1. What have the authors contributed in "A promoter-level mammalian expression atlas author" ?
Supplementary Information is available in the online version of the paper.. Online Content Any additional Methods, Extended Data display items and Source Data are available in the online version of the paper ; references unique to these sections appear only in the online paper.. The manuscript was written by A. R. R. F. and D. A. H. with help from A. Sandelin, J. K. B., M. Rehli, H. K., M. J. L. dH., V. H., I. V. K., M. T. and K. M. S. with contributions, edits and comments from all authors.. The project was managed by Y. Hayashizaki, A. R. R. F., P. C., M. I., M. S., J. Kawai, C. O. D., H. Suzuki, T. L. and N. K.. The authors declare no competing financial interests.. Readers are welcome to comment on the online version of the paper.. 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.. To this end, the FANTOM5 project has performed cap analysis of gene expression ( CAGE ) 3 across 975 human and 399 mouse samples, including primary cells, tissues and cancer cell lines, using single-molecule sequencing3 ( Fig. 1 ; see the full sample list in Supplementary Table 1 ).. Moreover, the authors show in an accompanying manuscript4 that the data can be used to locate active enhancers, and to provide numerous insights into cell-type-specific transcriptional regulatory networks ( see the FANTOM5 website http: // fantom.. The data extend and complement the recently published ENCODE5 data, and microarray-based gene expression atlases6 to provide a major resource for functional genome annotation and for understanding the transcriptional networks underpinning mammalian cellular differentiation.. To identify CAGE peaks across the genome the authors developed decomposition-based peak identification ( DPI ; described in Supplementary Methods ; Extended Data Fig. 1 ).. To minimize the fraction of peaks3 that map to internal exons ( which could exist due to post-transcriptional cleavage and recapping of RNAs12 ), and enrich for TSSs, the authors applied tag evidence thresholds to define robust and permissive subsets ( described in more detail in Supplementary Methods and summarized in Table 1 ).. A flow diagram showing the relationship between samples, peaks, thresholding and subsets used in each analysis is provided in the Supplementary Figure 1.. Supporting evidence that the peaks are genuine TSSs, based upon support from expressed sequence tags ( ESTs ), histone H3 lysine 4 trimethylation ( H3K4Me3 ) marks and DNase hypersensitive sites is provided in Supplementary Note 2.. In comparison to the previous FANTOM3 and 4 projects, FANTOM5 measured expression at an additional 4,721 human and 5,127 mouse RefSeq genes.. The inclusion of primary cells, cell lines and tissues in the atlas provided greater coverage than any of the sample types alone ( Fig. 1d ) and the primary cell samples in particular were a rich source of unannotated peaks ( Fig. 1e ).. To enable comparative analysis, the authors projected the expression patterns from one species to the other ( Extended Data Fig. 4 ) and provide the peak position and orthologous expression profile through a cross-species track in ZENBU10.. Examining CGI and non-CGI promoters separately the authors find that cell-type-specific promoters of both classes were enriched for binding of cell-type-specific transcription factors ( evidenced by over-representation of motifs and bound sites in public ChIP-seq data sets ).. For the human hepatocellular carcinoma cell line HepG2 the authors observed enrichment of liver-specific transcription factors ( HNF4, FOXA2, and TCF7L2 ) at both CGI and non-CGI HepG2 specific promoters ( Extended Data Fig. 6b, c ; similar examples are shown in Extended Data Figs 5d and 7 ).. To demonstrate their likely relevance the authors systematically reviewed phenotypes of transcription factor knockout mice at the MGI ( see Supplementary Note 7 ).. Introducing sample ontology enrichment analysis ( SOEA ), the authors show that expression profiles can also be associated with cell, anatomical and disease ontology terms by testing for overrepresentation of terms in ranked lists of systematically annotated samples expressing each peak ( Extended Data Fig. 11 and Supplementary Methods ).. The precise phasing was supported further by the pattern of H2A.. This suggests a more rapidly evolving immune system.. It also suggests contributions of relaxed constraint and positive selection to the remodelling of transcription initiation through the insertion and deletion of promoter sequences.. For example, in mouse inner ear hair cells, knockout of six of the top 20 most enriched transcription factor genes in mouse ( Pou3f4 ( ref. 24 ), Sox2 ( ref. 25 ), Egr2, Six1 ( ref. 26 ), Fos27, Tbx18 ( ref. 28 ) ) as well as patient mutations in a further four top transcription factor genes ( POU4F3 ( ref. 29 ), ZIC2 ( ref. 30 ), SOX10 ( ref. 31 ), FOXF2 ( ref. 32 ) ) resulted in hearing-related defects.
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2. How many promoters were present in the KEGG pathway?
Of 19 promoters in coexpression group 413, eight were present in the KEGG pathway, including RIG-I (DDX58), the gene encoding the receptor for the mitochondrial antiviral signalling pathway53.
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3. How many de novo motifs were found in the MACRO-APE database?
The remaining 1,221 de novo motifs that were not similar to known motifs were then clustered using MACRO-APE, resulting in 169 unique novel motifs.
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4. What is the KEGG pathway for influenza A?
Annotated expression profiles of alternative promoters Overlay of coexpression groups enriched for genes involved in the KEGG pathway for influenza A pathogenesis (hsa:05164; FDR < 0.1, n > 2).
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