Regulation of the MiTF/TFE bHLH-LZ transcription factors through restricted spatial expression and alternative splicing of functional domains

Abstract

The MiTF/TFE (MiT) family of basic helix-loop-helix leucine zipper transcription factors is composed of four closely related members, MiTF, TFE3, TFEB and TFEC, which can bind target DNA both as homo- or heterodimers. Using real-time RT-PCR, we have analyzed the relative expression levels of the four members in a broad range of human tissues, and found that their ratio of expression is tissue-dependent. We found that, similar to the MiTF gene, the genes for TFEB and TFEC contain multiple alternative first exons with restricted and differential tissue distributions. Seven alternative 5' exons were identified in the TFEB gene, of which three displayed specific expression in placenta and brain, respectively. A novel TFEC transcript (TFEC-C) encodes an N-terminally truncated TFEC isoform lacking the acidic activation domain (AAD), and is exclusively expressed in kidney and small intestine. Furthermore, we observed that a considerable proportion of the TFEC transcripts splice out protein-coding exons, resulting in transcription factor isoforms lacking one or more functional domains, primarily the basic region and/or the AAD. These isoforms were always co-expressed with the intact transcription factors and may act as negative regulators of MiTF/TFE proteins. Our data reveal that multiple levels of regulation exist for the MiTF/TFE family of transcription factors, which indicates how these transcription factors may participate in various cellular processes in different tissues.

Figure 1

Bar graphs showing the relative expression levels of the MiTF/TFE transcription factors in twenty human tissues, as analyzed by real-time RT–PCR. (A) The sum of the expression levels of TFE3, TFEB, TFEC and MiTF is set to 100% to enable comparison of the MiTF/TFE expression ratios in each tissue. (B) The same data set depicted as absolute levels to illustrate the variation in MiTF/TFE expression between tissues. The data were based on two independent in duplo experiments.

Figure 2

(A) The genomic organization of the human TFEB gene. The ATG start codon in exon 2 and TAG stop codon in exon 9 are indicated. Dotted lines depict alternative splicing. A 1600-bp CpG island colocalizes with exons 1b, 1c1 and part of exon 1d. (B) Alternative splice events in the 5′-regions of TFEB-C, TFEB-E and TFEB-G. Dotted lines depict alternative splicing. The largest of three TFEB-G derived products contained the complete exon 1g, including 1g1, 1g2 and the intermediate ‘intronic’ sequence. Accession numbers of the nine novel TFEB transcript fragments are AJ608786–AJ608794. (C) Tissue distribution of the human TFEB variants determined by RT–PCR analysis on 20 human tissues.

Figure 3

(A) Schematic representation of the human TFEC gene containing three alternative 5′ exons. For clarity reasons, exon sizes are depicted five times larger than the intron sizes. Relevant start and stop codons are indicated, as well as the regions encoding the AAD, the basic region, the bHLH-LZ and the proline-rich activation domain and serine-rich stretch (Pro-AD/Ser). Dotted lines depict alternative splicing. The accession number of the novel TFEC-C transcript fragment is AJ608795. (B) Western blot analysis of COS-1 cells transfected with empty vector (lane 1) or VSV-tagged TFEC-C cDNA (lane 2). (C) Tissue distribution of the TFEC transcripts determined by RT–PCR analysis. For each of the panels, the various PCR fragments were isolated and sequenced. Next to the full-length TFEC variants, alternatively spliced transcripts were amplified that lacked exon 2 (Δ2), exon 3 (Δ3) and/or exon 5 (Δ5). The lower two panels show RT–PCR products using a forward primer in exon 6, and reverse primers in either exon 8 or intron 7, which depict the presence of TFEC transcripts encoding the complete C-terminus of TFEC or a truncated C-terminus, respectively. In order to enrich for active cytoplasmic transcripts, we used oligo-dT priming in the reverse transcription reaction.