CTCF regulates global chromatin accessibility and transcription during rod photoreceptor development

Abstract

Chromatin architecture facilitates accurate transcription at a number of loci, but it remains unclear how much chromatin architecture is involved in global transcriptional regulation. Previous work has shown that rapid depletion of the architectural protein CTCF in cell culture alters global chromatin organization but results in surprisingly limited gene expression changes. This discrepancy has also been observed when other architectural proteins are depleted, and one possible explanation is that full transcriptional changes are masked by cellular heterogeneity. We tested this idea by performing multiomics analyses with sorted juvenile postmitotic mouse rods, which undergo synchronized development, and we identified CTCF-dependent regulation of global chromatin accessibility and gene expression. CTCF depletion leads to dysregulation of ~20% of the entire transcriptome (>3,000 genes) and ~41% of genome accessibility (>27,000 sites) before any prominent cellular or physiological phenotypes arise. Importantly, these changes are highly enriched for CTCF occupancy at euchromatin, suggesting direct CTCF binding and transcriptional regulation at these active loci. CTCF mainly promotes chromatin accessibility and frequently inhibits expression of these direct binding targets, which are enriched for binding motifs of transcription repressors. These findings provide different and sometimes opposite conclusions from previous studies, emphasizing the need to consider cellular heterogeneity and cell-type specificity when performing multiomics analyses. CTCF knockout rods undergo complete degeneration by adulthood, indicating an essential role for their viability. We conclude that the architectural protein CTCF binds chromatin and regulates global chromatin accessibility and transcription during rod development.

Keywords: CTCF; chromatin architecture; retinal development; rod; transcriptome.

Fig. 1.

CTCF protein localizes to euchromatin. (A) Nrl-Cre-labeled, newly born rods contain CTCF protein distributed throughout nuclei at P11 (yellow arrow heads) and becomes enriched at the nuclear periphery in developing P30 rods. In contrast, CTCF protein is broadly distributed in nonrod nuclei throughout development. (Scale bars, 10 µm.) (B) Dissociated P30 retinal cells contain rods that have small nuclei with a single central chromocenter surrounded by a thin ring of euchromatin (H3K4me3), where CTCF localizes. (Scale bars, 5 µm.) (C) Higher magnification images of dissociated retinal cells indicate strong segregation of chromatin in rods and various numbers of major chromocenters (numbered) among nonrod cells. Note that cells with single major chromocenters may represent rods or other retinal cells from the inner nuclear layer and the ganglion cell layer (22). (Scale bars, 2 µm.)

Fig. 2.

The cKO rods contain minimal CTCF protein at P30, display morphological phenotypes at P35, and undergo degeneration in adults. (A) CTCF cKO rods contain reduced but prominent CTCF protein at P23, which becomes minimally detectable at P30 and undetectable at P35. CTCF protein levels stay unchanged in cones and nonrod retinal cells. Note aggregates of synaptic ribbons between photoreceptors and bipolar cells (yellow arrows) and aggregated apical inner segments (white arrows). These morphological defects become prominent at P35 and more pronounced in single confocal planes. Note that some escaper rods express no Nrl-Cre and Tomato reporter, and maintain normal CTCF expression in P35 cKO retina (see merge). IS, inner segment; OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer. (Scale bars, 10 µm.) (B) CTCF is widely depleted in rods that display aggregated outer segments at P35. (Scale bars, 30 µm.) (C) Microglia are labeled by lba1 and reside specifically in OPL of a healthy retina but upon CTCF depletion, migrate and infiltrate to clear cell debris and aggregates that eventually leads to loss of rods. (Scale bars, 10 µm.)

Fig. 3.

CTCF cKO rods display physiological defects by P35. (A–C) ERG measures retinal response to light stimulation across a 25 d window. (D–F) Quantification of a and b waves indicates the first significant defects in b wave at P35. Each replicate animal is represented by a data point, and Student’s t test was performed to determine statistical significance.

Fig. 4.

Global CTCF-regulated transcription is significantly associated with CTCF-regulated accessibility at active chromatin. (A) Global transcriptional changes in P30 CTCF-depleted rods are highly enriched for CTCF-regulated accessibility. FET, Fisher’s exact test; tx, transcription. (B) Gene ontology analyses of CTCF-regulated gene expression in P30 rods. (C) Global chromatin accessibility changes in P30 CTCF-depleted rods. (D) Loci regulated by CTCF for both transcription and accessibility are highly enriched for CTCF occupancy, suggesting direct CTCF regulation. (E) Heatmaps of ATAC-seq signals that are regulated and occupied by CTCF in control and CTCF cKO P30 rods. Both opened and closed loci are included. CUT&Tag signals of CTCF and histone marks in control rods are also shown (one locus per row, sorted by H3K4me1 signal). CTCF CUT&Tag indicates loss of CTCF binding in CTCF cKO rods. Average signals across regions are color-annotated and correspond to box colors surrounding heatmaps. (F and G) Heatmaps and averages of ATAC-seq signals at active promoters and enhancers (H3K4me1 and H3K27ac) that are regulated and occupied by CTCF in (E). (H and I) Rescales of averaged ATAC-seq signals at enhancers in (G). Note stronger accessibility changes at enhancers than at promoters (F). (J) The floxed Ctcf locus indicates loss of transcription of all CTCF-coding sequences and the reduced expression of a juxtaposed gene upon CTCF depletion. (K–M) Cell type specificity of FACS purification is confirmed by high expression of the rod-specific gene Rho, no expression of the amacrine cell marker AP2α/TFAP2A, and no expression of the bipolar cell-specific gene GRM6/mGluR6 in the RNA-seq data. Transcriptional and cell type specificity is also reflected by the active and inactive histone marks at Rho, AP2α/TFAP2A, and GRM6/mGluR6.

Fig. 5.

CTCF promotion of chromatin accessibility corresponds to substantial transcription inhibition. (A) There are 220 solely closed genes with higher expression upon CTCF depletion. (B) Both the 220 genes and global targets of CTCF-inhibited accessibility are enriched with binding motifs of transcription repressors identified by motif enrichment analyses. (C and D) Representative loci that contain CTCF-promoted accessibility at active chromatin with CTCF-inhibited transcription.