Fig 1.
Endogenous expression of lysine methyltransferase mRNAs by smFISH in whole C. elegans.
Endogenous mRNA expression of transcripts in whole L1 larval stage C. elegans hermaphrodites of (A) ubiquitous set-16, (B) broadly-expressed set-17, (C) hypodermal and germline mes-2, (D) germline-specific set-24, (E) muscle-specific set-18 and (F) neuron-specific set-11. Scale bar, 10 μm. (D) (i) Inset of the primordial germ cells expressing set-24 mRNA.
Fig 2.
L1 tissue-specific expression patterns of all C. elegans KMT genes.
Most KMT gene were expressed with tissue-specificity. Expression in a tissue was defined as more than one transcript detected in multiple cells in all animals. Mam Orth, mammalian ortholog of the C. elegans protein; SET target, experimentally shown to catalyze histone lysine methylation at these targets.
Fig 3.
set-17 functions in the germline to promote sperm-production and fertility.
A) Broodsizes of mutants defective in KMT genes expressed in the germline. Progeny number was determined for single adult hermaphrodites over three days and progeny were scored as adults after 3–6 days. Grey shaded area indicates the range of the wild-type 95% confidence interval. n > 10 for all genotypes. set-23, set-24 and set-27 mutants were not available, and instead these genes were examined using RNAi treated wild-type animals; fertility was not affected. B) Broodsize of wild-type, set-17, set-17; Pset-17::set-17(+) (expressing wild-type set-17 from its endogenous promoter) and set-17; Pmex-5::set-17(+) (expressing wild-type set-17 from the mex-5 germline-specific promoter) hermaphrodites. n > 20; *** P < 0.0001, ** P < 0.0012, t-test. C) Progeny of mating single males (wild-type or set-17 mutant) and single females (wild-type or set-17 mutant). Strains carried the fog-2(q71) mutation, which feminizes hermaphrodites by suppressing hermaphrodite but not male sperm production and ensured that all progeny were cross progeny. After 24 hr the male was removed, and the female allowed to lay progeny until completion. Females were placed on fresh plates every 24 hr over 4 days. Cross progeny were scored as number of adult female progeny 3–7 days after mating; only females were scored because adult males burrow, crawl off plates and clump together, making their quantification unreliable. n > 15. *** P < 0.0001, t-test. D) Spermatid counts in individual spermathecas of the indicated genotypes. Spermatid counts were determined by imaging DAPI-stained spermatid nuclei in hermaphrodites fixed in 4% formaldehyde 12 hr post-L4 around the time of first fertilization. n: wild-type = 26, set-17(-) = 32, set-17(-); Pset-17::set-17(+) = 33, set-17(-); Pmex-5::set-17(+) = 15. *** P < 0.0001, t-test.
Fig 4.
set-17 promotes the number and size of major sperm protein vesicles in primary spermatocytes.
A) Schematic of the male germline. Meiotic germ cells differentiate into primary spermatocytes, which mature as they progress along the gonad into secondary spermatocytes and then spermatids. A, anterior; P, posterior; D, dorsal; V, ventral. B) Electron micrographs of spermatocyte differentiation in wild-type and set-17 males. All spermatocyte differentiation stages are present in set-17 mutants. Images were staged based on their relative positions along the germline. Scale bar, 2 μm. C) Electron micrographs of spermatids in wild-type and set-17 males. Spermatids appear normal in set-17 mutants. Scale bar, 500 nm. D) Electron micrographs of mature primary spermatocytes from wild-type and set-17 males. Left, schematic traces of the outlines of the left-most panels of wild-type and set-17 primary spermatocytes (black), respectively, indicating the areas and positions of FB-MOs (magenta). Fibrous-body membranous organelles (FB-MOs) are smaller in set-17 mutants. Scale bar, 2 μm. E) Percent cytoplasmic cross-sectional area taken up by FB-MOs per cell in wild-type, set-17 and set-17; Pset-17::set-17(+) male mature primary spermatocytes. n = 10; **** P < 0.0001, *** P < 0.001, t-test. F) Frequency distributions of FB-MO cross-sectional areas (size) in wild-type, set-17 and set-17; Pset-17::set-17(+) male primary spermatocytes. Arrowheads, FB-MOs in micrographs of representative wild-type and set-17 spermatocytes. n > 95; **** P < 0.0001, KS-test; Mean: wild-type = 0.41 μm2, set-17 = 0.24 μm2, set-17; Pset-17::set-17(+) = 0.35 μm2; Scale bar, 500 nm. G) Schematic of sperm production in wild-type and set-17 males. In set-17 mutants, defective FB-MO production in primary spermatocytes leads to smaller FB-MOs and a reduction in the production of otherwise normal spermatids. A, anterior; P, posterior; D, dorsal; V, ventral.
