Setdb1 regulates proper differentiation of adult intestinal stem cells via restraining permissive chromatin structure and transcriptional variability

The histone methylase Setdb1 plays a pivotal role in embryonic stem cell maintenance and developmental lineage specification. However, its function in adult stem cells remains elusive. Here we show that conditional inactivation of Setdb1 in Lgr5+ intestinal stem cells alters the transcriptional programs of the progeny cell types and results in increased cell-to-cell transcriptional variability. Loss of Setdb1 blocked differentiation towards the absorptive enterocyte lineage, while the generation of secretory cell types was only marginally affected due to the activation of alternative developmental trajectories. Setdb1 inactivation did not alter H3K9 methylation at large heterochromatin domains but led to reduced localized modifications at islands of gene-rich areas around transposase-accessible chromatin regions, which have doubled in number, became broader and more heterogeneous in size. The results demonstrate that Setdb1 regulates intestinal stem cell properties and epithelial lineage specification by limiting chromatin accessibility at open genomic areas and controlling cell-to-cell transcriptional variability. To study the role of Setdb1 in Lgr5 stem cell population we used FACS-sorted cells from mice (GFP_WT and GFP_Setdb1-KO) mice 5 days after Tamoxifen treatment. To study the role of Setdb1 in Lgr5 stem cell progeny, we used FACS-sorted cells from mice (GFP_WTnTnG and GFP_Setdb1-KOnTnG) mice 5 days after Tamoxifen treatment. Single cell RNA-seq and single cell ATAC-seq assays were performed in the samples, as indicated. In addition, FACS-sorted GFP_WT and GFP_Setdb1-KO cells we performed total RNA sequencing and mapped H3K9Me3 locations in the genome by CUT&Tag assays.