p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner.

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.

p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells.

It is well established that induction of the p53 tumor suppressor protein in cells can lead to either cell cycle arrest or apoptosis. To further understand features of p53 that contribute to these cell responses several p53-null Saos2 and H1299 cell lines were generated that express wild-type or mutant forms of p53, or the cyclin-dependent kinase inhibitor p21/WAF1, under a tetracycline-regulated promoter. Our results show that the cellular level of p53 can dictate the response of the cell such that lower levels of p53 result in arrest whereas higher levels result in apoptosis; nevertheless, DNA damage can heighten the apoptotic response to p53 without altering the protein level of p53 in cells. We also demonstrate that arrest and apoptosis are two genetically separable functions of p53 because a transcriptionally incompetent p53 can induce apoptosis but not arrest, whereas induction of p21/WAF1, which is a major transcriptional target of p53, can induce arrest but not apoptosis. Finally, we show that a full apoptotic response to p53 requires both its amino and carboxyl terminus, and our data suggest that there is synergism between transcription-dependent and -independent functions of p53 in apoptosis. Thus, there are multiple independent cellular responses to p53 that together may account for the extraordinarily high frequency of p53 mutations in diverse types of human tumors. The implications of these results are discussed and a model is proposed.

DNA-binding specificities of the GATA transcription factor family.

Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.

cis and trans regulation of tissue-specific transcription.

Analysis of both the cis-regulatory sequences which control globin gene switching as well as the trans-acting factors which bind to these sequences to elicit a differential, developmentally regulated response has lent insight into the general mechanisms responsible for tissue-specific gene regulation. We show here that the chicken adult beta-globin gene promoter sequences are intimately involved in competitive interaction with the beta/epsilon-globin enhancer to regulate differentially epsilon- versus beta-globin gene transcription. Secondly, we show that the family of GATA transcription factors directs gene regulation in a variety of discrete cell types, and describe potential cellular target genes for each member of the GATA factor family, as well as potential mechanisms whereby multiple GATA factors expressed in a single cell might be used to elicit differential transcriptional activities.

Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer.

A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation.

Activity and tissue-specific expression of the transcription factor NF-E1 multigene family.

NF-E1, a DNA-binding protein that recognizes the general consensus motif WGATAR, is the first tissue-specific factor to be identified in erythroid cells. Using a probe from the murine GF-1 (NF-E1) cDNA clone, we isolated three homologous chicken cDNAs: One of these corresponds to an mRNA (NF-E1a) that is abundantly and exclusively expressed in erythroid cells; a second mRNA (NF-E1b) is also expressed in all developmental stages of erythroid cells but is additionally found in a limited subset of other chicken tissues; mRNA representative of a third gene (NF-E1c) is expressed only in definitive (adult) erythrocytes within the red cell lineage but is also abundantly expressed in T lymphocytes and brain. All NF-E1 proteins are highly conserved within the DNA-binding domain and bind to the consensus motif with similar affinities in vitro; they are also all stimulatory trans-acting factors in vivo. The factors differ quantitatively in their ability to trans-activate reporter genes in which the number and position of cognate binding sites is varied relative to the transcriptional initiation site. These data suggest that the NF-E1 consensus motif directs a broader and more complicated array of developmental transcriptional regulatory processes than has been assumed and that NF-E1c may play a unique regulatory role in the developing chicken brain and in T lymphocytes.