alized DNA context, that frequently characterizes the DNA content of MARs. To test the MAR potential of this newly identified fragment, the core sequence of this fragment with predicted SATB1 binding sites was detected for the SATB1 binding capacity by EMSA. Compared with the well-known SATB1 binding sequence, the tested sequence showed similar binding capability. In addition, the ChIP result also showed an obvious in vivo binding of SATB1 to the two core sequences of this region. Therefore, both the in vitro and in vivo experiments proved that the newly identified fragment has SATB1 binding capability, suggesting that a potential MAR is enclosed in the interval region between HS4 and HS5. Here, we name it as MARHS4. MARs in b-globin gene cluster associate with each other In the QACT assay, MARHS2 was extensively associated with MARHS4, MARe and MARc. To further validate the associations among these MAR elements, 22315414 3C assay was performed using MARHS4, MARHS2, and MARc located restriction fragments as the fixed fragment respectively. The results showed that the four MAR elements in b-globin gene cluster can associate with each other at obviously high frequencies, suggesting that the four MAR elements are spatially close to each other. The 2 MAR Elements & Gene Expression PCR. Error bars represent the mean and standard errors from triplicate and averaged determinations. D. Quantified assay of the expressions of b-like globin genes during the hemin induction after normalizing to bactin. doi:10.1371/journal.pone.0004629.g002 association results in the looping out of the intermediate regions between different MARs. This observation is consistent with the hypothesis that genomic DNA is attached to the NM through MAR elements and the intermediate regions between two MAR elements are organized as separate units. Also, the positions of these four MAR elements on NM are spatially proximal which indicates a MAR corestructure in the b-globin gene locus in K562 cells. The functional significance of this MAR corecould be observed when the terminal differentiation of K562 cells was induced by hemin. More than 90% cells could be induced when measured by, the Benzidine staining. The comparative 3C assay in 12150697 uninduced and induced K562 cells showed the obvious increasing of the ligation frequencies among the MARs after hemin induction, implying that the MAR coreformation is also significantly increased accompanying the up-regulation of the b-like globin genes expression. Taken together, these data indicate that the association of the four MAR elements of b-globin gene cluster establishes a MAR-coreas the base to organize the intermediate regions. In addition, the association of the MAR elements can be induced by hemin and the association accompanies the activation of e and cglobin genes in K562 cells. SATB1 can specifically bind to these potential MARs and mediate the association among these MARs in b-globin gene cluster The b-like globin genes expressions are order AG-221 always accompanied by the establishment of specific 3D chromatin structure. The transcription factors are supposed to be responsible for this active process. SATB1 is one of the candidates that have been reported to regulate the b-globin genes expression by 
influencing the chromatin structure. We compared the varying binding status of SATB1 at the MAR elements in b-globin cluster during the erythroid differentiation. As expected, we detected the strong binding of SATB1 to at least three of the four MAR
