Chromosome-biased binding and gene regulation by the Caenorhabditis elegans DRM complex

Abstract

DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we describe new aspects of DRM binding and function revealed through genome-wide analyses of the Caenorhabditis elegans DRM subunit LIN-54. We show that LIN-54 DNA-binding activity recruits DRM to promoters enriched for adjacent putative E2F/DP and LIN-54 binding sites, suggesting that these two DNA-binding moieties together direct DRM to its target genes. Chromatin immunoprecipitation and gene expression profiling reveals conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find that LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, C. elegans DRM does not act uniformly throughout the genome: the DRM recruitment motif, DRM binding, and DRM-regulated embryonic genes are all under-represented on the X chromosome. However, germline genes down-regulated in lin-54 mutants are over-represented on the X chromosome. We discuss models for how loss of autosome-bound DRM may enhance germline X chromosome silencing. We propose that autosome-enriched binding of DRM arose in C. elegans as a consequence of germline X chromosome silencing and the evolutionary redistribution of germline-expressed and essential target genes to autosomes. Sex chromosome gene regulation may thus have profound evolutionary effects on genome organization and transcriptional regulatory networks.

Figure 1. LIN-54 binds DNA directly through its tesmin domains and recruits DRM to promoters.

(A) C. elegans lin-54 gene structure for wild-type isoforms (lin-54a and lin-54b), lin-54 mutant alleles, and yeast constructs used in this study. The lin-54 gene encodes a protein with two tesmin/CXC domains (black boxes). lin-54(n3423) is a null allele in which the 5′ end and most exons are deleted. lin-54(n2990) is a missense allele that harbors a mutation in the second tesmin domain, and lin-54(n2231) has both the tesmin domain mutation and an additional point mutation. Constructs equivalent to lin-54a, lin-54(n2990), and lin-54(n2231) were used in yeast one-hybrid (Y1H) assays, and are referred to as LIN-54a, LIN-54^G252E, and LIN-54^G252E/A442T, respectively. An additional LIN-54 construct containing a point mutation in each tesmin domain was created and is referred to as LIN-54^K186E/G252E. Gray box = exon, black box = tesmin domain, white box = 3′ untranslated region, asterisk = missense mutation. (B) Y1H assays using wild-type LIN-54a, LIN-54^G252E, LIN-54^G252E&A442T, and LIN-54^K186E/G252E mutant proteins with the promoters of the genes pos-1, lin-54, and vha-15. AD = Gal4 activation domain, P = permissive media, S = selective media. (C) DRM subunit binding in wild-type and lin-54(n2990) mutants, measured by ChIP-qPCR at the target promoters lin-9 and lin-54. Binding is shown as the amount of DNA amplified in each ChIP sample relative to input, with the ratio in wild-type set to 1.0. Standard deviations from three independent experiments are shown.

Figure 2. LIN-54 tesmin domain mutation does not disrupt its stability or association with DRM.

(A) Immunofluorescence of LIN-54 in embryos from wild-type and lin-54(n2990) animals. (B) Western blots of whole worm extracts from wild-type, lin-54(n2990), and lin-54(n3423) mutants, probed with antibodies against LIN-54, histone H3, and actin. Lanes contain protein from 25, 50, and 100 worms. (C) Yeast two-hybrid assay using either wild-type LIN-54 (top) or mutant LIN-54^G252E/A442T (bottom) as bait and LIN-9 as prey. DB = Gal4 DNA-binding domain. AD = Gal4 activation domain. P = permissive media, S = selective media. (D) Immunoprecipitation using antibodies against LIN-37 in lin-54(n2231) tesmin mutant extract, and probed with antibodies listed at left.

Figure 3. LIN-54 binding is enriched at promoters of genes involved in development, reproduction, and cell division that contain a putative E2F-LIN-54 binding motif.

