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. 2021 May 7;49(8):4506-4521.
doi: 10.1093/nar/gkab248.

Mechanism of REST/NRSF regulation of clustered protocadherin α genes

Yuanxiao Tang  1 , Zhilian Jia  1 , Honglin Xu  1 , Lin-Tai Da  1 , Qiang Wu  1
Affiliations

Mechanism of REST/NRSF regulation of clustered protocadherin α genes

Yuanxiao Tang et al. Nucleic Acids Res. .

Abstract

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 PCDHα genes. Thus, REST/NRSF represses PCDHα gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

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Figures

Figure 1.
Figure 1.
REST/NRSF binding sites in the human PCDH clusters. (A) Schematic of the REST/NRSF protein. REST/NRSF protein contains a DNA binding domain of eight ZFs (cylinders), two repression domains (RD1 and RD2), lysine-rich and proline-rich domains, two nuclear localization signals (NLS, shown in an orange diamond shape), a phosphodegron, and a C-terminal ZF domain. (B) Schematic of the three human PCDH gene clusters. The PCDH α and γ clusters share similar organization, with dozens of variable exons each of which is spliced to a single set of downstream constant exons. The variable exons are divided into alternate and C-type groups. The PCDHβ cluster contains only variable exons. The super-enhancer of the PCDHα cluster (red ellipses) is located between the PCDH α and β clusters. The super-enhancer of the PCDHβγ clusters (red ellipses) is located downstream of PCDHγ. The locations and orientations of REST/NRSF sites (NRSE) and CTCF sites (CBS) are shown under the genes. (C) Venn diagram of REST/NRSF ChIP-nexus peaks of HEC-1-B and SK-N-SH cells. (D) Schematic of the NRSE locations within the four subgroups of the clustered PCDH variable exons. (E–G) EMSA experiments using REST/NRSF with a site ‘a’ NRSE probe of each member of the alternate PCDHα(E), a site ‘b’ NRSE probe of each PCDHγa (F), or a site ‘c’ NRSE probe of each member of the alternate PCDHγ (G), using mock as a control (Ctr). Supershifted bands were detected with a specific antibody against human MYC (Ab) tag fused to the C-terminal of REST/NRSF.
Figure 2.
Figure 2.
Directional base-specific binding of REST/NRSF to the clustered PCDH NRSEs. (A, B) Schematics of truncated REST/NRSF, fusing with a MYC tag, with sequential deletions of ZFs from either N or C terminus. Purple ellipses denote zinc-finger domains. (C) Western blot of REST/NRSF with sequential deletions of ZFs. (D, E) EMSA using the truncated proteins with biotinylated probes of noncanonical HS5–1 NRSE (D) or of the right-half only PCDHα8 NRSE (E). The sequences of the motifs are shown above the gel. (F) Schematic of point mutations in each ZF domain. Two key cysteine residues were mutated to two arginine residues. (G) Sequence alignment of the eight tandem Cys2-His2 zinc fingers of REST/NRSF, with the residues involved in chelating Zn2+ highlighted in the red background and residues involved in base contacts with NRSE motifs highlighted in the blue background. The −1, 2, 3, and 6 positions of the α-helix of each ZF domain are indicated above the sequences. (H) Diagram of the REST/NRSF mutants in each ZF domain (highlighted by red ellipses). (I) Western blot of ZF-mutated proteins. (J, K) EMSA using ZF mutated proteins with a biotinylated probe of the noncanonical HS5–1 NRSE motif (J) or of the right-half only PCDHα8 NRSE motif (K). (L) EMSA of the wild-type (WT) PCDHα8 NRSE and its mutants using a set of ZF-deleted REST/NRSFs. (M) Sequences of the HS5–1 NRSE WT (highlighted by blue) and mutant highlighted by red) motifs. (N–P) EMSA for ZF-deleted proteins using a biotinylated probe of the HS5–1 NRSE Mut1-Mut6 (N, O) or Mut7-Mut10 (P), with the WT probe as a control. (Q) A model of base-specific contacts of ZF3–8 with DNA duplexes.
Figure 3.
Figure 3.
