Histone methyltransferases MES-4 and MET-1 promote meiotic checkpoint activation in Caenorhabditis elegans

Abstract

Chromosomes that fail to synapse during meiosis become enriched for chromatin marks associated with heterochromatin assembly. This response, called meiotic silencing of unsynapsed or unpaired chromatin (MSUC), is conserved from fungi to mammals. In Caenorhabditis elegans, unsynapsed chromosomes also activate a meiotic checkpoint that monitors synapsis. The synapsis checkpoint signal is dependent on cis-acting loci called Pairing Centers (PCs). How PCs signal to activate the synapsis checkpoint is currently unknown. We show that a chromosomal duplication with PC activity is sufficient to activate the synapsis checkpoint and that it undergoes heterochromatin assembly less readily than a duplication of a non-PC region, suggesting that the chromatin state of these loci is important for checkpoint function. Consistent with this hypothesis, MES-4 and MET-1, chromatin-modifying enzymes associated with transcriptional activity, are required for the synapsis checkpoint. In addition, a duplication with PC activity undergoes heterochromatin assembly when mes-4 activity is reduced. MES-4 function is required specifically for the X chromosome, while MES-4 and MET-1 act redundantly to monitor autosomal synapsis. We propose that MES-4 and MET-1 antagonize heterochromatin assembly at PCs of unsynapsed chromosomes by promoting a transcriptionally permissive chromatin environment that is required for meiotic checkpoint function. Moreover, we suggest that different genetic requirements to monitor the behavior of sex chromosomes and autosomes allow for the lone unsynapsed X present in male germlines to be shielded from inappropriate checkpoint activation.

Figure 1. H3K9me2 is enriched on unsynapsed chromosomes when either the DNA damage checkpoint or the synapsis checkpoint is activated.

Indirect immunofluorescence was performed on meiotic nuclei in wildtype, meDf2 homozygotes and meDf2 heterozygotes using antibodies against the axial element component HTP-3, the central element component SYP-1 and H3K9me2. Wildtype meiotic nuclei exhibit colocalization of HTP-3 and SYP-1 (A, B and D), and have areas of H3K9me2 enrichment (carets) and depletion (C and D). Meiotic nuclei in both meDf2 homozygotes and meDf2 heterozygotes have chromosomes with HTP-3 but not SYP-1 (arrows in E, F, H, I, J and L, magnified insets in I, J and L). These are unsynapsed X chromosomes and are highly decorated with H3K9me2 (G, H, K and L, magnified insets in K and L). Other regions of H3K9me2 enrichment are also observed in nuclei with synapsed chromosomes in meDf2 heterozygotes (carets in K and L). Scale bar represents 4 microns.

Figure 2. A chromosomal duplication with Pairing Center activity often does not undergo heterochromatin assembly.

A and B. In meDf2/+ hermaphrodites, it is difficult to determine if PCs of unsynapsed X chromosomes become enriched with H3K9me2. Indirect immunofluorescence was performed on meiotic nuclei in meDf2 heterozygotes using antibodies against the X chromosome PC protein HIM-8, the central element component SYP-1 and H3K9me2. The nucleus on the left contains an unsynapsed X chromosome, as identified by HIM-8, an enrichment of H3K9me2 (A) and the absence of SYP-1 (B); the PC appears enriched with H3K9me2 (arrow in A). The nucleus on the right also contains an unsynapsed X chromosome, as indicated by the absence of SYP-1 in B, and the PC of this chromosome does not exhibit robust H3k9me2 (caret in A). C. Quantification of the percentage of unsynapsed X chromosomes in meDf2 heterozygotes that were enriched for H3K9me2 at their PC and non-PC regions. D. A chromosomal duplication with Pairing Center activity (mnDp73) activates the synapsis checkpoint. A duplication that does not have Pairing Center activity (mnDp66/+) fails to activate the synapsis checkpoint and a duplication of the non-PC portion of the X chromosome (mnDp3) activates the DNA damage checkpoint. A ** indicates a p value of <0.0001. Error bars indicate 2XSEM. E–J. Indirect immunofluorescence was performed on meiotic nuclei from hermaphrodites carrying mnDp3 (non-PC end of the X chromosome) and mnDp73 (PC end of the X chromosome) with antibodies against H3K9me2, SYP-1 or HIM-8. mnDp3 is unsynapsed (arrow in F) and highly decorated with H3K9me2 (arrow in E). This was observed in 100% of meiotic nuclei (23 of 23) in which mnDp3 could unambiguously be identified as unsynapsed. mnDp73 is identified as a free DAPI staining body that recruits HIM-8 (caret in H and arrow in J) but that is often not enriched with H3K9me2 (caret in G). This was observed in 13 out of 17 in which mnDp73 could unambiguously be identified by HIM-8 recruitment. In the remaining four nuclei in which mnDp73 was identified by HIM-8 binding (arrow in J), the duplication was enriched for H3K9me2 (arrow in I) but was not the primary signal of H3K9me2 in the nucleus. Scale bar represents 4 microns.

