Mechanism of REST/NRSF Regulation of Clustered Protocadherin Alpha Genes

Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5-1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDH alpha genes. Thus, REST/NRSF represses PCDH alpha gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes. We performed ChIP-nexus experiments in HEC-1-B and SK-N-SH cells to pinpoint the NRSEs, and uncover the DNA-recognition code of NRSF. We performed ChIP-Seq experiments for REST, H3K4me3, H3K27ac after REST knockdown by shRNA or HS5-1 NRSE deletion in HEC-1-B cells and HEK293T cells by CRISPR/Cas9, and for REST, H3K4me3, CTCF after HS5-1 NRSE deletion in mice by CRISPR/Cas9. RNA-seq results revealed the influence on the expression of clustered Pcdh alpha by knockdown of NRSF or deletion of HS 5-1 NRSE in HEC-1-B and HEK293T cells, or in mouse cortex, and kidney, taking wild-type (WT) as control. QHR-4C experiments were used to supervise the DNA interactions between PCDH apha promoters and HS5-1 in the HEC-1-B cells after REST knockdown or HS5-1 NRSE deletion, and in the kidney of HS5-1 NRSE-deleted mice.