Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.     

Involvement of Intronic Sequences in Cell-Specific Expression of the Peripherin Gene

Peripherin is an intermediate filament protein expressed in restricted populations of neurons. Our previous study of the chromatin structure of the mouse peripherin gene in cells that do or do not express peripherin suggested that the region located between -1,500 and +800 bp of the gene could be involved in its cell specificity. In the present work, we performed an in vitro functional analysis of the 5' flanking region of the mouse peripherin gene and observed that this region up to 9 kb contained both enhancer and inhibiting activities; however, it was insufficient to achieve a complete extinction of reporter gene expression in peripherin-negative cells. Furthermore, analysis of the first three introns with the 5' flanking sequences of the gene showed that intron I greatly increased specificity of the gene expression. Intron I also conferred the same properties to thymidine kinase heterologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompasses an AP-2-like binding site that interacted with both neuroblast and fibroblast nuclear factors, as well as with the recombinant AP-2alpha protein. However, gel shift experiments suggested that the interacting nuclear factors are distinct from AP-2alpha itself and probably belong to the AP-2 family. Inl B perfectly matched the consensus binding site for Sp1 and specifically interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I indicated that the Inl A element alone is responsible for its enhancing properties, whereas a region located between +789 and +832 gives to intron I its silencer activity.