No evidence for generation of alternatively spliced isoforms from the mutated Trim24 allele lacking exon 4 in mouse liver
Authors: Benjamin Herquel, Céline Keime, Irwin Davidson
Journal: Journal of Hepatology (2015)

TRIM24 suppresses development of spontaneous hepatic lipid accumulation and hepatocellular carcinoma in miceJournal of HepatologyVol. 62Issue 2PreviewAberrantly high expression of TRIM24 occurs in human cancers, including hepatocellular carcinoma. In contrast, TRIM24 in the mouse is reportedly a liver-specific tumour suppressor. To address this dichotomy and to uncover direct regulatory functions of TRIM24 in vivo, we developed a new mouse model that lacks expression of all Trim24 isoforms, as the previous model expressed normal levels of Trim24 lacking only exon 4. Full-Text PDF The Losson and Davidson laboratories have investigated the role of the Trim24 gene in mouse physiology. Germ line knockout of the Trim24 gene involved targeted deletion of exon 4 [[1]Khetchoumian K. Teletin M. Tisserand J. Mark M. Herquel B. Ignat M. et al.Loss of Trim24 (Tif1alpha) gene function confers oncogenic activity to retinoic acid receptor alpha.Nat Genet. 2007; 39: 1500-1506Crossref PubMed Scopus (127) Google Scholar]. Trim24ex4−/− mice were viable and fertile, but developed hepatocellular carcinoma with 100% penetrance after 18 months of age. Hepatocarcinogenesis involved a progressive multi-step process; with altered cell cycle, clear-cell foci of altered hepatocytes, hepatoadenoma and finally hepatocarcinoma [[1]Khetchoumian K. Teletin M. Tisserand J. Mark M. Herquel B. Ignat M. et al.Loss of Trim24 (Tif1alpha) gene function confers oncogenic activity to retinoic acid receptor alpha.Nat Genet. 2007; 39: 1500-1506Crossref PubMed Scopus (127) Google Scholar]. A very similar series of events was seen when Trim24 was selectively inactivated in hepatocytes [[2]Herquel B. Ouararhni K. Khetchoumian K. Ignat M. Teletin M. Mark M. et al.Transcription cofactors TRIM24, TRIM28, and TRIM33 associate to form regulatory complexes that suppress murine hepatocellular carcinoma.Proc Natl Acad Sci U S A. 2011; 108: 8212-8217Crossref PubMed Scopus (152) Google Scholar]. Gene expression studies showed that the antiviral interferon response is the main pathway altered at an early age [3Tisserand J. Khetchoumian K. Thibault C. Dembele D. Chambon P. Losson R. Tripartite motif 24 (Trim24/Tif1alpha) tumor suppressor protein is a novel negative regulator of interferon (IFN)/signal transducers and activators of transcription (STAT) signaling pathway acting through retinoic acid receptor alpha (Raralpha) inhibition.J Biol Chem. 2011; 286: 33369-33379Crossref PubMed Scopus (49) Google Scholar, 4Herquel B. Ouararhni K. Martianov I. Le Gras S. Ye T. Keime C. et al.Trim24-repressed VL30 retrotransposons regulate gene expression by producing noncoding RNA.Nat Struct Mol Biol. 2013; 20: 339-346Crossref PubMed Scopus (52) Google Scholar]. The Barton laboratory [[5]Jiang S. Minter L.C. Stratton S.A. Yang P. Abbas H.A. Akdemir Z.C. et al.TRIM24 suppresses development of spontaneous hepatic lipid accumulation and hepatocellular carcinoma in mice.J Hepatol. 2015; 62: 371-379https://doi.org/10.1016/j.jhep.2014.09.026Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar] recently analyzed a Trim24 knockout mouse generated by deleting Trim24 exon 1. Trim24ex1−/− mice also spontaneously develop hepatocarcinoma with a time-course resembling that originally described. Attention is drawn to several differences with the Trim24ex4−/− model that Jiang et al. argue may be due to the persistence of alternatively spliced isoforms generated from our Trim24ex4−/− allele. Many potential Trim24 isoforms are reported in public databases (Fig. 1A) corresponding to alternative splicing events or use of intragenic promoters. A spliced Trim24ex4−/− transcript initiates from the most upstream transcription start site (TSS) as attested by the presence of trimethylated histone H3 lysine 4 (H3K4me3) and paused RNA polymerase II (Pol II) [[4]Herquel B. Ouararhni K. Martianov I. Le Gras S. Ye T. Keime C. et al.Trim24-repressed VL30 retrotransposons regulate gene expression by producing noncoding RNA.Nat Struct Mol Biol. 2013; 20: 339-346Crossref PubMed Scopus (52) Google Scholar]. No other sites of H3K4me3 and Pol II accumulation were seen within the gene body indicating no use of alternative intragenic promoters. This is confirmed in an independent data set [[6]Alpern D. Langer D. Ballester B. Le Gras S. Romier C. Mengus G. et al.TAF4, a subunit of transcription factor II D, directs promoter occupancy of nuclear receptor HNF4A during post-natal hepatocyte differentiation.eLife. 2014; 3: e03613Crossref Scopus (18) Google Scholar] where Pol II and the general transcription machinery are found only at the upstream TSS (Fig. 1A). There is therefore no evidence for use of alternative Trim24 intragenic promoters in mouse liver. We analysed the wild-type total RNA-seq data [[4]Herquel B. Ouararhni K. Martianov I. Le Gras S. Ye T. Keime C. et al.Trim24-repressed VL30 retrotransposons regulate gene expression by producing noncoding RNA.Nat Struct Mol Biol. 2013; 20: 339-346Crossref PubMed Scopus (52) Google Scholar] and found only exon-junction reads consistent with generation of the Trim24-001 and Trim24-002 isoforms in the wild-type liver. For Trim24ex4−/−, the Trim24-002 isoform is predominant, with junction fragments between exons 3 and 5 consistent with the absence of exon 4. In contrast, we found no exon-junction fragments indicative of other exon-skipping events and generation of other isoforms. The only reliable ORF produced from the Trim24ex4−/− allele lacking exon 4 would initiate at the first ATG, terminate prematurely at a nonsense codon following amino acid 214 and encode a protein comprising of the RING finger, but lacking all other known domains of the protein, coiled-coil homo/heterodimerization domain, the HP1 and nuclear receptor interaction domains and the chromatin binding PHD-finger-bromodomain unit. It is not known whether such a short truncated Trim24 protein is stable, but it is highly improbable that it could mimic any of the normal functions of Trim24. We thus believe that the minor differences between the Trim24ex4−/− and Trim24ex1−/− mouse models are more likely accounted for by differences in genetic backgrounds of the mice and the ages at which gene expression was analyzed rather than the existence of Trim24 isoforms arising from the Trim24ex4−/− allele that were overlooked in our previous studies and for which we find no evidence. The authors who have taken part in this letter to the editor declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.