Mechanistic dissection of theCHD4enhancers reveals cooperative functions among the homotypic ZNF410 clustered motifs

Abstract

SUMMARYTranscription factors often regulate numerous target genes. However, ZNF410 controls only a single gene,CHD4,in human erythroid cells by its highly restricted chromatin occupancy to theCHD4locus via two clusters of ZNF410 binding motifs. Here, we uncover that ZNF410 controls chromatin accessibility and activity of the twoCHD4enhancer regions. Combining CRISPR/Cas9 genomic deletion with CRISPRi approaches, we demonstrate that both enhancer regions additively contribute toCHD4gene expression. Mutations of the di-adenine nucleotides within the ZNF410 binding motif fully disrupt ZNF410-DNA interaction. Luciferase assays, ChIP-seq, and ATAC-seq studies reveal that the homotypic clustered motifs within theCHD4enhancers are recognized by ZNF410 in a collaborative fashion. Especially, the three ZNF410 motifs located in the 3’ end of the distal enhancer act as “switch motifs” to control the chromatin accessibility and activity of the whole distal enhancer region. Together, our findings expose a complex functional hierarchy of motifs, where clustered motifs bound by the same transcription factor cooperate to fine-tune the expression of target gene.HighlightsZNF410 maintains the accessibility of theCHD4enhancer regionsThe two enhancer elements additively modulateCHD4gene expressionThe dinucleotide “AA” is the key bases within ZNF410 binding motifZNF410 binds to homotypic clustered motifs in a cooperative manner