Fig 5.
SET-17 is expressed in the nuclei of primary spermatocytes and enriched in chromatin-associated foci.
A) Confocal imaging of a longitudinal section showing SET-17::GFP expression in germ cells and spermatocytes of an adult male germline. White lines delineate the gonad. Scale bar, 10 μm. A, anterior; P, posterior; D, dorsal; V, ventral. B) Representative Z-axis projection of confocal images of extruded and formaldehyde-fixed male germlines expressing SET-17::GFP and stained for GFP by immunofluorescence (yellow). DNA stained with DAPI (blue). Scale bar, 10 μm. Inserts, higher magnification views of individual representative nuclei. Scale bar, 1 μm. C) & D) Representative Z-axis projection of primary spermatocyte nuclei of males expressing SET-17::GFP. Nuclei were stained by immunofluorescence for GFP and H3K4me1 C) or H3K4me2 D), respectively. DNA, DAPI. Scale bar, 1 μm.
Fig 6.
set-17 promotes expression of msp genes in spermatogenic gene clusters.
A) Expression categories defined by Ortiz et al. (2014) of 123 transcripts misregulated in set-17 mutants based on RNAseq studies of whole L4 hermaphrodites. Sperm, enriched in sperm-producing germlines (60, red); sperm + oocyte, genes expressed similarly in sperm and oocytes (40, dark grey); non-germline, genes not detected in dissected germlines (23, light grey). We did not detect genes enriched in oocytes but not sperm. B) set-17 promotes expression of a subset of spermatogenic transcripts. Average log2-fold change in expression vs. the wild-type for all 123 set-17 misregulated transcripts (set-17 all) in set-17 mutants; for the 60 set-17 misregulated spermatogenic transcripts (set-17 sperm.) in set-17 mutants, in set-17; Pset-17::set-17(+) and in set-17; Pmex-5::set-17(+) mutants, respectively; and for all 2,306 previously identified spermatogenic transcripts (All sperm.) in set-17 mutants. **** P < 0.0001, MW exact or KS test. C) set-17 promotes msp expression in the germline. log2-fold change in expression vs. the wild type for all 28 msp gene transcripts (msp genes) in set-17, set-17; Pset-17::set-17(+) and set-17; Pmex-5::set-17(+) mutants. **** P < 0.0001, MW exact or KS test. D), E) set-17 spermatogenic genes cluster in the genome. Positional distributions of spermatogenic genes and msp genes on chromosomes II and IV and spermatogenic set-17-misregulated genes on chromosomes II and IV. Above: histograms of spermatogenic genes on chromosomes (D) II and (E) IV. Blue, spermatogenic gene clusters. Below: log2-fold change in expression in set-17 vs. the wild type from RNAseq studies in whole L4 hermaphrodites for the set-17 spermatogenic genes (red) on chromosomes (D) II and (E) IV. Yellow, msp genes. F) set-17 promotes expression of spermatogenic genes in a cluster on chromosome II. log2-fold change in expression vs. wild type from RNAseq studies of whole L4 hermaphrodites for all spermatogenic genes in the spermatogenic gene cluster on chromosome II (blue region in 5D) in set-17 (blue), set-17; Pset-17::set-17(+) (orange) and set-17; Pmex-5::set-17(+) (yellow) as well as for all spermatogenic genes outside of the blue region on chromosome II (grey). Mean +\-95% CI; **** P < 0.0001, MW exact or KS test. G) set-17 promotes expression of spermatogenic genes in clusters on chromosome IV. log2-fold change in expression vs. the wild type from RNAseq studies of whole L4 hermaphrodites for all spermatogenic genes in the spermatogenic gene clusters on chromosome IV (blue regions in 5E) in set-17 (blue), set-17; Pset-17::set-17(+) (orange) and set-17; Pmex-5::set-17(+) (yellow), as well as for all spermatogenic genes outside of the blue region on chromosome IV (grey). Mean +\-95% CI; **** P < 0.0001, MW exact or KS test. H) set-17 promotes expression of spermatogenic genes in a cluster on chromosome V. log2-fold change in expression vs. the wild type from RNAseq studies of whole L4 hermaphrodites for all spermatogenic genes in the newly identified spermatogenic gene cluster on chromosome V (blue region in S5K) in set-17 (pink), set-17; Pset-17::set-17(+) (orange) and set-17; Pmex-5::set-17(+) (yellow) as well as for all spermatogenic genes outside of the blue region on chromosome V (grey). Mean +\-95% CI shown. Cl. V (set-17(-)) vs. Non-Cl. V (set-17(-)), ** P < 0.005, MW exact; Cl. V (set-17(-)) vs. Cl. V (Pset-17::set-17(+)), *** P < 0.0005, Wilcoxon test; Cl. V (set-17(-)) vs. Cl. V (Pmex-5::set-17(+)), **** P < 0.0001, Wilcoxon test.
