Chromosome-level organization of the regulatory genome in the Drosophila nervous system

Abstract

Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations.

Keywords: Drosophila; TAD; chromosomal loop; gene regulation; genome architecture; genome organization; nervous system; neuron; transcription.

Figure 1.. Meta-domains harbor loops involving CNS-expressed genes. See also Figures S1-S2.

A. Top: Third instar larva wing disc (left) or CNS (right) Micro-C map of chromosome 3L. Dotted squares mark meta-domains. Bottom: Zoom-in on left (in x) and right (in y) anchors of a meta-domain (center) harboring meta-loops L35 and L36. Pseudo-bulk scATAC-seq on larval CNSs, DNase-seq on embryonic neurons and muscles , mRNA-seq on larval CNSs and gene tracks (with longest isoforms colored according to transcription direction) are shown. Zoom-ins of 10 kb regions around each meta-loop anchor are shown in dotted rectangles. B. Cartoon representation of panel A column 2. C. Normalized larval CNS Micro-C count distribution (at 6.4 kb resolution) versus genomic distance of all pairwise contacts on all chromosomes (except Y). Red dots show meta-loops, purple dots show intra- TAD loops involving meta-loop anchors, light and dark grey curves respectively show 1-99 or 25-75 percentile ranges of all contact pairs, and the black line shows the median. D. Sizes (in bp, in y) of all indicated loop types (points). E. Number of meta-domains (in y) each harboring the indicated numbers of loops involving meta-loop anchors (in x). F. Distances (in bp, in y) of all 79 meta-loop anchors (points) to the nearest protein-coding gene TSS. The 200 bp line marks the cut-off for defining promoter or intergenic anchors. G. Classification of all 58 meta-loops called in larval CNSs (I: intergenic, P: promoter). H. RNA-seq on adult fly tissues from FlyAtlas v2 .

Figure 2.. Meta-loop anchors specifically pair over flexible distances in evolution. See also Figure S3.

A. Loops observed by Hi-C in CNSs of adult D. melanogaster (D. mel., left) and D. virilis (D. vir., right). For each chromosome arm, conserved loops are uniquely colored (but colors are re-used between chromosome arms) whereas loops present only in D. mel. or D. vir. are gray. Scale is in Mb. B. Dot plot of aligned regions of D. mel. (in x) and D. vir. (in y) chromosome 3L. Aligned regions in the same orientation are blue, those that are inverted in one species are red. Conserved loops are colored as in A and loop anchors are highlighted (in x and y). C. Sizes of all 37 conserved loops involving meta-loop anchors (colored symbols) plotted in D. vir. (in y) versus D. mel. (in x). Different symbols represent conserved loops with different relative anchor orientations. The “complex” case is illustrated in Figs. S3I-S3J. D. Similar to Fig. 1A but showing Hi-C maps of a D. vir. meta-loop (Dvir_L48) homologous to an intra-TAD loop in D. mel. (circled). D. mel. adult CNS ATAC-seq from Janssens et al. 2022 or whole D. vir. embryo DNase-seq at the indicated hours of development from Liu et al. 2021 are shown below. Anchors of meta-loops called in D. vir. were assigned to underlying DNase-seq peaks in old (25-28h) D. vir. embryos.

Figure 3.. Meta-loop anchors come into enhanced proximity in a large fraction of neurons during neuronal differentiation. See also Figures S4-S5.

