MAR Elements Of SATB1 Mediated Interactions Involved In The Establishment Of The Human ¦Â-globin Gene Cluster Active Chromatin Structure

Cell and cell nucleus are both well-organized. Many important biological processes including gene transcription, DNA replication happen in the nucleus. As we have known, gene transcriptionis is exquisitely regulated. Disturbance of the gene regulation frequently results in severe diseases. In depth study of the mechanisms of gene regulation will provide the clues to know the mechanisms of many diseases, which will help to design effective drugs.Gene expression can be regulated at three levels including DNA, chromatin and nuclear level. As we have known, the three levels of regulation are interrelated to each other. The interaction between cis-regulatory elements and trans-regulatory factors reflects DNA level regulation. Many regulatory elements are far away from genes and they can contact genes by chromatin folding. The contacting and folding is chromatin level regulation. A basic and important question is how the remote regulatory elements bypass the long-range distance and regulate gene expression. Two models have been proposed, that is, the Contact Model and Non-contact model. A basic model in Contact-Model is Looping Model, which describes that the trans-factors binding to long-range enhancing elements can contact the target gene promoters through the formation of looping structure to overcome the spatial distance between them. Non-contact Model is represented by the Linking Model ,which suggests that LCR can not contact genes' promoters directly but direct transcriptional factors and enhancer facilitators to bind chromatin elements along with the gene locus.The regulation at both DNA level and chromatin level happen in the nucleus and are closely related to the nuclear structure. Many regulatory proteins are nuclear proteins and they have important roles in the initiation and termination of gene expression. The nucleus provides the space for the gene expression. Many studies suggested that the chromatin fragment can loop out of its chromosome territory (CT) when the gene actively expresses, which is associated with regulation machinery distributed in the interval of chromosomes. This is the famous CT-IC model. Since then, the gene localization has developed from simply being described as at the euchromatin region and heterochromatin region to more complicated nuclear localization. Therefore, the gene regulation attracted more attention now.Recent studies have showed that nuclear matrix is an important non-chromatin structure in the nucleus and the nuclear matrix can have influence on the gene expression, chromatin conformation and gene localization by recruiting the transcriptional factors and enzymes. Some studies have suggested that the binding between nuclear matrix and gene can affect the gene expression. A group of important proteins, called the matrix binding proteins can mediate the binding between the nuclear matrix and chromatin fragments. The DNA elements that associate with nuclear matrix were named matrix attachment regions (MAR). The binding between MARs and matrix is selective and the selectivity is related with the gene expression. MARs binding proteins have been confirmed including SATBl, cux, SMAR1 and PARP. We have studied the function of SATBl in regulating gene expression. The characteristic self-association of SATBl lead to the formation of a birdcage structure which provides a platform for gene expression regulation and chromatin fragments crosstalking. A previous report has confirmed that SATBl can up-regulate the expression ofε-globin gene and down-regulate the expression ofγ-globin gene. The regulation is related to the binding of SATBl to special MARs. In this study, we have explored the regulatory function of SATBl toβ-globin gene cluster at chromatin level.We used the QACT to capture possible associated fragments with a known MAR element in HS2 ofβ-globin LCR (HS2MAR, the leader). The results showed that there are three HS2MAR associated chromatin fragments including 8150 , a privously unknown element locates between HS4 and HS5, HBEMAR that locates upstream ofεgene promoter, HBGMAR that locates upstream ofγ~A gene promoter. HBEMAR and HBGMAR have been confirmed to be MARs. Our EMSA and ChIP results had suggested that 8150 is a potential MAR. 3C assay had showed that HBEMAR and HBGMAR can associated with HS2MAR and 8150 elements; also, the associations can be strengthened after induction of the cells by Hemin. These data indicated that a MAR base structon may exist. Our ChIP assay showed that HS2MAR,8150 and HBEMAR can bind SATB1 and the binding increase after Hemin induction . The ChIP-3C results showed that SATB1 can bind to HBEMAR,HS2MAR and 8150 in vivo and mediated the associations among these MARs in theβ-globin gene cluster, especially the association between HBEMAR and upstream regulatory fragments. We presumed that SATB1 function as core of the MARbase. To explore if SATB1 is necessary for the MARbase, RNA interference was used to knock down SATB1 expression in K562 cells . Expression assay of globin genes showed that embryonic gene likeεandζgenes were substantially decreased. For the fetal and adult stage globin genes, while the expression ofβ-globin gene showed no obvious change,γandαglobin genes been decreased upon STAB1 down-regulation, which is not consistent with previously reported results. We reasoned that this may result from cell differences. Our K562 cell is an early passage K562 cells that is different from the late passage K562 cells used in that the above mentioned report. Our K562 cells can be induced by Hemin into the erythroid direction with the up-regulation of bothεandγglobin genes. In the case of late passage K562 cells, the expression ofεglobin gene is down-regulated and the expression ofγgene is up-regulated with Hemin induction. Our results also showed that the expression of erythroid specific transcriptional factors had no obvious change. The ChIP assay showed that the binding of SATB1 to these MARs reduced . The 3C assay showed that the associations between HBEMAR and HS2MAR/8150 decreased substantially, but not the associations between HBGMAR and HS2MAR/8150. Another 3C assay showed that the association between HS2 core sequence and HBE gene promoter had also been reduced in the SATB1 RNAi cells. We concluded that SATB1 is necessary for theεspecific MARbase formation, forγspecific MARbase, SATB1 is not necessary and the influence onγ-globin gene expression may result from indirect effects .We next analyzed the acetylation status of SATB1 after hemin induction of the K562 cells. We found that the acetylation of SATB1 had been reduced after induction. A previous paper had showed that the acetylation of SATB1 can reduce the binding of SATB1 to DNA, so that the acetylation change of SATB1 can serve as a dynamic regulator of the MARbase. Finally, immunofluorenscence assay,GFP-SATB1 fusion protein localization analysis and ChIP assay were used to show that the binding of SATB1 to these MARs can be partially maintained on mitotic chromosome.Our results indicated that SATB1 can mediate the formation of aεgene specific MARbase to regulate the high-order chromatin structure ofβ-globin gene cluster and that the acetylation state of SATB1 may serve as a dynamic regulator of the MARbase. Meanwhile, the MARbase may be maintained to some degree during cell mitosis and facilitate the reestablishment of active chromatin structure in the next cell cycle.