Two Isoforms of Drosophila TRF2 Are Involved in Embryonic Development, Premeiotic Chromatin Condensation, and Proper Differentiation of Germ Cells of Both Sexes

Abstract

The Drosophila TATA box-binding protein (TBP)-related factor 2 (TRF2 or TLF) was shown to control a subset of genes different from that controlled by TBP. Here, we have investigated the structure and functions of the trf2 gene. We demonstrate that it encodes two protein isoforms: the previously described 75-kDa TRF2 and a newly identified 175-kDa version in which the same sequence is preceded by a long N-terminal domain with coiled-coil motifs. Chromatography of Drosophila embryo extracts revealed that the long TRF2 is part of a multiprotein complex also containing ISWI. Both TRF2 forms are detected at the same sites on polytene chromosomes and have the same expression patterns, suggesting that they fulfill similar functions. A study of the manifestations of the trf2 mutation suggests an essential role of TRF2 during embryonic Drosophila development. The trf2 gene is strongly expressed in germ line cells of adult flies. High levels of TRF2 are found in nuclei of primary spermatocytes and trophocytes with intense transcription. In ovaries, TRF2 is present both in actively transcribing nurse cells and in the transcriptionally inactive oocyte nuclei. Moreover, TRF2 is essential for premeiotic chromatin condensation and proper differentiation of germ cells of both sexes.To initiate transcription, each eukaryotic RNA polymerase requires a set of general transcription factors. TFIID, composed of the TATA box-binding protein (TBP) and TBP-associated factors (TAFs), recognizes the core promoter in a sequence-specific manner and is thought to be the only sequence-specific factor that operates with RNA polymerase II (4, 51). The C-terminal core domain of TBP is highly conserved among eukaryotes and contains two symmetrical repeats that fold into a saddle-like structure essential for interaction with the promoter sequences (24,25).A second gene encoding a protein with high homology to the core domain of TBP, TBP-like factor (TLF; also called TRF2 or TLP), was detected in metazoan species (11,23,30,34,38,39,40,41,52). Like TBP, most members of the TLF family have a bipartite structure with a variable N-terminal domain and the highly conserved C-terminal core domain containing two direct repeats (11). TLF was shown to mediate polymerase II transcription initiation and to interact with TFIIA and TFIIB to form a preinitiation complex. However, TLF does not bind to the classical TATA box elements and has been shown to control a set of genes different from those controlled by TBP (12,34,40,41,45,50).Sequence comparison of core domains in the TLF family reveals that they are less conserved in evolution (40 to 45% identity among the metazoan species) than the TBP core domains (about 80% identity between yeast and humans). Thus, while the role of TBP is similar in different species, the function of TLF may have evolved into different regulatory pathways in evolutionarily distant species (11). Studies on the physiological function of TLF in Caenorhabditis elegans, Xenopus laevis, and Danio rerio have demonstrated that TLF is essenti...

TRF2: TRansForming the view of general transcription factors

“…15,78 Moreover, Drosophila embryos with reduced TRF2 have previously shown homeotic and segmentation defects, as well as dorsal-ventral abnormalities. 68 Unlike TRF2 in Drosophila, zebrafish and Xenopus, mouse TRF2 is not required for embryonic development. 79,80 TRF2 is widely expressed in the adult animal.…”

“…61 Interestingly, Drosophila trf2 encodes 2 protein products -a short (632 aa) protein, and a protein of 1715 aa (which will henceforth be referred to as long TRF2), in which the same short amino acid sequence is preceded by an N-terminal domain composed of coil-coiled motifs. 68 Translation of the short isoform probably results from an internal translation initiation by an IRES mechanism. 68 Both proteins show similarity to the C-terminal core domain of TBP.…”

“…68 Translation of the short isoform probably results from an internal translation initiation by an IRES mechanism. 68 Both proteins show similarity to the C-terminal core domain of TBP. The long TRF2 has only been identified in Drosophila.…”

“…60,66,68,75,76 Interestingly, Xenopus TRF2 was shown to play a role in the expression of catabolic genes during embryonic development. 77 Recent analysis of Drosophila genes with high TRF2 occupancy revealed a strong relationship between TRF2 and processes such as cell differentiation and development.…”

“…82 Notably, Drosophila TRF2 has been shown to be involved in differentiation of germ cells of both male and female. 68 Interestingly, it was recently demonstrated that Taspase1-mediated proteolytic cleavage of the TFIIAa-b precursor (into the a and b subunits of TFIIA) is necessary for the activation TRF2-specified transcriptional spermiogenic program in the juvenile and adult mouse testes. 83 Hence, TRF2 may form tissue-specific preinitiation complexes that regulate transcription of specific subsets of genes necessary for germ cell differentiation.…”