Involvement of GTA protein NC2β in Neuroblastoma pathogenesis suggests that it physiologically participates in the regulation of cell proliferation

Transcription initiation, the most important and regulated event along the pathway connecting genotype to phenotype, is governed by the General Transcription Apparatus (GTA): GTA proteins constitute the PreInitiation Complex (PIC) and guide its assembly [1‐3]. Class II PIC, larger than 2 MDa, comprises more than fifty different polypeptides, including RNA polymerase II, GTFs, Mediator [4‐6]. GTF2D consists of the TATA Box – Binding Protein (TBP) and TBP – Associated Factors (TAFs), a group of evolutionarily conserved proteins that participate in determining the state of chromatin, contribute to promoter recognition, serve as coactivators, and post-translationally modify other GTA proteins to facilitate PIC assembly and transcription initiation [1, 5]. Its DNA binding activity is regulated by positive and negative cofactors such as heterodimeric NC2 (also named Dr1/Drap 1), comprised of α- and β-type subunits [7, 8]. The molecular actions of GTA proteins were clarified through an extensive series of studies [1‐3, 6, 9]. On the other hand, only scanty success was obtained in identifying their biological functions as well as in verifying their involvement in genetic pathology, including tumorigenesis [10, 11]. We examined the involvement of GTA proteins in the pathogenesis of Neuroblastoma (NB), exploiting our data and those from the literature on the genomics of GTA (see Additional file 1; reviewed in ref. [12]) to perform the positional and functional gene candidate approach. NB is a group of early childhood tumours with a complex molecular pathogenesis [13, 14]. It is generally believed that the abnormally proliferating cell in all types of NB is the neuroblast, a fleeting stem cell that transiently appears during the early stages of mammalian development [15]. NB clinical phenotype is remarkably heterogeneous, ranging from spontaneous regression to restless progression [14, 16]. This variability is associated to a high genetic heterogeneity. The most important genomic alterations in NB are interstitial deletions at 1p, 11q, 17q, and MYCN amplification [14, 17]. NB molecular phenotype is characterized by the altered expression of a plethora of genes belonging to different Gene Ontology categories: all of them are potential NB biomarkers [14, 18]. Our analysis was initially focused on human chromosome 1 short arm, where one or more NB master genes are thought to reside [17, 19]. In this region, we had previously mapped the genes encoding TAF13, GTF2B, NC2β, TAF12 to 1p13.3, 1p21-p22, 1p22.1, 1p35.3, respectively [20‐22].

