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

Meetei, Amom Ruhikanta; Ullas, Kolathur S.; M, Rao

doi:10.1074/jbc.m002734200

Cited by 28 publications

(23 citation statements)

References 34 publications

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“…TP2 appears to have two separate domains that contribute to its interaction with DNA. The amino-terminal three-quarters of the molecule has been reported to provide the specificity in the protein's binding to CpG sequences (21, 43), possibly through the formation of two or more zinc finger domains (44), while the carboxyl-terminal domain may play a major role in facilitating condensation (45). This hypothesis was confirmed for TP2 by condensation experiments conducted using only the carboxyl-terminal 25-mer of TP2: SKRKAVRRRKRTHRAKRRTSGRRYK.…”

Section: Dna Condensation Induced By Protamines P1 and P2-insupporting

confidence: 64%

Condensation of DNA by Spermatid Basic Nuclear Proteins

Brewer¹,

Corzett²,

Balhorn³

2002

Journal of Biological Chemistry

129

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Two transition proteins, TP1 and TP2, participate in the repackaging of the spermatid genome early in mammalian spermiogenesis, coincident with the first detectable changes in chromatin condensation. Using an optical trap and a two-channel flow cell to move single DNA molecules into buffer containing protein, we have measured the rates of DNA condensation and decondensation induced by the binding of Syrian hamster transition proteins TP1 and TP2 and protamines P1 and P2. The results show that both transition proteins condense free DNA, with rates similar to those of protamine 1 and 2. DNA molecules condensed with TP1 were significantly less stable than DNA condensed by protamine or by TP2. Experiments conducted with a peptide corresponding to the C-terminal 25 residues of TP2 showed that this domain is responsible for condensing DNA. Experiments conducted with two fragments of TP1 containing arginine and lysine residues demonstrated that DNA binding by TP1 must involve more than these basic sequences. Zinc facilitated the condensation of DNA by P2 but not by TP2. The dissociation rates of TP2 and P2 from DNA were not affected by the addition of zinc.The structure of chromatin is changed dramatically during the final stages of spermiogenesis in mammals as the spermatid's genome is condensed and inactivated by the sequential binding of several basic nuclear proteins (1). Although the most dramatic change in condensation occurs in late-step spermatids (2) when the protamines displace transition proteins TP1 and TP2 (3) and coil the DNA into toroidal subunits (4), the replacement of histones by these two transition proteins several days earlier coincides with the first appearance of a detectable change in the condensation state of chromatin (5, 6). Studies in the rat have demonstrated that the deposition of the two transition proteins in spermatid chromatin occurs sequentially, with TP2 appearing first in step 10 spermatids. TP1 was observed to appear ϳ24 h later in step 12 spermatids (7).Previous experiments have shown that TP2 binds preferentially to CG sequences, and the observation that TP2 appears early in spermatid chromatin (8) suggested that the function of TP2 may be to shut down transcription by binding to the CG islands that are associated with gene promoter domains. TP1, which appears a day later, has been reported to stimulate DNA repair of single-stranded breaks (9) and is suggested to function by binding to the breaks induced during the removal of the histones until they can be repaired (9, 10). Following the removal of the histones and the repair or ligation of the singlestranded breaks, protamines are synthesized and deposited on chromatin to complete the compaction of the chromatin and ensure the sperm genome remains inactive until it can be deposited inside an egg and reactivated.Protamines, on the other hand, are highly charged, argininerich proteins that bind to DNA in a nonspecific manner. Previous in vivo and in vitro studies have been carried out to determine how DNA is condensed by protamine...

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Section: Dna Condensation Induced By Protamines P1 and P2-insupporting

confidence: 64%

Condensation of DNA by Spermatid Basic Nuclear Proteins

Brewer¹,

Corzett²,

Balhorn³

2002

Journal of Biological Chemistry

129

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“…We had earlier shown that TP2 is a zinc metalloprotein (Baskaran and Rao 1991) and condenses DNA with a preference for GC-rich DNA in a zinc-dependent manner (Kundu and Rao 1995). We have also delineated the domain architecture of TP2 and shown it to possess two structural and functional domains (Meetei et al 2000). The N-terminal two thirds of TP2, upstream of the lone glutamate 86 residue, has two novel zinc finger modules comprising four histidine and four cysteine residues, respectively.…”

mentioning

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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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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“…TP2, by contrast, is a 13-kDa protein with distinct structural domains (39). The carboxyl third of the molecule is enriched in basic residues and is likely to be a major site of electrostatic DNA binding (14), whereas the amino-terminal region has two proposed zinc fingers (41). The preferential binding activity of TP2 to CpG sequences, which are often associated with promoter regions, is dependent on zinc (36).…”

mentioning

confidence: 99%

“…TP1 has been reported to decrease the melting temperature of DNA and decrease nucleosome compaction (55,56), whereas TP2 does the opposite (7), leading to the suggestion that TP1 is involved in histone removal and TP2 is involved in chromatin condensation. Finally, the preferential binding of TP2 to CpG islands may indicate a role for it in global repression of transcription (41).…”

mentioning

confidence: 99%

Targeted Disruption of the Transition Protein 2 Gene Affects Sperm Chromatin Structure and Reduces Fertility in Mice

Zhao¹,

Shirley²,

Yu³

et al. 2001

Molecular and Cellular Biology

180

127

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During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.

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

Cited by 28 publications

References 34 publications

Condensation of DNA by Spermatid Basic Nuclear Proteins

Condensation of DNA by Spermatid Basic Nuclear Proteins

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

Targeted Disruption of the Transition Protein 2 Gene Affects Sperm Chromatin Structure and Reduces Fertility in Mice

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