DNA Condensation by the Rat Spermatidal Protein TP2 Shows GC-Rich Sequence Preference and Is Zinc Dependent

Kundu, Tapas K.; M, Rao

doi:10.1021/bi00015a027

Cited by 30 publications

(39 citation statements)

References 25 publications

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“…We have shown previously that the transition protein TP2 binds and condenses GC-rich sequences preferentially (Kundu and Rao 1995). Our observation that TP2 also localizes to nucleolus in the transfected Cos-7 and HeLa cells (Ullas and Rao 2003) indicated that TP2 prefers GC-rich sequences, even in vivo.…”

Section: Resultssupporting

confidence: 50%

“…At present, molecular studies have shown clearly that TP1 stimulates DNA repair activity in vitro (Caron et al 2001), whereas there is strong evidence that TP2 is more closely associated with DNA and consequently the chromatin condensation process (Singh and Rao 1987;Kundu and Rao 1995;Zhao et al 2004a,b). In the TP1 2/2 and TP2 2/2 single-knockout mice, the chromatin condensation process is severely hampered, but still produces some sperm that are capable of fertilization (Shirley et al 2004).…”

Section: Discussionmentioning

confidence: 99%

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

Kolthur‐Seetharam

Pradeepa

Gupta

et al. 2009

J Histochem Cytochem.

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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...

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Section: Resultssupporting

confidence: 50%

Section: Discussionmentioning

confidence: 99%

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

Kolthur‐Seetharam

Pradeepa

Gupta

et al. 2009

J Histochem Cytochem.

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“…TP2 does not have much ␣-helicity as predicted by various algorithms as well as by circular dichroism spectroscopic experiments (8,15). It has a higher propensity to form ␤-turns due to the presence of large number of prolines.…”

Section: Discussionmentioning

confidence: 91%

“…The localization pattern of the two arginine mutants (R20A and R36A) behaved like wild type TP2 (panels l and n). As mentioned earlier, there is a single glutamate residue at position 86 based on which we have assigned the N-terminal two-thirds as the DNA recognition domain with its two zinc fingers and the C-terminal one-third having stretches of basic amino acid residues as the DNA condensation domain (8,9). When a TP2 construct in pCMX PL1 vector containing only the N-terminal two-thirds of the molecule-(1-86) was transfected into COS-7 cells, this partial TP2 was predominantly localized in the cytoplasm (panel p) indicating the presence of a potential nuclear localization signal in the C-terminal third of TP2 (Fig.…”

Section: Zinc Finger Modules Of Rat Spermatidal Protein Tp2 38504mentioning

confidence: 98%

“…TP2, in contrast with TP1, was shown to have DNA condensing properties (6). Rat TP2 is a zinc metalloprotein, containing two atoms of zinc per molecule (7), condenses GC-rich DNA more than other types of DNA sequences (8), and recognizes a human CpG island sequence in a zinc-dependent manner (9). Methylation of cysteine residue in the CpG doublet inhibited the recognition of the CpG island sequence by TP2.…”

Section: Gkvskrkav 95 Present In the C-terminal Third Of Tp2 As A Parmentioning

confidence: 99%

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Identification of Two Novel Zinc Finger Modules and Nuclear Localization Signal in Rat Spermatidal Protein TP2 by Site-directed Mutagenesis

Meetei

Ullas

2000

Journal of Biological Chemistry

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Spermatidal protein TP2, which appears transiently during stages 12-16 of mammalian spermiogenesis, is a DNA condensing zinc metalloprotein with a preference to GC-rich DNA. We have carried out a detailed sitedirected mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc. Two zinc fingers modules have been identified involving 4 histidine and 4 cysteine residues, respectively. The modular structure of the two zinc fingers identified in TP2 define a new class of zinc finger proteins that do not fall into any of the known classes of zinc fingers. Transfection experiments with COS-7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus. The nuclear localization signal in TP2 was identified to be 87 GKVSKRKAV 95 present in the C-terminal third of TP2 as a part of an extended NoLS sequence.Spermiogenesis is an elaborate process of cellular differentiation wherein the haploid spermatids produced following meiotic division finally mature into spermatozoa. In mammals, in particular, it is characterized by the transient appearance of a group of proteins, namely TP1, TP2, and TP4 which replace the somatic and testis-specific histones, before themselves being replaced by protamines (1). The biological significance of the role of transition proteins in chromatin remodeling, in mammals, is a matter of great importance to understand the final stages of sperm development and maturation in the testis. Among the three basic proteins, TP1, TP2, and TP4, TP1 was shown to be a DNA melting protein in vitro and has been speculated to be involved in the destabilization of nucleosome to facilitate displacement of histones (2, 3). More recently Yu et al. (4) have shown that TP1-deficient mice have abnormal spermatogenesis with reduced fertility. TP4 has been shown recently to stimulate SV40 DNA relaxing activity of eukaryotic DNA topoisomerase I suggesting a probable role in chromatin reorganization (5). On the other hand, more information is available regarding DNA binding properties of TP2. TP2, in contrast with TP1, was shown to have DNA condensing properties (6). Rat TP2 is a zinc metalloprotein, containing two atoms of zinc per molecule (7), condenses GC-rich DNA more than other types of DNA sequences (8), and recognizes a human CpG island sequence in a zinc-dependent manner (9). Methylation of cysteine residue in the CpG doublet inhibited the recognition of the CpG island sequence by TP2. Chromomycin A 3 interference experiments revealed that probably one of the modes of interaction of TP2 with GC-rich DNA is through its minor groove. These results suggested that CpG islands might serve as specific loci for initiation of chromatin condensation by TP2 through its zinc-binding domain.For a better understanding of the nature of interaction of TP2 with DNA, it is necessary to delineate the zinc-binding domains of TP2. A careful examination of the amino acid sequence of rat TP2 and its al...

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Stimulation of DNA repair by the spermatidal TP1 protein

2001

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The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single‐strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid‐spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV‐induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid‐spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis. Mol. Reprod. Dev. 58:437–443, 2001. © 2001 Wiley‐Liss, Inc.

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DNA Condensation by the Rat Spermatidal Protein TP2 Shows GC-Rich Sequence Preference and Is Zinc Dependent

Cited by 30 publications

References 25 publications

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

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

Identification of Two Novel Zinc Finger Modules and Nuclear Localization Signal in Rat Spermatidal Protein TP2 by Site-directed Mutagenesis

Stimulation of DNA repair by the spermatidal TP1 protein

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