Genomic organization of the human e1af gene, a member of Ets transcription factors

The E1AF protein belongs to the family of Ets transcription factors and is involved in the regulation of metastasis gene expression. It has recently been reported in an undifferentiated child sarcoma that part of this gene could be fused by translocation to the ews gene. We show here that the human e1af gene, which is located in the q21 region of chromosome 17, is organized in 13 exons distributed along 19 kb of genomic DNA. Its two main functional domains, the acidic domain and the DNA-binding ETS domain, are each encoded by three different exons. The 3'-untranslated region of e1af is 0.7 kb. The 5'-untranslated region is about 0.3 kb and is composed of a first exon upstream from the exon containing the first methionine. These data could possibly accelerate an understanding of the molecular basis of putative inherited diseases linked to E1AF. (C) 1999 Elsevier Science B.V. All rights reserved.

Characterization of the human and mouse ETV1/ER81 transcription factor genes: Role of the two alternatively spliced isoforms in the human

The Ets transcription factors of the PEA3 group - E1AF/PEA3, ETV1/ER81 and ERM - are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1α) or the absence (ETV1β) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1β) and the full-length isoform (human ETV1α) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1α and human ETV1β expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

Human E2F6 is alternatively spliced to generate multiple protein isoforms

E2F6 protein belongs to the family of the E2F transcription factors. Here, we showed that the human E2F6 gene contains nine exons distributed along 20.4kbp of genomic DNA on chromosome 2 leading to the transcription of six alternatively spliced E2F6 mRNAs that encode four different E2F6 proteins. Moreover, we identified an E2F6 pseudogene localized on chromosome 22 completely spliced and devoid of exons 2, 3, and 4, and part of exons 1 and 5. Definition of the transcriptional initiation site and sequence analysis show that the gene contains a TATA less, CAAT less, GC-rich promoter with multiple transcription start sites. Regulatory elements necessary for basal transcription reside within a 134bp fragment as determined by transient transfection experiments. © 2004 Elsevier Inc. All rights reserved.

Characterization of human 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase gene and its expression in mammalian cells

Three β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD) catalyze the oxidative conversion of Δ5-3β-hydroxysteroids to the Δ4-3-keto configuration and is therefore essential for the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Using human 3β-HSD cDNA as probe, a human 3β-HSD gene was isolated from a λ-EMBL3 library of leucocyte genomic DNA. A fragment of 3β-HSD genomic DNA was also obtained by amplification of genomic DNA using the polymerase chain reaction. The 3β-HSD gene contains a 5′-untranslated exon of 53 base pairs (bp) and three successive translated exons of 232, 165, and 1218 bp, respectively, separated by introns of 129, 3883, and 2162 bp. The transcription start site is situated 267 nucleotides upstream from the ATG initiating codon. DNA sequence analysis of the 5′-flanking region reveals the existence of a putative TATA box (ATAAA) situated 28 nucleotides upstream from the transcription start site while a putative CAAT binding sequence is located 57 nucleotides upstream from the TATA box. Expression of a cDNA insert containing the coding region of 3β-HSD in nonsteroidogenic cells shows that the gene encodes a single 42-kDa protein containing both 3β-hydroxysteroid dehydrogenase and Δ5-Δ4-isomerase activities. Moreover, all natural steroid substrates tested are transformed with comparable efficiency by the enzyme. In addition to its importance for studies of the regulation of expression of 3β-HSD in gonadal as well as peripheral tissues, knowledge of the structure of the human 3β-HSD gene should permit investigation of the molecular defects responsible for 3β-HSD deficiency, the second most common cause of adrenal hyperplasia in children.