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Teflon promotes chromosomal recruitment of homolog conjunction proteins during Drosophila male meiosis [1]

['Zeynep Kabakci', 'Department Of Molecular Life Science', 'Dmls', 'University Of Zurich', 'Zurich', 'Hiro Yamada', 'Luisa Vernizzi', 'Samir Gupta', 'Joe Weber', 'Michael Shoujie Sun']

Date: 2022-12 

Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. Here, we have analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, our findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes.

Sexual reproduction depends on meiosis, a special cell division that generates haploid cells. Haploid cells have only one set of chromosomes in contrast to the diploid precursor cell, which has two sets. Haploid cells can differentiate into gametes. Fusion of two gametes during fertilization recreates the diploid state. Meiosis is distinct in males and females to produce two distinct types of compatible gametes, sperm and egg. In the fly Drosophila melanogaster, sex-specific differences are particularly pronounced. While pairing of homologous chromosomes into bivalents early in meiosis proceeds in a canonical manner in females, males use an alternative system. This system maintains homolog pairing, replacing crossovers that result from homologous recombination during canonical meiosis. Four genes (snm, uno, mnm and tef) are known to be required specifically for alternative homolog conjunction in males. Here, we demonstrate that the TEF protein binds directly to MNM. Thereby, TEF promotes the recruitment of MNM and consequentially SNM and UNO to chromosomes. However, while SNM, UNO and MNM remain on bivalent chromosomes until they are separated apart during the first meiotic division, TEF disappears prematurely, suggesting that it is not part of the final physical linkage between homologous chromosomes.

The findings summarized above strongly support the notion that SNM, MNM and UNO function as proteinaceous glue that conjoins chromosomes into bivalents. However, it remains to be clarified how these proteins are recruited to chromosomes. SNM, MNM and UNO do not include known bona fide DNA-binding domains. They might therefore be recruited by other chromatin proteins. The zinc finger protein TEF is clearly an attractive candidate factor for chromosomal recruitment of the other AHC proteins. TEF’s pattern of expression and its subcellular localization during spermatogenesis have not yet been characterized. Here, we close this gap in understanding. Using transgenes encoding tagged functional versions of TEF, we observed that it is only transiently detectable in early spermatocytes. In contrast to the other known AHC proteins (MNM, SNM and UNO), TEF cannot be detected on bivalents at the start of M I, indicating that it is unlikely a stoichiometric component of the homolog-conjoining glue. However, we provide evidence that TEF can recruit MNM to chromosomes by direct protein-protein interaction. Indirectly, TEF can also recruit SNM-UNO, as they bind to MNM. Moreover, presumably by promoting AHC protein interactions, TEF stabilizes these proteins and controls their levels. AHC protein levels need to be controlled, as suggested by the consequences of simultaneous overexpression of all four AHC proteins in spermatocytes, which resulted in ectopic chromosome conjunction, failure of territory formation and segregation errors during M I. Ectopic expression of the four AHC proteins in somatic cells induced aberrant chromosome conjunction during mitosis, suggesting that AHC might not depend on additional spermatocyte-specific proteins beyond those already known.

MNM, SNM and UNO accumulate in early spermatocytes, eventually co-localizing during spermatocyte maturation in multiple subnucleolar foci [ 10 , 11 ]. At the start of M I, these foci coalesce into a single prominent spot on the chrXY bivalent [ 10 , 11 ]. In D. melanogaster, chrX and chrY are strongly heteromorphic, lacking extended euchromatic homology that could mediate specific pairing. However, both sex chromosomes harbor rDNA gene clusters in the centromere-proximal heterochromatin and these rDNA clusters function as pairing centers during male M I [ 15 , 16 ]. The prominent dot formed by MNM, SNM and UNO on the chrXY bivalent at the start of M I is localized on the paired rDNA loci of chrX and chrY [ 10 , 16 ]. Apart from the prominent dot on the chrXY pairing center, autosomal bivalents, which rely on euchromatic homology for pairing [ 17 , 18 ] display far weaker dot signals of co-localized MNM, SNM and UNO [ 10 , 11 ]. These autosomal dot signals were shown to be at least partially dependent on tef function [ 10 , 11 ]. Strikingly, MNM, SNM and UNO disappear rapidly from all the bivalents within minutes during the onset of anaphase I [ 10 , 11 , 19 ]. Separase, an endoprotease known to eliminate chromosomal cohesin at the metaphase to anaphase transition during mitotic and meiotic divisions, is required for the rapid disappearance of MNM, SNM and UNO from M I bivalents [ 19 ]. UNO includes a separase cleavage site [ 11 ]. Mutations that abolish this cleavage site prevent the rapid disappearance of MNM, SNM and UNO from M I bivalents and preclude homolog separation [ 11 ].