Recognition of tandem NRSE motifs by REST/NRSF. (A) Comparison of the REST/NRSF ChIP-nexus and ChIP-seq data of the human protocadherin CELSR3 gene containing four tandem NRSE motifs. (B–D) EMSA for probes of the first two NRSE motifs (B), the last two NRSE motifs (C), or all of the four tandem NRSE motifs (D) with a repertoire of truncated or mutated REST/NRSFs, using WT as a control. (E) EMSA for probes of the PCDHγa6 or PCDHγa7 tandem NRSE motifs with truncated REST/NRSFs, using WT as a control. (F) Schematic of the stereo-hindrance and proper configuration of REST/NRSF recognition of tandem NRSE motifs.
Figure 4.
Figure 4.
Flexible ZF6 determines two binding models of REST/NRSF with canonical and noncanonical NRSEs. (A) Six types of NRSEs and their motifs revealed by ChIP-nexus. (B, C) The molecular dynamics (MD) simulations for the base-specific contacts of ZF3–8 of REST/NRSF with site ‘c’ canonical NRSE of PCDHγa6 with 2-bp gaps (B), or with HS5–1 noncanonical NRSE with 8-bp gaps (C). In both (B) and (C), the upper panel is the initial structure of the MD simulations while the lower panel is the comparison of the initial and final structures. Base-specific contacts of ZF3–8 with the two NRSEs are shown on the right side of the panels.
Figure 5.
Figure 5.
REST/NRSF knockdown enhances HS5–1 activity and long-distance enhancer-promoter contacts. (A) Western blot showing the REST/NRSF knockdown efficiency of the three shRNAs in HEC-1-B and HEK293T cells. (B) RNA-seq showing the increased PCDHα expression upon REST/NRSF knockdown in HEC-1-B cells. Data are presented as mean ± SEM. * P < 0.05. (C–E) ChIP-seq with antibodies against REST/NRSF, H3K4me3 and H3K27ac in control and REST/NRSF knockdown cells. Note the decrease of REST/NRSF and the increase of H3K4me3 and H3K27ac upon REST/NRSF knockdown. (F, G) QHR-4C interaction profiles of the PCDHα locus using HS5–1 (F), or PCDHα12 (G) as a viewpoint (VP, arrowheads) for the control and REST/NRSF knockdown cells. Differences (shREST versus shGFP) are shown under the 4C profiles.
Figure 6.
Figure 6.
HS5–1 NRSE deletion results in increased HS5–1 enhancer activity and long-distance enhancer-promoter contacts. (A) RNA-seq results showing the increased PCDH α6 and α12 expression upon HS5–1 NRSE deletion in HEC-1-B cells. M46 and M47 are two CRISPR single-cell clones with HS5–1 NRSE deletion. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. (B–D) ChIP-seq with a specific antibody against REST/NRSF, H3K4me3, or H3K27ac in WT and HS5–1 NRSE deletion cells. (E, F) QHR-4C interaction profiles of the PCDHα locus using HS5–1 (E), or PCDHα12 (F) as a viewpoint (VP, arrowheads) for the WT and HS5–1 NRSE deletion single-cell clones. Differences (deletion vs WT) are shown under the 4C profiles.
Figure 7.
Figure 7.
NRSE deletion reshapes the Pcdhα 3D chromatin structure in mice in vivo via CTCF enrichments. (A) Schematic of the HS5–1 enhancer showing the precise deletion of NRSE through CRISPR DNA-fragment editing with dual sgRNAs. The PAM sites are highlighted. The two CTCF binding sites (HS5–1a and HS5–1b) are marked with cyan ovals. (B, C) The expression levels of REST/NRSF in the kidney and cortical tissues of mice tested by western blot (B) or RNA-seq (C). (D, E) RNA-seq showing the significant increase of the Pcdhα expression levels in mouse kidney (D), but not in cortical (E), tissues upon NRSE deletion. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. (F, G) ChIP-seq of H3K4me3 (F) and CTCF (G) in the Pcdhαcluster in the kidney and cortical tissues of WT and HS5–1 NRSE-deleted (ΔNRSE) mice. Note the significant increases of H3K4me3 and CTCF in kidney upon NRSE deletion. (H, I) QHR-4C interaction profiles of the Pcdhα locus using HS5–1 (H) or Pcdhα9 (I) as a viewpoint (VP, arrowheads) for the kidneys of WT and ΔNRSE mice. Differences (deletion versus wild-type) are shown under the 4C profiles. (J) A model depicting the DNA-recognition and mechanism of REST/NRSF repressing PCDHα gene expression through CTCF/cohesin-mediated higher-order chromatin structures.
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