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Tubulin Acetylation: a Novel Functional Avenue for CDYL in Sperm

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Tubulin Acetylation: a Novel Functional Avenue for CDYL in Sperm Received: 3 January 2017 | Revised: 8 June 2017 | Accepted: 28 June 2017 DOI: 10.1002/cm.21381 RESEARCH ARTICLE Tubulin acetylation: A novel functional avenue for CDYL in sperm Sweta Parab1 | Veena Dalvi1 | Sushma Mylavaram1 | Abhipriya Kishore2 | Susan Idicula-Thomas2 | Shobha Sonawane3 | Priyanka Parte1 1Department of Gamete Immunobiology, National Institute for Research in Abstract Reproductive Health (ICMR), Mumbai Motility in sperm is driven by the flagella, the principal component of which is the axoneme. The 400012, India microtubules which make up the 9 1 2 axoneme are composed of heterodimers of alpha and beta 2 Biomedical Informatics Centre, National tubulins and undergo several post-translational modifications. We have earlier reported that Institute for Research in Reproductive HDAC6 functions as tubulin deacetylase in sperm and has a role in sperm movement. While Health(ICMR), Mumbai 400012, India exploring the specific tubulin acetyltransferase (TAT) in sperm, we observed the presence of Chro- 3Department of Neuroendocrinology and Confocal Microscopy Lab, National Institute modomain Y-Like (CDYL), on the principal piece of rat spermatozoa which compelled us to explore for Research in Reproductive Health(ICMR), its function in sperm. CDYL was observed to be colocalized with acetylated alpha-tubulin (Ac a Mumbai 400012, India Tubulin) in sperm flagella. Sperm axonemal fraction showed the presence of CDYL protein indicat- ing its strong association with flagellar microtubules. Sequence alignment of CDYL chromo domain Correspondence Priyanka Parte, Department of Gamete and Alpha tubulin acetyltransferase (aTAT1) revealed that of the 10 residues of aTAT1 known to Immunobiology, National Institute for be involved in a-tubulin binding, 5 residues were identical and 1 was conserved between the two Research in Reproductive Health (ICMR), proteins. Docking of CDYL chromo domain and a-tubulin showed that 6 of the 11 important bind- Mumbai 400012, India. ing residues of a-tubulin showed an interaction with CDYL chromo domain. The putative CDYL Email: [email protected] chromodomain –a-tubulin interaction was further confirmed by Microscale Thermophoresis. We Funding information further asserted the ability of recombinant CDYL and Sperm CDYL to acetylate soluble tubulin Department of Science and Technology and microtubules in vitro. Acetylation of tubulin was increased over twofold in cells overexpress- Grant / Award Number: (D.O. No. SR/SO/ HS/112/2007), India; Indian Council of ing CDYL. Thus, our studies convincingly demonstrate the ability of CDYL to moonlight as a Medical Research (NIRRH - RA/390/06- tubulin acetyltransferase. 2016); Indian Council of Medical Research (Senior Research Fellowship to S.P.); DST KEYWORDS (JRF and SRF to S.P.) alpha-tubulin, Chromodomain Y-Like, microtubules, sperm, tubulin acetyltransferase 1 | INTRODUCTION and a C-terminal catalytic domain (Lahn & Page, 1999). A significant association has been demonstrated between testicular impairment/ Chromodomain Y like (CDYL) gene is an autosomal gene mapped on spermatogenic failure and the expression of CDY1 and CDY2 tran- human chromosome 6. Chromodomain Y (CDY) gene in the course of scripts in infertile men with DAZ deletions (Ferlin, Moro, Rossi, & For- primate evolution ascended by retropositioning of an mRNA from the esta, 2001; Kleiman et al., 2003), Potash (2006) in his thesis CDYL gene. Autosomal CDYL is ubiquitously expressed while CDY is demonstrated that mice lacking Cdyl produce spermatozoa with mis- testis specific and located on the Y chromosome and implicated in shapen heads and show significant germ cell death which in turn male infertility (Kuroda-Kawaguchi et al., 2001; Lahn et al., 2002). In affects spermatogonia, spermatocytes and spermatid number is mouse, where Y-linked CDY homologs are absent, Cdyl gene is exp- observed. ressed as 2 transcripts: a ubiquitous transcript, and a highly expressed Human CDY and mouse CDYL proteins have been reported to testis-specific transcript. Human CDYL shares 93% and 63% protein exhibit HAT activity in vitro and their expression during spermatogene- sequence identity with mouse Cdyl and human CDY, respectively. sis correlates with the occurrence of H4 hyperacetylation (Akella et al., CDYL protein has an N-terminal chromodomain (DNA binding domain) 2010). However, histone H4 hyperacetylation was detected equally in Cytoskeleton.2017;1–12. wileyonlinelibrary.com/journal/cm VC 2017 Wiley Periodicals, Inc. | 1 2 | PARAB ET AL. spermatids of both wild type and Cdyl knockout mice (Potash, 2006). epididymis of the adult male rat, CDYL expression is seen in the princi- These observations question