A boundary-defining protein facilitates megabase-scale regulatory chromosomal loop formation in Drosophila neurons

Abstract

Regulatory elements, such as enhancers and silencers, control transcription by establishing physical proximity to target gene promoters. Neurons in flies and mammals exhibit long-range three-dimensional genome contacts, proposed to connect genes with distal regulatory elements. However, the relevance of these contacts for neuronal gene transcription and the mechanisms underlying their specificity necessitate further investigation. Here, we precisely disrupt several long-range contacts in fly neurons, demonstrating their importance for megabase-range gene regulation and uncovering a hierarchical process in their formation. We further reveal an essential role for the chromosomal boundary-forming protein Cp190 in anchoring many long-range contacts, highlighting a mechanistic interplay between boundary and loop formation. Finally, we develop an unbiased proteomics-based method to systematically identify factors required for specific long-range contacts. Our findings underscore the essential role of architectural proteins such as Cp190 in cell type-specific genome organization in enabling specialized neuronal transcriptional programs.

Keywords: CTCF; Cp190; Drosophila; TAD; genome folding; genome organization; long-range gene regulation; meta-domain; meta-loop; neuron; transcription.

Figure 1.

Meta-loop requirements for meta-domain formation and neuronal gene expression. (A) Cartoon Hi-C maps at the chromosome level (left) and a zoomed-in view of a meta-domain (beat-IV; right). (B–D) Micro-C maps of WT third-instar larval CNSs showing the Mp (B), beat-IV (C), or GluRIA–GluRIB (D) meta-domains. (Bottom) Zoom-in on left (X-axis) and right (Y-axis) anchors of meta-loops (black for I-I anchors, green for I-P or P-P anchors; numbered as in Mohana et al. 2023) with DNA-FISH probes (gray), gene tracks (longest isoforms colored by transcription direction), published pseudobulk ATAC-seq in larval CNSs (Mohana et al. 2023), and anchor deletions. (E–G) Violin plots (central horizontal lines mark medians; boxes mark interquartile ranges) of distances (in micrometers; Y-axis) between meta-loop anchors measured by DNA-FISH in n ELAV⁺ neuronal nuclei (blue) and neighboring nonneuronal nuclei (gray) of nerve cords of N independent embryos for the indicated genotypes (X-axis). Percentages of nuclei with colocalized anchors (<250 nm apart) are indicated. (H–J) RT-qPCR fold changes in Mp (H), beat-IV (I), or GluRIA or GluRIB (J) mRNA levels (normalized to the RpL15 housekeeping internal control gene) in adult heads of the same genotypes as in E–G. See also Supplemental Figures S1 and S2 and Supplemental Table S1.

Figure 2.

The GluRIA–GluRIB meta-domain facilitates paralogous gene cotranscription. (A) smRNA-FISH images of GluRIA (green) and GluRIB (pink) mRNAs in WT (top) and L36^ΔA46 (bottom) embryonic nerve cords. Arrowheads mark colocalized GluRIA and GluRIB transcription foci. (B) The percentage of neurons exhibiting a nascent transcription site for GluRIA, GluRIB, or both, shown relative to either the total neuron count (left bar plot) or neurons with nascent foci for at least one of the two genes (right bar plot). Data are presented for WT and L36^ΔA46. (C) Violin plots of distances (in micrometers; Y-axis) between GluRIA and GluRIB transcription sites in embryonic nerve cord nuclei coexpressing both genes in the indicated genotypes (X-axis).

Figure 3.

