Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Aug 31:6:32069.
doi: 10.1038/srep32069.

TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression

Igor Martianov  1 , Amandine Velt  1 , Guillaume Davidson  1 , Mohamed-Amin Choukrallah  2 , Irwin Davidson  1
Affiliations

TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression

Igor Martianov et al. Sci Rep. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Generation of Trf2tag/tag mice.
(A) Schematic representation of WT and modified Trf2 alleles with those of the partially overlapping Slc2a12 gene. Exons (E) are indicated along with the Trf2 stop codon, the location of the Tap-Tag and the FRT sites. (B) Immunoblot on protein extracts from testis of WT and Trf2tag/tag testes. Molecular mass markers are indicated on the left of the panel. (C) Hoechst staining of histological sections from mice of the indicated genotypes. RS shows round spermatids with a prominent chromocenter, PS shows pachytene spermatocytes and ES elongate spermatids.
Figure 2
Figure 2. Trf2 is co-purified with TFIIA and ALF.
(A) Schematic of purification of testis into cytoplasmic (CE) and nuclear extract (NE) before gel filtration and tandem affinity purification. (B) Immunoblots of cytoplasmic and nuclear extracts from Trf2tag/tag and Trf2−/− testis. (C) Elution of molecular mass markers on the gel filtration column. (D) Immunoblots on fractions following gel filtration of cytoplasmic extracts from Trf2tag/tag and Trf2−/− testes. (E) Immunoblots on fractions following gel filtration performed at the indicated KCl concentrations. (F) Immunoblots on fractions following gel filtration of cytoplasmic extracts from Trf2tag/tag testes and murine embryonic stem cells. (G) Upper panel shows immunoblots on cytoplasmic extracts (Input, Inp.) from WT and Trf2+/tag heterozygous mice indicating the positions of the native and tagged proteins. The right hand lanes show the Flag immunoprecipitates from the corresponding Input fractions in the left lanes. The lower panel shows the immunoblots of the anti-ALF IP performed on the fractions eluted from the Flag IP in the upper panel.
Figure 3
Figure 3. Composition of the cytoplasmic Trf2 complex.
(A) Immunoblots of the fractions obtained by tandem affinity purification. (B) SDS-PAGE and silver nitrate staining of the immunoprecipiated fractions. The locations of tagged Trf2 and the TFIIA subunits are indicated. (C) The number of peptides obtained for each protein after mass-spectrometry analysis of the precipitated fractions. (D) Immunoblots of CE, NE and chromatin pellet from non-crosslinked testis and the MNase digested chromatin from formaldehyde crosslinked chromatin. (E) Input fractions and Flag IPs from formaldehyde crosslinked chromatin. Control 1 is no antibody IP on the chromatin, control 2 is antibody and sepharose beads, but no input chromatin.
Figure 4
Figure 4. Trf2 occupies active promoters.
(A) UCSC screen shot of ChIP-seq for the indicated factors over a 3-megabase region illustrating the colocalisation of Trf2, Tbp, Pol II, and Taf7l at active promoters. (B) Read density clustering showing, colocalisation of Trf2 with Pol II at a subset of promoters active in testis and co-localisation and proportionality of Trf2, Tbp and Taf7l occupancy at 6000 Trf2 bound sites and 6000 Tbp bound sites. (C) Meta-profiles of the ChIP-seq data for the indicated factors in Trf2tag/tag and Trf2−/− testis.
Figure 5
Figure 5. Trf2 promotes haploid cell gene expression.
(A) Comparison of RNA-seq profiles from 21 day old testes from WT and Trf2−/− mice. (B) Gene set enrichment analyses of the down-regulated genes in Trf2−/− testes. (C) Gene expression data from public data set GSE39970 was organised into 4 clusters representing genes with different expression dynamics relative to the onset of the first wave of spermatogenesis between post-natal days (PND) 7 and 28. The lower part of the panel shows the % of the genes deregulated in the Trf2−/− mice belonging to the different clusters. (D) Metaprofiles of the indicated factors at the promoters of the 711 down-regulated genes in WT and Trf2−/− testis.
Figure 6
Figure 6. Presence of Trf2 alters PIC organisation.
The figure shows a schematic summarising the results of this study. The Trf2-TFIIA/ALF complexes are weakly associated with the chromatin and readily extracted from the nucleus. They are chaperoned in the cytoplasm by heat shock factors. In haploid cells the PIC minimally comprises Tbp, Taf7l and Taf1l together with Pol II and the other general transcription factors (GTFs). As Trf2 is a subunit of TFIIA in haploid cells, Tbp cannot engage TFIIA in the canonical interaction seen in somatic cells. In contrast, in somatic cells in absence of Trf2, Tbp can interact with TFIIA. The presence of Trf2 as a subunit of TFIIA therefore modifies the organisation of the PIC in haploid cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kandiah E., Trowitzsch S., Gupta K., Haffke M. & Berger I. More pieces to the puzzle: recent structural insights into class II transcription initiation. Curr Opin Struct Biol 24, 91–97, doi: 10.1016/j.sbi.2013.12.005 (2014). - DOI - PubMed
    1. Liu X., Bushnell D. A. & Kornberg R. D. RNA polymerase II transcription: structure and mechanism. Biochim Biophys Acta 1829, 2–8, doi: 10.1016/j.bbagrm.2012.09.003 (2013). - DOI - PMC - PubMed
    1. Sainsbury S., Bernecky C. & Cramer P. Structural basis of transcription initiation by RNA polymerase II. Nat Rev Mol Cell Biol 16, 129–143, doi: 10.1038/nrm3952 (2015). - DOI - PubMed
    1. Grunberg S. & Hahn S. Structural insights into transcription initiation by RNA polymerase II. Trends Biochem Sci 38, 603–611, doi: 10.1016/j.tibs.2013.09.002 (2013). - DOI - PMC - PubMed
    1. Cler E., Papai G., Schultz P. & Davidson I. Recent advances in understanding the structure and function of general transcription factor TFIID. Cell Mol Life Sci 66, 2123–2134 (2009). - PMC - PubMed

Publication types

MeSH terms