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To date, reprogramming strategies for generating cell types of interest have been facilitated by detailed understanding of relevant developmental regulatory factors. However, identification of such regulatory drivers often represents a major challenge, as specific gene combinations may be required for reprogramming. Here we show that a computational systems approach can identify cell type specification genes (master regulators) that act synergistically, and demonstrate its application for reprogramming of fibroblasts to prostate tissue. We use three such master regulators (FOXA1, NKX3.1 and androgen receptor, AR) in a primed conversion strategy starting from mouse fibroblasts, resulting in prostate tissue grafts with appropriate histological and molecular properties that respond to androgen-deprivation. Moreover, generation of reprogrammed prostate does not require traversal of a pluripotent state. Thus, we describe a general strategy by which cell types and tissues can be generated even with limited knowledge of the developmental pathways required for their specification in vivo., The identification of master regulator genes that may be manipulated in vitro to regulate reprogramming has been difficult. Here, the authors use a computational systems approach to identify three genes (FoxA1, Nkx3.1 and the androgen receptor) that can reprogramme fibroblasts to prostate tissue.

Motivation Gene expression and regulation, a key molecular mechanism driving human disease development, remains elusive, especially at early stages. Integrating the increasing amount of population-level genomic data and understanding gene regulatory mechanisms in disease development are still challenging. Machine learning has emerged to solve this, but many machine learning methods were typically limited to building an accurate prediction model as a ‘black box’, barely providing biological and clinical interpretability from the box. Results To address these challenges, we developed an interpretable and scalable machine learning model, ECMarker, to predict gene expression biomarkers for disease phenotypes and simultaneously reveal underlying regulatory mechanisms. Particularly, ECMarker is built on the integration of semi- and discriminative-restricted Boltzmann machines, a neural network model for classification allowing lateral connections at the input gene layer. This interpretable model is scalable without needing any prior feature selection and enables directly modeling and prioritizing genes and revealing potential gene networks (from lateral connections) for the phenotypes. With application to the gene expression data of non-small-cell lung cancer patients, we found that ECMarker not only achieved a relatively high accuracy for predicting cancer stages but also identified the biomarker genes and gene networks implying the regulatory mechanisms in the lung cancer development. In addition, ECMarker demonstrates clinical interpretability as its prioritized biomarker genes can predict survival rates of early lung cancer patients (P-value < 0.005). Finally, we identified a number of drugs currently in clinical use for late stages or other cancers with effects on these early lung cancer biomarkers, suggesting potential novel candidates on early cancer medicine. Availabilityand implementation ECMarker is open source as a general-purpose tool at https://github.com/daifengwanglab/ECMarker. Supplementary information Supplementary data are available at Bioinformatics online.

SummaryIdentification of driver mutations in human diseases is often limited by cohort size and availability of appropriate statistical models. We propose a framework for the systematic discovery of genetic alterations that are causal determinants of disease, by prioritizing genes upstream of functional disease drivers, within regulatory networks inferred de novo from experimental data. We tested this framework by identifying the genetic determinants of the mesenchymal subtype of glioblastoma. Our analysis uncovered KLHL9 deletions as upstream activators of two previously established master regulators of the subtype, C/EBPβ and C/EBPδ. Rescue of KLHL9 expression induced proteasomal degradation of C/EBP proteins, abrogated the mesenchymal signature, and reduced tumor viability in vitro and in vivo. Deletions of KLHL9 were confirmed in > 50% of mesenchymal cases in an independent cohort, thus representing the most frequent genetic determinant of the subtype. The method generalized to study other human diseases, including breast cancer and Alzheimer’s disease.

The p53 family member p73 is essential for brain development, but its precise role and scope remain unclear. Global p73 deficiency determines an overt and highly penetrant brain phenotype marked by cortical hypoplasia with ensuing hydrocephalus and hippocampal dysgenesis. The ΔNp73 isoform is known to function as a prosurvival factor of mature postmitotic neurons. In this study, we define a novel essential role of p73 in the regulation of the neural stem cell compartment. In both embryonic and adult neurogenesis, p73 has a critical role in maintaining an adequate neurogenic pool by promoting self-renewal and proliferation and inhibiting premature senescence of neural stem and early progenitor cells. Thus, products of the p73 gene locus are essential maintenance factors in the central nervous system, whose broad action stretches across the entire differentiation arch from stem cells to mature postmitotic neurons.

