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.

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.

The methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. Because aberrant hypermethylation may be used as a marker for disease, a sensitive method for the global detection of DNA methylation events is of particular importance. We describe a novel and robust technique, called methyl-CpG immunoprecipitation, which allows the unbiased genome-wide profiling of CpG methylation in limited DNA samples. The approach is based on a recombinant, antibody-like protein that efficiently binds native CpG-methylated DNA. In combination with CpG island microarrays, the technique was used to identify >100 genes with aberrantly methylated CpG islands in three myeloid leukemia cell lines. Interestingly, within all hypermethylation targets, genes involved in transcriptional regulation were significantly overrepresented. More than half of the identified genes were absent in microarray expression studies in either leukemia or normal monocytes, indicating that hypermethylation in cancer may be largely independent of the transcriptional status of the affected gene. Most individually tested genes were also hypermethylated in primary blast cells from acute myeloid leukemia patients, suggesting that our approach can identify novel potential disease markers. The technique may prove useful for genome-wide comparative methylation analysis not only in malignancies. (Cancer Res 2006; 66(12): 6118-28)

Aberrant DNA methylation at CpG islands is thought to contribute to cancer initiation and progression, but mechanisms that establish and maintain DNA methylation status during tumorigenesis or normal development remain poorly understood. In this study, we used methyl-CpG immunoprecipitation to generate comparative DNA methylation profiles of healthy and malignant cells (acute leukemia and colorectal carcinoma) for human CpG islands across the genome. While searching for sequence patterns that characterize DNA methylation states, we discovered several nonredundant sequences in CpG islands that were resistant to aberrant de novo methylation in cancer and that resembled consensus binding sites for general transcription factors (TF). Comparing methylation profiles with global CpG island binding data for specific protein 1, nuclear respiratory factor 1, and yin-yang 1 revealed that their DNA binding activity in normal blood cells correlated strictly with an absence of de novo methylation in cancer. In addition, global evidence showed that binding of any of these TFs to their consensus motif depended on their co-occurrence with neighboring consensus motifs. In summary, our results had two major implications. First, they pointed to a major role for cooperative binding of TFs in maintaining the unmethylated status of CpG islands in health and disease. Second, our results suggest that the majority of de novo methylated CpG islands are characterized by the lack of sequence motif combinations and the absence of activating TF binding.