A promoter-level mammalian expression atlas.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

Estimating the costs of genomic sequencing in cancer control.

BACKGROUND: Despite the rapid uptake of genomic technologies within cancer care, few studies provide detailed information on the costs of sequencing across different applications. The objective of the study was to examine and categorise the complete costs involved in genomic sequencing for a range of applications within cancer settings. METHODS: We performed a cost-analysis using gross and micro-costing approaches for genomic sequencing performed during 2017/2018 across different settings in Brisbane, Australia. Sequencing was undertaken for patients with lung, breast, oesophageal cancers, melanoma or mesothelioma. Aggregated resource data were captured for a total of 1433 patients and point estimates of per patient costs were generated. Deterministic sensitivity analyses addressed the uncertainty in the estimates. Estimated costs to the public health system for resources were categorised into seven distinct activities in the sequencing process: sampling, extraction, library preparation, sequencing, analysis, data storage and clinical reporting. Costs were also aggregated according to labour, consumables, testing, equipment and 'other' categories. RESULTS: The per person costs were AU$347-429 (2018 US$240-297) for targeted panels, AU$871-$2788 (2018 US$604-1932) for exome sequencing, and AU$2895-4830 (2018 US$2006-3347) for whole genome sequencing. Cost proportions were highest for library preparation/sequencing materials (average 76.8% of total costs), sample extraction (8.1%), data analysis (9.2%) and data storage (2.6%). Capital costs for the sequencers were an additional AU$34-197 (2018 US$24-67) per person. CONCLUSIONS: Total costs were most sensitive to consumables and sequencing activities driven by commercial prices. Per person sequencing costs for cancer are high when tumour/blood pairs require testing. Using the natural steps involved in sequencing and categorising resources accordingly, future evaluations of costs or cost-effectiveness of clinical genomics across cancer projects could be more standardised and facilitate easier comparison of cost drivers.

Mincle polarizes human monocyte and neutrophil responses to Candida albicans.

The distribution and function of the C-type lectin Mincle has not previously been investigated in human cells, although mouse models have demonstrated a non-redundant role for Mincle in the host response to fungal infections. This study identified an unusual pattern of reciprocal expression of Mincle on peripheral blood monocytes or neutrophils isolated from the same donor. Expression on monocytes was inversely correlated with phagocytosis and yeast killing, but was necessary for the induction of inflammatory cytokines in response to ex vivo Candida challenge. In contrast, Mincle expression on neutrophils was associated with phagocytic and candidacidal potential of those cells. Candida challenge upregulated Mincle expression but only in Mincle+ cells. These data highlight species-specific differences between the regulation of Mincle expression in mouse and man. Reciprocal expression of Mincle modified the candidacidal potential of monocytes or neutrophils, suggesting it may also polarize the type of host response to fungal infection.

Colony-stimulating factor-1 promotes kidney growth and repair via alteration of macrophage responses.

Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.

Diversification of TOLLIP isoforms in mouse and man.

The Toll-interacting protein TOLLIP is an ubiquitin-binding protein that interacts with several components of the Toll-like receptor signaling cascade. The canonical protein consists of three annotated domains: an N-terminal TBD-loop-coil domain that mediates protein-protein interactions, a C2 domain that targets TOLLIP to the endosome, and a CUE domain at the C-terminus that binds monoubiquitin. TOLLIP has been described primarily in trafficking of the interleukin-1 receptor (IL1R) and turnover of the interleukin-1 receptor-associated kinase (IRAK), so it is an essential regulator of inflammatory signaling. Here we describe the expression of numerous alternate transcripts from mouse and human TOLLIP, which are predicted to generate at least five variant proteins between the two species. Most of the variant proteins are predicted to have altered N-terminal domains, altered TBD-loop-coil domains, or a truncated C2 domain. A mouse-specific variant arises from an alternate termination exon, and the resulting protein lacks the CUE domain. Two transcripts arising from alternate initiating exons are highly conserved between mouse and human but exhibit different patterns of expression. The consequent protein isoforms retain (TOLLIP.A) or lack (TOLLIP.D) the protein-binding TBD, so are predicted to traffic monoubiquitinated proteins to alternate protein complexes within the endosomal compartment. In summary, the widespread and inducible expression of Tollip isoforms predicts diversification of its function in rodent and human immune systems. Alternate splicing of critical signaling molecules such as Tollip may provide one mechanism behind the broad repertoire of responses generated by cells of the innate immune system in response to infection.

