Chromatin structure and transcriptional regulation of the stem cell leukaemia (SCL) gene in mast cells

Abstract

The stem cell leukaemia (SCL) gene is a member of the basic helix-loop-helix family of transcription factors and is essential for the development of all haematopoietic lineages. SCL is expressed in pluripotent haematopoietic stem cells and also following commitment to the erythroid, mast and megakaryocytic lineages. The mechanisms responsible for this pattern of expression are poorly understood, but are likely to illuminate the molecular basis for stem cell development and lineage commitment. Here we present the first description of the regulation of the SCL gene in mast cells. In this study we systematically analysed the chromatin structure of a 45 kb region of the murine SCL locus in mast cells. The pattern of DNase 1 and restriction endonuclease hypersensitive sites in mast cells was distinct from, but overlapped with, the pattern previously described in erythroid and primitive myeloid cells. Each potential regulatory element was tested using transient reporter assays to assess their functional significance in mast cells. These studies identified two potent enhancers, one of which was downstream of the SCL gene. Further characterisation of this 3Ј enhancer demonstrated that it required the presence of two distinct DNase 1 hypersensitive sites for full activity, and that it was capable of stimulating transcription from both promoter 1a and 1b. Since the 3Ј enhancer is active in both erythroid and mast cells, it will now be important to see whether it is independently activated in these lineages, or whether it is also active in haematopoietic stem cells.

The scl +18/19 Stem Cell Enhancer Is Not Required for Hematopoiesis: Identification of a 5′ Bifunctional Hematopoietic-Endothelial Enhancer Bound by Fli-1 and Elf-1

“…Three regions of accessible chromatin were identified ( Fig. 3B; data not shown), all of which coincided with the regions previously mapped in murine hematopoietic cell lines (24). The positions of the accessible restriction endonuclease sites were identified in the mouse sequence and compared to a human/ mouse homology profile.…”

“…However, a 6.3-kb fragment from the 5Ј region of the mouse scl gene directed expression to the vast majority of embryonic endothelial cells, together with rare round cells in the fetal liver (62). Moreover, the same fragment had also been shown to contain three regions of open chromatin in murine myeloid cell lines (24,32), suggesting that the rare round fetal liver cells may be of hematopoietic origin. We therefore investigated whether the scl 5Ј region contained an enhancer that might ensure scl expression during hematopoietic differentiation of scl ⌬19/⌬19 ES cells.…”

Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites

“…Within this well-characterized region, two novel DNaseI hypersensitive sites were identified as significantly enriched (+29 and +32) and investigated in functional assays. These sites are outside the region previously mapped with Southern blotting, although restriction enzyme ac- (Fordham et al 1999;Göttgens et al 1997Göttgens et al , 2004Sinclair et al 1999) Promoter 1a Promoter 1a 113402105-113402405 Yes Yes No Yes (Aplan et al 1990;Bockamp et al 1995Bockamp et al , 1997Bockamp et al , 1998Fordham et al 1999;Göttgens et al 1997Göttgens et al , 2001Sinclair et al 1999) Promoter 1b Promoter 1b 113402565-113402725 Yes Yes No Yes (Bockamp et al 1995(Bockamp et al , 1998Fordham et al 1999;Göttgens et al 1997Göttgens et al , 2001Sinclair et al 1999) cessibility mapping around the Pdzk1ip1 gene in 416B cells has now identified both regions as hypersensitive using conventional Southern blotting approaches (data not shown). Consistent with its location immediately upstream of Pdzk1ip1 exon 1, the +32 element functioned as a promoter in transient and stable reporter assays (Fig.…”

Regulation of the stem cell leukemia ( SCL ) gene: A tale of two fishes

“…Both human and mouse SCL are transcribed from two lineage-specific promoters (20)(21)(22)(23)(24). In addition, a panel of DNase I hypersensitive sites associated with enhancer or silencer activity have been identified within the murine SCL locus (25,26). Studies using transgenic mice have subsequently defined a 30-kb region containing five distinct murine enhancers that are capable of directing reporter gene expression to hemopoietic progenitor cells, endothelium, and specific regions of the brain and spinal cord (6,27,28).…”