A novel homeobox protein which recognizes a TGT core and functionally interferes with a retinoid-responsive motif.

We describe here a novel homeobox gene, denoted TGIF (5"TG3' interacting factor), which belongs to an expanding TALE (three amino acid loop extension) superclass of atypical homeodomains. The TGIF homeodomain binds to a previously characterized retinoid X receptor (RXR) responsive element from the cellular retinol-binding protein II promoter (CRBPII-RXRE), which contains an unusual DNA target for a homeobox. The interactions of both the homeprotein TGIF and receptor RXR alpha with the CREBPII-RXRE DNA motif occur on overlapping areas and generate a mutually exclusive binding in vitro. Transient cellular transfections demonstrate that TGIF inhibits the 9-cis-retinoic acid-dependent RXR alpha transcription activation of the retinoic acid responsive element. TGIF transcripts were detected in a restricted number of tissues. The canonical binding site of TGIF is conserved and is an integral part of several responsive elements which are organized like the CRBPII-RXRE. Hence, a novel auxiliary factor to the steroid receptor superfamily may participate in the transmission of nuclear signals during development and in the adult, as illustrated by the down-modulation of the RXR alpha activities.

Rb may act as a transcriptional co-activator in undifferentiated F9 cells.

The reversible interaction of the retinoblastoma susceptibility gene product (Rb) with the cellular transcription factor E2F has recently been demonstrated. Activation of the adenovirus E2a promoter by the products of the viral E1a gene correlates with the ability of both early E1a proteins to sequester Rb, thereby releasing E2F from inactive complexes with this protein. The E2a promoter is also efficiently stimulated by a product (17.5 kDa) of the viral E4 gene. The specific interaction of this E4 protein with E2F results in the formation of complexes that bind cooperatively to the two neighboring E2F binding sites in the E2a promoter. We have previously shown that in undifferentiated F9 cells (F9EC) the E2a promoter is refractory to E2F-mediated activation by E1a, but not by E4. Using both band-shift and transfection experiments, we now demonstrate (i) that in F9EC cells the E4 product, in combination with E2F, recruits Rb into a stable multiprotein complex and (ii) that in these undifferentiated cells, as opposed to their differentiated counterpart, Rb is actively involved in the transcriptional stimulation of the E2a promoter by E4. Our results suggest that, depending on the cell state, Rb may behave either as a transcriptional activator (F9EC cells) or as a transcriptional inhibitor (differentiated F9 cells).

Transcriptional activation by the adenovirus larger E1a product is mediated by members of the cellular transcription factor ATF family which can directly associate with E1a.

We recently isolated three cDNA clones encoding closely related proteins (ATFa1, ATFa2, and ATFa3) that belong to the activating transcription factor-cyclic AMP-responsive element family of cellular transcription factors. Using cotransfection experiments, we showed that these proteins mediate the transcriptional activation induced by the adenovirus E1a 13S mRNA gene product and that the zinc-binding domains present in both E1a conserved region 3 and the most N-terminal portion of the ATFa proteins play crucial roles in this activity. Reciprocal coimmunoprecipitation experiments demonstrated direct interactions between these proteins. Neither the conserved region 3 domain of E1a nor the N-terminal metal-binding element of ATFa is essential for these interactions. The simultaneous alteration of both the N-terminal and the C-terminal domains of ATFa abolished E1a binding, while either mutation alone failed to impair these interactions.

Nucleotide sequence of a pregnancy-specific beta 1 glycoprotein gene family member. Identification of a functional promoter region and several putative regulatory sequences.