Figure 3. mes-4 is required for the synapsis checkpoint and prevents heterochromatin assembly on a chromosomal duplication with PC activity.

A. Mutation of mes-4 reduces apoptosis in meDf2/+ but not in meDf2 homozygotes. A ** indicates a p value of <0.0001. B. Mutation of mes-2 does not reduce apoptosis in meDf2/+ or meDf2 homozygotes. Error bars indicate 2XSEM. C and D. Indirect immunofluorescence was performed on meiotic nuclei in hermaphrodites carrying mnDp73 in which mes-4 was knocked down by RNAi with antibodies against H3K9me2 and HIM-8. mnDp73 is identified as a free duplication bound by HIM-8 (arrow in C). In 14 out of 16 in which the duplication could be identified, mnDp73 is enriched with H3K9me2 (arrow in D). Scale bar represents 4 microns. E. Quantification of the percentage of duplications that were enriched with H3K9me2 in mnDp73 bearing hermaphrodites and mnDp73 bearing hermaphrodites in which mes-4 was inactivated by RNAi.

Figure 4. mes-4 and met-1 are redundant for synapsis checkpoint activation when all chromosomes are unsynapsed.

A. Inactivation of both mes-4 (by mutation) and met-1 (by RNA interference) is required to reduce apoptosis in syp-1 and spo-11; syp-1 mutants but does not affect apoptosis in pch-2; syp-1 mutants. A * indicates a p value of <0.05 and a ** indicates a p value of <0.0001. Error bars indicate 2XSEM. B–G. Heterochromatin assembly occurs on unsynapsed chromosomes in syp-1 and spo-11; syp-1 mutants. Indirect immunofluorescence was performed on meiotic nuclei in wildtype, syp-1 and spo-11; syp-1 double mutants with an antibody against H3K9me2. Wildtype nuclei exhibit regions of enrichment or depletion while both syp-1 and spo-11; syp-1 mutants exhibit dispersed H3K9me2. Scale bar represents 4 microns.

Figure 5. met-1 is not required for the synapsis checkpoint when only X chromosomes are unsynapsed.

A. Mutation of met-1 does not reduce apoptosis in meDf2/+ or meDf2 homozygotes. B. Inactivation of met-1 by RNAi in meDf2; syp-1 double mutants does not reduce apoptosis. Error bars indicate 2XSEM.

Figure 6. mes-4's role in the synapsis checkpoint is independent of met-2 mediated heterochromatin assembly on unsynapsed X chromosomes.

meDf2/+; mes-4 double mutants have nearly wild-type levels of apoptosis when met-2 is inactivated by RNA interference. A * indicates a p value of <0.05. Error bars indicate 2XSEM.

Figure 7. Is H3K36me3 the relevant chromatin modification for synapsis checkpoint activation?

A–L. Indirect immunofluorescence was performed on meiotic nuclei in wildtype and meDf2 heterozygotes against SYP-1, HIM-8 and H3K36me3. H3K36me3 is depleted from X chromosomes in both of these genetic backgrounds except for a single dot near the end (carets in D, E, F, J, K and L) that does not colocalize with HIM-8 (E and K). Scale bar represents 4 microns. M–P. Mutation of mes-4 results in a reduction of H3K36me3 at the left end of X chromosomes. Indirect immunofluorescence was performed on meiotic nuclei in mes-4 single mutants against HIM-8 and H3K36me3. Q. Quantification of the percentage of meiotic nuclei in wildtype, mes-4 mutants, meDf2 heterozygotes and meDf2/+; mes-4 double mutants with H3K36me3 at the left end of X chromosomes. Mutation of mes-4 reduces but does not eliminate the percentage of X chromosomes that exhibit this chromatin modification. X chromosomes in meDf2 heterozygotes, whether synapsed or unsynapsed, exhibit this chromatin modification with a frequency similar to that observed in wildtype animals.