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

Date Published: March 1, 2012

Publisher: Public Library of Science

Author(s): George A. Follows, Rita Ferreira, Mary E. Janes, Dominik Spensberger, Francesco Cambuli, Amy F. Chaney, Sarah J. Kinston, Josette R. Landry, Anthony R. Green, Berthold Göttgens, Ferenc Mueller. http://doi.org/10.1371/journal.pone.0031484

Abstract

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.

Partial Text

The Stem Cell Leukemia (Scl) gene, also known as Tal1, encodes a basic helix-loop-helix transcription factor with important roles in the development of the haematopoietic [1]–[3] vascular [4], [5] and central nervous systems [4], [6], [7]. Ectopic expression of Scl is detrimental for the normal differentiation of the affected lineages. For example, SCL overexpression in T-cells is one of the most common molecular abnormalities found in T-ALL [8]. Appropriate spatio-temporal transcription of Scl is therefore essential for the normal development and homeostasis of the haematopoietic system and this focuses attention on the mechanisms controlling Scl transcription.

We have used array-based DNaseI hypersensitive site mapping to identify a novel regulatory element 45 kb downstream of the mouse Scl promoter 1a. We have shown that this element binds CTCF using both in vivo and in vitro assays, and that protein-DNA contacts extend to 57 base pairs around a CTCF-consensus binding site. Furthermore, we have shown that this element functions as an insulator in enhancer-blocking assays when tested in haematopoietic cell lines and we have used a novel transgenic reporter assay to demonstrate that the element can both enhance the efficiency and specificity of midbrain-specific expression compared to conventional transgenic analysis. Moreover, analysis of genome-wide data from the ENCODE consortium confirmed that the +45 element is a ubiquitous CTCF-bound insulator region. This element is identified as a DNaseI HSS bound by CTCF in a variety of Scl/Map17 expressing and non-expressing tissues, including haematopoietic tissues (bone marrow and spleen), brain, kidney, liver, heart and lung.

Source:

http://doi.org/10.1371/journal.pone.0031484