Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express human β-globin gene. However the molecular mechanisms by which the expression of β-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681～ -10971 bp , HS3 core sequence -14991～ -14716 bp and HS4 core sequence -18586～ -18306 bp) of DNase I hypersensitive sites in the human/3-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of human β-like globin genes through their interaction with HSs (HS2,HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cells; in addition, HS2 core DNA sequence was capable of binding to the nuclear matrix prepared from Hu-induced HEL cells, while both HS3 and HS4 core DNA sequences could not do so. Results indicated that the HS2 core DNA sequence may be a functional MAR (matrix attachment region). We suggest that the HS2 core DNA sequence binding to the nuclear matrix in Hu-induced HEL cells may open the structure of chromatin to make the LCR accessible to the promoter of β-globin gene and to promote its transcription.
SHU BING ZHANG, Ruo LAN QIANState Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China
Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mMand 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic 'beads-on-a-string' structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution ofnucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5'flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.