The TEF protein includes three C2H2-type zinc fingers and is therefore predicted to bind to DNA [ 9 ]. The SNM protein is a distant relative of the stromalins (SCC3/SA/STAG protein family) [ 10 ]. Stromalins are subunits of cohesins, complexes of crucial importance for chromosome organization during interphase and M phase in somatic and meiotic cells. However, SNM is not co-localized with core components of cohesin, indicating that it does not function as a cohesin subunit [ 10 ]. MNM is encoded by one of many differentially spliced mRNAs transcribed from the highly complex mod(mdg4) locus [ 10 , 12 ]. MNM has an N-terminal BTB/POZ motif that is shared among almost all of the more than 30 distinct protein products expressed from the mod(mdg4) locus [ 10 , 12 , 13 ]. In addition, MNM has a unique C-terminal zinc finger motif of the FLYWCH type. These N- and C-terminal motifs of MNM are predicted to mediate protein-protein interactions [ 14 ]. UNO does not have obvious similarities to functionally characterized proteins [ 11 ].

The mechanisms that break up non-homologous chromosome associations during territory formation disrupt also homolog pairing and sister chromatid cohesion, presumably because of inevitable side effects. However, normally, homolog separation does not proceed to completion already during spermatocyte maturation. Complete premature homolog separation is prevented by residual homolog conjunction maintained by a dedicated special system that serves as an alternative to canonical homolog linkage by crossovers. Large-scale mutant screening has led to the identification of three genes (tef, mnm, and snm) that are specifically required for this alternative homolog conjunction (AHC) [ 8 – 10 ]. A proteomic approach has recently uncovered an additional AHC gene (uno) [ 11 ]. Loss-of-function mutations in these four genes result in chromosome missegregation during M I, but exclusively in males. In mnm, snm and uno mutant males, both sex chromosomes and autosomes are distributed randomly during M I [ 10 , 11 ]. In contrast, only autosomes are missegregated in tef mutant males during M I [ 8 , 9 ].

The drastic loss of homolog pairing and sister cohesion in mid-stage spermatocytes starts concomitantly with the process of territory formation, which separates three major chromosome territories apart within the interphase nucleus. One of the major territories contains the chromosome (chr) 2 bivalent, another the chr3 bivalent and the third the chrXY bivalent. The additional bivalent of chr4, a small dot chromosome, is often associated with the chrXY territory. Territory formation breaks up all non-homologous associations between the large chromosomes. Such non-homologous associations are extensive in S1 spermatocytes. They arise from a coalescence of large blocks of pericentromeric heterochromatin into a chromocenter. Similarly, centromeres are clustered initially. Disrupting these non-homologous associations during territory formation at the S2b stage depends on condensin II activity and additional unidentified forces [ 6 , 7 ]. Failure of territory formation leads to persistence of non-homologous associations until prometaphase I and consequential chromosome segregation errors [ 6 , 7 ].

In D. melanogaster spermatocytes, not only MR but also SC formation does not occur. Nevertheless, soon after the last spermatogonial mitosis, homologous chromosomes are paired all along their length, according to analyses with a lacO/lacI-GFP system and FISH [ 3 , 4 ]. It remains to be clarified whether the pairing of homologous chromosomes in early spermatocytes during the S1 stage is driven by the same mechanisms that are responsible for the pervasive somatic homolog pairing in D. melanogaster [ 5 ]. Importantly, the extensive pairing of homologs in spermatocytes lasts only a few hours. During the S2b/S3 stages, homolog pairing was no longer detectable at any of the analyzed 14 distinct locations with euchromatic lacO array insertions [ 3 ]. Moreover, even sister chromatid cohesion appeared to be lost except at centromeres [ 3 ].

Meiosis is a key innovation that evolved before the eukaryotic radiation into the extant domain. The canonical program of this conserved process relies on meiotic recombination (MR). MR contributes to the initial pairing of homologous chromosomes and generates crossovers that maintain homologs linked as bivalent chromosomes until the onset of anaphase during the first meiotic division (M I). MR proceeds usually in concert with synapsis, which achieves close homolog pairing all along the chromosomes via formation of the synaptonemal complex (SC). In spite of the eminent significance of MR, diverse meiosis variants have evolved that do not rely on MR [ 1 ]. A most thoroughly studied example of such an achiasmate meiosis occurs in Drosophila melanogaster. While meiosis is largely canonical in D. melanogaster females and includes MR, it is achiasmate in the heterogametic males. This sex-specific difference in meiosis is characteristic among higher dipterans. Its evolution is poorly understood, but may be linked to the suppression of recombination between sex chromosomes [ 2 ].

Results

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010469

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