MEIOSIN directs initiation of meiosis and subsequent meiotic prophase program during spermatogenesis

which underlie meiosis-specific events such as meiotic recombination and homolog synapsis. In spermatocytes, meiotic prophase is accompanied by robust alterations of gene expression programs and chromatin status for subsequent sperm production. The mechanisms regulating meiotic initiation and subsequent meiotic prophase programs are enigmatic. Recently, we discovered MEIOSIN (Meiosis initiator), a DNA-binding protein that directs the switch from mitosis to meiosis. This review mainly focuses on how MEIOSIN is involved in meiotic initiation and the meiotic prophase program during spermatogenesis. Further, we discuss the downstream genes activated by MEIOSIN, which are crucial for meiotic prophase-specific events, from the viewpoint of chromosome dynamics and the gene expression program.


INTRODUCTION
During spermatogenesis, initiation of meiosis is one of the most noteworthy events that coincides with spermatocyte differentiation. Meiosis is a specialized cell cycle that produces haploid gametes from diploid cells. Meiotic entry occurs concomitantly with pre-meiotic S phase and is followed by meiotic prophase. Meiotic prophase is equivalent to G2 phase, but is prolonged to ensure the completion of numerous meiosis-specific chromosome events. During meiotic prophase, chromosomes are reorganized into axis-loop structures, which provide the structural framework for meiosis-specific events. Homologous chromosomes (homologs) then undergo pairing, synapsis and meiotic recombination, yielding crossovers called chiasmata that are physical linkages between the homologs (Baudat et al., 2013;Keeney et al., 2014;Zickler and Kleckner, 2015;Cahoon and Hawley, 2016). During these processes, chromosomes undergo dynamic movement to facilitate homolog pairing and synapsis, driven by telomeres attached to the nuclear envelope . In this way, the chromosome architecture and dynamics during meiotic prophase are markedly different from those in mitosis.
Completion of meiotic prophase is regulated by sexually dimorphic mechanisms, so the transcription and chromatin status are altered in the subsequent postmeiotic developmental program for sperm production and oocyte arrest/maturation. In spermatocytes, the completion of meiotic prophase is monitored under several layers of regulation such as the pachytene checkpoint and meiotic sex chromosome inactivation (Burgoyne et al., 2009;Ichijima et al., 2012;Turner, 2015). Male meiotic prophase is accompanied by robust alterations of gene expression programs (Schultz et al., 2003;Shima et al., 2004;Namekawa et al., 2006;Green et al., 2018;Grive et al., 2019) and epigenetic status (Kota and Feil, 2010;Sin et al., 2015;Maezawa et al., 2020) for post-meiotic spermiogenesis. However, the precise mechanisms by which this gene expression and epigenetic status are controlled under the meiotic prophase program is unknown.
It has been a longstanding enigma what triggers the initiation of meiosis upon spermatocyte differentiation, and activates the subsequent meiotic prophase program. Recently, we discovered MEIOSIN, which directs the switching from mitosis to meiosis . This review mainly focuses on the mechanism of meiotic initiation and the downstream meiotic prophasespecific events controlled by MEIOSIN.

INITIATION OF MEIOSIS DURING SPERMATOCYTE DIFFERENTIATION
Meiotic initiation coincides with spermatocyte differentiation. In mouse, retinoic acid (RA) and BMP signaling, at least in oocytes, synergistically induce meiotic transcription (Bowles et al., 2006;Koubova et al., 2006;Miyauchi et al., 2017;Nagaoka et al., 2020). Upon stimulation by RA, STRA8 (stimulated by retinoic acid gene 8) is transiently expressed in postnatal testis prior to the entry into meiosis (Oulad-Abdelghani et al., 1996) (Fig.  1). Since Stra8 knockout (KO) germ cells fail to undergo normal meiosis in male and female (Baltus et al., 2006;Anderson et al., 2008;Mark et al., 2008;Dokshin et al., 2013), it has been assumed that STRA8 plays an essential role in the progression of meiosis. However, whereas Stra8 KO germ cells fail to undergo meiosis in the C57BL/6 background (Baltus et al., 2006;Anderson et al., 2008), those in a mixed genetic background initiate but fail to complete meiotic prophase I (Mark et al., 2008). Thus, whether STRA8 is involved in the initiation of meiosis depends on genetic background. Furthermore, STRA8 is also transiently expressed in differentiating spermatogo-nia long before their differentiation into spermatocytes (Mark et al., 2008;Zhou et al., 2008aZhou et al., , 2008bEndo et al., 2015) (Fig. 1). This indicates that Stra8 expression alone is not sufficient for the induction of meiosis in spermatogonia, which raises the question of why meiosis is induced only in pre-leptotene spermatocytes, the phase of the second expression of STRA8, but not in spermatogonia despite the expression of STRA8. Thus, whether STRA8 is indeed required for meiotic initiation has been controversial.
