Description
Histone modifications are precisely controlled within the developing germ line. During first meiotic prophase in
C. elegans
, H3K9me2 immunolabeling detects a relatively bright focus of signal on unsynapsed chromosomes and a relatively weak, diffuse signal on synapsed chromosomes (Bean
et al.
2004, Kelly
et al.
2002, Reuben and Lin 2002). The naturally occurring unsynapsed chromosome is the single male X. Examples of other chromatin that is enriched for H3K9me2 at pachytene stage include single-copy free chromosomal duplications, chromosomes that fail to pair due to certain mutations, and multicopy extrachromosomal arrays. In all cases, the enriched H3K9me2 signal decreases sharply as nuclei move into diplotene stage, and H3K9me2 is not detected in diakinesis nuclei. As the naturally occurring example of H3K9me2 enrichment, the male X is commonly used to evaluate this meiotic targeting phenomenon (Fig. 1). We previously showed that meiotic H3K9me2 distribution is altered in males lacking CSR-1 Argonaute or factors required for accumulation of CSR-1-associated 22G RNAs, including EGO-1 RdRP, DRH-3 helicase,
and the Tudor domain protein, EKL-1 (Maine
et al
. 2005, She
et al.
2009). In mutants lacking any of these factors, pachytene H3K9me2 signal intensity is reduced on unsynapsed chromosomes and, in all cases except
ego-1(null)
mutants, ectopic H3K9me2 foci are detected on synapsed chromosomes. Interestingly, as in wildtype, H3K9me2 signal is not detected in diakinesis nuclei in these mutants. CSR-1 pathway activity is essential for germ line development where it is thought to license the correct pattern of gene expression (Yigit
et al.
2006, Ashe
et al.
2012, Shirayama
et al
. 2012). EGO-1, DRH-3, and EKL-1 are critical for biogenesis and stability of 22G RNAs that associate with CSR-1 (reviewed in Billi
et al.
2014).
RRF-3 RdRP is essential for biogenesis of 26G RNAs that participate in the ALG-3 and ALG-4 Argonaute pathway during spermatogenesis and the ERGO-1 Argonaute pathway during oogenesis (Billi
et al.
2014). We previously observed an essentially wildtype H3K9me2 immunolabeling pattern in
rrf-3(pk1426)
males during first meiotic prophase that then persists (presumably on the X chromosome) beyond diplotene and is visible on karyosomes in the late spermatogenesis condensation zone as described by Shakes
et al.
(2009) (Maine
et al.
2005). We observed wildtype H3K9me2 distribution in
ergo-1(tm1860)
single mutant,
alg-3(tm1155)
single mutant, and
alg-4(ok1041)
single
mutant males (She
et al.
2009), but we did not test
alg-4(ok1041);alg-3(tm1155)
double mutants at that time. We subsequently hypothesized that the
rrf-3(pk1426)
H3K9me2 phenotype may reflect impaired ALG-3 and ALG-4 pathway activity, since these Argonaute proteins together are critical during spermatogenesis (Han
et al.
2009, Conine
et al.
2010, 2013) at the time when the H3K9me2 signal typically decreases.
Here, we follow up on our earlier results to report meiotic H3K9me2 distribution in
eri-1(mg366)
single mutant,
eri-5(tm2528)
single mutant, and
alg-4(ok1041);alg-3(tm1155)
double mutant males. ERI-1, an exoribonuclease, and ERI-5, a Tudor domain protein, are both required for 26G RNA synthesis. We also evaluated H3K9me2 distribution in
pup-1(tm1021)
single mutant,
pup-2(tm4344)
single mutant, and
pup-1/-2(om129)
double mutant males. PUP-1 (aka CDE-1, CID-1) and PUP-2 are members of the poly(U) polymerase family responsible for adding non-templated uridine(s) to the 3’ end of RNA. PUP-1 is implicated in adding 3’ uridine to 22G RNAs that associate with CSR-1 and WAGO-4 Argonautes (van Wolfswinkel
et al.
