Phillip Shaw

A wild-type p53 gene under control of the metallothionein MT-1 promoter was stably transfected into human colon tumor-derived cell line EB. Repeated inductions of the metallothionein wild-type p53 gene with zinc chloride results in progressive detachment of wild-type p53 cells grown on culture dishes. Examination at both the light and electron microscopic level revealed that cells expressing wild-type p53 developed morphological features of apoptosis. DNA from both attached and detached cells was degraded into a ladder of nucleosomal-sized fragments. Expression of wild-type p53 inhibited colony formation in soft agar and tumor formation in nude mice. Furthermore, established tumors in nude mice underwent regression if wild-type p53 expression was subsequently induced. Regressing tumors showed histological features of apoptosis. Thus, regression of these tumors was the result of apoptosis occurring in vivo. Apoptosis may be a normal part of the terminal differentiation program of colonic epithelial cells. Our results suggest that wild-type p53 could play a critical role in this process.

Imprinting is defined as the parental allele-specific expression of a very limited set of genes (about 50-80). This regulation depends upon an epigenetic marking of parental alleles during gametogenesis. Monoallelic expression ensures that the levels of the proteins encoded by imprinted genes, important factors of embryonic growth, placental growth or adult metabolism, are assured. Without precise control of their expression, developmental abnormalities result, as is shown by a number of hereditary over-growth syndromes, including Beckwith-Wiedemann syndrome. The regulation of imprinted genes is largely dependent on methylation marks, which are laid down during embryological development of germ cells. Once in place, the methylation status of precise chromosomal regions, Imprinting Control Regions (ICRs), is read by either of two mechanisms, chromatin barrier formation or untranslated RNAs, thereby ensuring that only the maternal or paternal allele is expressed. Each imprinted gene is classified as maternal or paternal according to the expressed allele. The stability of the marked regions in somatic cells is maintained through each cellular replication by a methylation enzyme complex containing Dnmt1. Although the major reading mechanisms of imprinted status are known, chromatin boundary formation by CTCF and untranslated RNAs, the molecules elaborating the initial ICR methylation, are just being uncovered. Mis-regulation of imprinted gene expression (loss of imprinting [LOI]) is seen frequently and precociously in a large variety of human tumours, making LOI a potentially valuable tool for both diagnosis and treatment. In fact, LOI is presently considered the most abundant and most precocious alteration in cancer. The present review proposes a mechanism responsible for LOI, as well as its eventual value in tumour diagnosis and prognosis.

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation-DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.