SATB1 By Changing The Regulation Of Nuclear Localization Of ¦Â-globin Gene Expression

The nucleus is a compartmentalized organelle;the localization of many genes in the nucleus is specific.Understanding the basic principles of nuclear architecture and the changes in nuclear organization is important to study the profound differences in gene activities established and maintained to ensure the development and function of a complex organism.Every single chromosome occupies a certain territory in nucleus,with smaller one situating towards the interior and larger one towards the periphery.Gene content is also a key determinant of CT positioning.The nucleus also contains a largely chromatin-free space lined by chromatin-domain surfaces.A ribonucleo-protein network is located in this space.These non-chromatin domains may represent storage sites of proteins or protein complexes.The CT-IC model hypnotizes that partial transcription complexes are pre-established in the IC to fulfill its role as a functionally defined compartment; Regulatory and coding sequences of these active genes can interact with the transcription machinery only when they are positioned at the surface of chromatin domains that line the IC,or on chromatin loops that extend into the IC;Long-term or permanently silenced genes should be located within the interior of compact chromatin domains that are nonaccessible to the transcription machinery.In more general terms,genes that require long-term silencing should be physically separated from permanently active genes to an extent that allows their positioning in different chromatin compartments.Nuclear organization may be cell type-specific,because the chromatin organization was found to be similar in cells from the same cell linage and maintained during cell division.Co-regulated gene clusters may be proximally positioned to facilitate their regulation through the creation of expression hubs with shared concentrations of regulatory proteins.The combined effect of the association of domains from throughout the genome would yield a cell-specific nuclear topology,resulting in definable patterns of chromosome organization.Nuclear organization may be also developmentally specific.Recent studies suggested that many genes had the ability to mobilize to specific locations within the interphase nucleus according to their state of transcription during development.As for theβ-globin locus,the nuclear relocation has been realized to be one of the mechanisms in the regulation of expression.It was reported that moving away from centromeric heterochromatin is essential for the activation ofβ-globin locus;and extrusion from the chromosomal territory in the interphase nucleus is associated with high level expression ofβ-like globin genes.Both cis-regulatory elements and transacting proteins have been implied involved in the repositioning ofβ-globin locus.But the complex mechanism is far from clear and other factors participating in the relocation ofβ-globin locus need elucidating.The nuclear matrix,composed of lamin and internal network made of proteins and RNAs,can organize nuclear structure by attaching with chromatin periodically. Additionally,this attachment is mobile and associated with the expression state of related genes.So the nuclear matrix is one of the factors that regulate the position of genes according to its expression activity.Furthermore,a MAR binding protein-SATB1 was lately found to improve embryonicβ-like globin gene expression in K562 cells.SATB1 proteins can form cage-like structure and provide a regulatory platform for genes,and this structure is associated with the nuclear matrix.Though the previous study suggested that SATB1 regulatesβ-like globin genes through remodeling chromatin,we assume that SATB1 may also play regulatory role in the level of nuclear structure and the regulation of relocation may be related with nuclear matrix.We constructed normal and mutant(with matrix binding domain deleted) SATB1 expression vectors,and transfected them into K562 cells.Different primers were designed to confirm the expression of both endogenous and exogenous,both normal and mutant SATB1.Western Blot was applied to detect the expression in the protein level.After the construction of SATB1/K562 cells and m SATB1/K562 cells,the expression level of[3-like globin genes were detected by RT-PCR.The results showed that SATB1 over expression increasedεglobin gene expression and decreasedγ-globin gene expression,leavingβ-globin gene still not expressed,which is consistent with the previous results.But the mutant SATB1 didn't have this regulatory effect,suggesting the regulatory mechanism of SATB1 is associated with nuclear matrix.DNA FISH exploring the nuclear location ofβ-globin locus showed that both normal SATB1 and mutant SATB1 could induceβ-globin locus to loop out of chromosomal territory,but only the normal SATB1 can anchor the locus on the nuclear matrix.We also detcted that binding situation ofβ-globin locus with transacting factors by ChIP assay using the antibodies to PolⅡand GATA1.The results showed that over expression of normal SATB1 increased the binding of HS2 core sequence andε-globin gene promoter with RNAPolⅡand decreased the binding ofε-globin gene upstream regulatory elements with GATA1,while the mutant SATB1 didn't.The results suggested that anchoring with the nuclear matrix is important for the proper binding situation to improve the expression ofεglobin gene.Based on our and previous results,we proposed that SATB1 regulatingβ-1ike globin genes is a multi-step process.SATB1 proteins form cage-like structure,inducing the associatedβ-globin locus loop out of CT and anchored it on the nuclear matrix.Because the binding sites of SATB1 locate in the HS2 andεgene promoter,the regulatory factors on the nuclear matrix mainly affect the chromatin of the LCR andε-globin gene.But the relocation is the first step of regulation;it brings not only direct effect but also the regulation of next step.During binding with SATB1,the LCR andε-globin gene interact with each other.LCR causes the further opening of the chromatin nearε-globin gene and enhances the expression ofε-globin gene.In K562 cells,bothε-globin gene and T globin gene have the potential to express,and compete for the LCR.SATB1 improve the interaction between LCR andε-globin gene,the competition mechanism will decrease the interaction between LCR andγ-globin gene and cause the decreae of theγ-globin gene expression.In conclusion,our results suggested thatβ-like globin genes are regulated by SATB1 through relocating the cluster in the nuclear;and looping out of the chromosomal territory is necessary but not suffitient forβ-like globin genes regulation,attachment with nuclear matrix is also very important.Furthermore,SATB1 regulatesβ-like globin genes in all the levels of DNA,chromatin and nuclear organization.