Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Ospedale Policlinico San Martino

N3-methyladenine (3-mA), generated by the reaction of methylating agents with DNA, is considered a highly toxic but weakly mutagenic lesion. However, due to its intrinsic instability, some of the biological effects of the adduct can result from the formation of the corresponding depurination product [an apurinic (AP)-site]. Previously, we exploited Me-lex, i.e. {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2 carboxamido}propane, a minor groove equilibrium binder with selectivity for A/T rich sequences that efficiently reacts with DNA to afford 3-mA as the dominant product, to probe the biology of this lesion. Using human p53 cDNA as a target in a yeast system, a weak increase in mutagenicity was observed in the absence of Mag1 (3-methyladenine-DNA glycosylase 1, mag1), the enzyme devoted to remove 3-mA from DNA. Moreover, a significant increase in mutagenicity occurred in the absence of the enzymes involved in the repair of AP-sites (AP endonucleases 1 and 2, apn1apn2). Since methyl methanesulfonate (MMS) has been extensively used to explore the biological effects of 3-mA, even though it produces 3-mA in low relative yield, we compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast. A mutagenesis reporter plasmid was damaged in vitro by MMS and then transformed into wild-type and Translesion Synthesis (TLS) Polζ (REV3) and Polη (RAD30) deficient strains. Furthermore, a mag1rad30 double mutant strain was constructed and transformed with the DNA plasmid damaged in vitro by Me-lex. The results confirm the important role of Polζ in the mutagenic bypass of MMS and Me-lex induced lesions, with Polη contributing only towards the bypass of Me-lex induced lesions, mainly in an error-free way. Previous and present results point towards the involvement of AP-sites, derived from the depurination of 3-mA, in the observed toxicity and mutagenicity.

Comparison of the biological effects of MMS and Me-lex, a minor groove methylating agent: clarifying the role of N3-methyladenine.

The observation that human transcription factors (TFs) can function when expressed in yeast cells has stimulated the development of various functional assays to investigate (i) the role of binding site sequences (herein referred to as response elements, REs) in transactivation specificity, (ii) the impact of polymorphic nucleotide variants on transactivation potential, (iii) the functional consequences of mutations in TFs and (iv) the impact of cofactors or small molecules. These approaches have found applications in basic as well as applied research, including the identification and the characterisation of mutant TF alleles from clinical samples. The ease of genome editing of yeast cells and the availability of regulated systems for ectopic protein expression enabled the development of quantitative reporter systems, integrated at a chosen chromosomal locus in isogenic yeast strains that differ only at the level of a specific RE targeted by a TF or for the expression of distinct TF alleles. In many cases, these assays were proven predictive of results in higher eukaryotes. The potential to work in small volume formats and the availability of yeast strains with modified chemical uptake have enhanced the scalability of these approaches. Next to well-established one-, two-, three-hybrid assays, the functional assays with non-chimeric human TFs enrich the palette of opportunities for functional characterisation. We review ∼25 years of research on human sequence-specific TFs expressed in yeast, with an emphasis on the P53 and NF-кB family of proteins, highlighting outcomes, advantages, challenges and limitations of these heterologous assays.

Human transcription factors in yeast: the fruitful examples of P53 and NF-кB.

In contrast to TP53, cancer development is rarely associated with mutations in the TP63 and TP73 genes. Recently, next generation sequencing analysis revealed that TP63 mutations are frequent, specifically in cutaneous melanomas. Cutaneous melanoma represents 4% of skin cancers but it is responsible for 80% of skin cancer related deaths. In the present study, we first determined whether all three members of the P53 family of transcription factors were found mutated in cutaneous melanomas by retrieving all TP53, TP63 and TP73 mutations from cBioPortal (http://www.cbioportal.org/). TP53 and TP63 were frequently mutated [15.0% (91/605) and 14.7% (89/605), respectively], while TP73 [1.5% (9/605)] was more rarely mutated (p<0.0001). A UV-mutation fingerprint was recognized for TP63 and TP73 genes. Then, we tried to evaluate the potential role of TP63 mutations as drivers or passengers in the tumorigenic process. In the former case, the amino acid substitutions should cause significant functional consequences on the main biochemical activity of the P63 protein, namely transactivation. The predicted effects of specific amino acid substitutions by two bioinformatics tools were rather different. Using a yeast-based functional assay, the observed hotspot mutant R379CP63 protein exhibited a substantial residual activity compared to the wild-type (>70%). This result does not support a major role of the mutant P63 protein in melanomagenesis while it is still consistent with the TP63 gene being a recorder of UV exposure. The TP63 mutation spectrum from cutaneous melanomas, when compared with that observed at the germinal level in patients affected by P63-associated diseases [ectodermal dysplasia syndromes, (EDs)], revealed significant differences. The TP63 mutations were more frequent at CpGs sites (p<0.0001) in EDs and at PyPy sites (p<0.0001) in cutaneous melanomas. The two spectra differed significantly (p<0.0001). We conclude that TP63 mutations are frequent in cutaneous melanoma, support UV etiology, but their role in melanomagenesis is unclear.

Gilberto Fronza

Mutagenesis and Cancer Prevention Unit

Ospedale Policlinico San Martino

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Gilberto Fronza

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Mutagenesis and Cancer Prevention Unit

Ospedale Policlinico San Martino

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Mutagenesis and Cancer Prevention Unit