Recently, information on a second isoform of TIPT (isoform 1) was independently reported [39
]. These authors describe expression in male germ cells, interaction with TBPL1 (TRF2), DNA binding and association with chromatin, in line with our findings on isoform 2. However, they did not detect the widespread expression we saw on the RNA and protein level for isoform 2, although probe and antibodies would be expected to crossreact.
In our study, we describe the synergistic function of two interacting factors, TIPT2 and geminin, in the activation of transcription. TIPT2 and geminin have in common that they can interact with polycomb group members (PcGs), which are known for their function in the maintenance of gene repression [47
]. Therefore, it came unexpected that PcGs were found together with the basal transcriptional machinery on repressed promoters [49
]. It lead to the hypothesis that PcG proteins maintain silencing by inhibiting general transcription factor-mediated activation of transcription by interfering with the formation of the preinitiation complex [54
]. Our findings suggest that TIPT2 and geminin could be involved in the transition from inactive to active transcription via association with basal transcription as well as Polycomb factors. We found both geminin and TIPT2 in the chromatin of transcribed genes. TIPT2 was significantly enriched in nucleoli, a subcellular localization shared by the bHLH transcription factor Hand1, the basal transcription factor TBPL1, by Drosophila
testis-specific TAF homologs and, albeit as an exception, by some PcGs [40
]. For the nuclear geminin, on the other hand, a nucleolar localization was not specifically described. If we observe a cooperation of these proteins in Pol II-dependent transcription, their functional interactions must occur in the nucleoplasm. Nucleoli might represent a site to keep transcription factors unavailable for gene regulatory functions, a hypothesis which may also apply to TIPT2. However, we have preliminary evidence that the nucleolar localization of TIPT2 also reflects a role in the activation of ribosomal RNA by polymerase I. In addition, our unpublished data indicate that TIPT2 nucleolar localization is regulated by phosphorylation.
Sequences flanking the core TATA box can influence the assembly of complexes, affect the basal level of transcription and the response to activators [58
]. Our results show that TIPT2 contacts the AdMLP on a BRE site immediately upstream of the TATA box, without a need for TFIIB or TBP. In band-shift assays TIPT2/AdMLP complexes run in two or more bands (Figure , lanes 2,3; 3D, lane 2; 3F, lane2). The faster migrating band represents TIPT2 associated with the BREu
site of the oligonucleotide. The slower band(s) could represent more than one TIPT2 protein per oligonucleotide, with only one monomer engaged in DNA interactions. The slower migrating complex becomes more prominent than the faster complex after addition of cold oligonucleotides with BREu
mutations (m2, m3, m4; Figure ). This might be indicative of a second DNA binding site on the TIPT2 protein interacting with the competitor.
The binding of TIPT2 to the BRE site interfered significantly with the binding of TBP to the TATA box. Both proteins on one oligonucleotide providing both binding sites were not clearly demonstratable. But some material of lower electrophoretic mobility may indicate the formation of a triple complex, containing AdMLP, TIPT2 and TBP. This disappeared upon addition of anti-TBP antibodies. ChIP experiments indicated that TBP and TIPT2 could be associated in vivo with chromatin in the TATA box containing promoter region of the GAPDH gene. While this finding does not formally prove the simultaneous presence, it appears very unlikely that the active GAPDH promoter exists in two different configurations, being occupied with either TBP or TIPT2.
Our data suggest that transcriptional activation by TIPT2 is optimal in the presence of a BREu
element (AdMLP), but occurs also with a mutated (AdMLPm3) BREu
element or in its absence (TK promoter). DNA binding of TIPT2 appears not to be essential, and association just by protein-protein contact seems to be sufficient. A recent bioinformatic study revealed that 24.5% of core promoters from the EPD (Eukaryotic Promoter Database: http://www.epd.isb-sib.ch
), and 25.5% from the DBTSS (Database of Transcriptional Start Sites; http://dbtss.hgc.jp/index.html
), respectively contain a BREu
]. More precisely, 28,1% of TATA-less promoters and 11.8% of TATA-containing promoters present a BREu
. It is therefore conceivable that TIPT2 plays a role in the activation of transcription from many promoters.
The TIPT2 binding protein, geminin, activated transcription in several reporter assays. The simultaneous transfection of geminin and TIPT2 expression vectors boosted the activation of TATA-containing promoters. A mutant of TIPT2 that could not interact with geminin was clearly less active, again underlining the importance of the geminin/TIPT2 interaction in transcriptional activation. Our results concerning AdML promoter mutations analysis extends the previous findings that disruption of BREu
decreased activated transcription [60
]. Our data indicate that a similar mechanism for transcriptional activation could hold true in the case of the TATA-less NF1 promoter [61
]. Mammalian TBPL1 does not stimulate transcription in vitro
from TATA box-containing E4, AdML and Hsp70 promoters [24
]. We showed here that TIPT2, TBPL1 and NF1 promoter form a complex in vitro
. The synergistic transcriptional activation of the NF1 promoter by TBPL1, TIPT2 and geminin is similar to the synergistic activation we observed for the TATA box-containing promoters (AdML, TK) by TBP, TIPT2 and geminin. It may, however, be too simplistic to correlate exclusively the TATA box promoters with TBP, and the TATA-less promoters with TBPL1 [29
Down-regulation of geminin levels in human U2OS cells decreased the activity obtained from activated TK and NF1 promoters, indicating that already the endogenous geminin plays a significant role in our transcriptional assays. Geminin was reported to be involved in different, transcription-related complexes as a negative regulator of transcription, either directly or indirectly. In this study we show geminin's function as transcriptional co-activator. Other studies have shown that another chromatin remodeler, the SWI/SNF complex, is able to act in a gene-dependent manner either as activator or repressor [65
]. Our results suggest that geminin may interact with distinct protein complexes that exhibit cell-type or gene-specific functions. These interactions may decide if geminin affects transcription as a transcriptional repressor or activator.
In agreement with in vitro
and reporter assay data, the chromatin analysis revealed geminin and TIPT2 on TATA-containing and TATA-less, active gene promoters. On the active endogenous chromatin, TIPT2 and geminin were present near the TATA box of the GAPDH
gene, but not of the c-fos
or the HSP-70
gene on a distance of two-three nucleosomes. The active TATA-less NF1 promoter is associated with geminin and TIPT2 in the region where TBPL1 was shown to bind. Our own ChIP evidence does not allow to conclude for the presence of TBP in the chromatin of the NF1 promoter in U2OS cells. However, recently the presence of TBP was described in the chromatin of the NF1 promoter in HeLa cells [29
A genome-wide analysis of TRF2 recognition sites in Drosophila
indicates that TRF2, a protein closely related to vertebrate TBPL1, plays a more general role than previously thought, being required for the expression of more than 1,000 genes, some involved in regulation of chromatin organization and cell growth (Histone H1 linker
and ribosomal protein genes) [30
]. This may suggest also that in mammals TBPL1 plays an important role regulating essential cell functions not only in testis, but also in other tissues. In this new perspective, our study might indicate new factors binding to both TBP and TBPL1, which might constitute co-regulatory proteins common for both core promoter corresponding complexes.