By a proteomic approach, we screened STRA8interacting factors from mouse testes and identified MEIOSIN (Meiosis initiator), which was encoded by a hypothetical gene, Gm4969 . As the gene information and the exon-intron prediction for Gm4969 in the database were yet to be correctly annotated, even extensive studies in the field with transcriptome approaches may have failed to recognize Gm4969. MEIOSIN protein possesses basic helixloop-helix (HLH) and high mobility group (HMG) box domains, implying a role as a DNA-binding protein ( Fig. 2A). Meiosin mRNA showed a specific expression pattern in adult testis and embryonic ovary. Indeed, MEIOSIN and STRA8 were transiently co-expressed in pre-leptotene stage spermatocytes (Fig. 2B, 2C), which coincides with the S phase prior to meiotic prophase.
Whereas STRA8 is expressed both in differentiating spermatogonia and in pre-leptotene spermatocytes (  shown along the developmental stages. STRA8 protein is expressed in differentiating spermatogonia and in pre-leptotene spermatocytes. MEIOSIN protein is expressed at the transition toward meiotic initiation in pre-leptotene spermatocytes. The pre-leptotene stage is defined as the time point that coincides with the S phase starting shortly before meiotic prophase. RA is secreted from Sertoli cells in the stage VII-VIII seminiferous tubules, which contain differentiating spermatogonia and pre-leptotene spermatocytes. AE: axial element. meiotic entry in spermatogonia by negatively regulating RA-dependent transcription (Matson et al., 2010). Since chromatin immunoprecipitation followed by sequencing (ChIP-seq) data showed that DMRT1 binds to the 5′ region upstream of the Meiosin locus (Murphy et al., 2015), DMRT1 may repress Meiosin expression in spermatogonia, which would explain why RA does not induce MEIOSIN expression and subsequent meiosis in differentiating spermatogonia. Although the precise mechanism of Meiosin expression is yet to be fully investigated, the temporal expression of MEIOSIN ensures the proper timing of meiotic initiation in testis.

MEIOSIN DIRECTS THE SWITCH FROM MITOSIS TO MEIOSIS
In Meiosin-deficient testes, spermatocytes later than pre-leptotene stage do not appear, and consequently postmeiotic spermatids or sperm are absent, resulting in severe infertility (Fig. 3A, 3B). In Meiosin KO testes, STRA8 is still expressed and vice versa, implying that Meiosin and Stra8 expression are regulated independently of each other. Notably, despite the expression of STRA8, Meiosin KO spermatocytes fail to enter meiotic prophase (Fig. 3C). It is also worth noting that in Meiosin KO, STRA8 largely remains in the cytosol rather than in the nucleus, suggesting that MEIOSIN is required for nuclear localization of STRA8 in preleptotene spermatocytes. In differentiating spermatogonia, where MEIOSIN is not expressed, STRA8 localizes to the nucleus (Fig. 2C). This suggests that the nuclear localization of STRA8 is independent of MEIOSIN in differentiating spermatogonia, which further emphasizes that the expression of both MEIOSIN and STRA8 is required for the initiation of meiosis.  (C) WT neonatal male mice were subjected to consecutive injection of the RA synthesis inhibitor WIN 18,446 from postnatal day 5 (P5) to P11, followed by RA injection at P12 to enrich pre-leptotene spermatocytes. Seminiferous tubule sections at P13 were stained as indicated. Pre-lep: pre-leptotene spermatocyte. Arrowheads: STRA8-positive/MEIOSIN-negative differentiating spermatogonia. Boundaries of the seminiferous tubules are indicated by dashed lines. Scale bars: 25 μm. Samples for the photo images (B, C) were prepared and the images acquired as described previously .
