Developmental Genetics, Department of Biomedicine, University of Basel, 4058 Basel, Switzerland.
Functional Genomics Department, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Development. 2021 Dec 1;148(23). doi: 10.1242/dev.200182. Epub 2021 Dec 3.
SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction, and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously downregulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in upregulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are upregulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH- and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.
SMAD4 调节基因表达以响应 BMP 和 TGFβ信号转导，并需要进行多种形态发生过程，但它的靶基因仍然很大程度上难以捉摸。在这里，我们使用表位标记的 Smad4 等位基因进行 ChIP-seq 分析，结合转录谱分析，鉴定了小鼠肢芽中的 SMAD4 靶基因。该分析表明，SMAD4 在早期肢芽发育过程中主要介导 BMP 信号转导。出乎意料的是，胆固醇生物合成酶的表达过早地下调，并且 Smad4 缺陷的肢芽间充质祖细胞中的细胞内胆固醇水平降低。最重要的是，我们的分析揭示了 SMAD4 在上调前肢芽间充质中的靶基因方面的主要功能。在 Smad4 和 Shh 缺陷的肢芽之间共享的差异表达基因的分析证实了 SMAD4 的这一功能，并且还揭示了 SMAD4 对在后因的抑制作用，在后因中，对 SHH 信号的反应而上调。该分析揭示了在早期肢芽的指模式形成和生长过程中，来自 SHH 和 SMAD4 介导的 BMP 信号转导的相反的转录调节输入，对前基因和后基因的表达产生影响。
