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A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

Sethi I, Romano RA, Gluck C, Smalley K, Vojtesek B, Buck MJ, Sinha S - BMC Genomics (2015)

Bottom Line: Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions.We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63.Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Center of Excellence in Bioinformatics and Life Sciences, State University of New York, 701 Ellicott Street, Buffalo, NY, 14203, USA.

ABSTRACT

Background: The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, β, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field.

Results: Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions.

Conclusions: In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

No MeSH data available.


Related in: MedlinePlus

Protein expression profiles of p63 isoforms. Western blot analysis of whole cell extracts demonstrates p63 expression using (a) a pan-p63 antibody (b) and an alpha specific antibody (H-129). TAp63 and ∆Np63 isoform specific expression is shown in panels (c) and (d) respectively. Blue and red arrows mark the TAp63 and ∆Np63 protein bands, respectively. Beta-tubulin serves as a loading control. se: short exposure and le:long exposure
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Fig2: Protein expression profiles of p63 isoforms. Western blot analysis of whole cell extracts demonstrates p63 expression using (a) a pan-p63 antibody (b) and an alpha specific antibody (H-129). TAp63 and ∆Np63 isoform specific expression is shown in panels (c) and (d) respectively. Blue and red arrows mark the TAp63 and ∆Np63 protein bands, respectively. Beta-tubulin serves as a loading control. se: short exposure and le:long exposure

Mentions: We ran the first analysis pipeline with the goal to examine the six major p63 isoforms that are annotated and well-defined in the literature (TAp63α, β, and γ, ΔNp63α, β, and γ) as well as to detect novel transcripts. We found that the longest C-terminal α variant was the predominant isoform expressed across all p63 positive cell-lines, irrespective of their source. Thus in all normal and tumorigenic cell lines, in which the ΔNp63 transcripts were detectable at appreciable levels, ΔNp63α was several log fold higher than ΔNp63β and ΔNp63γ. Of the two shorter isoforms, ΔNp63β displayed a relatively widespread distribution as evident from its low to modest expression (0–15 FPKM) in 11 out of 15 cell-lines, whereas ΔNp63γ on the other hand was detected only at low levels (<5 FPKM) in a small subset of HNSCC cell lines (Additional file 3: Table S2). Interestingly ΔNp63β levels remained consistent in differentiating keratinocytes unlike the ΔNp63α isoform (Additional file 4: Figure S2). The dynamic range of expression of the ΔNp63 isoforms suggests that there might exist a distinct functional role for each isoform, especially the β isoform. In most TAp63 expressing cell-lines, only the TAp63α isoform was detected at appreciable levels (Fig. 2). It is important to note that in oocytes, where TAp63α is highly expressed, it is thought to exist in an inactive conformation due to complex domain-domain interactions [49, 50]. Whether similar structural mechanism of TAp63α inhibition also operates in BL cells is an interesting question that needs to be addressed. Interestingly, a novel p63 isoform, TAp63δ was identified in the GUMBUS BL cell-line by the de novo method (as implemented in Analysis Pipeline 1), albeit at very low levels (data not shown). This is a shorter p63 isoform that is generated by exon skipping in the 3’ end and was only recently discovered [51].Fig 2


A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

Sethi I, Romano RA, Gluck C, Smalley K, Vojtesek B, Buck MJ, Sinha S - BMC Genomics (2015)

Protein expression profiles of p63 isoforms. Western blot analysis of whole cell extracts demonstrates p63 expression using (a) a pan-p63 antibody (b) and an alpha specific antibody (H-129). TAp63 and ∆Np63 isoform specific expression is shown in panels (c) and (d) respectively. Blue and red arrows mark the TAp63 and ∆Np63 protein bands, respectively. Beta-tubulin serves as a loading control. se: short exposure and le:long exposure
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4528692&req=5

Fig2: Protein expression profiles of p63 isoforms. Western blot analysis of whole cell extracts demonstrates p63 expression using (a) a pan-p63 antibody (b) and an alpha specific antibody (H-129). TAp63 and ∆Np63 isoform specific expression is shown in panels (c) and (d) respectively. Blue and red arrows mark the TAp63 and ∆Np63 protein bands, respectively. Beta-tubulin serves as a loading control. se: short exposure and le:long exposure
Mentions: We ran the first analysis pipeline with the goal to examine the six major p63 isoforms that are annotated and well-defined in the literature (TAp63α, β, and γ, ΔNp63α, β, and γ) as well as to detect novel transcripts. We found that the longest C-terminal α variant was the predominant isoform expressed across all p63 positive cell-lines, irrespective of their source. Thus in all normal and tumorigenic cell lines, in which the ΔNp63 transcripts were detectable at appreciable levels, ΔNp63α was several log fold higher than ΔNp63β and ΔNp63γ. Of the two shorter isoforms, ΔNp63β displayed a relatively widespread distribution as evident from its low to modest expression (0–15 FPKM) in 11 out of 15 cell-lines, whereas ΔNp63γ on the other hand was detected only at low levels (<5 FPKM) in a small subset of HNSCC cell lines (Additional file 3: Table S2). Interestingly ΔNp63β levels remained consistent in differentiating keratinocytes unlike the ΔNp63α isoform (Additional file 4: Figure S2). The dynamic range of expression of the ΔNp63 isoforms suggests that there might exist a distinct functional role for each isoform, especially the β isoform. In most TAp63 expressing cell-lines, only the TAp63α isoform was detected at appreciable levels (Fig. 2). It is important to note that in oocytes, where TAp63α is highly expressed, it is thought to exist in an inactive conformation due to complex domain-domain interactions [49, 50]. Whether similar structural mechanism of TAp63α inhibition also operates in BL cells is an interesting question that needs to be addressed. Interestingly, a novel p63 isoform, TAp63δ was identified in the GUMBUS BL cell-line by the de novo method (as implemented in Analysis Pipeline 1), albeit at very low levels (data not shown). This is a shorter p63 isoform that is generated by exon skipping in the 3’ end and was only recently discovered [51].Fig 2

Bottom Line: Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions.We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63.Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Center of Excellence in Bioinformatics and Life Sciences, State University of New York, 701 Ellicott Street, Buffalo, NY, 14203, USA.

ABSTRACT

Background: The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, β, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field.

Results: Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions.

Conclusions: In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

No MeSH data available.


Related in: MedlinePlus