Research Article: Genomic Approaches Uncover Increasing Complexities in the Regulatory Landscape at the Human SCL (TAL1) Locus

Date Published: February 5, 2010

Publisher: Public Library of Science

Author(s): Pawandeep Dhami, Alexander W. Bruce, Johanna H. Jim, Shane C. Dillon, Amanda Hall, Jonathan L. Cooper, Nicolas Bonhoure, Kelly Chiang, Peter D. Ellis, Cordelia Langford, Robert M. Andrews, David Vetrie, Laszlo Tora.

Abstract: The SCL (TAL1) transcription factor is a critical regulator of haematopoiesis and its expression is tightly controlled by multiple cis-acting regulatory elements. To elaborate further the DNA elements which control its regulation, we used genomic tiling microarrays covering 256 kb of the human SCL locus to perform a concerted analysis of chromatin structure and binding of regulatory proteins in human haematopoietic cell lines. This approach allowed us to characterise further or redefine known human SCL regulatory elements and led to the identification of six novel elements with putative regulatory function both up and downstream of the SCL gene. They bind a number of haematopoietic transcription factors (GATA1, E2A LMO2, SCL, LDB1), CTCF or components of the transcriptional machinery and are associated with relevant histone modifications, accessible chromatin and low nucleosomal density. Functional characterisation shows that these novel elements are able to enhance or repress SCL promoter activity, have endogenous promoter function or enhancer-blocking insulator function. Our analysis opens up several areas for further investigation and adds new layers of complexity to our understanding of the regulation of SCL expression.

Partial Text: Understanding the molecular events which occur as stem cells differentiate into committed cell lineages is a fundamental issue in cell biology. It has been shown that the SCL (TAL1) gene is central to the mechanisms whereby pluripotent stem cells differentiate into haematopoietic stem cells (HSCs) that give rise to the various blood lineages. While this process is thought to be tightly regulated at the level of gene expression, the exact ways in which SCL helps direct this process are not well understood.

We constructed a tiling microarray across 256,636 bp of the genomic region containing the human SCL locus at an average resolution of 458 bp. The tiling array encompassed SCL and all of its known regulatory sequences, and the neighbouring genes MAP17 (PDZK1IP1) and SIL (STIL), and the 5′ end of the housekeeping KCY (CMPK, UCK) gene, all of which are expressed during haematopoiesis [14]. We also included genomic regions covering the genes for CYP4A22 and most of CYP4AZ1 located downstream of MAP17, which are not appreciably expressed during haematopoiesis [14]. The hybridization of fluorescently-labelled unamplified ChIP DNAs, coupled with a highly sensitive and quantitative array platform [19], allowed us to identify reproducibly (Figure S1), and validate with real-time PCR (Figure S2), a wide range of regulatory features across the SCL locus as described below.

Over the last twenty years, a number of approaches have been used to characterise regulatory elements at the SCL locus. In many of these approaches, a variety of human and murine haematopoietic cell lines have been used to identify regions capable of mediating regulatory activities on SCL [12], [14], [32], [33], [49], [52]. We describe here the first comprehensive analysis using ChIP-chip approaches and downstream analyses to facilitate the characterization of a further set of genomic cis-acting sequences which underlie the regulation of gene expression at the human SCL locus. Patterns of histone modifications, chromatin accessibility/nucleosome density and the binding of regulatory/transcriptional proteins were obtained across 256 kb of the locus in the K562 haematopoietic cell line. Based on this, six novel human regulatory elements (named −31, −13, −10, −7, +53, +57) found either upstream or downstream of SCL are characterised here, and one existing element, the SCL erythroid enhancer (+51), has been re-defined. This study effectively more than doubles the number of regulatory elements localised to the human SCL locus, and emphasises the impact that such approaches have at rapidly defining the regulatory architecture of gene loci. In addition to these, we identified a further 3 regions of interest (−26, +39, +45) by virtue of their association with peaks of histone modifications; we have not examined these in this study but they warrant further investigation. Our analysis also redefines the size of what we consider to be the SCL regulon – to a region encompassing 88 kb between the −31 element upstream of SCL and the +57 element downstream of the +51 erythroid enhancer. Both −31 and +57 bind CTCF – consistent with the role of CTCF-binding elements defining regulatory domains [35], [36]. Previous studies have suggested that the SCL regulon encompasses 65 kb [57], but this figure seems to be an underestimate given that, while it includes −31 within its boundaries, it excludes the +51 erythroid enhancer, and the +53 and +57 elements. Whilst the exact roles of +53 and +57 with respect to SCL activity have yet to be determined, we cannot exclude them from the SCL regulon until further studies are performed (see also discussion below).