indicates that meiosis-associated genes, including those involved in the processes of meiotic chromosome organization, the meiotic cell cycle and spermatogenesis, are downregulated in Meiosin KO. This accounts for the failure of progression into meiotic prophase and subsequent maintenance of the meiotic cell cycle observed in Meiosin KO spermatocytes. Altogether, MEIOSIN plays a crucial role in the initiation of meiosis and the subsequent meiotic program. In Stra8 KO, some spermatocytes still have the ability to enter meiosis but fail to complete meiotic prophase, indicating a phenotypic difference between Meiosin KO and Stra8 KO testes. A subset of meiotic genes is more strongly downregulated in Meiosin KO than in Stra8 KO, which may account for the phenotypic difference of a more severe pre-leptotene block in Meiosin KO than in Stra8 KO. Since their spermatocytes do not enter meiosis, what happens to Meiosin KO testes? In Meiosin KO tes-tes, mitotic prometaphase-like cells marked by histone H3Ser10 phosphorylation accumulate to a high level. Moreover, the mitotic cyclin A2 is ectopically expressed in Meiosin KO spermatocytes, as if Meiosin KO spermatocyte-like cells are in the mitotic cell cycle, which are consequently eliminated by apoptosis. These observations suggest that Meiosin KO spermatocyte-like cells acquire precocious mitotic status soon after reaching the pre-leptotene-like stage. In summary, MEIOSIN is required for cell cycle switching from mitosis to meiosis.

MEIOSIN IN COLLABORATION WITH STRA8 ACTIVATES THE MEIOTIC PROPHASE PROGRAM
It has been suggested that the gene expression program for meiotic prophase is executed under RA signaling through either STRA8-dependent or -independent path- Pachy. (C) Seminiferous tubule sections from eight-week-old WT and Meiosin KO testes were stained for SYCP3, STRA8 and DAPI. Spermatocytes that enter meiotic prophase are absent in Meiosin KO testes. Pre-lep: pre-leptotene spermatocyte; Pachy: pachytene spermatocyte. Round Sp.: round spermatid. Note that STRA8 is localized in the nuclei in WT testes, and mostly remains in the cytoplasm in Meiosin KO testes. Scale bar: 25 μm. Samples for the photo images (B, C) were prepared and the images acquired as described previously  ways (Koubova et al., 2014;Soh et al., 2015). However, it remained elusive how STRA8 might be involved in the expression program for meiotic prophase. MEIOSIN possesses the HMG box DNA-binding domain ( Fig. 2A), and interacts with STRA8. Indeed, ChIP-seq analysis indicates that MEIOSIN and STRA8 share binding sites at promotor regions on the mouse genome (exemplified in Fig. 4A) with common DNA-binding motifs (Fig. 4B). MEIOSIN-and STRA8-binding sites overlap well at the transcription start site (TSS) regions, as revealed by CAGE-seq (cap analysis gene expression with sequencing) in the testis. Since meiotic gene promoters in spermatogonia are often poised by the enrichment of H3K4me2 and the loading of RNA polymerase II , one possibility is that at the target TSSs, MEIOSIN and STRA8 promote the release of paused RNA polymerase II, or stimulate the activity of the polymerase II-associated basal transcription machinery, for a rapid and synchronous burst of transcription. Indeed, it should be mentioned that some, if not all, of these MEIOSIN/STRA8-bound genes start to show weak expression just prior to meiotic entry, which is con- sistent with the observed upregulation of a broad range of genes after RA signaling (Koubova et al., 2014;Kojima et al., 2019). BMP signaling may also synergistically induce meiotic transcription through ZGLP1 (Nagaoka et al., 2020). Although the precise mechanism is yet to be investigated, MEIOSIN and STRA8 may amplify the transcription of these primed genes for meiotic entry. Importantly, genes bound by both MEIOSIN and STRA8 are downregulated in Meiosin KO testes. Many of these genes have functions associated with meiotic prophase processes such as meiotic chromosome dynamics and recombination (Fig. 4C), which is consistent with the STRA8 ChIP data (Kojima et al., 2019). Notably, MEIOSIN and STRA8 bind to their own promoter regions, which may realize rapid expression during the short period of meiotic initiation by an autoactivation feedback loop. Interestingly, among the MEIOSIN-and STRA8-bound genes are the Meioc and Ythdc2 genes, whose products are suggested to destabilize mitotic cell cycle-associated transcripts (Abby et al., 2016;Soh et al., 2017). In Meioc KO and Ythdc2 KO mice, germ cells initiate but fail to maintain the meiotic prophase, showing