Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5 untranslated region (5UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth-and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals.
These homodimers, however, have little activity on myogenic enhancers (Weintraub, H., Genetta, T., and Kadesch, T. (1994) Genes Dev. 8, 2203-2211). We report here the identification of a novel cis-acting transcriptional repression domain in the E protein family of bHLH transcription factors. This domain, the Rep domain, is present in each of the known vertebrate E proteins. Extensive mapping analysis demonstrates that this domain is an acidic region of 30 amino acids with a predicted loop structure. Fusion studies indicate that the Rep domain can repress both of the E protein transactivation domains (AD1 and AD2). Physiologically, the Rep domain plays a key role in maintaining E protein homodimers in an inactive state on myogenic enhancers. In addition, we demonstrate that Rep domain mediated repression of AD1 is a necessary for the function of MyoD-E protein heterodimeric complexes. These studies demonstrate that the Rep domain is important for modulating the transcriptional activity of E proteins and provide key insights into both the selectivity and mechanism of action of E protein containing bHLH protein complexes.The basic helix-loop-helix (bHLH) 1 family of transcription factors plays an important role in embryonic patterning, cell fate determination, cellular differentiation, and proliferation decisions (reviewed in Ref. 1). Structurally, the bHLH domain is a 60-amino acid region containing two helices separated by a loop segment preceded by a region rich in basic residues. The basic region is responsible for DNA binding and the HLH region is important for dimerization with other members of the family (2-4). The bHLH family has been divided into three major classes. Class I consists of the ubiquitously expressed E proteins. There are three E protein family members in mammals, E2A (with three major splice products: E12, E47, and E2-5), E2-2 (also called ITF2) and HEB (2, 5-9). Class II bHLH proteins, such as MyoD, are expressed in a tissue-specific manner (10) and require E proteins as obligate heterodimeric partners for DNA binding and transcriptional activation (11). Cell fate decisions are often regulated by alterations in the expression of these tissue-specific proteins. Finally, class III members include the dominant negative Id proteins that heterodimerize with the E proteins but, because they lack a basic domain, form heterodimeric complexes incapable of binding DNA (12,13).In addition to their role as heterodimeric partners for tissue-specific bHLH proteins, E proteins are also capable of forming homodimeric complexes, which can activate a variety of B cell-specific genes and are necessary for B cell differentiation (14 -20). The E proteins have two transcriptional activation domains, activation domain1 (AD1) and 2 (AD2) (21-23). AD1 is contained within the first 100 amino acids of the protein and contains a putative ␣-helix. AD2, also termed the loop-helix domain, is located midway between the AD1 and bHLH domains. The transcriptional activation domains of the E proteins play a critical role in ce...
We have shown previously that E2A helix-loop-helix proteins spontaneously form an intermolecular disulfide cross-link that is required for stable homodimer binding to DNA (Benezra, R. (1994) Cell 79, 1057-1067). These homodimers are important for the development of B lymphocytes but are not present in other cell lineages. We have purified two proteins that are capable of regulating the formation of this disulfide bond and found them to be members of the protein disulfide isomerase (PDI) family. By regulating the formation of the disulfide cross-link, these proteins are capable of regulating the dimerization state of E proteins. PDI-mediated reduction appears to dissociate E protein homodimers and favors heterodimer formation with other basic helixloop-helix proteins in both a purified protein system and in cellular extracts. These studies suggest that PDI may play an important role in the regulation of E2A transcription factor dimerization and the development of the B lymphocyte lineage.The basic helix-loop-helix (bHLH) 1 proteins are a family of lineage-and development-regulated transcription factors that appear to regulate cell type-specific transcription and differentiation (for reviews, see Refs. 2 and 3). HLH proteins are distinguished by a dimerization domain consisting of two amphipathic ␣ helices linked by a loop segment. The basic domain is responsible for DNA binding and is characteristically rich in the basic amino acids lysine and arginine (4 -6). The bHLH family is divided into several groups. In mammals, the E proteins, consisting of the E2A gene products (the alternate splice products E12, E47, and E2-5), E2-2, and HEB (5, 7-12), are expressed in most tissues during all stages of development and usually function as heterodimers with a second class of bHLH proteins (13, 14) such as MyoD and MASH-1, whose expression is tissue-restricted (15, 16). MyoD-E protein and MASH-1-E heterodimers activate the transcription of genes specific for muscle and neural lineages, respectively (5,14,(17)(18)(19). These dimeric interactions are inhibited by a third class of HLH proteins that act as dominant negative factors. This class is exemplified by the Id proteins, which bind to E proteins and prevent dimerization with other bHLH proteins and DNA binding (20 -22). This heterodimeric interaction prevents tissuespecific transcription and differentiation (21,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33).Although E2A proteins can form functional heterodimers with tissue-specific factors, during B cell differentiation E proteins form homodimers and bind to the E2, E4, E5, and E2 regions of the immunoglobulin enhancer heavy and light chain enhancers (34 -39). These dimers form at the transition between the pro-B cell and pre-B cell stages of development and play a critical role in immunoglobulin rearrangement and B cell differentiation as evidenced by the loss of function mutations in mice (33,37,40,41). In addition, overexpression of E47 is sufficient to stimulate sterile transcription from the IgH enhancer in fibroblas...
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