Masahide Goto

cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs.

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.

The Role of Human MBF1 as a Transcriptional Coactivator

Multiprotein bridging factor 1 (MBF1) is a coactivator which mediates transcriptional activation by interconnecting the general transcription factor TATA element-binding protein and gene-specific activators such as the Drosophilanuclear receptor FTZ-F1 or the yeast basic leucine zipper protein GCN4. The human homolog of MBF1 (hMBF1) has been identified but its function, especially in transcription, remains unclear. Here we report the cDNA cloning and functional analysis of hMBF1. Two isoforms, which we term hMBF1α and hMBF1β, have been identified. hMBF1α mRNA was detected in a number of tissues, whereas hMBF1β exhibited tissue-specific expression. Both isoforms bound to TBP and Ad4BP/SF-1, a mammalian counterpart of FTZ-F1, and mediated Ad4BP/SF-1-dependent transcriptional activation. While hMBF1 was detected in the cytoplasm by immunostaining, coexpression of the nuclear protein Ad4BP/SF-1 with hMBF1 induced accumulation of hMBF1 in the nucleus, suggesting that hMBF1 is localized in the nucleus through its binding to Ad4BP/SF-1. hMBF1 also bound to ATF1, a member of the basic leucine zipper protein family, and mediated its activity as a transcriptional activator. These data establish that the coactivator MBF1 is functionally conserved in eukaryotes.

Transcriptional activation through the tetrameric complex formation of E4TF1 subunits.

Transcription factor E4TF1 is composed of two types of subunit, an ets‐related DNA binding protein, E4TF1‐60, and its associated proteins with four tandemly repeated Notch‐ankyrin motifs, E4TF1‐53 and E4TF1‐47. To determine the functional domains, we constructed various mutants of the subunits. E4TF1‐60 bound to DNA as a monomer. The ets domain and its N‐terminal flanking region were necessary to recognize the specific DNA sequence. The 48 amino acids at the E4TF1‐60 C‐terminus were required for interaction with the other type of subunit. E4TF1‐53 and E4TF1‐47 share the N‐terminal 332 amino acids but differ at the C‐termini. They interacted with E4TF1‐60 through the N‐terminal flanking region to form a heterodimer. E4TF1‐53 dimerized with itself, whereas E4TF1‐47 did not. The C‐terminal region specific for E4TF1‐53 was required for the dimerization. Therefore, heterodimers composed of E4TF1‐53 and E4TF1‐60 were further dimerized, resulting in the formation of a tetrameric complex, which stimulated transcription in vitro. Heterodimers of E4TF1‐47 and E4TF1‐60 weakly stimulated transcription in vitro. The results indicated that the tetrameric complex formation of E4TF1 subunits was necessary to activate transcription efficiently in vitro.

FUNCTIONAL INTERACTIONS OF TRANSCRIPTION FACTOR HUMAN GA-BINDING PROTEIN SUBUNITS

The transcription factor human GA-binding protein (hGABP) is composed of two subunits, the Ets-related hGABPα, which binds to a specific DNA sequence, and either one of two hGABPα-associated subunits, hGABPβ or hGABPγ. The DNA-binding protein hGABPα cannot affect transcription by itself, but can modify hGABP-dependent transcription in vitro andin vivo in the presence of its associated subunits. In this study, co-transfection assays showed that the ratio of hGABPβ to hGABPγ affected transcription from a promoter containing hGABP binding sites. Biochemical analysis showed that they bind to hGABPα competitively, indicating that the ratio of hGABPβ to hGABPγ is important for hGABP complex formation. Kinetic analysis of the protein-protein interaction using the surface plasmon resonance system showed that hGABPα binds to hGABPβ or hGABPγ with similar equilibrium constants. Kinetic analysis of the DNA-hGABP interaction showed that the binding of hGABPγ to hGABPα stabilized the interaction of hGABPα with its DNA binding site. In addition, the kinetic analysis revealed that this was due to a slower dissociation of the protein complex from the DNA. These results suggest that hGABPα-associated subunits influence the DNA binding stability of hGABPα and regulate hGABP-mediated transcription by competing with each other.

Synergistic Transcriptional Activation by hGABP and Select Members of the Activation Transcription Factor/cAMP Response Element-binding Protein Family

The Ets-related DNA-binding protein human GA-binding protein (hGABP) α interacts with the four ankyrin-type repeats of hGABPβ to form an hGABP tetrameric complex that stimulates transcription through the adenovirus early 4 (E4) promoter. Using co-transfection assays, this study demonstrated that the hGABP complex mediated efficient activation of transcription from E4 promoter synergistically with activating transcription factor (ATF) 1 or cAMP response element-binding protein (CREB), but not ATF2/CRE-BP1. This synergy also partially occurred when hGABPα was used alone in place of the combination of hGABPα and hGABPβ. hGABP activated an artificial promoter containing only ATF/CREB-binding sites under coexistence of ATF1 or CREB. Consistent with these results, physical interactions of hGABPα with ATF1 or CREB were observed in vitro. Functional domain analyses of the physical interactions revealed that the amino-terminal region of hGABPα bound to the DNA-binding domain of ATF1, which resulted in the formation of ternary complexes composed of ATF1, hGABPα, and hGABPβ. In contrast to hGABPα, hGABPβ did not significantly interact with ATF1 and CREB. Taken together, these results indicate that hGABP functionally interacts with selective members of the ATF/CREB family, and also suggest that synergy results from multiple interactions which mediate stabilization of large complexes within the regulatory elements of the promoter region, including DNA-binding and non-DNA-binding factors.

Transcriptional activation through interaction of MBF2 with TFIIA

Background: Transcriptional activation of the Drosopohila melanogaster fushi tarzu gene by FTZ‐F1 or its silkworm counterpart BmFTZ‐F1 requires two cofactors MBF1 and MBF2 which do not directly bind to DNA. MBF1 is a bridging molecule that connects FTZ‐F1 (or BmFTZ‐ F1), MBF2 and TATA binding protein TBP. MBF2 is a positive cofactor that activates transcription.

Resonance assignments, secondary structure and 15N relaxation data of the human transcriptional coactivator hMBF1 (57-148).

Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator that is thought to bridge between the TATA box-binding protein (TBP) and DNA binding regulatory factors, and is conserved from yeast to human. Human MBF1 (hMBF1) can bind to TBP and to the nuclear receptor Ad4BP, and is suggested to mediate Ad4BP-dependent transcriptional activation. Here we report the resonance assignments and secondary structure of hMBF1 (57–148) that contains both TBP binding and activator binding residues. 15N relaxation data were also obtained. As a result, hMBF1 (57–148) was shown to consist of flexible N-terminal residues and a C-terminal domain. The C-terminal domain contains four helices and a conserved C-terminal region.

Assignment of the E4TF1-60 gene to human chromosome 21q21.2–q21.3 ☆

The gene encoding human transcription factor E4TF1-60 was previously mapped to chromosome 21q21. We analyzed the localization of the E4TF1-60 gene in more detail by genomic Southern hybridization and determined the sequence of the exons and the regions surrounding the intron boundaries. We report here that E4TF1-60 locates in the long arm of chromosome 21 at q21.2-q21.3 and contains a total of ten exons.