A. Top: Hours in embryogenesis, embryonic stages and major events in neurogenesis. Bottom: Whole-genome Hi-C maps in neuroblasts (row 1), glia (row 2) or neurons (row 3) FACS-purified from embryos of the indicated ages (columns). Dotted squares show zoom-ins around example meta-loops. 3D genome configurations deduced by Hi-C are schematized below and bounded by colored boxes. B. For each meta-loop observed in WT larval CNSs (rows), loop strength (color, measured as the observed-over-expected normalized Hi-C count) is shown in each sample (columns). Loop types are color-coded on the right. C. Similar to Fig. 1A but showing Hi-C maps and DNase-seq profiles in neurons isolated from staged embryos by Reddington et al. 2020 at the beat-IV meta-domain, in neurons of 6-8 (left), 10-12 (middle) or 14-16 (right) hour old embryos. Arrowheads mark emerging TAD boundaries near meta-loop anchors. DNA-FISH probes used in panel D are highlighted in green or red in the gene tracks. D. Violin plot of distances (in y, in μm) measured by DNA-FISH with probes against beat-IV and its intergenic meta-loop anchor, in Elav-immunolabeled (pink) or neighboring (blue) nuclei of embryos of the indicated stages (in x). Below, lateral views of embryos of the indicated stages stained with a probe against beat-IV mRNA (green), α-Elav (pink) and DAPI (anterior, left; posterior, right). P values and D-statistics from two-sided Kolmogorov-Smirnov tests are indicated.

Figure 4.. Meta-domains are formed by meta-loop anchors and delimited by TAD boundaries.

A. Similar to Fig. 1A but showing Hi-C maps of the nolo meta-domain in WT (left) or nolo^ΔA23 (right) larval CNSs. Filled/empty arrowheads point to meta-loop L22 presence/absence in each genotype. The extents of meta-loop anchor A23 deletion or testing for enhancer activity in Fig. S6G are indicated below. B. Same as A but for the beat-IV meta-domain in WT (left) or beat-IV^ΔA65 mutants (right). C. Similar to A but also showing CTCF ChIP-seq profiles and called peaks at the GluRIA-GluRIB meta-domain in WT (left) or CTCF⁰ (middle) larval CNSs . Filled/empty arrowheads mark normal/weakened TAD boundaries in each genotype. A differential (CTCF⁰ minus WT) Hi-C map is shown on the right (scale is log2 fold change of contacts). Differentially expressed genes defined by RNA-seq in CTCF⁰ relative to WT larval CNSs (padj<0.05 and ∣fold change∣>1.5) are highlighted in pink and labeled with a letter on the gene track in the middle, and the fold changes are indicated in the table.

Figure 5.. An intergenic anchor enables distal activation of nolo. See also Figure S6.

A. Combined scRNA-seq datasets generated in WT, nolo^ΔA23 and beat-IV^ΔA65 third instar larval CNSs. Cell clusters are numbered, and major cell types color-coded. B. Same as A but coloring cells expressing nolo (red) or beat-IV (blue). C. For each cell cluster, the percentage (circle size) of cells expressing the indicated genes (columns) and the average scaled expression (color) are shown in WT (1), nolo^ΔA23(2) or beat-IV^ΔA65 (3) larval CNSs. Cells from cluster 11 were variably recovered between samples and thus excluded, pros, repo and wor are respectively neuronal, glial and neuroblast markers. Asterisks mark cell clusters that significantly (padj≤0.01) differentially expressed the indicated gene in nolo^ΔA23 (red asterisk) or beat-IV^ΔA65 (orange asterisk) mutants relative to WT. D. RNA-FISH with a probe against nolo mRNA (green) in dissected nerve cords of stage 16 WT and nolo^ΔA23 mutant embryos immunostained with α-Elav (pink) and labeled with DAPI (anterior, left; posterior, right). Reduced nolo expression shown here was observed in 100% of nolo^ΔA23 mutants (≥10 embryos imaged per genotype). E. Percentage (in y) of nuclei with nolo promoter DNA-FISH signal that overlapped nascent nolo RNA-FISH signal in nerve cords of stage 17 WT (left) or nolo^ΔA23 (right) mutant embryos (dots). Horizontal lines show means. N, number of embryos; n, number of nuclei with DNA-FISH signal scored. F. Schematic of the longest Nolo protein isoform (Uniprot accession Q0E8N3). Protein domains and positions of premature stop codons present in nolo^KO alleles are marked. G. Percentages of indicated genotypes that successfully developed from pupae to fully hatched adults in three independent batches of 30-40 animals (dots). Horizontal lines show means. H. Immunostaining of synaptic boutons (α-vGlut) and the whole neuromuscular junction (α-HRP) in muscle 6/7 terminals in WT and the indicated nolo mutant third instar larvae. Representative images are shown, and results from several images are quantified in panel I. I. Quantification of neuromuscular junction (NMJ) area (left) and numbers of synaptic boutons (right) from n≥8 larvae (data points) of the indicated genotypes. P values from one-way ANOVA (for wildtype versus mutant comparisons) or unpaired two-sided t tests (for luciferase to nolo knock-down comparisons) are indicated.

