Distinctive regulatory architectures of germline-active and somatic genes in C. elegans

Abstract

RNA profiling has provided increasingly detailed knowledge of gene expression patterns, yet the different regulatory architectures that drive them are not well understood. To address this, we profiled and compared transcriptional and regulatory element activities across five tissues of Caenorhabditis elegans, covering ∼90% of cells. We find that the majority of promoters and enhancers have tissue-specific accessibility, and we discover regulatory grammars associated with ubiquitous, germline, and somatic tissue-specific gene expression patterns. In addition, we find that germline-active and soma-specific promoters have distinct features. Germline-active promoters have well-positioned +1 and -1 nucleosomes associated with a periodic 10-bp WW signal (W = A/T). Somatic tissue-specific promoters lack positioned nucleosomes and this signal, have wide nucleosome-depleted regions, and are more enriched for core promoter elements, which largely differ between tissues. We observe the 10-bp periodic WW signal at ubiquitous promoters in other animals, suggesting it is an ancient conserved signal. Our results show fundamental differences in regulatory architectures of germline and somatic tissue-specific genes, uncover regulatory rules for generating diverse gene expression patterns, and provide a tissue-specific resource for future studies.

Figure 1.

Tissue-specific profiling of chromatin accessibility and gene expression in C. elegans tissues. (A) Procedure to perform tissue-specific nuclear RNA-seq and ATAC-seq experiments. Representative results at known tissue-specific loci are shown on the right. (B, top) Heatmap of normalized accessibility (log₂ RPM) for 25,205 classified sites. (Bottom) Classification of the accessible sites into tissue-specific, tissue-restricted, or ubiquitous classes. Protein-coding promoters are in dark colors, enhancers are lighter, and other accessible sites (e.g., noncoding promoters, unassigned promoters, other elements) are lightest. (C, top) Heatmap of normalized gene expression (log₂ TPM) for 12,301 classified protein-coding genes. (Bottom) Classification of genes into tissue-specific, tissue-restricted, or ubiquitous classes. For classification procedure, see Methods. Unclassified sites and genes are not shown.

Figure 2.

Regulatory architectures of ubiquitous, germline, and soma-restricted genes have distinctive features. (A) Percentage of genes organized in an operon for each gene class. (B) Percentage of genes with one, two, or three or more promoters for each gene class. (C) GO terms from Biological Process ontology enriched in ubiquitous genes with one, two, or three or more annotated promoters. (D) Percentage of genes with zero, one, two, or three or more enhancers associated with genes of each expression class. Only genes with at least one annotated promoter are considered. (E) Percentage of unidirectional or bidirectional protein-coding promoters for each gene class. (F) Percentage of genes with the indicated number of introns for each gene class. (G) Intron length for each gene class. (H) Classes of promoters associated with genes of each expression class. Only the major promoter classes are displayed. For all results, see Supplemental Table S2. (I) Concordance of promoter classes for genes with two promoters. (J) Gene expression levels in whole young adults for ubiquitous genes with one, two, or three or more promoters (left) or with zero, one, two, or three or more enhancers (right). (K) Gene expression levels of tissue-specific genes with one promoter or two promoters specifically active in the same tissue. In panels B through K, only first genes in operons and nonoperon genes were considered. (L, left) Examples of the simple regulatory architecture shared by ubiquitous genes and germline-specific genes. (Right) Examples of more complex architectures found at developmental ubiquitous genes (e.g., lin-45) or somatic tissue–specific genes (e.g., mlt-10).

Figure 3.