2009, Xu
et al.
2018) and to microRNAs (Vieux
et al.
bioRxiv preprint). PUP-2 promotes germline development redundantly with PUP-1 under conditions of heat stress (Li and Maine 2018) and has been implicated in regulating stability of
let-7
miRNA (Lehrbach
et al.
2009). U-tailing of small RNAs may influence their stability and/or Argonaute associations, as has been suggested (Billi
et al.
2014, Xu
et al.
2018).
We immunolabeled H3K9me2 in adult
him-8
control males raised at 20°C and 25°C and in various mutant backgrounds. We tested
alg-4(ok1041);alg-3(tm1155)
double mutant,
eri-1(mg366)
single mutant,
eri-5(tm2528)
single mutant,
pup-1(tm1021)
single mutant,
pup-2(tm4344)
single mutant, and
pup-1/-2(om129)
double mutant males. The
alg-4(ok1041);alg-3(tm1155)
double mutant,
pup-1(tm1021)
single mutant,
pup-2(tm4344)
single mutant, and
pup-1/-2(om129)
double mutant phenotypes are all temperature sensitive, and these mutants were therefore tested at restrictive temperature (25°C). The
pup
single and double mutants were F1 (M+Z-) offspring (where M indicates maternal genotype and Z indicates embryonic genotype) of heterozygous hermaphrodites (see Methods). We note that
pup-1/-2(om129)
M+Z- males all produce sperm, although many of those sperm have abnormal morphology and are fertilization defective (Li and Maine 2018). H3K9me2 immunolabeling in pachytene nuclei appears wildtype in
eri-1(mg366)
single mutant,
alg-4(ok1041);alg-3(tm1155)
double mutant,
pup-1(tm1021)
single mutant, and
pup-2(tm4344)
single mutant males; in each case, a strong signal is visible in one region of the genome, and weak, diffuse signal is visible elsewhere across the genome (Fig. 1A)
.
In contrast, multiple discrete H3K9me2 foci are visible in pachytene nuclei of
pup-1/-2(om129) M
+Z- double mutant and
eri-5(tm2528)
single mutant males (Fig. 1A). In spermatogenic nuclei, H3K9me2 is not detected in
pup-1
or
pup-2
single mutants, as it is not in the control (Fig. 1B). In contrast, H3K9me2 is detected in karyosomes in
alg-4(ok1041);alg-3(tm1155)
double mutant,
eri-1(mg366)
single mutant,
eri-5(tm2528)
single mutant,
and
pup-1/-2(om129)
M+Z- double mutant males (Fig. 1B). We note that H3K9me2 appears wildtype
pup-1/-2(om129)
F1 (M+Z-) double
mutants at 20°C, consistent with the temperature-sensitivity of other aspects of the
pup
phenotype (Spracklin
et al.
2017, Li and Maine 2018).
Our working model is that the delayed H3K9me2 turnover in
rrf-3(pk1426)
single mutant,
alg-4(ok1041);alg-3(tm1155)
double mutant,
eri-1(mg366)
single mutant, and
eri-5(tm2528)
single
mutant males
reflects a role for the ALG-3 and ALG-4 pathway in chromatin regulation during spermatogenesis. The broader H3K9me2 phenotype in
pup-1/-2(om129)
M+Z- 25°C
mutants may reflect impaired activity of both the CSR-1 pathway and the ALG-3 and ALG-4 pathway, perhaps due to misrouting of siRNAs that would typically associate with these Argonaute proteins. CSR-1 promotes spermatogenesis, and it is implicated as functioning downstream of ALG-3 and ALG-4 to regulate gene expression during spermatogenesis (Conine
et al.
2010, 2013; Charlesworth
et al.
2021) although CSR-1 does not appear to act directly downstream of ALG-3 and ALG-4 in all cases (Nguyen and Phillips 2021). Misrouting of siRNAs may occur, at least in part, due to absence of 3’ U-tailing as has been proposed by van Wolfswinkel
et al.
. (2009), Xu
et al.
(2018), and others.