misexpression of mitotic cyclin A2 and metaphase-like chromosome condensation (Abby et al., 2016;Soh et al., 2017;Jain et al., 2018). Since a similar mitotic status is also observed in Meiosin KO and Stra8 KO mice (Mark et al., 2008;Ishiguro et al., 2020), the cell cycle switching program from mitosis to meiosis may be executed through the activation of Meioc and Ythdc2. This implies that a MEIOSIN-STRA8 complex directly activates the transcription of a subset of critical meiotic genes (Fig.  4D). Given that meiosis starts from the S phase of the cell cycle (Pratto et al., 2021), the meiotic prophase program may be installed on S phase by robust activation of MEIOSIN/STRA8 target genes.  Fig. 5. Chromosome structure and dynamics during meiotic prophase. (A) Meiotic prophase I is a prolonged G2 phase divided into four substages according to the chromosome morphology. During meiotic prophase I, sister chromatids form an axial element (AE). Cohesins are loaded along sister chromatids. DSBs are generated at leptotene. Homologous chromosomes undergo pairing and synapsis through leptotene to zygotene. The synaptonemal complex (SC) is assembled between the homologous chromosomes during this process. When the SC is assembled between homologous chromosomes, the AE is called the lateral element (LE). Transverse filaments link two LEs. Cohesin locates at the inner side of the LE. Meiotic recombination machineries are loaded on the AE to repair DSBs and generate crossovers between homologous chromosomes, yielding physical linkages called chiasmata. At diplotene, the SC is disassembled. At metaphase I, sister kinetochores are mono-oriented, and chromosomes are condensed. (B) Telomeres are anchored to the nuclear envelope by meiosis-specific telomere-binding proteins (MAJIN, TERB1, TERB2, KASH5 and SUN1). By the movement of telomeres along the nuclear envelope, which is mediated by dynein on microtubules, chromosomes undergo dynamic movement to facilitate homolog pairing and synapsis. Chromosomes transiently exhibit extensive telomere clustering, called the bouquet. (C) Sister chromatids are held together by cohesin complexes in mitosis and meiosis. The cohesin complex consists of four core subunits that differ between meiosis and mitosis. In mitosis, it consists of SMC1α, SMC3, the kleisin family protein RAD21/SCC1, and STAG1 or STAG2. In meiosis of mammalian germ cells, it consists of SMC1β, SMC3, meiosis-specific kleisin subunits REC8 or RAD21L, and STAG3. Meiotic cohesin plays crucial roles in meiosis-specific chromosomal events during meiotic prophase I.
During these processes, telomeres are anchored to the nuclear envelope (NE) by meiosis-specific telomere and NE proteins (TERB1 , TERB2, MAJIN (Shibuya et al., 2015), KASH5/CCDC155 (Morimoto et al., 2012) and SUN1 (Ding et al., 2007)) that are also encoded by MEIOSIN/STRA8 target genes (Fig. 5B). By telomere-driven force, chromosomes undergo dynamic movement to facilitate homolog pairing and synapsis . It should be mentioned that some well-known meiotic genes such as Hei10, Msh4, Six6OS and Rec8 are not bound by the MEIOSIN-STRA8 complex, suggesting that a subset of the meiotic genes is regulated independently of MEIOSIN and STRA8, as in the case of Rec8 (Koubova et al., 2014).

GENE EXPRESSION OF CHROMATIN COMPONENTS IS ALTERED BY ZFP541 REPRESSOR COMPLEX PRIOR TO THE COMPLETION OF MEIOTIC PROPHASE
In spermatocytes, meiotic prophase is accompanied by robust alterations of gene expression programs (Schultz et al., 2003;Shima et al., 2004;Namekawa et al., 2006;Green et al., 2018;Grive et al., 2019) and chromatin status (Kota and Feil, 2010;Sin et al., 2015;Maezawa et al., 2020) as well as by reorganization of the chromatin structure (Alavattam et al., 2019;Patel et al., 2019;Wang et al., 2019), for sperm production. At the pachytene stage of spermatocytes, the transcriptional program for post-meiotic development starts to take place (da Cruz et al., 2016;Ernst et al., 2019). The germ cell-specific Polycomb protein SCML2 is, at least in part, responsible for the suppression of somatic genes and the activation of late-spermatogenesis-specific genes in spermatocytes and in round spermatids Maezawa et al., 2018aMaezawa et al., , 2018b. However, gene activation mechanisms during pachytene and late spermatogenesis largely remain unknown. Thus, for spermatocytes, pachytene exit is a critical developmental event for the subsequent spermatid differentiation, but how the completion of meiotic prophase is ensured prior to post-meiotic differentiation remained elusive.