Ubiquitous and germline-specific promoters have a stereotypical architecture with well-positioned nucleosomes. (A) Interpretation of two ATAC-seq fragment density plots (also known as “V-plots”). The dense cluster of short fragments at the promoter centers represents the nucleosome-depleted region (NDR), whereas the dense clusters of longer fragments located −100 and +100 bp away from the promoter centers are indicative of aligned −1/+1 flanking nucleosomes. (B) ATAC-seq fragment density plots (V-plots) over different classes of promoters. The x-axis represents the distance between the fragment midpoint and the promoter center. The y-axis represents ATAC-seq fragment length. The color scale indicates the normalized density of ATAC-seq fragments. (C) Tissue-specific nucleosome occupancy probability over different classes of promoters aligned at their TSS. Only promoters with experimentally defined forward and reverse TSSs are considered. Rows are ordered by the distance between TSS and +1 nucleosome. (D, left) Schematic of the distance metrics measured in promoters: (d₁) distance between the mode TSS and the +1 nucleosome edge; (d₂) distance between modes of divergent TSSs within the same promoter; (w) width of the NDR. (Right) d₁, d₂, and w distance metrics for different classes of promoters. The metrics for ubiquitous promoters were measured using nucleosome occupancy probability track derived from whole young adult ATAC-seq data (Jänes et al. 2018).

Figure 4.

Ubiquitous and germline-specific promoters have strong 10-bp WW periodicity correlated with nucleosomes. (A) Motifs enriched in different classes of promoters. Sequences from −75 to +105 bp around the promoter centers were considered. (B, top) Normalized distribution of pairwise distances between WW dinucleotides found in the sequences from −50 bp to +300 bp relative to TSSs, for different classes of promoters. (Bottom) Associated WW power spectral densities (PSDs). (C) Metaplots of WW, TT, and AA 10-bp periodicity scores at different classes of promoters, aligned at TSSs. The +1 nucleosome position observed at ubiquitous and germline promoters (∼20–167 bp downstream from the TSS) is displayed by the shaded orange area delimited by dotted lines. (D) WW (red) and TT (green) dinucleotide occurrences observed at +1 nucleosomes of ubiquitous promoters (400-bp window centered at nucleosome dyads). Rows were shifted up to 5 bp to highlight the phased 10-bp periodic patterns. Summed dinucleotide occurrences are represented on top of each heatmap by a line plot. The average TSS positions of ubiquitous promoters (∼20 bp upstream of the +1 nucleosome edge) are displayed by the shaded gray area. (E) Correlation between +1 nucleosome occupancy and 10-bp WW periodicity in ubiquitous and germline-specific promoters. The +1 nucleosomes were binned by their nucleosome occupancy score, and the overall 10-bp WW periodicity was assessed in each bin (approximately 20 200-bp long nucleosomal sequences centered at nucleosome dyads). The y-axis represents the average nucleosome occupancy in each bin.

Figure 5.

Ten-base-pair WW periodicity at ubiquitous promoters is a feature of nonmammalian genomes. (A) Nucleosome occupancy probability scores (red; left axis) and 10-bp WW periodicity (blue; right axis) at worm, fly, zebrafish, mouse, and human TSSs. (B) Normalized distribution of pairwise distances between WW dinucleotides found in the sequences from −50 bp to +300 bp relative to TSSs, for genes with broad expression (top row; 20% lowest gene expression CV scores) or regulated expression (bottom row; 20% highest gene expression CV scores) in worm, fly, zebrafish, mice, and human. (C) Associated WW power spectral density values at a 10-bp period.

Figure 6.

Two models of preinitiation complex (PIC) positioning at promoters. The nucleosome organization and sequences features found in ubiquitous, germline-specific, and somatic tissue–specific promoters suggest that two models of PIC recruitment exist. (A) In ubiquitous and germline-specific promoters (i.e., germline-active promoters), nucleosomes flank a narrow 120- to 140-bp-wide NDR. Positioning of these nucleosomes is facilitated by the underlying DNA sequence, which harbors highly periodic WW (mainly TT) dinucleotides. Thus, the PIC assembling at the NDR is physically constrained by the +1 nucleosome edge, resulting in transcription initiation ∼20 bp upstream of the +1 nucleosome edge. Many of these promoters lead to bidirectional elongative transcription. Otherwise, upstream-antisense RNA (uaRNA) are transcribed. (B) In soma-restricted promoters, NDRs are wider (>200 bp), and flanking nucleosomes are weakly positioned and not reproducibly aligned relative to the TSS. Core and transcription factors recruited to the NDR facilitate assembly and positioning of the PIC, resulting in transcription initiation −45 to −50 bp downstream.