RNA Pol I activity is required for meiotic chromatin organization and the H3K4me3 gradient essential for oogenesis, independent of ribosome synthesis

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 Pol I catalytic subunits or ribosome assembly factors, 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 Pol I activity led to widespread changes in chromatin accessibility, a dampening of the distal-proximal H3K4me3 gradient required for oogenesis, reduced synapsis, and elevated ATM/ATR phosphorylation, 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.. Auxin-inducible depletion of RNA Pol I subunits and ribosome assembly factors reveal that nucleolar structure is dependent on pre-rRNA synthesis but not downstream ribosome maturation. See also Figure S1.

(A-D) Germline expression of fluorescently tagged RPOA-2 (A), RPOA-1 (B), GRWD-1 (C), and TSR-2 (D) proteins after control or auxin treatment. Dissected germ lines are outlined in white. Each strain’s control/auxin images were taken with identical exposure settings. Scale bar = 50 μm. (E) Distribution of germ nuclei GFP intensity after auxin-inducible depletion of GFP-tagged RPOA-1/RPOA-2/GRWD-1/TSR-2. Statistical comparisons performed using left-tailed Welch’s two-sample t-tests. (F) Comparisons of counts per million (CPM) reads across a simplified single copy of the ITS1 and ITS2 loci based on gonadal RNA-seq (total RNA) after auxin-inducible depletion of RPOA-2/GRWD-1/TSR-2. Points represent biological replicates (3 per condition, each consisting of 20 gonads); bars and error bars represent the mean and SEM, respectively. (G-H) Germline nucleolar morphology (endogenous NUCL-1::mKate2) after auxin-inducible depletion of RPOA-2 (G) or GRWD-1 (H). Germ lines are outlined in white; zoomedin nucleoli are shown in the right column. Scale bars = 50 μm (whole gonad) and 5 μm (zoomed nucleoli).

Figure 2.. Oocyte morphology and organization are impaired by reduced RNA Pol I activity, independent from RNA Pol I’s role in ribosome production. See also Figure S2.

(A) Representative DIC images of adult germ lines after auxin-inducible depletion of RPOA-1/RPOA-2/GRWD-1/TSR-2. Dotted lines highlight the proximal arm; triangles point to proximal oocytes. Scale bar = 50 μm. (B) Graphical representation of proximal arm length and oocyte positions quantified in (C-E). (C-E) Proximal arm lengths (C), proximal oocyte counts (D), and oocyte sizes (E) after auxin-inducible depletion of RPOA-1/RPOA-2/GRWD-1/TSR-2. Each point represents a measurement from an individual animal, normalized to the mean of each strain’s control group. Crossbars represent SEM. All comparisons were performed using two-tailed Welch’s two-sample t-tests followed by Bonferroni corrections.

Figure 3.. Synaptonemal complex formation during the mitotic-to-meiotic germ cell transition is dependent on Pol I activity, but not other ribosome processing steps. See also Figure S3 and S4.

(A) Confocal images of transition zone nuclei expressing degron::GFP::RPOA-2, EmGFP::SYP-3, and NUCL-1::mKate2 following control or auxin-inducible depletion of RPOA-2 (top two rows). Additionally, we show non-treated nuclei from a wild-type genetic background (middle row) and zoomed-in nuclei from RPOA-2-depleted and wild-type conditions (bottom row, labeled a-e). Scale bar = 5 μm (transition zone) and 0.5 μm (zoomed-in nuclei). (B) Confocal images of transition zone nuclei expressing EmGFP::SYP-3 and NUCL-1::mKate2 48 hours post RNAi knockdown of a non-target locus (wrmScarlet), nucl-1, grwd-1, rpoa-2, and fib-1 (top), as well as zoomed-in nuclei from each condition (bottom). Scale bar = 5 μm (transition zone) and 0.5 μm (zoomed-in nuclei). All images were taken with identical laser intensity; due to decreased SYP-3 expression under knockdown of grwd-1, rpoa-2, and fib-1, we have increased the brightness of these conditions to be more comparable to wild type.

Figure 4.. Disrupting RNA Pol I activity prematurely primes autosomal promoters in an accessible state associated with oogenesis. See also Figure S5.

(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. SMA regions after RPOA-2 or GRWD-1 depletion are predominantly located within 1 kb of the nearest promoter. (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. (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. (E) Overlap between peaks that are SMA after RPOA-2 depletion and peaks near genes involved in oogenesis (Hypergeometric test). (F) Chromosomal distribution of genes involved in oogenesis and 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 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 5.. Chromatin regions that become more accessible after reducing RNA Pol I activity display patterns of H3K4me3 remodeling characteristic of the germ-to-oocyte transition. See also Figure S6.

(A) Log₂ fold-change estimates of chromatin accessibility after RPOA-2 depletion in peaks annotated to genes that undergo H3K4me3 remodeling during oogenesis, compared to a random subset of peaks. The number of peaks in each set is specified by n, and means are denoted by an x. Statistical comparisons were performed using right-tailed Welch’s two-sample t-tests with Bonferroni corrections. (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 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).

Figure 6.. Schematic model illustrating the non-ribosomal function of RNA Pol I during C. elegans oogenesis.

Under normal conditions (left), RNA Pol I activity maintains nucleolar integrity, which supports the establishment of a proper H3K4me3 gradient from the distal to proximal germ line. This gradient facilitates meiotic synapsis and the progression of oogenesis, resulting in normal oocyte formation. When RNA Pol I activity is reduced (right), nucleolar disruption leads to premature and ectopic H3K4me3 deposition and aberrant oogenesis chromatin remodeling. This dysregulation interferes with the mitotic-to-meiotic transition, resulting in decreased synapsis that leads to abnormal oogenesis, characterized by fewer and enlarged oocytes. The model proposes an epigenetic feedback mechanism that links nucleolar function to germline genome integrity. Created in BioRender. https://BioRender.com/oq1fz8k.