Figure 6:. The GAF BTB domain is required to form the sns-hbs meta-domain.

A. Similar to Fig. 1A but also showing GAF ChIP-seq profiles in larval tissues including the CNS at the sns-hbs meta-domain in WT (left) and GAF^ΔBTB (right) larval CNSs. Filled/empty arrowheads point to WT/reduced meta-loop strength in each genotype. Differential analysis of strengths of loops involving meta-loop anchors (dots) measured by Micro-C in WT versus GAF^ΔBTB larval CNSs. Labeled loops (pink dots) were significantly weaker (padj≤0.01) in GAF^ΔBTB compared to WT. C. Distribution of GAF ChIP-seq signal in larval tissues including CNS in 1-kb bins ±25 kb around all anchors of meta-loops (lane 1) or loops previously defined in tissue culture cells (lane 2) or embryos (lanes 3 and 4) ranked by GAF ChIP occupancy. Average signals over all loop anchors are shown above. D. HCR RNA-FISH with co-hybridized probes against sns (magenta) and hbs (yellow) mRNAs in WT (left) and GAF^ΔBTB (right) larval CNSs. Filled/empty arrowheads mark a stripe with WT/reduced sns expression in each genotype. This phenotype was observed in 100% of GAF^ΔBTB mutants (≥10 larval CNSs imaged per genotype). E. Fold change of hbs and sns mRNA levels measured by RT-qPCR with two independent primer pairs each, in GAF^ΔBTB relative to WT larval CNSs. Dots represent individual biological triplicate values, thick horizontal bars represent average values, and error bars indicate standard deviation. P values from two-sided unpaired t tests are shown.

Figure 7:. CTCF is required to form a different set of meta-loops than GAF. See also Figure S7.

A. Same as Fig. 4C but also showing Cp190 ChIP-seq profiles and called peaks at the beat-IV meta-domain in WT (left) or CTCF⁰ (right) larval CNSs. B. Differential analysis of strengths of loops involving meta-loop anchors (dots) measured by Hi-C in WT versus CTCF⁰ larval CNSs. Labeled loops (pink dots) were significantly weaker (padj≤0.01) in CTCF⁰ compared to WT. C. Distribution of DNase-seq signal from Reddington et al. 2020 around the anchors of CTCF-dependent meta-loops shown in B, in 500-bp bins ±25 kb around these anchors ordered by genomic position, in 10-12 hour old embryonic neurons (top) or muscle (bottom). D. Distribution of CTCF ChIP-seq peaks and differential Cp190 ChIP-seq occupancy (colored by average best.logFC) in CTCF⁰ versus WT larval CNSs (data from Kaushal et al. 2021 ) in 1-kb bins ±25 kb around all meta-loop anchors ordered by genomic position. Percentages of meta-loop anchors with at least one CTCF ChIP peak or differential Cp190 binding region in WT versus CTCF⁰ mutants over all anchors are shown on top. Pink filled/empty arrowheads indicate anchors of meta-loops weakened in CTCF⁰ that are bound/not bound by CTCF. These anchors are labeled on the left and meta-loops are labeled on the right. E. Ventral views of embryos of indicated genotypes (rows) and stages (columns) stained with a probe against beat-IV mRNA (green), α-Elav (pink) and DAPI (anterior, left; posterior, right; merged images on top). Specific RNA-FISH signal appears as dots; string-like and broad green non-specific signal is visible in trachea and epidermis. Filled/empty arrowheads point to WT/reduced beat-IV RNA-FISH signal in each genotype. Phenotypes shown here were observed in 100% of embryos (≥ 6 embryos imaged per genotype and stage).