Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Abstract

S U M M A R Y Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12-15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GCpreference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4′,6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC-and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis. (J Histochem Cytochem 57:951-962, 2009) K E Y W O R D S spermiogenesis DNA-binding dyes TP1 and TP2 colocalization THE MAMMALIAN GENOME of ?3-5 3 10 9 bp is packaged very tightly inside the sperm nucleus with the help of protamines (protamine P1 and P2 in mice and humans), facilitated by charge neutralization and intermolecular disulfide linkages. In mouse, this packaging results in the sperm nucleus adopting a volume ?40-fold smaller than a normal somatic interphase nucleus (Wyrobek et al. 1976). Although it was originally believed that the entire genome is packaged into nucleoprotamine fibers, more-recent evidence indicates that 2% and 15% of the genome is still associated with histones in mouse and human sperm, respectively (Gatewood et al. 1987(Gatewood et al. ,1990. Recently, Nazarov et al. (2008) have demonstrated that chromatin released from human spermatozoa following nuclease digestion exhibits a nucleosomal periodicity of ?195 bp. Specific structural and functional features have been attributed to this nucleohistone fraction, including sequence-specific DNA packaging (Pittoggi et al. 2001) and a role in the regulation of gene expression (Garden et al. 1998). This has been supported by reports showing that parts of the telomeric DNA (Zalenskaya et al. 2000;Li et al. 2008) and retroposon DNA (Pittoggi et al. 1999) are found in the nucleohistone fraction. These unexpected observations and the presence of transcription factors in sperm chromatin (Pittoggi et al. 2001) raise an interesting question as to the nature of the chromatin domains that remain in nucleosomal context and the possible significance of DNA sequences embedded in these domains in...

Insights into Role of Bromodomain, Testis-specific (Brdt) in Acetylated Histone H4-dependent Chromatin Remodeling in Mammalian Spermiogenesis

“…With the peptide antibody raised in our laboratory, the detection of Brdt was possible even in the elongating spermatids when used at the appropriate concentration. Additionally, the cells were swollen in DTT before performing immunocytochemistry, which enhances the accessibility of the antibody to nuclear proteins in the condensing spermatids (24). This result is further corroborated by a recent report demonstrating the presence of Brdt in humans from spermatocytes to mature spermatozoa (62).…”

“…The released cells were washed in PBS and swollen with dithiothreitol according to the method standardized in our laboratory (24). Briefly, total testicular cells were resuspended in decondensation buffer (0.05 mg/ml heparin and 10 mM dithiothreitol in PBS) and incubated on ice for 40 min.…”

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

“…For better appreciation of the post-translational modifications of transition proteins, TP1 and TP2, sequence conservation of the modified residues across selected mammals was analyzed. Most of the post-translationally modified amino acid residues of TP1 are conserved across the analyzed mammals except for the phosphorylation sites Ser 36 , Ser 37 , and Ser 48 (Fig. 3C).…”

“…Decondensation and Immunofluorescence of Testicular Cells-Total testicular cells were decondensed by resuspending in decondensation buffer (PBS, 10 mM dithiothreitol) and incubated at 4°C for 1 h in an end to end rotor (36). Cells were obtained after centrifuging at 1,500 ϫ g for 10 min at 4°C and were washed twice with ice-cold PBS.…”

“…These include serine phosphorylation at Ser 36 41 . In addition, K32ac was observed only in association with R34me ( Fig.…”