Cp190 is essential for forming CTCF-dependent and additional meta-loops. (A, right) Distribution of TAD boundaries called in WT larval CNSs (first lane), Cp190 or CTCF ChIP-seq peaks or differential Cp190 ChIP-seq occupancy in CTCF⁰ versus WT larval CNSs (second through fourth lanes, colored by average best.logFC), and expressed transcription start sites in WT larval CNSs (fifth lane), in 1 kb bins ±25 kb around all meta-loop anchors (rows) ordered by distance to the nearest TAD boundary (data are from Kaushal et al. 2021). Average signals across all anchors are shown above. (Left) Classification of anchors as intergenic (I; gray), promoter (P; blue), or Cp190-dependent (pink) or Cp190-independent (gray). (B) Differential analysis of strengths of loops involving meta-loop anchors (dots) measured by Hi-C in CTCF⁰ (top) or Cp190⁰ (bottom) versus WT 16–24 h embryo neurons. Labeled loops (red dots) were significantly weakened (Padj ≤ 0.05, |fold change| ≥ 2.5). (C) Overlap between all meta-loops and those dependent on CTCF and/or Cp190. (D,E) Same as Figure 1, B–D, but showing Hi-C maps of neurons from 16–24 h embryos of the indicated genotypes, with published CTCF and Cp190 ChIP-seq tracks and called peaks in larval CNSs (Kaushal et al. 2021) and single-cell ATAC-seq (sci-ATAC-seq3) in 16–18 h embryonic nerve cords (Calderon et al. 2022), showing a CTCF- and Cp190-dependent meta-domain (Mp; D) and a Cp190-dependent, CTCF-independent meta-domain (E). See also Supplemental Figure S3 and Supplemental Table S2.

Figure 4.

Lola-I copurifies with certain meta-loop anchors and is required for axon guidance. (A) Workflow for unbiased identification of meta-loop-associated proteins. (B,C) Volcano plots showing fold change (X-axis) and P-value (Y-axis) of proteins enriched at intergenic anchors of the CTCF- and Cp190-dependent meta-loop L34 (B) or the Cp190-dependent, CTCF-independent meta-loop L24 (C) compared with the Phs control bait in triplicate pull-down experiments. (D) lola gene structure with common BTB-encoding exons (orange) spliced to ZnF-encoding exons (green) in isoforms (rows; gray indicates noncoding exons). Blue lines mark peptides enriched (>10-fold) in any meta-loop anchor pull-down relative to the Phs control. Red shading indicates deleted regions in lola-I^KO and lola-G^KO. Published DNA-binding motifs for different isoforms (Enuameh et al. 2013) are shown at the right. (E) Percentages of the indicated genotypes (X-axis) that completed the indicated developmental transitions across biological replicates (dots). Horizontal lines indicate means. (F) α-Tropomyosin (green) and α-Fasciclin II (red) immunostaining of old (stage 16) embryos of the indicated genotypes (columns) and merged images with DAPI-stained DNA (blue). See also Supplemental Figure S4 and Supplemental Table S3.

Figure 5.

Lola-I is required to form a Cp190-dependent meta-domain. (A) Similar to Figure 3A but comparing Lola-I ChIP peaks with published Cp190 and CTCF ChIP peaks (Kaushal et al. 2021) around meta-loop anchors in WT larval CNSs. (B) Overlap between Lola-I (white), Cp190 (blue), and CTCF (gray) peaks in WT larval CNSs, with some peaks split for three-way comparisons (see the Materials and methods). (C) Percentages (Y-axis) of mCherry⁺ neuronal nuclei FACS-purified from dissociated 16–24 h embryos of the indicated genotypes (X-axis). (D) Differential analysis of strengths of loops involving meta-loop anchors (dots) measured by Hi-C in lola-I^KO versus WT embryonic neurons. Labeled loops (red dots) were significantly weakened (Padj ≤ 0.01, |fold change| ≥ 2.5). (E) Same as Figure 3D but showing Hi-C maps of neurons from 16–24 h embryos of the indicated genotypes, with Lola-I and published Cp190 (Kaushal et al. 2021) ChIP-seq tracks in larval CNSs, showing a meta-domain dependent on Lola-I and Cp190. See also Supplemental Figure S5 and Supplemental Table S4.