Tom Burdon

Signalling, cell cycle and pluripotency in embryonic stem cells

Pluripotent mouse embryonic stem (ES) cells can be expanded in large numbers in vitro owing to a process of symmetrical self-renewal. Self-renewal entails proliferation with a concomitant suppression of differentiation. Here we describe how the cytokine leukaemia inhibitory factor (LIF) sustains self-renewal through activation of the transcription factor STAT3, and how two other signals - extracellular-signal-related kinase (ERK) and phosphatidylinositol-3-OH kinase (PI3K) - can influence differentiation and propagation, respectively. We relate these observations to the unusual cell-cycle properties of ES cells and speculate on the role of the cell cycle in maintaining pluripotency.

Analysis of Oct4‐Dependent Transcriptional Networks Regulating Self‐Renewal and Pluripotency in Human Embryonic Stem Cells

The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference‐mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4‐dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4‐associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans.

Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus

A major barrier to research on Parkinsons disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding α-synuclein, causes a fully penetrant, aggressive form of Parkinsons disease with dementia. α-Synuclein dysfunction is the critical pathogenic event in Parkinsons disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of α-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinsons disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of α-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by α-synuclein dysfunction.

Signaling Mechanisms Regulating Self-Renewal and Differentiation of Pluripotent Embryonic Stem Cells

An ability to propagate pluripotent embryonic cells in culture is the foundation both for defined germline modification in experimental rodents and for future possibilities for broad-based cellular transplantation therapies in humans. Yet, the molecular basis of the self-renewing pluripotent phenotype remains ill-defined. The relationship between factors that influence embryonic stem cell propagation in vitro and mechanisms of stem cell regulation operative in the embryo is also uncertain. In this article we discuss the role of intracellular signalling pathways in the maintenance of pluripotency and induction of differentiation in embryonic stem cell cultures and the mammalian embryo.

Stat5 as a Target for Regulation by Extracellular Matrix

Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine β-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of β-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with -interferon activation site-related motifs within the β-lactoglobulin promoter, which we show are required for matrix dependence of β-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.

An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination‐mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase‐deficient embryonic stem cells.

Differential activation of STATs 3 and 5 during mammary gland development

We have investigated the activity of STAT family members throughout a mammary developmental cycle. Transcripts for Stat 5 were upregulated during pregnancy whilst STAT1 and STAT3 mRNAs were expressed at constant levels. DNA binding complexes containing both STAT5a and 5b showed differing affinities for two naturally occurring STAT5 binding sites. In the involuting mammary gland STAT5 activity decreased whereas STAT3 was specifically activated. These observations reveal a complex pattern of activation of STAT factors during mammary growth, differentiation and remodelling and provide the first evidence for the involvement of STAT3 in development of the mammary gland.

Stat3-induced apoptosis requires a molecular switch in PI(3)K subunit composition.

Physiological apoptosis is induced by a switch from survival to death signalling. Dysregulation of this process is frequently associated with cancer. A powerful model for this apoptotic switch is mammary gland involution, during which redundant milk-producing epithelial cells undergo apoptosis. Signal transducer and activator of transcription 3 (Stat3) is an essential mediator of this switch but the mechanism has not yet been defined. Stat3-dependent cell death during involution can be blocked by activation of Akt/protein kinase B (PKB), a downstream effector of the phosphoinositide-3-OH kinase (PI(3)K) pathway. Here we show that expression of the PI(3)K regulatory subunits p55α and p50α is induced by Stat3 during involution. In the absence of Stat3 in vivo, upregulation of p55α and p50α is abrogated, levels of activated Akt are sustained and apoptosis is prevented. Chromatin immunoprecipitation assays show that Stat3 binds directly to the p55α and p50α promoters in vivo. Overexpression of either p55α or p50α reduces levels of activated Akt. We propose a novel mechanism in which Stat3 regulates apoptosis by inducing expression of distinct PI(3)K regulatory subunits to downregulate PI(3)K-Akt-mediated survival signalling.

Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype

The whey acidic protein (WAP) gene is expressed in mammary epithelial cells at late pregnancy and throughout lactation. We have generated transgenic mice in which a mouse WAP transgene is expressed precociously in pregnancy. From 13 founder mice bearing WAP transgenes, two female founders and the daughters from a male founder failed to lactate and nurture their offspring. We named this phenotype milchlos. Mammary tissue from postpartum milchlos mice was underdeveloped, contained too few alveoli and resembled the glands of non-transgenic mid-pregnant mice. The hypothesis that alveolar development in milchlos mice was functionally arrested in a prelactational state is consistent with low levels of alpha-lactalbumin mRNA, and an unidentified keratin RNA in mammary tissue from postpartum mice. Defects in alveolar function in milchlos mice were detected at mid-pregnancy; in non-transgenic mice, WAP was secreted into the alveolar lumen but remained preferentially in the cytoplasm of the alveolar epithelial cells in the milchlos mice. Since deregulated WAP expression resulted in impaired mammary development, it is possible that WAP plays a regulatory role in the terminal differentiation and development of mammary alveolar cells.

The mammary factor MPBF is a prolactin-induced transcriptional regulator which binds to STAT factor recognition sites

Site‐directed mutagenesis of the three binding sites for the mammary factor MPBF in the β‐lactoglobulin (BLG) promoter demonstrates that MPBF is a transcriptional activator of the BLG gene in mammary cells. MPBF requires phosphorylation on tyrosine for maximum binding activity and binds to GAS (interferon γ‐activation site) elements which are similar to the MPBF binding sites. Prolactin induces MPBF binding activity in CHO cells and is not antigenically related to Stat1 (p91) and Stat2 (p113), suggesting that this transcription factor is likely to be another member of the STAT family of cytokine/growth factor‐induced transcription factors.