Tcf7l1协同共激活靶基因，最终诱导谱系标志物的表达。与此同时，雌激素相关受体β（Esrrb）可维持高细胞密度特异性的多能性相关基因的表达，但低接种密度的分化过程会降低其在靶基因位点的结合占有率。本研究鉴定出可转录调控细胞密度响应基因的调控因子，有助于深化我们对干细胞基因调控机制的认知；同时由于不同实验室及实验方案间细胞密度差异显著，本研究结果对提升实验重复性亦具有重要参考价值。整体实验设计：检测不同接种密度（高、低）下小鼠胚胎干细胞的基因表达水平及转录因子结合占有率。