its role as a histone acetyltransferase. We pal epithelial cells of caput and caudal region of epididymis. The pres- observed the localization of CDYL in the principal piece of rat caudal ence of CDYL is also observed in the luminal sperm of the caput and sperm flagella. CDYL co-localized with acetyl alpha tubulin (Ac a-Tubu- caudal region indicating its presence in mature sperm (Figure 1b). The lin). This encouraged us to explore whether it acted as a tubulin acetyl expression of the transcript for Cdyl in rat testicular- and caudal-sperm transferase (TAT) in light of its controversial role as a histone acetyl- was investigated by RT-PCR using primers to the full length (1.9 kb) transferase. MEC-17, a protein related to Gcn5 has also been identified and partial cDNA (214 bp). Our observations demonstrate prominent as a TAT in Tetrahymena, C. elegans and Zebrafish. However, in Zebra- presence of the CDYL transcript in testicular- and caudal sperm. ‘Rea- fish ablation of MEC-17 displays lack of a-tubulin acetylation in the gent’ control as well as ‘No RT’ control showed no contamination from neurons but not in the cilia (Akella et al., 2010). Interestingly, mec17or- reagents used or from genomic DNA (Figure 1c). Western blot analysis tholog atat1knockout mice show defect in sperm morphology, and for CDYL and AC a-tubulin in rat testicular- and caudal- sperm motility and litter size suggesting that tubulin acetylation is involved in revealed a 70 kDa band for CDYL and 55 kDa for Ac a-tubulin in regulating sperm fertility. Surprisingly, development of brain which is both (Figure 1d). known to have high amounts of acetylated a tubulin, and neurological behaviour are not affected, neither are any other ciliated organelles. 2.2 | Co-localization of CDYL and Ac a-tubulin However, these mice show increased microtubule stability (Kalebic, in sperm Sorrentino, et al., 2013). Studies around the same time by Kim, Gor- bani, You, and Yang (2013) have observed that atat1 KO mice are via- Co-localization of CDYL and Ac a-tubulin was investigated in ble, testicular function and fertility of these mice is normal, but display testicular-, caput- and caudal-sperm. CDYL was localized mainly on the a-tubulin acetylation deficiency and abnormal dentate gyrus Kim et al. head region in testicular-, caput- and caudal-sperm. Flagellar localiza- (2013). They conclude that atat1 is not required for normal develop- tion of CDYL seen along the complete length of testicular- and caput- ment but may regulate more advanced functions such as memory and sperm flagella differs from the localization in caudal sperm where it is learning. Whilst both these authors agree that atat1 is the tubulin ace- restricted to the principal piece. Ac a-tubulin was observed to be local- tyltransferase, there is a disagreement on its significance in sperm func- ized throughout the sperm flagella of testicular-, caput- and caudal tion. Interestingly, the fraction shown by Maruta, Greer, and sperm (Figure 2a). Statistical analysis of signal intensities for CDYL and Rosenbaum (1986) to possess tubulin acetyltransferase activity impli- Ac a-tubulin along the length of sperm flagella demonstrates an cates a protein with a molecular weight of 67 kDa. This prompted us increase in the levels of Ac a-tubulin in caput- and caudal- sperm with to investigate the possibility that there may be another protein with respect to testicular sperm whereas no change was observed in the tubulin acetyltransferase activity on the sperm flagella. levels of CDYL protein from testicular- to epididymal-sperm (Figure 2b) The molecular size of Cdyl, its controversial role as a HAT and its although as can be appreciated from Figure 2a, its distribution along expression in the principal piece region of the rat sperm flagella the flagella is altered. CDYL and Ac a-tubulin are co-localized along the prompted us to investigate whether Cdyl had a role beyond spermato- length of the flagella. The overlap coefficient of CDYL and Ac a-tubulin genesis and possessed a-tubulin acetyltransferase activity. The present show a significant increase in the caudal sperm as compared to testicu- study was undertaken to explore this possibility. We observed a co- lar- and caput sperm indicating increased co-localization of the two localization of CDYL with Ac a-tubulin in sperm flagella. Enrichment of proteins in mature sperm flagella (Figure 2c). Similar observations were CDYL in sperm flagellar axoneme fraction strengthens the possibility of reported by us for the tubulin deacetylase HDAC6 and Ac a-tubulin its interaction with flagellar microtubules. The sequence and structural (Parab et al., 2015). Whereas there HDAC6 expression was predomi- alignment of CDYL and aTAT1 and molecular docking analysis sug- nantly on midpiece, for CDYL we see it strongly on the principal piece. gested that CDYL chromo domain
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