SUMMARY Enhancers control the correct temporal and cell type-specific activation of gene expression in higher eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. We use the FANTOM5 panel of samples covering the majority of human tissues and cell types to produce an atlas of active, in vivo transcribed enhancers. We show that enhancers share properties with CpG-poor mRNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, identify disease-associated regulatory single nucleotide polymorphisms, and classify cell type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell type-specific enhancers and gene regulation.

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The protein product of the CHI3L1 gene, human cartilage 39-kDa glycoprotein (HC-gp39), is a tissue-restricted, chitin-binding lectin and member of glycosyl hydrolase family 18. In contrast to many other monocyte/macrophage markers, its expression is absent in monocytes and strongly induced during late stages of human macrophage differentiation. To gain insights into the molecular mechanisms underlying its cell typerestricted and maturation-associated expression in macrophages, we initiated a detailed study of the proximal HC-gp39 promoter. Deletion analysis of reporter constructs in macrophage-like THP-1 cells localized a region directing high levels of macrophage-specific reporter gene expression to ϳ300 bp adjacent to the major transcriptional start site. The promoter sequence contained consensus binding sites for several known factors, and specific binding of nuclear PU.1, Sp1, Sp3, USF, AML-1, and C/EBP proteins was detectable in gel shift assays. In vivo footprinting assays with dimethyl sulfate demonstrate that the protection of corresponding sequences was enhanced in macrophages compared with monocytes. Mutational analysis of transcription factor binding sites indicated a predominant role for a single Sp1 binding site in regulating HC-gp39 promoter activity. In addition, gel shift assays using nuclear extracts of monocytes and macrophages demonstrated that the binding of nuclear Sp1, but not Sp3, markedly increases during macrophage differentiation. Our results further highlight the important role of Sp1 in macrophage gene regulation.

Cellular differentiation is orchestrated by lineage-specific transcription factors and associated with cell type-specific epigenetic signatures. In the present study, we used stage-specific, epigenetic "fingerprints" to deduce key transcriptional regulators of the human monocytic differentiation process. We globally mapped the distribution of epigenetic enhancer marks (histone H3 lysine 4 monomethylation, histone H3 lysine 27 acetylation, and the histone variant H2AZ), describe general properties of marked regions, and show that cell type-specific epigenetic "fingerprints" are correlated with specific, de novo-derived motif signatures at all of the differentiation stages studied (ie, hematopoietic stem cells, monocytes, and macrophages). We validated the novel, de novo-derived, macrophage-specific enhancer signature, which included ETS, CEBP, bZIP, EGR, E-Box and NF-B motifs, by ChIP sequencing for a subset of motif corresponding transcription factors (PU.1, C/EBP␤, and EGR2), confirming their association with differentiationassociated epigenetic changes. We describe herein the dynamic enhancer landscape of human macrophage differentiation, highlight the power of genome-wide epigenetic profiling studies to reveal novel functional insights, and provide a unique resource for macrophage biologists. IntroductionHuman monocyte-to-macrophage differentiation is a process involving marked morphologic, functional, and transcriptional changes that proceed in the absence of proliferation. The mechanisms controlling this transition are not well understood on the molecular level, in part because both human monocytes and macrophages are hard to manipulate without triggering defense programs that interfere with normal differentiation.Recent global epigenetic and transcription factor profiling studies in various cell types have provided ample evidence for a tight relationship between transcription factor binding and the local deposition/removal of some epigenetic marks, including histone methylation or acetylation, the appearance of histone variants, or DNA demethylation. 1 Cell type-specific epigenetic signatures are particularly evident at promoter-distal sites, where histone H3K4 monomethylation/dimethylation, 2,3 histone H3K27 acetylation, 3,4 the histone variant H2AZ, 2 or DNA demethylation 5,6 indicate the presence of poised or activated lineage-specific enhancer elements. These distal regulatory elements are often cell type-specific, are correlated with gene expression, and are bound by combinations of common and cell type-specific key regulators. 1,7 For example, in the murine hematopoietic system, macrophage-specific putative enhancer elements are characterized by PU.1, C/EBP␣/␤, and AP-1 binding, whereas putative enhancer elements in a related blood-cell type (murine B cells) that are also characterized by PU.1 binding associate with a distinct set of B cell-specific factors, including E2A, EBF, and OCT2. 8 Observations of correlating transcription factor binding and epigenetic patterns were also made in other cellul...