RNA Pol I activity maintains chromatin condensation and the H3K4me3 gradient essential for oogenesis, independent of ribosome production

Abstract

Oogenesis requires extensive and dynamic chromatin remodeling that primes gene promoters for later transcriptional activation during embryonic development. Here, we uncover a pivotal, non-canonical role for RNA Polymerase I (Pol I) in driving these chromatin state transitions during Caenorhabditis elegans oogenesis. Using the auxin-inducible degron system to selectively deplete either a Pol I-specific catalytic subunit or a ribosome assembly factor, we disentangle the consequences of impaired nucleolar integrity from reductions in ribosome biogenesis. Strikingly, although disrupting ribosome assembly caused minimal effects on oocyte production, loss of nucleolar structure via Pol I depletion led to severe meiotic chromosome abnormalities, widespread changes in chromatin accessibility, and a dampening of the typical distal-proximal H3K4me3 gradient required for oogenesis, resulting in fewer but significantly larger oocytes. Despite their promoters becoming more accessible, oogenesis genes did not show large changes in steady-state mRNA, consistent with transcriptional repression prior to fertilization. Instead, Pol I depletion prematurely remodeled oogenic chromatin, through a misdirection of H3K4me3 deposition towards promoters normally primed for zygotic genome activation. These findings reveal an epigenetic gating function for nucleolar integrity in oocyte maturation: Pol I preserves three-dimensional chromatin organization and maintains proper spatiotemporal regulation of histone modifications, independent of ribosome production. Given the evolutionary conservation of nucleolar dynamics and histone modifications during gametogenesis, our work suggests that nucleolar stress, whether from environmental factors, aging, or genetic disorders, could broadly compromise fertility by disrupting oogenic chromatin priming.

Figure 1.. Nucleolar integrity remains intact during germ cell maturation until the penultimate mature oocyte.

(A) 3-D confocal/T-PMT projection of an adult gonad arm with an endogenous NUCL-1::mKate2 nucleolar marker and an EmGFP::SYP-3 meiosis marker. Nucleoli are present throughout the mitotic, pachytene, and diakinesis zones, as indicated by the localization of EmGFP::SYP-3. Scale bar = 50 μm. (B) 3-D confocal/T-PMT projection of an adult gonad arm with an endogenous NUCL-1::mKate2 nucleolar marker. NUCL-1 forms droplets in the “−2” oocyte and is absent in the most proximal and mature “−1” oocyte. Scale bar = 50 μm. (C) 3-D confocal/T-PMT projection of an adult gonad arm with an endogenous GFP::DAO-5 nucleolar marker. Similar to NUCL-1, DAO-5 forms droplets in the “−2” oocyte and disappears entirely in the “−1” oocyte. Scale bar = 50 μm.

Figure 2.. Reducing RNA Pol I activity, but not ribosome assembly, disrupts nucleolar structure in the germline.

(A) Fluorescent images of degron::GFP::RPOA-2 and GRWD-1::degron::GFP in dissected gonads from young adults with 18 hours of control (EtOH) or 1 mM auxin (IAA) treatment at the L4 stage. Dotted lines indicate the outline of the gonad arm. Scale bar = 50 μm. (B) Average log10 counts per million (CPM) reads from gonadal RNA-seq (without polyA selection) mapped to a simplified single-copy 45S rDNA locus. Averages are composed of three biological replicates per condition, each consisting of 20 gonads. RPOA-2-depleted germlines show reduced coverage across internally transcribed spacer (ITS) regions compared to GRWD-1-depleted germlines. (C) Live 3-D confocal projection (left) of an endogenous NUCL-1::mKate2 nucleolar marker in the late pachytene zone of young adults treated with or without auxin to deplete degron::GFP::RPOA-2 or GRWD-1::degron::GFP. A zoomed-in 2-D layer (right) shows that nucleoli lose their spherical shape and form nucleolar caps (circled in white) after RPOA-2 depletion, whereas GRWD-1 depletion leads to vacuolar nucleoli formation with persistent spherical volume. Scale bar = 50 μm.

Figure 3.. Oocyte morphology and organization are impaired by reduced RNA Pol I activity, independent from RNA Pol I’s role in ribosome production.