Based on TAF13, GTF2B, NC2β, TAF12 genomic location and molecular functions, we considered these four GTA genes as positional and functional candidates for involvement in NB pathogenesis (see Additional file 1, Table 1 and Figure 1). As reference marker, we used the GTA protein NC2α (the molecular partner of NC2β), that is encoded by a gene located at 11q13.3 (see Additional file 1, Table 1) [32]. Expression of these genes was analyzed through RT – PCR in fifty eight NB biopsies (see Additional file 2, Table 1 and Figure 2). As a preliminary step, we characterized their mRNA phenotype in ten peripheral blood samples from normal individuals and we did not observe significant variations of their transcription level (Table 1, Figure 2). On the other hand, in NB we found a clear-cut decrease (from 3 to 8 times) of the mRNAs encoding TAF13, NC2β, TAF12 with respect to peripheral blood from the same individuals in 49% (21/43), 74% (39/53), 43% (19/44) samples, respectively (Figures 2 and 3). Statistical analysis demonstrated a significant positive correlation between NC2β transcripts reduction and NB (p = 0.0039), whereas this was not obtained for TAF13 and TAF12 (Table 3). NC2β mRNA levels are significantly decreased in both NB advanced stages III and IV (p = 0.0039); their reduction also in stage I (p = 0.0039) could suggest a peculiar natural history of NB (Figure 4). In the same set of samples, we did not detect any change of the levels of the mRNAs encoding GTF2B and NC2α (Figure 2). By using at least two closely flanking microsatellite markers for each gene, we then analyzed the genome of the same NB samples for deletions at the loci TAF13, GTF2B, NC2β, TAF12, NC2α (Table 2, Figures 1 and 3): these were found in more than 60% of samples with diminished levels of the mRNAs encoding either TAF13 (62.5%), NC2β(65%), or TAF12 (67%), but were never detected in NB samples with normal levels of mRNA for these three genes (Figures 3A, B, 5A). Similarly, we never found deletions at the GTF2B and NC2α loci both in NB biopsies as well as in controls (not shown). Due to aneuploidy, we suggest that for NB as for other tumours GI (Genomic Imbalance) may be more appropriate than LOH (Loss of Heterozygosity). In NB samples with GI for NC2β, we never detect it for TAF12 or TAF13 (Figure 5B): the anticorrelation between NC2β and TAF13 reached statistical significance (p = 0.043). TAF 12 and TAF 13 seemed to show a reciprocal positive correlation since in 80% of samples GI was absent in both. Besides genomic deletions, the reduced levels of the mRNAs encoding NC2β, TAF12, TAF13 in NB samples could be due to: (i) epigenetic modifications of promoter DNA, such as methylation [33]; (ii) mutations in the promoter or other regulatory regions [34]; (iii) increased mRNA turnover [35]; (iv) negative regulation by miRNAs [36, 37]. To test the first of these possibilities, we analyzed a panel of NB biopsies and NB cell lines with low levels of mRNAs for these three genes and checked the methylation status of the 4 to 7 CpG doublets found in their bona fide promoter (see Additional file 3, Figure 6). In general, very low or no methylation was observed both in the tumor samples as in the cell lines (see Additional file 3, Figure 6). We did observe higher levels of DNA methylation in the NC2β gene, that was however not correlated to its expression with the exception of sample NB41 (Figures 2 and 6): this indicates that other mechanisms, such as negative regulation by microRNAs, should be considered to account for the low levels of expression of these genes in NB [36, 37]. Our data allow us to speculate on the biological role of GTA proteins NC2, TAF12, TAF13, GTF2B. NC2 is an evolutionarily conserved transcriptional regulator for which an intriguing general role was proposed: it could repress basal transcription in the absence of activators or alternatively stimulate it in their presence [9]. Analogously to p53 role as Guardian of the Genome, NC2 could represent the Guardian of the Transcription Machinery and control its balanced functioning, performing a role as tumor suppressor possibly not restricted to NB: reduced levels of the protein could be responsible for the inappropriate activation of genes promoting cellular proliferation, thus contributing to cell transformation. Its two subunits NC2α and NC2β could perform distinct roles in the regulation of gene expression and determination of cell phenotype: binding of holoNC2 marks repressed promoters, while occupancy by NC2α correlates with active promoters [38]. TAF12 and TAF13 encode two small subunits of GTF2D. TAF12 interacts directly with TBP [39]. Similar to TAF4b, TAF6 and TAF9, it contains a histone fold domain (HFD): the polypeptide is part of a histone-like TAF complex that was shown to be critically important for GTF2D architecture [40]. TAF13, that interacts with TBP, TAF10 and TAF11, is associated with only a subset of GTF2D complexes [39]. Reduced levels of TAF12 and TAF13 could alter GTF2D structure and function and deregulate the expression of different genes, such as those involved in cell cycle control: different mutations of yTAF25 (the yeast ortholog of TAF12) are known to induce distinct phenotypes and affect the regulation of different subsets of genes [41]. GTF2B is a highly conserved member of GTA [42]: it forms a complex with GTF2D and GTF2A (the DAB complex), bridging RNA polymerase II and the promoter [43]. The protein is also a target of Mediator and gene – specific transcriptional activators [44]. Its critical molecular and biological role could explain our failure to detect GTF2B deletions in NB, since they would expose the cells to a strong negative selection. A similar argument could be put forward to explain the absence of NC2α deletions in our dataset. Cell proliferation and specularly tumorigenesis are very complex phenomena. Aneuploidy and GI are common features of neoplastic genomes [45, 46]. The ensuing haploinsufficiency may cause loss of the cell ability to autonomously control its proliferation and to coordinate it with that of other cells of the same organism. Unexpected odd partners seem to be involved in the process. Our approach has allowed us to demonstrate that reduced expression and GI at the GTA locus NC2β is frequently and specifically present in the genome of NB tumour cells, possibly as a result of mutations of caretaker genes involved in DNA repair or chromosomal segregation and of complex selective mechanisms [14, 46, this paper]. The possible anticorrelation between NC2β on the one side and TAF12 and TAF13 on the other may suggest that these proteins could perform analogous biological roles and as such they may reciprocally complement each other as negative regulators of proliferation.

The data presented in this paper experimentally confirm our hypothesis that at least some GTA proteins may also be physiologically involved in the control of cell proliferation, at the same time underscoring the importance of natural selection within complex biopathological processes [22, 47]. They also suggest possible ways to exploit molecular omic profiling to determine biological functions and design rational anticancer therapies.