An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability.

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes.Research in the authors’ laboratories was supported by Bloodwise, The Wellcome Trust, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, the National Institute of Health Research, the Medical Research Council, the MRC Molecular Haematology Unit (Oxford) core award, a Weizmann-UK “Making Connections” grant (Oxford) and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research (100140) and Wellcome Trust–MRC Cambridge Stem Cell Institute (097922).This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.1146

Opportunities and challenges in sustainable treatment and resource reuse of sewage sludge: A review

Sludge or waste activated sludge (WAS) generated from wastewater treatment plants may be considered a nuisance. It is a key source for secondary environmental contamination on account of the presence of diverse pollutants (polycyclic aromatic hydrocarbons, dioxins, furans, heavy metals, etc.). Innovative and cost-effective sludge treatment pathways are a prerequisite for the safe and environment-friendly disposal of WAS. This article delivers an assessment of the leading disposal (volume reduction) and energy recovery routes such as anaerobic digestion, incineration, pyrolysis, gasification and enhanced digestion using microbial fuel cell along with their comparative evaluation, to measure their suitability for different sludge compositions and resources availability. Furthermore, the authors shed light on the bio-refinery and resource recovery approaches to extract value added products and nutrients from WAS, and control options for metal elements and micro-pollutants in sewage sludge. Recovery of enzymes, bio-plastics, bio-pesticides, proteins and phosphorus are discussed as a means to visualize sludge as a potential opportunity instead of a nuisance

Inhibition studies on hypoxia-inducible factor (HIF) hydroxylases

The hypoxia-inducible factor (HIF) is a key regulator of transcriptional responses to hypoxia in animals. As part of the cellular response to decreased oxygen concentrations, the transcriptional activity of a heterodimeric complex consisting of HIF&alpha; and HIF&beta; activates the expression of hundreds of target genes, including those involved in cellular growth, apoptosis, energy metabolism and angiogenesis. HIF&alpha; prolyl hydroxylation, as catalysed by the HIF prolyl hydroxylases (PHDs), leads to subsequent HIFα polyubiquitination and proteasomal degradation. HIF&alpha; asparaginyl hydroxylation as catalysed by factor inhibiting HIF (FIH), blocks the binding of HIF&alpha; to the co-activators CBP/p300, leading to reduced HIF transcriptional activity. The activities of the HIF hydroxylases (the PHDs and FIH) can be suppressed under limiting oxygen, resulting in the stabilisation of HIF&alpha; and activation of the HIF pathway. The development of PHD inhibitors in order to mimic aspects of the natural hypoxic response has been reported, although the selectivity of the inhibitors has not been investigated. Identification of selective small molecule inhibitors for the PHDs would enable further investigations into the differential role of the HIF hydroxylases in mediating the transcriptional response to hypoxia. Both the PHDs and FIH are part of the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenase family, which includes histone and nucleic acid demethylases that are involved in gene regulation. The transcriptional response to hypoxia and/or the effects of non-selective PHD inhibitors could thus be mediated by their effects on these closely related enzymes. In the work described in this thesis, in vitro hydroxylation assays for PHD2 were developed for the identification of PHD inhibitors and determination of their inhibitory potencies. The development of a cellular assay for HIF levels is also described and used to measure the efficacy of PHD inhibitors. The utilisation of these assays led to the identification of potent, selective and cell-permeable PHD inhibitors suitable for use as chemical probes to study the biological roles of the PHDs. To aid in selectivity studies with the PHD isoforms, a cellular model system was developed by the re-expression of individual PHD isoforms in PHD-null mouse embryonic fibroblast cells. PHD inhibitors, including one of the PHD chemical probes identified, were used in a pan-genomic study of the transcriptional response by hypoxia in human breast cancer MCF-7 cells. The results reveal that inhibition of the PHDs, together with FIH, does not fully induce the full set of hypoxia upregulated genes, suggesting that the activation of HIF transcriptional activity alone may not be sufficient to invoke the transcriptional response to hypoxia.</p

Inhibition studies on hypoxia-inducible factor (HIF) hydroxylases

The hypoxia-inducible factor (HIF) is a key regulator of transcriptional responses to hypoxia in animals. As part of the cellular response to decreased oxygen concentrations, the transcriptional activity of a heterodimeric complex consisting of HIFα and HIFβ activates the expression of hundreds of target genes, including those involved in cellular growth, apoptosis, energy metabolism and angiogenesis. HIFα prolyl hydroxylation, as catalysed by the HIF prolyl hydroxylases (PHDs), leads to subsequent HIFα polyubiquitination and proteasomal degradation. HIFα asparaginyl hydroxylation as catalysed by factor inhibiting HIF (FIH), blocks the binding of HIFα to the co-activators CBP/p300, leading to reduced HIF transcriptional activity. The activities of the HIF hydroxylases (the PHDs and FIH) can be suppressed under limiting oxygen, resulting in the stabilisation of HIFα and activation of the HIF pathway. The development of PHD inhibitors in order to mimic aspects of the natural hypoxic response has been reported, although the selectivity of the inhibitors has not been investigated. Identification of selective small molecule inhibitors for the PHDs would enable further investigations into the differential role of the HIF hydroxylases in mediating the transcriptional response to hypoxia. Both the PHDs and FIH are part of the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenase family, which includes histone and nucleic acid demethylases that are involved in gene regulation. The transcriptional response to hypoxia and/or the effects of non-selective PHD inhibitors could thus be mediated by their effects on these closely related enzymes. In the work described in this thesis, in vitro hydroxylation assays for PHD2 were developed for the identification of PHD inhibitors and determination of their inhibitory potencies. The development of a cellular assay for HIF levels is also described and used to measure the efficacy of PHD inhibitors. The utilisation of these assays led to the identification of potent, selective and cell-permeable PHD inhibitors suitable for use as chemical probes to study the biological roles of the PHDs. To aid in selectivity studies with the PHD isoforms, a cellular model system was developed by the re-expression of individual PHD isoforms in PHD-null mouse embryonic fibroblast cells. PHD inhibitors, including one of the PHD chemical probes identified, were used in a pan-genomic study of the transcriptional response by hypoxia in human breast cancer MCF-7 cells. The results reveal that inhibition of the PHDs, together with FIH, does not fully induce the full set of hypoxia upregulated genes, suggesting that the activation of HIF transcriptional activity alone may not be sufficient to invoke the transcriptional response to hypoxia.</p

An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability

Abstract Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes

Hydroxylation of HIFα and the chemical structures of IOX4 and other PHD inhibitors used in this study.

<p><b>(a)</b> Prolyl-hydroxylation (as catalyzed by the PHDs) of HIFα. <b>(b)</b> Structures of the dihydropyrazoles (<b>1</b> and <b>IOX4</b>) in comparison to structures of 2-oxoglutarate (<b>2OG</b>), <i>N</i>-oxalylglycine (<b>NOG</b>) (a catalytically inactive analogue of 2OG), dimethyloxalylglycine (<b>DMOG</b>) (a cell-permeable ester derivative of NOG) and <b>IOX2</b> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref009" target="_blank">9</a>]. Chemical structures of previously reported PHD inhibitors (compound <b>2</b>, bicyclic isoquinolinyl inhibitor <b>IOX3</b> and bicyclic naphthalenylsulfone hydroxythiazole <b>BNS</b>) used in this study are also shown.</p