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.

Long non-coding RNAs (lncRNAs) are largely heterogeneous and functionally uncharacterized. Here, using FANTOM5 cap analysis of gene expression (CAGE) data, we integrate multiple transcript collections to generate a comprehensive atlas of 27,919 human lncRNA genes with high-confidence 5' ends and expression profiles across 1,829 samples from the major human primary cell types and tissues. Genomic and epigenomic classification of these lncRNAs reveals that most intergenic lncRNAs originate from enhancers rather than from promoters. Incorporating genetic and expression data, we show that lncRNAs overlapping trait-associated single nucleotide polymorphisms are specifically expressed in cell types relevant to the traits, implicating these lncRNAs in multiple diseases. We further demonstrate that lncRNAs overlapping expression quantitative trait loci (eQTL)-associated single nucleotide polymorphisms of messenger RNAs are co-expressed with the corresponding messenger RNAs, suggesting their potential roles in transcriptional regulation. Combining these findings with conservation data, we identify 19,175 potentially functional lncRNAs in the human genome.

Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from non-target tissues is critical. Here, we achieve highly specific in vivo cancer visualization by employing a newly-designed targeted "activatable" fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pHactivatable probes based on the BODIPY fluorophore were synthesized, and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex and in vivo imaging of HER2-positive lung cancer cells in mice were performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energyconsuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific cell-surface-targeting molecules that result in cellular internalization.Genetic cell labeling techniques show the possibility of detecting or tracing a single cell in vivo [1][2][3] , however, currently available injectable molecular imaging probes are limited in their ability to detect small volumes of viable cancer because of low target-to-background ratios. Generally, small molecular probes lacks specificity and low target accumulation, in contrast, larger molecules shows prolonged high retention and background 4 . However, such largemolecular complexes are cleared slowly, so a considerable amount of unbound probe remains. These pharmacokinetic characteristics result in high background signal (Scheme 1a).In order to overcome this problem, we developed an activatable fluorescence probe consisting of: 1) a cancer targeting macromolecule and 2) a small-molecular fluorescent moiety activated only within cancer cells to minimize the background signal and maximize tumor-to-normal tissue (T/N) ratio (Scheme 1b).We targeted the human epidermal growth factor type 2 (HER2) receptor with the monoclonal antibody, trastuzumab which, after binding to HER2, is internalized via the endosomsallysosomal degradation pathway 5 .The lysosome is distinct from other cellular organelles because of its low pH (pH 5-6) relative to the cytoplasm (pH ∼7.4). By designing a probe that activates in an acidic environment, the agent yields a highly tumor specific signal with greatly reduced background signal (Scheme 1c). Results Development of tunable, acidic pH-activatable fluorescent moietyTo achieve signal activation within the acidic environment of the lysosome, we required smallmolecular fluorescent molecules with the following characteristics: 1) They should be almost non-fluorescent in the extracellular environment, i.e. at pH 7.4. 2) They should become highly fluorescent under acidic conditions, i.e. pH < 6. 3) They need to be excited by long-wavelength light (≥ 500 nm) and emit a fluorescent signal which overcomes autofluorescence. 4) ...

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.