Smooth muscle-specific transcriptional regulation by SRF and homeodomain proteins

虽然血清反应因子(SRF)/CArG盒相互作用已被充分记录在各种转录系统中，包括即时早期基因和肌肉基因，但它不能单独负责各自组织的转录。在这里，除了CArG盒子，我们还在α整合素启动子区域发现了两个顺式元件：一个TAAT序列，同源蛋白的一致结合位点，和一个GATA家族结合盒子。我们进一步克隆了一个主要在平滑肌组织和骨骼结构中表达的同源盒cDNA Nkx-3.2。凝胶位移实验显示SRF与Nkx-3.2或GATA-6及其对应的顺式元件形成三元配合物。此外，Nkx-3.2、SRF和GATA-6或其各自的功能域协同激活血管smc衍生细胞系和异源细胞中的α -整合素基因。我们得出结论，血管SMCs中α -整合素的转录受Nkx-3.2、SRF、GATA-6及其相应顺式元件的协同相互作用调控。此外，我们表征了- tm基因在SMCs中的转录机制。启动子和凝胶迁移分析显示，血清反应因子（SRF）及其与CArG盒序列的相互作用在分化的SMCs中β - tm基因的SMCs特异性转录中起着强制性作用。我们进一步从鸡胗中分离出Barx同源蛋白家族的一个新的同源物Barx1b。Barx1b仅局限于上消化器官及其附属动脉的SMCs和颅面结构。SRF与Barx1b直接结合，在分化的SMCs和异源细胞中协调反激活β - tm基因，并与CArG探针形成初级复合物。综上所述，这些结果表明SRF、同源结构域蛋白和/或GATA家族转录因子协同参与了平滑肌基因的组织特异性转录。

While serum response factor (SRF)/CArG box interaction has been well documented for a variety of transcription systems, including immediate early and muscle genes, it cannot solely be responsible for transcription in respective tissues. Here, we identified two cis-elements in the alphal-integrin promoter region in addition to CArG box : a TAAT sequence, a consensus-binding site for homeoproteins, and a GATA family-binding box. We further cloned a homeobox cDNA, Nkx-3.2, which is mainly expressed in smooth muscle tissues and skeletal structures. Gel-shift assays showed a ternary complex formation of SRF and Nkx-3.2 or GATA-6 with their corresponding cis-elements. Further, Nkx-3.2, SRF, and GATA-6 or their respective functional domains transactivate synergistically the alphal-integrin gene in vascular SMC-derived cell line and heterologous cells. We conclude that transcription of alphal-integrin in vascular SMCs is regulated by coordinated interactions between Nkx-3.2, SRF, GATA-6 and their corresponding cis-elements. In addition, we characterized the transcriptional machinery of the beta-TM gene in SMCs. Promoter and gel mobility shift analyses revealed an obligatory role for serum response factor (SRF) and its interaction with the CArG box sequence in the SMC-specific transcription of the beta-TM gene in differentiated SMCs. We further isolated a novel homologue of the Barx homeoprotein family, Barx1b, from chicken gizzard. Barx1b was exclusively localized to SMCs of the upper digestive organs and their attached arteries and to craniofacial structures. SRF and Barx1b bound each other directly, coordinately transactivated the beta-TM gene in differentiated SMCs and heterologous cells, and formed a temary complex with a CArG probe. Taken together, these results suggest that SRF, homeodomain proteins, and/or GATA family transcription factors are coordinately involved in the tissue-specific transcription of the smooth muscle genes.