(A) Representative DIC images of adult germlines after 18 hours of control or auxin treatment. The dotted line highlights the proximal arm, and arrows point to proximal oocytes. Scale bar = 50 μm. (B) Principal component analysis and density contour estimates of various animal and gonadal measurements (body area, length of the proximal arm, length and area of the three most proximal oocytes, and the number of proximal oocyte counts). Each point represents an individual worm. RPOA-2-depleted germlines cluster more distinctly from their controls compared to GRWD-1-depleted germlines. (C) Distribution of the ratios of proximal arm length to body area in adult germlines after 18 hours of control or auxin treatment. When accounting for body area, germlines developing under reduced RNA Pol I activity have smaller proximal arms relative to their controls, while germlines developing under similarly reduced levels of ribosome production show an insignificant difference in proximal arm size relative to their controls. (D) Distribution of the number of proximal oocytes in adult germlines after 18 hours of control or auxin treatment. The smaller proximal arm size observed in RPOA-2-depleted germlines is explained by a reduction in the number of proximal oocytes compared to controls. (E) Distribution of the ratio of oocyte length to proximal arm length for the three most proximal oocytes (“−1”, ”−2”, “−3”) in adult germlines after 18 hours of control or auxin treatment. The three-most proximal oocytes of RPOA-2-depleted germlines occupy a significantly larger proportion of the proximal arm length compared to their controls, suggesting that oocyte morphology is significantly altered by reducing RNA Pol I activity. Each point in (C-E) represents a measurement from an individual animal. All comparisons were performed using two-tailed Welch’s two-sample t-tests followed by Bonferroni correction, and are based on measurements from the following number of animals: RPOA-2 control = 29, RPOA-2 depletion = 35, GRWD-1 control = 26, GRWD-1 depletion = 30.

Figure 4.. Irregular chromosome morphology during meiotic entry is nucleolar-cap dependent and independent from reduced ribosome assembly.

(A) 3-D confocal average projections of late pachytene nucleoli identifiable by endogenously tagged NUCL-1::mKate2 (left), along with average projections of dissected gonad arms with a SPY555-DNA probe (right). Images were taken following control or 1 mM auxin (IAA) treatment to deplete RPOA-2 or GRWD-1. Left scale bar = 15 μm, right scale bar = 50 μm. (B) Effects of auxin concentration [IAA] on mean gonadal degron::GFP::RPOA-2 intensity (left). Intensity scaled with 0 μM treated maximum set to 1, and 1000 μM treated minimum set to 0. The mean and 95% confidence intervals for each distribution are represented in red, and each point represents the mean intensity measurement from a gonad. Sample sizes are 39, 31, 72, 58, and 23 gonads for 0, 0.5, 10, 500, and 1000 μM treatments, respectively. (C) Live 3-D confocal projection of an endogenous NUCL-1::mKate2 nucleolar marker in late pachytene nucleoli of young adults treated with varying concentrations of auxin (IAA) to deplete RPOA-2 (column 1). Scale bar = 15 μm. Zoomed in representative nuclei within the mitotic, transition zone (TZ), or meiotic germline zones, imaged from dissected adult gonad arms with a SPY555-DNA probe after varying concentrations of auxin (IAA) treatment to deplete RPOA-2 (columns 2–4). Scale bar = 5 μm. Asterisks highlight nuclei with typical TZ or meiotic chromosome morphology.

Figure 5.. Disrupting RNA Pol I activity prematurely primes autosomal promoters in an accessible state associated with oogenesis.

