Deborah J. Mays

The ΔNp63α phosphoprotein binds the p21 and 14-3-3σ promoters in vivo and has transcriptional repressor activity that is reduced by Hay-Wells syndrome-derived mutations

ABSTRACT p63 is a recently identified homolog of p53 that is found in the basal layer of several stratified epithelial tissues such as the epidermis, oral mucosa, prostate, and urogenital tract. Studies with p63−/− mice and analysis of several human autosomal-dominant disorders with germ line p63 mutations suggest p63 involvement in maintaining epidermal stem cell populations. The p63 gene encodes six splice variants with reported transactivating or dominant-negative activities. The goals of the current study were to determine the splice variants that are expressed in primary human epidermal keratinocytes (HEKs) and the biochemical activity p63 has in these epithelial cell populations. We found that the predominant splice variant expressed in HEKs was ΔNp63α, and it was present as a phosphorylated protein. During HEK differentiation, ΔNp63α and p53 levels decreased, while expression of p53 target genes p21 and 14-3-3σ increased. ΔNp63α had transcriptional repressor activity in vitro, and this activity was reduced in ΔNp63α proteins containing point mutations, corresponding to those found in patients with Hay-Wells syndrome. Further, we show that ΔNp63α and p53 can bind the p21 and 14-3-3σ promoters in vitro and in vivo, with decreased binding of p63 to these promoters during HEK differentiation. These data suggest that ΔNp63α acts as a transcriptional repressor at select growth regulatory gene promoters in HEKs, and this repression likely plays an important role in the proliferative capacity of basal keratinocytes.

Mitotic Phosphorylation of Bcl-2 during Normal Cell Cycle Progression and Taxol-induced Growth Arrest

There is increasing evidence that prolonged mitotic arrest initiates apoptosis; however, little is known about the signaling pathways involved. Several studies have associated deregulated Cdc2 activity with apoptosis. Herein, we report that the anti-apoptotic protein, Bcl-2, undergoes cell cycle-dependent phosphorylation during mitosis when there is elevated Cdc2 activity. We found that paclitaxel (Taxol®) treatment of epithelial tumor cells induced a prolonged mitotic arrest, elevated levels of mitotic kinase activity, hyperphosphorylation of Bcl-2, and subsequent cell death. The Taxol-induced Bcl-2 phosphorylation was dose-dependent. Furthermore, phosphorylated Bcl-2 remained complexed with Bax in Taxol-treated cells undergoing apoptosis. Immunoprecipitation experiments revealed a Bcl-2-associated kinase capable of phosphorylating histone H1 in vitro. However, the kinase was likely not cyclin B1/Cdc2, since cyclin B1/Cdc2 was not detectable in Bcl-2 immunoprecipitates, nor was recombinant Bcl-2 phosphorylatedin vitro by cyclin B1/Cdc2. The results of this study further define a link between mitotic kinase activation and the apoptotic machinery in the cell. However, the role, if any, of prolonged Bcl-2 phosphorylation in Taxol-mediated apoptosis awaits further definition of Bcl-2 mechanism of action. Taxol may increase cellular susceptibility to apoptosis by amplifying the normal downstream events associated with mitotic kinase activation.

Kinetics of p53 Binding to Promoter Sites In Vivo

ABSTRACT Downstream target genes of p53 are thought to mediate its tumor-suppressive activity, but it is unknown whether differential transactivation of these genes is regulated at the level of p53 binding to their promoters. To address this issue, p53 binding in vivo to consensus sites in the p21Waf1, MDM2, and PIG3 promoters was investigated in cells exposed to adriamycin (ADR) or ionizing radiation as well as in an inducible p53 cell line. p53-DNA complexes were cross-linked in vivo by treating the cells with formaldehyde and processed by chromatin immunoprecipitation-PCR. This methodology allowed for the analysis of relevant p53-DNA complexes by preventing redistribution of cellular components upon collection of cell extracts. Increased p53 binding to the p21Waf1, MDM2, and PIG3 promoters occurred within 2 h after p53 activation; however, significant increases in PIG3 transcription did not occur until 15 h after p53 binding. Gel shift analyses indicated that p53 had lower affinity for the consensus binding site in the PIG3 promoters compared to its consensus sites in the p21 and MDM2 genes, which suggests that additional factors may be required to stabilize the interaction of p53 with the PIG3 promoter. Further, acetylated p53 (Lys382) was found in chemically cross-linked complexes at all promoter sites examined after treatment of cells with ADR. In summary, the kinetics of p53 binding in vivo to target gene regulatory regions does not uniformly correlate with target gene mRNA expression for the p53 target genes examined. Our results suggest that target genes with low-affinity p53 binding sites may require additional events and will have delayed kinetics of induction compared to those with high-affinity binding sites.

