Transcription Factor E47 Research Articles

DNA binding properties of basic helix-loop-helix fusion proteins of Tal and E47.

The basic helix-loop-helix (bHLH) transcription factor Tal has been shown to form heterodimers with the ubiquitously expressed bHLH transcription factor E47 and thereby modulate gene expression. The absence of homodimeric Tal-DNA complexes had been attributed to the inability of Tal to homodimerize, but subsequent studies have shown that the bHLH region of Tal does homodimerize. In order to correlate the contributions of both the basic region and the helix-loop-helix (HLH) domain to the lack of DNA binding by Tal homodimers, mutant and fusion proteins based on Tal and E47 were designed and synthesized. Size-exclusion chromatography established that all mutant and fusion proteins were dimeric. Point mutations were made within the basic region of Tal based on residues within E47 that are essential for DNA binding, but an affinity for DNA was not observed. Even complete replacement of the basic region in Tal with the basic region of E47, in an E47-Tal fusion protein, did not confer DNA binding upon the protein. However, when the dimerization domain in Tal was replaced with its E47 counterpart, in a Tal-E47 fusion protein, sequence specific DNA binding was observed with an apparent dissociation constant of 3.6 x 10(-9) M2. Furthermore, circular dichroism studies showed that the basic region of Tal in the Tal-E47 fusion protein underwent a random coil to helix transition in the presence of a specific DNA probe. These experimental observations indicate that the inability of Tal homodimers to recognize DNA stems from a misalignment of its basic region with respect to the HLH domain, rather than an intrinsic inability of the Tal basic region to bind DNA.

Serine/Threonine Kinases 3pK and MAPK-activated Protein Kinase 2 Interact with the Basic Helix-Loop-Helix Transcription Factor E47 and Repress Its Transcriptional Activity

In the search for physiological substrates of MAPK-activated protein (MAPKAP) kinases, we identified the basic helix-loop-helix (bHLH) transcription factor E47 as an interaction partner of chromosome 3p kinase (3pK) and MAPKAP-K2 (MK2). The E2A protein E47 is known to be involved in the regulation of tissue-specific gene expression and cell differentiation. E47 is a phosphoprotein, and we identified 3pK and MK2 as E47 kinases in vitro. Furthermore, the expression of either kinase results in a repression of the transcriptional activity of E47 on an E-box containing promoter. In summary, the MAPK-activated protein kinases 3pK and MK2 were identified to form an assembly with the bHLH protein E47 suggesting that these kinases are regulators of E47 activity and E47-dependent gene expression.

An alternating selection strategy for cloning phage display antibodies

Phage display is a powerful technique that can be used to develop antibodies to target molecules. One approach for antibody phage display is to select phage from a large naive library of antibody immunoglobulin variable region fragments (Fv) expressed on the surface of phage. Phage that display antibody fragments of interest are selected by their ability to bind the target antigen immobilized on a solid support surface. A major difficulty often encountered with this approach is that phage that bind to additional antigens that are present during the phage selection steps are also selected. We have developed an alternating selection approach to minimize selection of unwanted phage. In the alternating selection approach, two selection methods are used. Each selection method contains different contaminating antigens. This approach was used to select phage that bind a phosphoryated form of the E47 transcription factor. Phage were selected based on their ability to bind a phospho-peptide in solution and alternatively a phospho-protein coated on a polyvinyl micortiter plate. This approach proved significantly better than selection with only one method. With one selection technique, 2 of 48 (4%) selected clones bound to the target antigen. With another selection technique, 15 of 48 (31%) selected clones bound to the target antigen. With alternating selection, 71 of 93 (76%) of the clones bound to the target antigen.

Structure–Function Relationship in a β-Sheet Peptide Inhibitor of E47 Dimerization and DNA Binding

A β-sheet peptide inhibitor, 2H10, has been developed that inhibits the dimerization of the transcription factor E47. Inhibition of E47 dimerization has been demonstrated to also inhibit the DNA binding of this transcription factor. Truncated peptides based on 2H10 have demonstrated that the β-sheet content of these peptides directly correlates with their inhibitory properties. Individual residues within 2H10 were identified that were responsible for the β-sheet secondary structure by employing an alanine replacement strategy. The β-sheet character of the alanine mutants also correlated well with their inhibition of E47 DNA binding. These results provide further evidence that interactions between the interfacial peptide inhibitors of E47 and the transcription factor itself are mediated by a β-sheet structure.

Consequences of Notch-mediated inhibition of the transcription factor E47.

Consequences of Notch-mediated inhibition of the transcription factor E47.