Udgivelsesdato: 2009-Aug p63, an ancestral transcription factor of the p53 family, has three C-terminal isoforms whose relative in vivo functions are elusive. The p63 gene is essential for skin and limb development, as vividly shown by two independent global knockout mouse models. Both strains, although constructed differently, have identical and severe phenotypes, characterized by absent epidermis and hindlimbs and only rudimentary forelimbs at birth. Here we show that mice from one model, Brdm2, express normal levels of truncated p63 proteins that contain the DNA binding and oligomerization domain but lack the long carboxy-terminal SAM (sterile alpha-motif) and post-SAM domains that are specific for the alpha and beta isoforms. As such, transcriptionally active p63 proteins from Brdm2 mice resemble the naturally occurring p63gamma isoforms, which of all the p63 isoforms most closely resemble p53. Thus, Brdm2 mice are p63alpha/beta isoform-specific knockout mice, gaining unexpected new importance. Our studies identify that p63alpha/beta but not p63gamma are absolutely required for proper skin and limb development.

In response to our recent report on the Brdm2 mouse model for p63 function1, Mikkola et al.2 raise concerns on three of our statements: 1) They maintain their initial claim that no functional p63 protein of any kind is synthesized in mice homozygous for the Brdm2 allele; 2) they report the occurrence of spontaneous wild-type allelic reversions in these mice, leading to patches of completely normal epidermis, and propose that these normal reverted skin patches are in fact the multilayered epithelia we described; 3) they insist that the Brdm2 allele is phenotypically indistinguishable from a global p63 knockout.

The ancient p53 paralogs p63 and p73 regulate specific tissue formation, cell survival and cell death via their TA and ΔN isoforms. Targeted disruption of the p73 locus leads to severe defects in the development of the central nervous system (CNS), and p73 has recently been shown to be an essential regulator of neural stem cell maintenance and differentiation in both embryonal and adult neurogenesis. In contrast, global p63-/- mice lack skin and limbs. Moreover, p63 is detectable in embryonic cortex. It has previously been proposed to also play critical pro-death and pro-survival roles in neural precursors of the developing sympathetic and central nervous system, respectively, based on experimental overexpression and siRNA-mediated knockdown of p63. Here we perform an extensive analysis of the developing central nervous system in global p63-/- mice and their wildtype littermates. Brain and spinal cord of embryos and newborn mice were assessed in vivo for neuroanatomy, histology, apoptosis, proliferation, stemness and differentiation, and in vitro for self-renewal and maturation in neurosphere assays. None of these analyses revealed a detectable phenotype in p63-/- mice. Hence, despite the profound impact of p63 on the development of stratified epithelia and limbs, p63 is completely dispensable for proper development of the central nervous system. Thus, despite their strong homology, the non-overlapping tissue specificity of p63 and p73 functions appears more pronounced than previously anticipated.

Previous studies showed that p53 plays a central role in G1 and DNA damage checkpoints, thus contributing to genomic stability. We show here that p73 also plays a role in genomic integrity but this mechanism is manifest only when p53 is lost. Isolated p73 loss in primary cells does not induce genomic instability. Instead, it results in impaired proliferation and premature senescence due to compensatory activation of p53. Combined loss of p73 and p53 rescues these defects, but at the expense of exacerbated genomic instability. This leads to rapid increase in polyploidy and aneuploidy, markedly exceeding that of p53 loss alone. Constitutive deregulation of cyclin-Cdk activities and excess failure of the G2/M DNA damage checkpoint appear to fuel increased ploidy abnormalities upon p53/p73 loss, while primary mitotic defects do not play a causal role. These data indicate that p73 is essential for suppressing polyploidy and aneuploidy when p53 is inactivated.