We have used a novel approach to investigate the control of initiation of replication of sperm nuclei in a Xenopus cell‐free extract. Nascent DNA was labelled with biotin by supplementing the extract with biotin‐11‐dUTP, and isolated nuclei were then probed with fluorescein‐conjugated streptavidin. Flow cytometry was used to measure the biotin content of individual nuclei and their total DNA content. This showed that incorporation of the biotinylated precursor increases linearly with DNA content. Haploid sperm nuclei replicate fully to reach the diploid DNA content over 2‐6 h in the extract. Synthesis stops once the diploid DNA content is reached. Different nuclei enter S phase at different times over greater than 1.5 h, although they share the same cytoplasmic environment. Nuclei reach their maximum rates of synthesis soon after entry into S phase and some replicate fully in less than 0.5 h, resembling the rates of replication observed in the intact egg. These results indicate that initiations are coordinated within each nucleus such that the nucleus is the fundamental unit of replication in the cell‐free system.

Mammary epithelial regeneration implies the existence of cellular progenitors with retained replicative capacity, prolonged lifespan and developmental potency. Evidence exists that DN-p63 isoforms preserve these features by modulating p53 activity in basal epithelia. DN-p63 mRNA levels decline at the onset of differentiation suggesting that its transcriptional regulation may contribute to the initiation of differentiation. To study transcriptional regulation of DN-p63, a 10.3 kbp fragment containing the DN-p63 promoter was isolated. We report here that DN-p63 is a positive and negative transcriptional target of p53 and DN-p63-a, respectively. Disruption of p53 activity or expression abolishes the expression of DN-p63-a. This regulation is mediated by a p53-binding element sufficient to confer these activities to a heterologous promoter. Chromatin immune-precipitation indicates that, in asynchronously growing cells, p53 occupies this element. In response to DNA damage, DN-p63-a is recruited to this element as transcription of DN-p63 declines. Disruption of DN-p63-a expression had differential effects on the transcriptional regulation of several p53-target genes. These findings indicate that p53 contributes to the preservation of basal epithelia by driving the expression of DN-p63 isoforms. These studies also suggest that in response to genotoxic stress, DN-p63-a mediates the silencing of its own promoter thereby altering the pattern of p53-target gene expression.

Patients diagnosed with lung squamous cell carcinoma (LUSC) have limited targeted therapies. We report here the identification and characterisation of BCL11A, as a LUSC oncogene. Analysis of cancer genomics datasets revealed BCL11A to be upregulated in LUSC but not in lung adenocarcinoma (LUAD). Experimentally we demonstrate that non-physiological levels of BCL11A in vitro and in vivo promote squamous-like phenotypes, while its knockdown abolishes xenograft tumour formation. At the molecular level we found that BCL11A is transcriptionally regulated by SOX2 and is required for its oncogenic functions. Furthermore, we show that BCL11A and SOX2 regulate the expression of several transcription factors, including SETD8. We demonstrate that shRNA-mediated or pharmacological inhibition of SETD8 selectively inhibits LUSC growth. Collectively, our study indicates that BCL11A is integral to LUSC pathology and highlights the disruption of the BCL11A–SOX2 transcriptional programme as a novel candidate for drug development.

The NM23 (non-metastatic 23) family is almost universally conserved across all three domains of life: eubacteria, archaea and eucaryotes. Unicellular organisms possess one NM23 ortholog, whilst vertebrates possess several. Gene multiplication through evolution has been accompanied by structural and functional diversification. Many NM23 orthologs are nucleoside diphosphate kinases (NDP kinases), but some more recently evolved members lack NDP kinase activity and/or display other functions, for instance, acting as protein kinases or transcription factors. These members display overlapping but distinct expression patterns during vertebrate development. In this review, we describe the functional differences and similarities among various NM23 family members. Moreover, we establish orthologous relationships through a phylogenetic analysis of NM23 members across vertebrate species, including Xenopus laevis and zebrafish, primitive chordates and several phyla of invertebrates. Finally, we summarize the involvement of NM23 proteins in development, in particular neural development. Carcinogenesis is a process of misregulated development, and NM23 was initially implicated as a metastasis suppressor. A more detailed understanding of the evolution of the family and its role in vertebrate development will facilitate elucidation of the mechanism of NM23 involvement in human cancer.