Comparative study of SATB family proteins

Abstract

SATB1 and SATB2 are members of special AT-rich binding protein family, a class of chromatin organizers and gene regulators. SATB1 has been shown to play seminal roles in development and differentiation of T cells whereas SATB2 has been mainly implicated in development of neurons and bone. These two have also been shown to play important roles in development and progression of various kinds of tumors. Recently SATB1/2 have been implicated in the regulation of Nanog, a pluoripotency-associated gene. Here we study the distinct roles of SATB1 and SATB2 using cell line and human stem cell models. We show that various cells and tissues differentially express SATB1 and SATB2. Using IgH-MAR reporter system we show that SATB1 and SATB2 both bind and regulate the target genes in MAR- dependent manner in association with various corepressors and activators. Our study further reveals that SATB1 and SATB2 interact in vivo to form heterodimeric complexes and cooperatively regulate transcription. Intriguingly, both SATB1 and SATB2 regulate each other’s expression by directly binding to respective promoters. We further dissected their roles during retinoic acid (RA) mediated differentiation of NT2D1, a pluripotent human testicular embryonal carcinoma cell line, and that of human embryonic stem cells. RA mediated differentiation of NT2D1 cells resulted in upregulation of SATB1 and SATB2 along with various differentiation markers while various pluripotency markers were downregulated. Knockdown of SATB1 and SATB2 resulted in contrasting effects to that of RA mediated differentiation. Interestingly, we observed that Wnt signaling is upregulated upon RA mediated differentiation. Activation of Wnt signaling via Wnt3a in these cells mimicked the RA mediated differentiation. Knockdown of SATB1 and SATB2 resulted in the downregulation of Wnt responsive genes in NT2D1 cells indicating that SATB1 and STAB2 might target Wnt-responsive genes during differentiation.We extended our studies to human embryonic stem cell line (hES) where we used two hES cell lines H9 and HS360. We showed that SATB1 is upregulated during differentiation whereas SATB2 is transiently upregulated but is then quickly downregulated. Activation of Wnt signaling results in differentiation of hES cells while inhibition of Wnt signaling keeps cells in pluripotent state. Gene expression analysis showed a number of dysregulated genes upon RA-induced differentiation. Genome-wide ChIP-seq analysis revealed specific targets of SATB1 and SATB2 distributed all over genome. Interestingly, we found highly enriched occupancy of SATB1 and SATB2 binding on X and Y chromosomes. We also derived consensus binding motif for SATB1 and SATB1 at various stages of differentiation. We could find specific motif for SATB1 and SATB2 homodimer as well as SATB1/SATB2 heterodimer. We propose that in ES cells SATB1 and SATB2 can bind to their specific targets as homodimer or heterodimer which then might regulate gene expression in a differential manner. Collectively, this study provides novel insights into distinct roles played by SATB family proteins during stem cell maintenance and differentiation.