A Gene Signature-Based Approach Identifies mTOR as a Regulator of p73

ABSTRACT Although genomic technologies have advanced the characterization of gene regulatory networks downstream of transcription factors, the identification of pathways upstream of these transcription factors has been more challenging. In this study we present a gene signature-based approach for connecting signaling pathways to transcription factors, as exemplified by p73. We generated a p73 gene signature by integrating whole-genome chromatin immunoprecipitation and expression profiling. The p73 signature was linked to corresponding signatures produced by drug candidates, using the in silico Connectivity Map resource, to identify drugs that would induce p73 activity. Of the pharmaceutical agents identified, there was enrichment for direct or indirect inhibitors of mammalian Target of Rapamycin (mTOR) signaling. Treatment of both primary cells and cancer cell lines with rapamycin, metformin, and pyrvinium resulted in an increase in p73 levels, as did RNA interference-mediated knockdown of mTOR. Further, a subset of genes associated with insulin response or autophagy exhibited mTOR-mediated, p73-dependent expression. Thus, downstream gene signatures can be used to identify upstream regulators of transcription factor activity, and in doing so, we identified a new link between mTOR, p73, and p73-regulated genes associated with autophagy and metabolic pathways.

p63 consensus DNA-binding site: identification, analysis and application into a p63MH algorithm.

p53 and p63 belong to a family of sequence-specific transcription factors regulating key cellular processes. Differential composition of the p53 and p63 DNA-binding sites may contribute to distinct functions of these protein homologues. We used SELEX (systematic evolution of ligands by exponential enrichment) methodology to identify nucleic acid ligands for p63. We found that p63 bound preferentially to DNA fragments conforming to the 20 bp sequence 5′-RRRC(A/G)(A/T)GYYYRRRC(A/T)(C/T)GYYY-3′. Relative to the p53 consensus, the p63 consensus DNA-binding site (DBS) was more degenerate, particularly at positions 10 and 11, and was enriched for A/G at position 5 and C/T at position 16 of the consensus. The differences in DNA-binding site preferences between p63 and p53 influenced their ability to activate transcription from select response elements (REs) in cells. A computer algorithm, p63MH, was developed to find candidate p63-binding motifs on input sequences. We identified genes responsive to p63 regulation that contain functional p63 REs. Our results suggest that the sequence composition of REs could be one contributing factor to target gene discrimination between p63 and p53.

Chromatin Immunoprecipitation-Based Screen To Identify Functional Genomic Binding Sites for Sequence-Specific Transactivators

ABSTRACT In various human diseases, altered gene expression patterns are often the result of deregulated gene-specific transcription factor activity. To further understand disease on a molecular basis, the comprehensive analysis of transcription factor signaling networks is required. We developed an experimental approach, combining chromatin immunoprecipitation (ChIP) with a yeast-based assay, to screen the genome for transcription factor binding sites that link to transcriptionally regulated target genes. We used the tumor suppressor p53 to demonstrate the effectiveness of the method. Using primary and immortalized, nontransformed cultures of human mammary epithelial cells, we isolated over 100 genomic DNA fragments that contain novel p53 binding sites. This approach led to the identification and validation of novel p53 target genes involved in diverse signaling pathways, including growth factor signaling, protein kinase/phosphatase signaling, and RNA binding. Our results yield a more complete understanding of p53-regulated signaling pathways, and this approach could be applied to any number of transcription factors to further elucidate complex transcriptional networks.

ISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy

BackgroundAutophagy is characterized by the sequestration of cytoplasm and organelles into multimembrane vesicles and subsequent degradation by the cells lysosomal system. It is linked to many physiological functions in human cells including stress response, protein degradation, organelle turnover, caspase-independent cell death and tumor suppression. Malignant transformation is frequently associated with deregulation of autophagy and several tumor suppressors can modulate autophagic processes. The tumor suppressor p53 can induce autophagy after metabolic or genotoxic stress through transcriptionally-dependent and -independent mechanisms. In this study we expand on the former mechanism by functionally characterizing a p53 family target gene, ISG20L1 under conditions of genotoxic stress.ResultsWe identified a p53 target gene, ISG20L1, and show that transcription of the gene can be regulated by all three p53 family members (p53, p63, and p73). We generated an antibody to ISG20L1 and found that it localizes to the nucleolar and perinucleolar regions of the nucleus and its protein levels increase in a p53- and p73-dependent manner after various forms of genotoxic stress. When ectopically expressed in epithelial cancer-derived cell lines, ISG20L1 expression decreased clonogenic survival without a concomitant elevation in apoptosis and this effect was partially rescued in cells that were ATG5 deficient. Knockdown of ISG20L1 did not alter 5-FU induced apoptosis as assessed by PARP and caspase-3 cleavage, sub-G1 content, and DNA laddering. Thus, we investigated the role of ISG20L1 in autophagy, a process commonly associated with type II cell death, and found that ISG20L1 knockdown decreased levels of autophagic vacuoles and LC3-II after genotoxic stress as assessed by electron microscopy, biochemical, and immunohistochemical measurements of LC3-II.ConclusionsOur identification of ISG20L1 as a p53 family target and discovery that modulation of this target can regulate autophagic processes further strengthens the connection between p53 signaling and autophagy. Given the keen interest in targeting autophagy as an anticancer therapeutic approach in tumor cells that are defective in apoptosis, investigation of genes and signaling pathways involved in cell death associated with autophagy is critical.

p73 Is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network

We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus; hippocampal dysgenesis; sterility; and chronic inflammation/infection of lung, middle ear, and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells and suggest that p73 marks these cells for MCC differentiation. In summary, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation, and, like p63, has an essential role in development of tissues.

Differential regulation of the p73 cistrome by mammalian target of rapamycin reveals transcriptional programs of mesenchymal differentiation and tumorigenesis

The transcription factor p73 plays critical roles during development and tumorigenesis. It exhibits sequence identity and structural homology with p53, and can engage p53-like tumor-suppressive programs. However, different pathways regulate p53 and p73, and p73 is not mutated in human tumors. Therefore, p73 represents a therapeutic target, and there is a critical need to understand genes and noncoding RNAs regulated by p73 and how they change during treatment regimens. Here, we define the p73 genomic binding profile and demonstrate its modulation by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) and inducer of p73. Rapamycin selectively increased p73 occupancy at a subset of its binding sites. In addition, multiple determinants of p73 binding, activity, and function were evident, and were modulated by mTOR. We generated an mTOR-p73 signature that is enriched for p73 target genes and miRNAs that are involved in mesenchymal differentiation and tumorigenesis, can classify rhabdomyosarcomas by clinical subtype, and can predict patient outcome.

Cell cycle‐dependent transduction of cell‐permeant Cre recombinase proteins

Protein transduction has been widely used to analyze biochemical processes in living cells quantitatively and under non‐steady‐state conditions. The present study analyzed the effects of cell cycle on the uptake and activity of cell‐permeant Cre recombinase proteins. Previous studies had suggested that the efficiency of recombination and/or protein transduction varied among individual cells, even within a clonal population. We report here that cells in the G1 phase of the cell cycle undergo recombination at a lower rate than cells at other phases of the cell cycle, and that this variation results largely from differences in protein uptake, associated with differences in cell size. These results have implications regarding the mechanism of protein transduction and identify a source of heterogeneity that can influence the response of individual cells to cell‐permeant proteins. J. Cell. Biochem. 89: 674–687, 2003.