An ISWI-related chromatin remodeler tunes MORC DNA binding and insulates gene expression in a densely packed genome

ATP-dependent nucleosome remodeling complexes, also known as chromatin remodelers, are specialized multiprotein machines involved in genome organization and accessibility. They alter the structure, composition and positioning of nucleosomes by histone displacement, rejection or incorporation of histone variants. These actions control the accessibility of regulatory regions such as promoters and enhancers for transcription factors, RNA and DNA polymerases as well as coactivators or repressors. Phylogenetic studies have shown that Toxoplasma has evolved two divergent ATP-dependent remodeler proteins of the ISWI family, TgSNF2h and TgSNF2L. These proteins differ not only in their internal structure, but also in their partnerships. They associate with different AP2 transcription factors and unique scaffold proteins, each characterized by specific folded domains. ChIP-seq data show significant colocalization of TgSNF2h and MORC throughout the genome. Depletion of TgSNF2h leads to a marked decrease in the binding of MORC to chromatin, but does not affect its binding to telomeres. When TgSNF2h is released from chromatin at highly active genes (approximately 572 genes), there is a corresponding decrease in chromatin accessibility near the transcription start site (TSS), as shown by ATAC-seq, which in turn triggers a marked drop in mRNA levels of these genes, many of which are essential for the tachyzoite lytic cycle (e.g., AMA1, MIC1, SAG1, and GRA15). In TgSNF2h-depleted zoites, release of MORC from chromatin leads to activation of the 1,017 genes associated with the sexual stages regulated by MORC. In this context, TgSNF2h functions as a chromatin remodeler that insulates neighboring genes by potentially blocking the influences of transcriptional activators and repressors. At highly transcribed loci, TgSNF2h and MORC appear to act as barriers to demarcate active chromatin regions typified by unique levels of acetylation or to polarize promoter regions. This action likely sets the stage for the establishment and maintenance of specific gene expression patterns. Together, TgSNF2h and MORC serve as a safeguard against the unintended spreading of transcriptionally active euchromatin from a gene active in tachyzoites to a neighboring developmental gene that is silenced. Thus, TgSNF2h, having an epistatic effect over MORC, plays a critical role in the partitioning of developmental genes in Toxoplasma.