Abstract
Mammalian genomes encode two genes related to the TATA-box binding protein (TBP), TBP-related factors 2 and 3 (TRF2 and TRF3). Male Trf2−/− mice are sterile and characterized by arrested spermatogenesis at the transition from late haploid spermatids to early elongating spermatids. Despite this characterization, the molecular function of murine Trf2 remains poorly characterized and no direct evidence exists to show that it acts as a bona fide chromatin-bound transcription factor. We show here that Trf2 forms a stable complex with TFIIA or the testis expressed paralogue ALF chaperoned in the cytoplasm by heat shock proteins. We demonstrate for the first time that Trf2 is recruited to active haploid cell promoters together with Tbp, Taf7l and RNA polymerase II. RNA-seq analysis identifies a set of genes activated in haploid spermatids during the first wave of spermatogenesis whose expression is down-regulated by Trf2 inactivation. We therefore propose that Trf2 is recruited to the preinitiation complex as a testis-specific subunit of TFIIA/ALF that cooperates with Tbp and Taf7l to promote haploid cell gene expression.
Highlights
Regulated initiation of transcription by RNA polymerase II (Pol II) involves formation of a preinitiation complex (PIC) over the transcription start site (TSS)
TATA-box binding protein (TBP) is a bipartite protein with a variable N-terminal domain, but a highly conserved C-terminal domain shared with the TBP related factors (TRFs) and forming a saddle-like structure with a concave surface that binds the consensus TATA element DNA and a convex surface that interacts with TBP-associated factors (TAFs) as well as the TFIIA and TFIIB components of the PIC13–16
Elongate spermatids were readily visible in sections from Trf2tag/tag mice showing that spermatogenesis was not arrested in these animals
Summary
Regulated initiation of transcription by RNA polymerase II (Pol II) involves formation of a preinitiation complex (PIC) over the transcription start site (TSS). We previously showed that Trf[2] interacts with TFIIA and its testis expressed paralogue ALF (TFIIA-like factor), but that unlike many other transcription factors, Trf[2] was readily extracted from the nucleus under low salt conditions[35] Despite these observations, the presence of Trf[2] in the chromatin-associated PIC at promoters in testis has not been demonstrated. RNA-seq analysis identifies a set of testis genes activated around post-natal day 21 when the haploid spermatids are formed in the first wave of spermatogenesis and whose expression is down-regulated by Trf[2] inactivation. We propose that Trf[2] is recruited to the PIC together with Tbp and Taf7l as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
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