Mapping and Functional Characterisation of a CTCF-Dependent Insulator Element at the 3′ Border of the Murine Scl Transcriptional Domain

The Scl gene encodes a transcription factor essential for haematopoietic development. Scl transcription is regulated by a panel of cis-elements spread over 55 kb with the most distal 3′ element being located downstream of the neighbouring gene Map17, which is co-regulated with Scl in haematopoietic cells. The Scl/Map17 domain is flanked upstream by the ubiquitously expressed Sil gene and downstream by a cluster of Cyp genes active in liver, but the mechanisms responsible for delineating the domain boundaries remain unclear. Here we report identification of a DNaseI hypersensitive site at the 3′ end of the Scl/Map17 domain and 45 kb downstream of the Scl transcription start site. This element is located at the boundary of active and inactive chromatin, does not function as a classical tissue-specific enhancer, binds CTCF and is both necessary and sufficient for insulator function in haematopoietic cells in vitro. Moreover, in a transgenic reporter assay, tissue-specific expression of the Scl promoter in brain was increased by incorporation of 350 bp flanking fragments from the +45 element. Our data suggests that the +45 region functions as a boundary element that separates the Scl/Map17 and Cyp transcriptional domains, and raise the possibility that this element may be useful for improving tissue-specific expression of transgenic constructs

The SCL transcriptional network and BMP signaling pathway interact to regulate RUNX1 activity

Hematopoietic stem cell (HSC) development is regulated by several signaling pathways and a number of key transcription factors, which include Scl/Tal1, Runx1, and members of the Smad family. However, it remains unclear how these various determinants interact. Using a genome-wide computational screen based on the well characterized Scl +19 HSC enhancer, we have identified a related Smad6 enhancer that also targets expression to blood and endothelial cells in transgenic mice. Smad6, Bmp4, and Runx1 transcripts are concentrated along the ventral aspect of the E10.5 dorsal aorta in the aorta–gonad–mesonephros region from which HSCs originate. Moreover, Smad6, an inhibitor of Bmp4 signaling, binds and inhibits Runx1 activity, whereas Smad1, a positive mediator of Bmp4 signaling, transactivates the Runx1 promoter. Taken together, our results integrate three key determinants of HSC development; the Scl transcriptional network, Runx1 activity, and the Bmp4/Smad signaling pathway

The 350 bp core region of the +45 element insulates transgenic constructs and improves tissue-specific expression from the Scl promoter.

<p>The total number of F0 transgenic embryos obtained with each construct was determined by PCR using specific primers. Upon ß-galactosidase staining, transgenic embryos were visually inspected and scored as either LacZ+ or LacZ- and the percentage of LacZ+ embryos calculated.</p>*<p>p<0.05 compared to SV/LacZ. The expression pattern of Scl/LacZ and 350/Scl/LacZ/350 transgenic embryos is considered ectopic if beyond the mid and hind brain, the expected expression pattern for the Scl promoter alone.</p>**<p>p<0.01 compared to Scl/LacZ.</p

The 350 kb core region of the +45 element improves tissue-specific expression of transgenes.

<p>The 350/Scl/LacZ/350 transgenic embryos consistently expressed LacZ in mid and hind brain (white arrow), reflecting endogenous expression pattern of Scl in brain, Scl/LacZ transgenic embryos show ectopic expression (grey arrow). The transgenic constructs are depicted diagrammatically on the left panel, with photographs of 3 representative transgenic embryos presented on the right.</p