Testis-specific expression and genomic multiplicity of the rat Rtdpoz genes that encode bipartite TRAF- and POZ/BTB-domain proteins
Introduction
TDPOZ is a hypothetical family of bipartite proteins that carry the tumor necrosis factor receptor-associated factor (TRAF) domain (TD) and POZ/BTB derived by in silico data mining (Choo et al., 2001, Huang et al., 2004). TD and POZ domains seldom co-exist in the same protein but are frequently found in association with other DNA-binding or protein–protein interacting domains. TD may be located at the amino or carboxyl terminus, or internally, in a TRAF-domain protein. TD protein diversity is achieved when TD is combined with functional protein segments such as the C2H2 and RBCC-type zinc fingers, POZ and ubiquitin-specific protease domains (Zapata et al., 2001). Biochemically, TD proteins, such as the TRAFs, are adaptor proteins that bind to the tumor necrosis factor receptors, or other adaptor molecules, kinases and apoptosis antagonists (reviewed in Uren and Vaux, 1996, Arch et al., 1998, Wajant et al., 2001, Bradley and Pober, 2001). Many TD proteins participate in cellular proliferation and survival, and in cell death signaling. On the other hand, POZ/BTB is a hydrophobic domain originally found at the amino end of many C2H2-type zinc finger proteins (reviewed in Bardwell and Treisman, 1994, Zollman et al., 1994, Collins et al., 2001, Stogios et al., 2005). The POZ domain is structurally folded into alternating α-helices with intervening β-sheets. Most of the conserved residues in the POZ domain are hydrophophic suggesting the importance of conserving the POZ conformation despite differences in the primary amino acid sequences. POZ domains are involved in oligomerization, a process that contributes to correct protein conformation (Hoatlin et al., 1999, Melnick et al., 2000, Stogios et al., 2005). POZ domain transcription factors, such as PLZF and FBI-1, act as transcriptional repressors by inhibiting DNA-binding (Melnick et al., 2000, Lee et al., 2002). Chromosomal translocations of the PLZF and another POZ-domain protein gene, LAZ/Bcl6, result in novel dominant negative POZ fusion proteins that act to sequester their fusion partners into inactive complexes (Ye et al., 1993, Bardwell and Treisman, 1994).
Presently, the only mammalian TDPOZ bipartite protein that has begun to be biochemically characterized is the nuclear speckle-type protein SPOP first identified as a novel antigen recognized by the serum of a scheroderma patient (Nagai et al., 1997). Both the TD and POZ domains of SPOP are shown to contribute to the nuclear speckled accumulation of the protein. In a biochemical analysis of TD-encompassing factors (TEF), Zapata et al. (2001) further show that while TDs of other TEF members interact strongly with one another, SPOP interacts only weakly with the TRAFs analyzed. Furthermore, SPOP does not self-associate nor does it inhibit NF-κB induction. Taken together, data from the study of Zapata et al. first suggest that the TD of SPOP is unique and behaves differently from other TD proteins. In another report, SPOP (also called PCIF1 by the authors) is found to interact with PDX-1, a homeodomain transcription factor essential for normal pancreatic development (Liu et al., 2004). SPOP and PDX-1 interactions lead to repression of the PDX-1 transactivation functions, resulting in the modulation of normal pancreatic development. SPOP is further shown to modulate Daxx-mediated transcriptional repression of the MMP1 gene expression (La et al., 2004). Besides being involved in transcriptional regulation, other works reveal that SPOP complexes with CULLIN3 to constitute an E3 ubiquitin ligase that, in association with the histone MACROH2A1, contributes to the inactivation of the X chromosome (Hernandez-Munoz et al., 2005). This finding echoes the reports that the C. elegans TDPOZ protein, MEL26, and the mammalian SPOP are also substrate-specific adaptors of the CUL3 ubiquitin ligase in the ubiquitin-proteosome protein degradation scheme (Pintard et al., 2003, Xu et al., 2003, Kwon et al., 2006).
Based on BLAST and BLAT algorithm queries of genome sequence databases of a number of model organisms, our previous report describes the presence of at least thirty-four Tdpoz orthologues in the genomes of a wide spectrum of animal and plant species including human, C. elegans, Drosophila, Arabidopsis and rice (Huang et al., 2004). Amongst them are eight rat Tdpoz homologs and nine mouse genes. The primary goal of this work was to obtain evidence to indicate that members of the hypothesized Tdpoz gene family are expressed. For this purpose, the rat was used as our working model since our previous work has indicated multiplicity of Tdpoz genes in the rat genome. We show here that at least two rat Tdpoz genes, now designated as Rtdpoz's, are transcribed specifically in the rat testis. We further identify twenty-four other putative TDPOZ-encoding genes in the rat genome many of which are expected to be shown to be expressed in future works.
Section snippets
RNA preparation and expression profile analysis
Total RNA was prepared from major organs of the rat strain Sprague–Dawley using the TRI REAGENT® (MRC, Cincinnati, OH) following manufacturer's instructions. In the initial phase of the derivation of the Rtdpoz cDNA sequences, the consensus primers used were Rtdpoz-f (5′-CCAAGATCTGTGGCTACACACATA-3′) and Rtdpoz-r (5′-TTGGTACAGCATTCTTCACAGAGA-3′) that generated a 800-bp amplicon. Northern blot analysis was performed using a commercial rat MTN Multiple Tissue Northern Blots obtained from BD (cat.
Testis-specific expression of Rtdpoz-T1 and -T2
We previously identified by data mining eight putative rat Tdpoz genes, now designated as Rtdpoz's (Huang et al., 2004). To determine if members of the predicted Rtdpoz genes are expressed, consensus Rtdpoz primers were designed based on conserved sequences located in the Rtdpoz open reading frames (ORFs) for use in reverse transcription (RT)-PCR expression profiling of Rtdpoz in major rat organs. The experiments detected amplification products predominantly in the testis (Fig. 1A). The
Discussion
We demonstrate in this work testis-specific expression of two new members of the previously hypothesized TDPOZ family first derived by data mining and bioinformatics analysis (Huang et al., 2004). The result establishes that members of the hypothetical gene family are, indeed, expressed. We also report here further expansion of the repertoire of Rtdpoz genes in the rat genome. The clustered Rtdpoz genes and pseudogenes, present in both transcriptional orientations, are all intronless in the
Acknowledgements
This work was supported by a Taipei Veterans General Hospital intramural grant (VGH94-315) awarded to KBC. We thank C.-Y. Tao for technical assistance.
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Evolutionary expansion of SPOP and associated TD/POZ gene family: Impact of evolutionary route on gene expression pattern
2010, GeneCitation Excerpt :Involvement of a SPOP ortholog, the Roadkill (rdx) gene, in Hedgehog signaling in Drosophila has also been reported (Kent et al., 2006). In the mouse and rat genomes, we have previously reported the presence of multiple TD/POZ retro-sequences (Choo et al., 2007; Huang et al., 2009). The mouse Tdpoz1 retrogene (GenBank accession number AF290198), re-designated as mTdpoz1 in this work for species distinction, is transcribed in the egg and the pre-implantation-stage embryo while other mTdpoz retrogenes are transcribed in low levels in the pre-implantation embryo and in the testis (Huang et al., 2004).
Retrogenes in Preimplantation Embryo Development: A Unique Mode of Transcriptional Regulation
2009, Journal of the Chinese Medical Association