(A) Representative MA2C derived peaks from two biological replicates of LIN-54 ChIP-chip from mixed-stage worms. Arrows indicate genes and direction of transcription. (B) Relative locations of LIN-54 ChIP peaks. The distance between the mode of each LIN-54 ChIP peak and the translational start site (TSS) of neighboring genes was calculated, and the percentages of four classes of LIN-54 locations are indicated. Enriched gene ontology (GO) terms among genes with peaks within 1 kb of their TSS include development, reproduction, and cell cycle/cell division. (C) The numbers of intergenic LIN-54 peaks relative to their distance from the nearest TSS. (D) Conservation of orthologous LIN-54 binding targets between worms, flies, and humans. (E) An overrepresented motif in LIN-54-bound promoters (Motif 1, top). Aligned below are previously defined motifs: the C. elegans EFL-1 consensus , an extended Drosophila dE2F2 motif enriched among dE2F2, dLIN-9 and dLIN-54 co-regulated genes and the human CDE/CHR motif from the cdc2 promoter . Dotted lines outline regions bound by human E2F4 and LIN-54 at cdc2 and their homologous motif sequences in other organisms. (F) Examples of LIN-54 binding (ChIP peaks shown by black bars representing MA2C score) and location of Motif 1 (orange square) at promoters of two genes (mrt-2 and C29E4.12, arrows = TSS; green boxes = exons).

Figure 4. LIN-54 can function as a transcriptional activator or repressor.

(A) Microarray gene expression profiling analysis of lin-54(n2990) embryos and lin-54(n3423) germlines. Genes that change expression in lin-54 mutant animals are grouped into four classes: “up in embryo”, “down in embryo”, “up in germline” and “down in germline”. Overlap with LIN-54 ChIP peaks is indicated. (B) Cartoon indicating the inferred regulation by wild-type LIN-54 in embryo (left) or germline (right) and the major Gene Ontology (GO) terms associated with each class of regulated genes. p-value<0.05 for all GO terms.

Figure 5. LIN-54 shows autosome-enriched binding and chromosome-biased gene regulation.

(A) LIN-54 ChIP peaks along the entire X chromosome (top) and chromosome IV (bottom). (B–C) Number of LIN-54 ChIP peaks per mega base (B) and percentage of promoters bound by LIN-54 (C) on each C. elegans chromosome. LIN-54 ChIP peaks occur less frequently on the X chromosome, independent of chromosome size and gene density. (D) Occurrence of putative E2F/DP-LIN-54 binding Motif 1 and other transcription factor binding motifs in promoter regions (1 kb upstream from translational start site) of autosomal genes and X-linked genes. Motif 1 is under-represented in X-linked promoters. (E) Chromosome distribution of genes up-regulated or down-regulated in lin-54(n2990) embryos (left), lin-54(n3423) germline (middle), or commonly co-regulated by cluster analysis of lin-54(n3423), efl-1(n3639), dpl-1(n3316), and lin-35(n745) germlines (right). Overlap with LIN-54 ChIP peaks for an average autosome or X chromosome is indicated below. Commonly up is group E, commonly down is group B from Figure S4. Data for efl-1, dpl-1, and lin-35 are from . (F) The percentage of genes located on the five autosomes (gray) or the X chromosome (black). Expected values are presented both for all genes in the genome, and for all genes normally expressed (expressed genes) in embryo or germline, and compared to observed percentages of genes up-regulated (genes up in mut) or down-regulated (genes down in mut) in lin-54 mutants. Asterisks indicate p-value<10⁻³ by Fisher's Exact test orG-test.

Figure 6. DRM complex members localize to germline autosomes.

Shown are nuclei in the meiotic pachytene stage in the hermaphrodite germline. Arrowheads indicate a chromosome in the nucleus with less LIN-54 staining (A–D) or less staining of other DRM subunits (E). (A) Immunofluorescence with anti-LIN-54 antibody (red) and DNA dye (green, merge in yellow). (B) Antibodies against a histone modification associated with active transcription (H4K12Ac, blue) show enrichment on autosomes, and co-localize with LIN-54 (red, DNA in green). (C) LIN-54 (red, DNA in green) staining in the him-8(e1489) mutant in which X chromosomes do not pair and acquire the histone modification H3K9me2 (blue). (D) Co-staining of LIN-54 (red) with DPL-1 (blue, DNA in green). Both are under-represented on the X chromosome (arrowhead). (E) Immunofluorescence of DRM complex subunits (red) on wild-type germline nuclei (DNA, green; merge yellow). Images in A and B represent deconvolved confocal stacks. Scale bar represents 5 µm (A) or 1 µm (B–E).