Study Of Protein-DNA Interactions Of Important Regulatory Elements Of Mouse β-globin Gene Cluster By In Vivo Footprinting

β-globin gene cluster has been one of the favorite model systems for analyzing the control of gene expression,particularly developmental regulation. During erythroid development, the β-like globin genes are expressed sequentially from 5' to 3' according to the genome orientation in a erythroid-specific and developmental stage-specific manner. Although clustered in distinct chromosomal loci, the expression of α-like and β-like globin chains is balanced.Gene expression is regulated by the complex interaction of many cis-acting elements and trans-acting factors. The correct expression of β-globin genes mainly depends on two kinds of regulatory elements: the locus control region located 5' of this cluster and promoter regions of individual genes. The β-globin gene LCR consists of four erythroid-specific DNase I hypersensitive sites (HS1-HS4),every HS activity is defined to its core sequences. It is known now that: the HS core sequences of the LCR and the promoter of individual globin genes have many binding sites for erythroid -specific as well as ubiquitous proteins. The involvement of specific DNA-protein interaction in the formation of hypersensitive sites, in the assembly of basal transcription apparatus and in the contact between LCR and downstream gene promoter (eg. looping) is suggested by a large body of evidence. Therefore, studying DNA-protein interaction is important and significant to elucidate the mechanism of LCR action and globin gene switching.In vivo footprinting is a method of studying DNA-protein interaction. It can reflect the authentic status of DNA-protein in vivo, and can also detect the change of DNA conformation. The introduction of Ligation-Mediated PCR(LM-PCR) greatly improves the sensitivity and specificity of in vivo footprinting study, and has facilitated the executation of in vivo footprinting in the regulation of eucaryotic gene expression.In this article, we have used in vivo footprinting and LM-PCR to study DNA - Protein interaction at HS2 of LCR and β-globin gene promoter of MEL cells with or without induction, of mouse bone marrow-derived and fetal liver-derived erythroid cells, and of bone marrow-derived erythroid cells of mice treated with myleran or hydroxyurea.(1) MEL cells are grown in DMEM medium,induction with 2%DMSO for three days.collect cells and resuspend in DMEM medium without serum,adjust cell concentration to 5×10~7.(2) Healthy kunming mice mate to produce embryo. sacifice prognant mice at...