(A) Venn diagram comparing the number of significantly more accessible (SMA) regions following RPOA-2 depletion versus GRWD-1 depletion, based on an adjusted p < 0.05. (B) Genomic feature annotations of ATAC-seq peaks. Regions with significantly increased accessibility after RPOA-2 or GRWD-1 depletion are predominantly located within 1 kb of the nearest promoter. (SMA = significantly more accessible). (C) Log₂ fold-change estimates of chromatin accessibility based on gonadal ATAC-seq data from at least three biological replicates per condition, each composed of 20 gonads. Each segment along the x-axis represents a genomic region. The number of differentially accessible regions are noted in the plot legend based on an adjusted p < 0.05. Percentages represent the proportion of differentially accessible peaks relative to the total number of called peaks per chromosome. Depleting RPOA-2 increases autosomal chromatin accessibility, but does not affect the X chromosome. (D) Enrichment of gene ontology (GO) biological processes in genes exhibiting increased accessibility after depletion of RPOA-2 or GRWD-1 (Hypergeometric test, FDR < 0.001). The term “gene” refers to those associated with the nearest genomic feature annotation of each peak. Both reductions in RNA Pol I activity and ribosome assembly increase the accessibility of translation-related genes, while reducing RNA Pol I activity distinctly increases the accessibility of genes involved in biological processes such as sexual reproduction. (E) Overlap between peaks that are significantly more accessible (SMA) after RPOA-2 depletion and peaks near genes involved in oogenesis (Hypergeometric test). (F) Chromosomal distribution of genes involved in oogenesis and significantly more accessible (SMA) peaks after RPOA-2 depletion. (G) Alignment of the C. elegans EFL-1-binding site, human E2F-binding site, and the two motifs enriched in significantly more accessible (SMA) peaks after RPOA-2 depletion. (H) Genomic feature annotations of peaks containing an EFL-1-binding site. (I) GO enrichment analysis for biological processes among genes with promoter peaks that contain an EFL-1-binding site (Hypergeometric test, FDR < 0.05).

Figure 6.. Chromatin regions that become more accessible after reducing RNA Pol I activity display patterns of H3K4me3 remodeling characteristic of the germ-to-oocyte transition.

(A) Log₂ fold-change estimates of chromatin accessibility after RPOA-2 depletion in a random set of peaks, along with peaks annotated to genes that undergo H3K4me3 remodeling during oogenesis. The number of peaks in each set is specified by n, and means are denoted by an x. One-tailed Welch’s two-sample t-tests with Bonferroni corrections were performed to test whether the average accessibility of regions near H3K4me3-remodeled genes is greater than the accessibility of a random set of regions in RPOA-2-depleted germlines. (B) Metagene analysis of mean log₂ ATAC signal (RPOA-2 depletion/control) within 1 kb of genes that undergo H3K4me3 remodeling during oogenesis. In gonads with reduced RNA Pol I activity, the shape of chromatin accessibility signal within each cluster mirrors the shape of H3K4me3 remodeling associated with each gene cluster. (C) Distribution of average H3K27me3, H3K36me3, and H3K4me3 ChIP signals from wild-type germ cells or oocytes within ATAC-seq peaks that are significantly more accessible (SMA) after RPOA-2 depletion, compared to signals within an identically-sized random subset of regions. Means are denoted by an x. (D) Metagene plot displaying the average H3K27me3, H3K36me3, and H3K4me3 ChIP coverage from wild-type germ cells or oocytes within 1 kb of genomic regions that are significantly more accessible (SMA) after RPOA-2 depletion, compared to coverage within an identically-sized random subset of promoter ATAC-seq peaks that are not SMA. (E) Representative 3-D confocal projections of adult gonad arms with H3K4me3 immunostaining and DAPI after control treatment or RPOA-2 depletion. Dotted lines represent gonad boundaries determined from T-PMT. Scale bar = 50 μm. (F) Using the example gonads from (E), we show germ cell position along a linear gonad axis following implementation of a gonad linearization algorithm (see Methods). Each point represents a germ cell detection; however, note that not all gonadal cells are detected by the algorithm. The distal tip is represented by zero on the gonad axis, whereas the most proximal tip of the gonad is variable based on biological size, gonad disruption during immunostaining, or gonad placement on Z-plane. H3K4me3 intensity measurements are normalized to DAPI to account for differences in permeability across gonads. (G) Linear relationship between germ cell position along the gonadal axis (from distal to proximal end) and H3K4me3 signal intensity (relative to DAPI intensity or scaled per-gonad), aggregated across all gonads. Spearman correlation coefficients are represented on the plot. Linear models and overall correlations are based on data from 3,933 control cell detections and 8,134 depletion cell detections from 50 and 77 gonads, respectively. (H) Distribution of Spearman coefficients estimating per-gonad positive correlations between cell position on the gonad axis and H3K4me3 intensity (relative to DAPI intensity or scaled per-gonad). Mean and standard error are represented by red points and crossbars. The number of gonads analyzed per condition are represented by n. A one-tailed Welch’s two-sample t-test suggests that on average, control gonads have a higher magnitude correlation than gonads with reduced RNA Pol I activity (Benjamini-Hochberg adjusted p-values).