title: Mapping of Post-translational Modifications of Transition Proteins, TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2.
abstract: In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg(71), Arg(75), and Arg(92) residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys(88) and Lys(91) residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome.
authors.affiliation: From the Chromatin Biology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Gupta
authors.affiliation: From the Chromatin Biology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India.
authors.forename: M Pradeepa
authors.initials: MP
authors.lastname: Madapura
authors.affiliation: From the Chromatin Biology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India.
authors.forename: U Anayat
authors.initials: UA
authors.lastname: Bhat
authors.affiliation: From the Chromatin Biology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India mrsrao@jncasr.ac.in.
authors.forename: M R Satyanarayana
authors.initials: MR
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D004252:DNA Mutational Analysis; D044127:Epigenesis, Genetic; D005260:Female; D057809:HEK293 Cells; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry*; D006657:Histones / Q000737:chemistry; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D013091:Spermatogenesis
chemical_list: D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D009707:Nucleosomes; D010455:Peptides; C053886:spermatid transition proteins; D001120:Arginine; D011484:Protein-Arginine N-Methyltransferases; C119966:coactivator-associated arginine methyltransferase 1; D011495:Histone-Lysine N-Methyltransferase
keywords: chromatin remodeling; epigenetics; mass spectrometry (MS); post-translational modification (PTM); protein methylation
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