Pancreatic β-Cell-specific Repression of Insulin Gene Transcription by CCAAT/Enhancer-binding Protein β: INHIBITORY INTERACTIONS WITH BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTOR E47

Chronic exposure of beta-cells to supraphysiologic glucose concentrations results in decreased insulin gene transcription. Here we identify the basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta), as a repressor of insulin gene transcription in conditions of supraphysiological glucose levels. C/EBPbeta is expressed in primary rat islets. Moreover, after exposure to high glucose concentrations the beta-cell lines HIT-T15 and INS-1 express increased levels of C/EBPbeta. The rat insulin I gene promoter contains a consensus binding motif for C/EBPbeta (CEB box) that binds C/EBPbeta. In non-beta-cells C/EBPbeta stimulates the activity of the rat insulin I gene promoter through the CEB box. Paradoxically, in beta-cells C/EBPbeta inhibits transcription, directed by the promoter of the rat insulin I gene by direct protein-protein interaction with a heptad leucine repeat sequence within activation domain 2 of the basic helix-loop-helix transcription factor E47. This interaction leads to the inhibition of both dimerization and DNA binding of E47 to the E-elements of the insulin promoter, thereby reducing functionally the transactivation potential of E47 on insulin gene transcription. We suggest that the induction of C/EBPbeta in pancreatic beta-cells by chronically elevated glucose levels may contribute to the impaired insulin secretion in severe type II diabetes mellitus.

The E47 transcription factor binds to the enhancer sequences of recombinant murine leukemia viruses and influences enhancer function

The genomes of most recombinant murine leukemia viruses (MuLVs) inherit pathogenic U3 region sequences from the endogenous xenotropic provirus Bxv-1. However, the U3 regions of about one-third of recombinant MuLVs from CWD mice, such as CWM-T15, have nonecotropic substitutions that are probably derived from an endogenous polytropic provirus. The CWM-T15 U3 region sequences contain five nucleotide substitutions compared with the less pathogenic sequences of the endogenous ecotropic virus parent, Emv-1. Three of these substitutions are located immediately 3' of the enhancer core, and two form part of an E-box motif that is also found in the Bxv-1 sequence. A series of electromobility shift assays revealed that nuclear extracts from S194 cells and the basic helix-loop-helix transcription factor E47 could distinguish between oligonucleotides that contained the core region sequences of CWM-T15 or Emv-1. The E47 homodimers appeared to bind to the CWM-T15 E-box motif and when expressed at high levels in cells transactivated the CWM-T15 but not the Emv-1 enhancer. Taken together, these results suggest that E47 or related basic helix-loop-helix proteins that are expressed in lymphoid cells bind to and transactivate the CWM-T15 enhancer in vivo. This transactivation may explain why the CWM-T15 and Bxv-1 U3 regions accelerate the onset of lymphoid neoplasms and why related enhancer core region sequences are preferentially incorporated into the genomes of recombinant MuLVs and are found in other leukemogenic mammalian retroviruses.

Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer.

A large group of transcription factors regulating cell growth and differentiation share a dimeric alpha-helical DNA-binding domain termed the basic region helix-loop-helix (bHLH). bHLH proteins associate as homodimers and heterodimers having distinctive DNA-binding activities and transcriptional activities that are central to the regulated differentiation of a number of tissues. Some of the bHLH residues specifying these activities have been identified, but a full understanding of their function has awaited further structural information. We report here the crystal structure of the transcription factor E47 bHLH domain bound to DNA. The bHLH of E47 is a parallel, four-helix bundle with structural features that distinguish it from the bHLH-zipper protein Max. The E47 dimer makes nonequivalent contacts to each half of the -CACCTG- binding site. Sequence discrimination at the center of the E box may result from interaction with both the DNA bases and the phosphodiester backbone.

Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning.

The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.

Helix-loop-helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line.

E47 is a helix-loop-helix transcription factor that binds to sites in the immunoglobulin heavy-chain and kappa light-chain gene enhancers. Other proteins of this type are involved in cell-type determination. A possible role for E47 in B-cell development was tested by overexpressing a cDNA encoding E47 in the pre-T-cell line 2017. We found a dramatic activation of a germ-line heavy-chain gene transcript in these stable transfectants and an equally large induction of immunoglobulin D-to-J rearrangement, the first recognized step in B-cell development. Germ-line kappa light-chain gene transcription and rearrangement were unaffected, but transcription of the recombination-activating genes RAG-1 and RAG-2 and the lymphoid-specific transcription factor Oct-2 was increased. These T cells did not transcribe their rearranged DJ alleles, however, and failed to progress to the next stage of heavy-chain gene assembly, V-to-DJ rearrangement. Because transcription factor E47 can induce pre-T cells to carry out events of B-cell differentiation, it may be a crucial determinant of the earliest stages of B-cell development.