Zinc starvation induces a stress response in Saccharomyces cerevisiae that is mediated by the Msn2p and Msn4p transcriptional activators.

During the production of wine and beer, the yeast Saccharomyces cerevisiae can encounter an environment that is deficient in zinc, resulting in a 'sluggish' or a 'stuck' ferment. It has been shown that the Zap1p-transcription factor induces the expression of a regulon in response to zinc deficiency; however, it was evident that a separate regulon was also activated during zinc deficiency in a Zap1p-independent manner. This study discovered the Msn2p and Msn4p (Msn2/4p) transcriptional activator proteins to be an additional control mechanism inducing the stress response during zinc deficiency. Promoter sequence analysis identified the stress-response element (STRE) motif, recognized by Msn2/4p, and was significantly enriched in the promoters of genes induced by zinc deficiency. An investigation using genome-wide analyses revealed a distinct regulon consisting of STRE-containing genes whose zinc-responsive expression was abolished in an msn2 msn4 double mutant. An STRE-driven lacZ reporter construct confirmed that expression of the genes within this regulon was perturbed by the deletion of MSN2 and MSN4 and also implicated Hog1p as a contributing factor. This research provides a better understanding of the molecular mechanisms involved in the yeast response to zinc deficiency during fermentation.

Human and mouse macrophage-inducible C-type lectin (Mincle) bind Candida albicans.

Candida albicans is a causative agent in mycoses of the skin, oral cavity, and gastrointestinal tract. Identification of receptors, and their respective ligands, that are engaged by immune cells when in contact with C. albicans is crucial for understanding inflammatory responses leading to invasive candidiasis. Mincle is a recently identified macrophage-expressed receptor that is important for host responses to C. albicans. The carbohydrate-recognition domain of human and mouse Mincle were expressed, purified under denaturing conditions, and successfully refolded. In addition to oligomers, there are isolatable monomeric and dimeric forms of the protein that occur under two different buffer solutions. The human and mouse homologues bound yeast extract, and the isolated dimeric and monomeric species also demonstrated the recognition of whole C. albicans yeast cells. The data are indicative of several functional states mediating the interaction of Mincle and yeast at the surface of the macrophage.

FANTOM5 CAGE profiles of human and mouse samples.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

Disease-specific, neurosphere-derived cells as models for brain disorders.

There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson's disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson's disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery.

Essential role of one-carbon metabolism and Gcn4p and Bas1p transcriptional regulators during adaptation to anaerobic growth of Saccharomyces cerevisiae.

The transcriptional activator Gcn4p is considered the master regulator of amino acid metabolism in Saccharomyces cerevisiae and is required for the transcriptional response to amino acid starvation. Here it is shown that Gcn4p plays a previously undescribed role in regulating adaptation to anaerobic growth. A gcn4 mutant exhibited a highly extended lag phase after a shift to anaerobiosis that was the result of l-serine depletion. In addition, the one-carbon metabolism and purine biosynthesis transcriptional regulator Bas1p were strictly required for anaerobic growth on minimal medium, and this was similarly due to l-serine limitation in bas1 mutants. The induction of one-carbon metabolism during anaerobiosis is needed to increase the supply of l-serine from the glycine and threonine pathways. Using a number of experimental approaches, we demonstrate that these transcription regulators play vital roles in regulating l-serine biosynthesis in the face of increased demand during adaptation to anaerobiosis. This increased l-serine requirement is most likely due to anaerobic remodeling of the cell wall, involving de novo synthesis of a large number of very serine-rich mannoproteins and an increase in the total serine content of the cell wall. During anaerobic starvation for l-serine, this essential amino acid is preferentially directed to the cell wall, indicating the existence of a regulatory mechanism to balance competing cellular demands.

