Identification of long range regulatory elements of mouse α-globin gene cluster by quantitative associated chromatin trap (QACT)

Chromatin from different regions of the genome frequently forms steady associations that play important roles in regulating gene expression. The widely used chromatin conformation capture (3C) assay allows determination of the in vivo structural organization of an active endogenous locus. However, unpredicted chromatin associations within a given genomic locus can not be identified by 3C. Here, we describe a new strategy, quantitative associated chromatin trap (QACT), which incorporates a modified 3C method and a quantitative assay tool, to capture and quantitatively analyzes all possible associated chromatin partners (ACPs) of a given chromatin fragment. Using QACT, we have analyzed the chromatin conformation of the mouse a-globin gene cluster and proved the extensive interaction between HS26 and a-globin genes. In addition, we have identified a candidate alpha 1-globin gene specific silencer 475A8 which shows the differentiation-stage specific DNase I hypersensitivity. Functional analysis suggests that 475A8 may regulate the alpha 1-globin gene during terminal differentiation of committed erythroid progenitor cells. ChIP (chromatin immunoprecipitation) and cotransfection assays demonstrate that GATA-1, a hemopoietic specific transcriptional factor, may increase alpha 1-globin gene expression by suppressing the function of 475A8 in terminally differentiated erythroid cells.