PML depletion disrupts normal mammary gland development and skews the composition of the mammary luminal cell progenitor pool.

Nuclear domains of promyelocytic leukemia protein (PML) are known to act as signaling nodes in many cellular processes. Although the impact of PML expression in driving cell fate decisions for injured cells is well established, the function of PML in the context of tissue development is less well understood. Here, the in vivo role of PML in developmental processes in the murine mammary gland has been investigated. Data are presented showing that expression of PML is tightly regulated by three members of the Stat family of transcription factors that orchestrate the functional development of the mammary secretory epithelium during pregnancy. Developmental phenotypes were also discovered in the virgin and pregnant Pml null mouse, typified by aberrant differentiation of mammary epithelia with reduced ductal and alveolar development. PML depletion was also found to disturb the balance of two distinct luminal progenitor populations. Overall, it is shown that PML is required for cell lineage determination in bi-potent luminal progenitor cells and that the precise regulation of PML expression is required for functional differentiation of alveolar cells.

Restricted co-expression of Dlk1 and the reciprocally imprinted non-coding RNA, Gtl2: implications for cis-acting control.

Dlk1 and Gtl2 are reciprocally imprinted neighboring genes located within a 1 Mb imprinted domain on murine distal chromosome 12. The two genes are expressed and developmentally regulated during mammalian embryogenesis. Dlk1/Pref1 encodes a transmembrane protein with homology to members of the Notch/Delta developmental signaling pathway and Gtl2 generates alternatively spliced poly-adenylated transcripts lacking a conserved open reading frame. An intergenic differentially methylated region (IG-DMR) located 13 kb upstream of Gtl2 has been shown to regulate imprinting throughout the domain by an as yet unknown mechanism. In order to gain insights into regulation at this domain and to compare it with imprinting control at other loci, we compared the expression profile of Dlk1 with Gtl2 during mouse embryogenesis in normal conceptuses and in those with uniparental disomy for chromosome 12. The expression profile of these genes suggests a causative role for Dlk1 and Gtl2 in the pathologies found in uniparental disomy animals, characterized by defects in skeletal muscle maturation, bone formation, placenta size and organization and prenatal lethality. Here, we show restricted overlap in cellular expression of these two genes throughout development. Dlk1 is imprinted and expressed in cell types within the lung, liver and placenta where Gtl2 is not expressed. Gtl2 is highly expressed in the central nervous system (CNS), whereas Dlk1 is found localized to specific regions such as the hypothalamus. Co-expression is observed in most of the mesodermal-derived tissues, notably the skeletal muscle where both genes are strongly co-expressed. In this tissue, Dlk1 shows a relaxation of imprinting with some expression from the maternal allele. These findings indicate that the general mechanism of imprinting at the stages analyzed is not through the co-ordinate non-coding RNA or insulator mechanisms observed for other imprinted domains, and suggest that the two genes have independent tissue-specific functions.

The beginning of the end: death signaling in early involution.

Mammary gland involution occurs in two distinct phases: an early, reversible phase, involving extensive apoptosis of the secretory alveolar epithelium without major changes in gland architecture, and a later, irreversible phase, involving remodelling of the gland to its pre-pregnancy state. Multiple signalling pathways are known to be important during early involution, however the precise triggers remain elusive. This review summarizes the roles of a number of key pathways (NF-kappaB, PI(3)K, Stat3, and TGFbeta) in the first phase of involution.

Caspase-dependent proteolytic cleavage of STAT3alpha in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines.

BACKGROUND: The STAT (Signal Transducers and Activators of Transcription) transcription factor family mediates cellular responses to a wide range of cytokines. Activated STATs (particularly STAT3) are found in a range of cancers. Further, STAT3 has anti-apoptotic functions in a range of tumour cell lines. After observing a proteolytic cleavage in STAT3alpha close to a potential apoptotic caspase protease cleavage site we investigated whether STAT3alpha might be a caspase substrate. METHODS: STAT3alpha status was investigated in vitro in several cell systems:- HM-1 murine embryonic stem (ES) cells following various interventions; IOUD2 murine ES cells following induction to differentiate along neural or adipocyte lineages; and in a number of breast cancer cell lines. STAT3alpha status was also analysed in vivo in wild type murine mammary glands undergoing controlled, forced involution. RESULTS: Immunoblotting for STAT3alpha in HM-1 ES cell extracts detected amino and carboxy terminal species of approximately 48 kDa and 43 kDa respectively--which could be diminished dose-dependently by cell treatment with the nitric oxide (NO) donor drug sodium nitroprusside (SNP). UV irradiation of HM-1 ES cells triggered the STAT3alpha cleavage (close to a potential caspase protease cleavage site). Interestingly, the pan-caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-FMK) and the JAK2 tyrosine kinase inhibitor AG490 both inhibited cleavage dose-dependently, and cleavage was significantly lower in a heterozygous JAK2 knockout ES cell clone. STAT3alpha cleavage also occurred in vivo in normal murine mammary glands undergoing forced involution, coinciding with a pulse of phosphorylation of residue Y705 on full-length STAT3alpha. Cleavage also occurred during IOUD2 ES cell differentiation (most strikingly along the neural lineage) and in several human breast cancer cell lines, correlating strongly with Y705 phosphorylation. CONCLUSION: This study documents a proteolytic cleavage of STAT3alpha into 48 kDa amino and 43 kDa carboxyl terminal fragments in a range of cell types. STAT3alpha cleavage occurs close to a potential caspase site, and can be inhibited dose-dependently by SNP, AG490 and z-VAD-FMK. The cleavage seems to be caspase-dependent and requires the phosphorylation of STAT3alpha at the Y705 residue. This highly regulated STAT3alpha cleavage may play an important role in modulating STAT3 transcriptional activity.

Genomic imprinting--insights from studies in mice.

A subset of mammalian genes is controlled by genomic imprinting. This process causes a gene to be expressed from only one chromosome homologue depending on whether it originally came from the egg or the sperm. Parental origin-specific gene regulation is controlled by epigenetic modifications to DNA and chromatin. Genomic imprinting is therefore a useful model system to study the epigenetic control of genome function. Here we consider the value of the mouse as an experimental organism to address questions about the role of imprinted genes, about the regulation of mono-allelic gene expression and about the evolution of the imprinting function and mechanism.

Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19.

Dlk1 and Gtl2 are reciprocally imprinted genes located 80 kb apart on mouse chromosome 12. Similarities between this domain and that of the well characterized Igf2-H19 locus have been previously noted. Comparative genomic and epigenetic analysis of these two domains might help identify allele-specific epigenetic regulatory elements and common features involved in aspects of imprinting control. Here we describe a detailed methylation analysis of the Dlk1-Gtl2 domain on both parental alleles in the mouse. Like the Igf2-H19 domain, areas of differential methylation are hypermethylated on the paternal allele and hypomethylated on the maternal allele. Three differentially methylated regions (DMRs), each with different epigenetic characteristics, have been identified. One DMR is intergenic, contains tandem repeats and is the only region that inherits a paternal methylation mark from the germline. An intronic DMR contains a conserved putative CTCF-binding domain. All three DMRs have both unique and common features compared to those identified in the Igf2-H19 domain.