The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line.

Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.

The adaptive response of anaerobically grown Saccharomyces cerevisiae to hydrogen peroxide is mediated by the Yap1 and Skn7 transcription factors.

The molecular mechanisms involved in the ability of cells to adapt and respond to differing oxygen tensions are of great interest to the pharmaceutical, medical and fermentation industries. The transcriptional profiles reported in previous studies of cells grown under anaerobic, aerobic and dynamic growth conditions have shown significantly altered responses including induction of genes regulated by the oxidative stress transcription factor Yap1p when oxygen was present. The present study investigated the phenotypic changes that occur in cells when shifted from anaerobic to aerobic growth conditions and it was found through mutant analyses that the elevated activity of Yap1p during the shift was mediated by the phospholipid hydroperoxide-sensing protein encoded by GPX3. Cell viability and growth rate were unaffected even though anaerobically grown cells were found to be hypersensitive to low doses of the oxidative stress-inducing compound hydrogen peroxide (H(2)O(2)). Adaptation to H(2)O(2) treatment was demonstrated to occur when anaerobically grown wild-type cells were aerated for a short time that was reliant on the Yap1p and Skn7p transcription factors.

The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans.

The recognition of carbohydrate moieties by cells of the innate immune system is emerging as an essential element in antifungal immunity, but despite the number and diversity of lectins expressed by innate immune cells, few carbohydrate receptors have been characterized. Mincle, a C-type lectin, is expressed predominantly on macrophages, and is here shown to play a role in macrophage responses to the yeast Candida albicans. After exposure to the yeast in vitro, Mincle localized to the phagocytic cup, but it was not essential for phagocytosis. In the absence of Mincle, production of TNF-alpha by macrophages was reduced, both in vivo and in vitro. In addition, mice lacking Mincle showed a significantly increased susceptibility to systemic candidiasis. Thus, Mincle plays a novel and nonredundant role in the induction of inflammatory signaling in response to C. albicans infection.

Resistance to hydrogen peroxide in Helicobacter pylori: role of catalase (KatA) and Fur, and functional analysis of a novel gene product designated 'KatA-associated protein', KapA (HP0874).

Helicobacter pylori infection elicits an aggressive inflammatory response that the bacterium is able to resist by virtue of its well-adapted antioxidant defence mechanisms. Catalase (KatA) appears to be a key enzyme in this resistance. Upstream of katA, a low-affinity ferric uptake regulator (Fur)-box has been identified. Downstream of katA, an ORF (HP0874) with no known function has also been identified. Non-polar isogenic mutants of katA, fur and HP0874 were constructed by allelic exchange. The impact of these mutations on the catalase activities and bacterial viability following exposure to hydrogen peroxide was studied. Concurrently, the effect of variation in the iron content of the media used to grow the cells was determined. The data showed that catalase-deficient isolates of H. pylori were hypersensitive to hydrogen peroxide, whereas wild-type cells could resist approximately approximately 100 mM hydrogen peroxide. Fur-deficient mutants and cells grown on low-iron-containing medium showed a distinct reduction in catalase activity and increased sensitivity to hydrogen peroxide. The data suggest a direct or indirect effect of Fur and iron on the activity of catalase. HP0874-deficient mutants showed no reduction in catalase activity but showed an increased sensitivity to hydrogen peroxide. That is, the protein encoded by HP0874 appears to have a role in resistance to hydrogen peroxide not directly related to catalase activity. This is the first report of a functional relationship of the product of this ORF. There is evidence of protein-protein interaction between KatA and the product encoded by HP0874, and the name 'KatA-associated protein' (KapA) is proposed.

Yeast genome-wide expression analysis identifies a strong ergosterol and oxidative stress response during the initial stages of an industrial lager fermentation.

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.