Fig 7.
SET-17 promotes transcription at spermatogenic gene clusters.
A) Endogenous msp mRNA expression determined by smFISH using a probe-set that recognizes transcripts from 28 msp genes in wild-type or set-17 sperm-producing germlines of L4 hermaphrodites. White lines delineate the gonad. Scale bar, 10 μm. B) Levels of endogenous msp mRNA as measured by smFISH in mature primary spermatocytes in wild-type and set-17 L4 hermaphrodites. n > 10, P < 0.001, t-test. C) Left, endogenous msp RNA transcription sites (TSs) in the nuclei of wild-type and set-17 primary spermatocytes in L4 hermaphrodites; scale bar, 5 μm. Right, percent of primary spermatocyte nuclei with at least one msp TS (positive). Wild type n = 177, set-17 n = 199. *** P < 0.0001, t-test. D) Frequency distribution of TSs in primary spermatocyte nuclei of wild-type or set-17(-) sperm-producing L4 hermaphrodites. Avg. TSs per nucleus: wild type, 1.75, n = 141; set-17, 0.82, n = 153; P < 0.0001, KS or MW test. E) Probability that a single msp cluster is transcribed in a wild-type or set-17 L4 hermaphrodite. Calculated from the TS distributions for wild-type and set-17 animals shown in (D). P < 0.0001, t-test. F) Co-localization of endogenous msp RNA TSs (as visualized by smFISH) and SET-17::GFP (as visualized by GFP protein fluorescence) in the nuclei of primary spermatocytes in the male germline. White circles, nuclear circumference; scale bar, 1 μm. G) Individual frames from an in vivo FRAP time series study of two primary spermatocytes and a hypodermal nucleus. White lines, nuclear circumference; scale bar, 1 μm; red circle, focal area of laser bleaching and region of measurement. H) Normalized fluorescence intensity measurement in the area of bleaching for the three experiments shown in (G). t = 0, pre-bleaching intensity. Recovery of SET-17::GFP is 10-fold slower in spermatocyte foci than in hypodermal nuclei. I) Normalized FRAP data for 16 experiments examining spermatocyte foci. Black dotted line indicates average half-maximal recovery time, 61 s, calculated from exponential fits for each experiment. J) Average half-maximal recovery times from FRAP studies of SET-17::GFP in spermatocyte foci (Foci t1/2) or hypodermal nuclei (Hyp t1/2). Error bars: SEM; P < 0.05, t-test.
Fig 8.
ELT-1 acts with SET-17 to promote spermatogenic gene cluster expression and fertility.
A) Overlays among the sets of the 44 predicted spermatogenic elt-1 targets (from data of del Castilles-Olivera et al., 2009), the 60 spermatogenic set-17 misregulated genes (from our Fig 6A) and the 28 msp genes. B) Average log2-fold change in expression from RNAseq studies compared to the wild type for the 44 predicted elt-1 target genes in set-17 (purple), set-17; Pset-17::set-17(+) (orange) and set-17; Pmex-5::set-17(+) (yellow) mutant whole L4 hermaphrodite. **** P < 0.0001, MW or KS-test. C) Endogenous msp mRNA detection by smFISH in primary spermatocytes of elt-1 partial loss-of-function (rf) L4 hermaphrodites. Scale bar, 10 μm. D) Endogenous msp mRNA levels as visualized by smFISH in mature primary spermatocytes in wild-type (black), elt-1 (purple), set-17 (red) and set-17; elt-1 (magenta) L4 hermaphrodites. n > 15. E) Number of progeny of wild-type (black), elt-1 (purple), set-17 (red) and set-17; elt-1 mutants (magenta). n > 20. F) Fertility correlates with endogenous msp mRNA levels as visualized by smFISH (data from Fig 7C and 7D). Pearson-correlation r = 0.9981, P < 0.002 (2-tailed). G) Model of SET-17 foci composition and function in msp gene expression in primary spermatocyte nuclei at spermatogenic gene clusters. See text for details.