The pregnancy-specific beta 1 glycoprotein (PSG) genes encode a group of heterogeneous proteins produced in large amounts by the human syncytiotrophoblast. Their expression seems to be regulated at the transcriptional level during normal pregnancy. In the present work, we isolated from a human placental library a 17 kb genomic fragment corresponding to a member of the PSG multigene family. DNA sequence analysis of 1190 nucleotides upstream of the translational start and of the first intron, revealed the presence of several putative regulatory sequences. In a transient chloramphenicol acetyltransferase expression assay, 5' flanking sequences within 123 nucleotides upstream to the first major transcription initiation site, functioned as a strong promoter in COS-7 cells. Meanwhile, sequences 5' further upstream had the ability to abolish this promoter activity. The sequence analyzed did not contain any obvious TATA-like boxes or G+C-rich regions, suggesting the existence of unique promoter elements implicated in transcription initiation and regulation of this PSG gene family member.

Two distinct cellular proteins interact with the EIa-responsive element of an adenovirus early promoter.

EIa-dependent transactivation of the adenovirus EIIa early (EIIaE) promoter is correlated with the activation of the cellular transcription factor E2F. In this study we identified a cellular protein, C alpha, that is distinct from E2F and that binds two sites in the EIIaE promoter, one of which overlaps with the proximal E2F binding site of the EIIaE promoter. The possible involvement of C alpha in the EIa responsiveness of this promoter is discussed.

Differential activation of the E2F transcription factor by the adenovirus EIa and EIV products in F9 cells.

Expression studies of the early EIIa transcription unit (EIIaE) of the adenovirus EIa-deletion mutant dl312 in murine embryonal carcinoma stem cells suggested that these cells contain an activity that substitutes for the viral EIa. To further characterize this cellular EIa-like activity, we analyzed expression of the EIIaE promoter as well as the binding activity of the cognate E2F transcription factor after infection of F9 embryonal carcinoma cells and their differentiated derivatives with wild-type adenovirus, EIa (dl312), or EIV (dl808) deletion mutants. We show that, in contrast to the viral EIa proteins that transactivate the EIIaE promoter in F9 cells only after differentiation, the viral EIV products activate the EIIaE promoter most efficiently in undifferentiated F9 cells. We also show that the EIV products induce a specific modification of the E2F transcription factor leading to its cooperative binding to the EIIaE promoter. Although the EIa-dependent transactivation of EIIaE in differentiated cells is also in part mediated by E2F, it does not by itself correlate with the simultaneous binding of two E2F molecules. In these cells E2F dimer binding only occurs as a secondary effect of EIa that also stimulates EIV expression. Our results suggest that EIa and EIV act through separate pathways, inversely regulated during cell differentiation, with the so-called "EIa-like" activity contributing to this modulation.

Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ; primary structure of the precursor of the glycolipid-anchored dimeric form.

In this paper, we show the existence of alternative splicing in the 3' region of the coding sequence of Torpedo acetylcholinesterase (AChE). We describe two cDNA structures which both diverge from the previously described coding sequence of the catalytic subunit of asymmetric (A) forms (Schumacher et al., 1986; Sikorav et al., 1987). They both contain a coding sequence followed by a non-coding sequence and a poly(A) stretch. Both of these structures were shown to exist in poly(A)+ RNAs, by S1 mapping experiments. The divergent region encoded by the first sequence corresponds to the precursor of the globular dimeric form (G2a), since it contains the expected C-terminal amino acids, Ala-Cys. These amino acids are followed by a 29 amino acid extension which contains a hydrophobic segment and must be replaced by a glycolipid in the mature protein. Analyses of intact G2a AChE showed that the common domain of the protein contains intersubunit disulphide bonds. The divergent region of the second type of cDNA consists of an adjacent genomic sequence, which is removed as an intron in A and Ga mRNAs, but may encode a distinct, less abundant catalytic subunit. The structures of the cDNA clones indicate that they are derived from minor mRNAs, shorter than the three major transcripts which have been described previously (14.5, 10.5 and 5.5 kb). Oligonucleotide probes specific for the asymmetric and globular terminal regions hybridize with the three major transcripts, indicating that their size is determined by 3'-untranslated regions which are not related to the differential splicing leading to A and Ga forms.