The Zfp541 gene, which encodes a zinc finger protein, has been identified as one of the MEIOSIN/STRA8 target genes (Horisawa-Takada et al., 2021). ZFP541 is also an interactor of KCTD19, which was discovered in KO screening of spermatogenic genes (Oura et al., 2021). ZFP541 is expressed in spermatocytes and in round spermatids (Fig. 6A, 6B), and interacts with HDAC1/2 and germ cellspecific KCTD19. Disruption of Zfp541 leads to defects in the completion of meiotic prophase, with a severe impact on male fertility (Fig. 6C). Thus, ZFP541 plays a critical role in promoting developmental progression of meiotic prophase toward completion in spermatocytes. Chromatin binding analysis of ZFP541 combined with transcriptome analysis demonstrates that ZFP541 binds to and represses a broad range of genes whose biological functions are associated with the processes of transcriptional regulation and covalent chromatin modification (Fig. 7A). It is worth noting that most of these genes are generally expressed in broad cell types rather than being germ cell-specific. For example, these include Dnmt1, Dnmt3A (DNA methyltransferase), Ino80D, Chd1, Chd2, Chd3, Chd6 (chromatin remodeling), Kat6A, Kmt2C,Kmt2B,Kdm2A,Kdm5B,Ash1L,Bmi1,Jarid2,Ezh1,Ezh2,Ehmt1,Rnf2,Suz12 (histone modification) and Ctcfl (chromatin binding) (Fig. 7B). Notably, expression of these ZFP541-bound genes declines overall in prophase spermatocytes and post-meiotic round spermatids, compared to spermatogonia. This, at least in part, leads to reconstruction of chromatin status for spermiogenesis. Thus, an HDAC1/2-containing ZFP541-KCTD19 complex represses the transcription of a subset of critical genes that are involved in transcriptional regulation and chromatin modification prior to the completion of meiotic prophase. ZFP541 may trigger the reconstruction of the transcription network to promote the completion of prophase, finalize meiotic divisions, and proceed into spermatid production. Since ZFP541 and KCTD19 are conserved only in mammals, it will be interesting to investigate whether a similar mechanism exists for the reconstruction of the transcription network that promotes the completion of prophase in other species.

CONCLUSION
MEIOSIN in collaboration with STRA8 directs meiotic entry upon spermatocyte differentiation. The initiation of meiosis occurs after the second induction of STRA8 when both MEIOSIN and STRA8 are co-expressed. The temporal expression of MEIOSIN ensures proper timing of meiotic initiation in testis. The MEIOSIN-STRA8 complex directly binds and activates the transcription of a subset of meiotic genes that are required for meiotic chromosome dynamics. Furthermore, the MEIOSIN-STRA8 complex activates Zfp541, which plays a critical role in the regulation of the transcription network during meiotic prophase. Therefore, MEIOSIN together with STRA8 acts as a master transcription factor for establishing and maintaining the meiotic prophase program. Given that MEIOSIN and STRA8 bind to TSSs rather than to upstream enhancers, it will be interesting to investigate the molecular mechanism that triggers meiotic gene transcription. Since MEIOSIN and STRA8 are conserved in vertebrates, it is possible that the MEIOSIN/STRA8-mediated system acts for meiotic gene activation in other vertebrate species.
It should be mentioned that some targets of the MEIOSIN-STRA8 complex are hypothetical genes. These uncharacterized MEIOSIN/STRA8 target genes may play crucial roles in meiosis. Indeed, we have newly identified BRME1/4930432K21Rik among the MEIOSIN/ STRA8 target genes, which acts for the recruitment of BRCA2-RAD51 during meiotic recombination (Takemoto et al., 2020). Further investigation of MEIOSIN/STRA8 target genes will shed light on new players in meiosis.
This work was supported in part by KAKENHI grants (#19H05743, #20H03265, #20K21504, #JP 16H06276) from MEXT, Japan; a grant from AMED PRIME (21gm6310021h0001); and grants from the Sumitomo Foundation, the Naito Foundation, the Astellas Foundation for Research on Metabolic Disorders, the Daiichi Sankyo Foundation of Life Science, the Uehara Memorial Foundation, the NOVARTIS Foundation (Japan) for the Promotion of Science, and the Takeda Science Foundation.