Abstract
We present a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunoprecipitate the tagged chromatin from specific cell types. We applied this approach to Distalless (Dll), a gene required for limb development in Drosophila. We show that the local chromatin conformation at Dll depends on the cell type: in cells that express Dll, the 5' regulatory region is in close proximity to the Dll promoter. In Dll-nonexpressing cells this DNA is in a more extended configuration. In addition, transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type-specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and nonexpressing cell types, respectively.
Highlights
The regulation of transcription in higher eukaryotes depends on cis-regulatory modules (CRMs), DNA sequences that integrate temporal and spatial information by binding groups of transcription factors (Istrail and Davidson, 2005)
Several transcription factors, such as GAGA factor (GAF) and CTCF, have been implicated in mediating such long-range interactions, which are thought to underlie much of gene regulation in eukaryotes (Ling et al, 2006; Mahmoudi et al, 2002; Ohtsuki and Levine, 1998)
chromatin immunoprecipitation (ChIP) assays suffer from the problem that it is difficult to determine if a DNA element is immunoprecipitated because of an interaction with another element or because both elements have a binding site for the immunoprecipitated transcription factor
Summary
We present a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunoprecipitate the tagged chromatin from specific cell types. We applied this approach to Distalless (Dll), a gene required for limb development in Drosophila. Transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type-specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and nonexpressing cell types, respectively
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