SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity [RNA-seq]. SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity [RNA-seq]

Spalt-Like Transcription Factor 1 (Sall1) is a critical regulator of organogenesis and microglia identity. Despite its known biological importance, mechanisms that specify the cell-specific expression of Sall1 and its transcriptional functions remain poorly understood. Here, we demonstrate that targeted deletion of a conserved microglia-specific super enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia, thereby identifying an essential regulatory element that transduces brain environmental signals required for microglia-specific gene expression. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knock out (EKO) mice to probe how SALL1 shapes the regulatory landscape of microglia, we provide evidence that SALL1 functions to both directly activate microglia-specific genes and repress genes that are associated with inflammation and aging. Unexpectedly, motifs for SMAD proteins that mediate transcriptional effects of TGFb signaling were enriched within the set of enhancers predicted to be directly activated by SALL1, suggesting that collaborative interactions between SALL1 and SMADs are required to establish microglia-specific gene expression. To test this hypothesis, we determined the transcriptional consequences of a conditional knockout of the common co-SMAD Smad4 and defined the genome-wide locations of SMAD4 in wild type and EKO microglia. These studies revealed two layers of functional interdependence. First, we found that SMAD4 binds directly to the Sall1 super enhancer and is required for Sall1 expression, consistent with the requirement of the TGFb and SMAD homologues Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Second, we extend this paradigm by demonstrating that SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in EKO microglia. Collectively, these results suggest molecular mechanisms by which SALL1 enforces microglia-specific functions of the TGFb-SMAD signaling axis that may be relevant to roles of SALL1 in other developmental contexts. Overall design: Poly-A RNA-seq, ATAC-seq, and H3K27ac-ChIP-seq of whole cell microglia or PU.1-positive nuclei isolated from mouse brains (Control, SALL1-Enhancer knockout, SMAD4 knockout).

类Spalt转录因子1（Spalt-Like Transcription Factor 1，Sall1）是调控器官发生与小胶质细胞身份确立的关键因子。尽管其生物学重要性已被广泛认知，但调控Sall1细胞特异性表达的分子机制及其转录功能仍有待深入阐明。
本研究证实，靶向删除与Sall1启动子相互作用的保守小胶质细胞特异性超级增强子，可导致小胶质细胞中Sall1表达完全且特异性缺失，由此鉴定出一种可转导脑源性信号的必需调控元件，该元件是小胶质细胞特异性基因表达的必要条件。
通过解析SALL1的全基因组结合位点，并利用Sall1增强子敲除（Enhancer Knock Out，EKO）小鼠探究SALL1如何塑造小胶质细胞的调控网络，本研究证实SALL1兼具双重功能：既可直接激活小胶质细胞特异性基因，又能抑制与炎症及衰老相关的基因表达。
出乎意料的是，介导转化生长因子β（TGFβ）信号转录效应的SMAD蛋白结合基序，在SALL1直接调控激活的增强子预测集合中显著富集，这提示SALL1与SMAD家族蛋白的协同互作是建立小胶质细胞特异性基因表达程序的必要环节。
为验证这一假说，我们对共同协同SMAD蛋白Smad4进行条件性敲除，并解析了野生型与EKO小胶质细胞中SMAD4的全基因组结合位点。
这些研究揭示了两层功能性互作调控网络：其一，我们发现SMAD4可直接结合Sall1超级增强子，且对Sall1的表达至关重要，这与果蝇翅中Spalt基因细胞特异性表达所需的TGFβ同源物Dpp及其下游效应因子Mad的功能范式一致。
其二，我们进一步拓展了这一调控范式，证实SALL1可促进SMAD4在小胶质细胞特异性增强子处的结合与功能活性，同时抑制SMAD4结合至在EKO小胶质细胞中异常激活的基因的增强子区域。
综上，本研究结果阐明了SALL1强化TGFβ-SMAD信号轴小胶质细胞特异性功能的分子机制，该机制或同样适用于SALL1在其他发育场景中的调控作用。
整体实验设计：对从小鼠脑组织中分离的全细胞小胶质细胞或PU.1阳性细胞核进行聚腺苷酸RNA测序（Poly-A RNA-seq）、转座酶可及性染色质测序（ATAC-seq）以及H3K27乙酰化染色质免疫共沉淀测序（H3K27ac-ChIP-seq），实验分组包括对照组、SALL1增强子敲除组、SMAD4敲除组。