Michael Rehli

An atlas of active enhancers across human cell types and tissues

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here 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 messenger RNA 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, to identify disease-associated regulatory single nucleotide polymorphisms, and to 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.

miRNA Expression Profiling in Melanocytes and Melanoma Cell Lines Reveals miRNAs Associated with Formation and Progression of Malignant Melanoma

Although deregulated expression of microRNAs (miRNAs) demonstrably contributes to the development and progression of all types of human cancers, little data are available about the changes in miRNA expression levels in malignant melanoma. In our study, we performed microarray-based miRNA profiling of melanocytes and melanoma cell lines derived from either primary tumors or metastatic melanomas. In addition, we analyzed miRNA expression patterns of melanoma cell clones in which the expression of melanoma specific genes was stably knocked down by antisense techniques. We also generated miRNA expression profiles for two derivatives of a melanoma cell line that differ in their invasive potential. Comparing miRNA expression patterns of melanocytes and subsets of melanoma cell lines, we identified large cohorts of miRNAs associated with malignant transformation as well as with the progression of the disease and with metastatic colonization. Surprisingly, the bulk of miRNAs that deregulated most strongly was not described to be of importance in tumor development before. The results of our study, therefore, not only provide insights into alterations in the miRnomes of melanocytes and melanoma cell lines during melanoma progression but also present a large assortment of miRNAs to be analyzed for their potential as diagnostic markers or targets for therapies in the future.

Functional Analysis of Promoter CPG-Methylation using a CpG-Free Luciferase Reporter Vector

Methylation of CpG-dinucleotides within proximal promoters is often associated with transcriptional silencing. Methylation-dependent repression is well established for hypermethylated CpG-island promoters that are characterized by a high density of CpG residues. The effect of CpG DNA methylation on CpG-poor promoters is less well characterized, probably due to the lack of convenient assay systems to test promoter activities in vitro. In this report, we describe a novel luciferase reporter vector, pCpGL, which completely lacks CpG dinucleotides and can be used to study the effect of promoter DNA methylation in transfection assays. Whereas a traditional reporter vector that contains a large number of backbone CpG residues significantly represses a CpG-free promoter when methylated, our new reporter vector is only repressed due to the presence of functionally important, methylated CpG residues. The pCpGL vector provides a useful tool to study the effects of CpG-methylation on CpG-rich and CpG-poor promoters.

Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity

DNA methylation participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. With few exceptions, research thus far has focused on gene promoters, and little is known about the extent, functional relevance, and regulation of cell type-specific DNA methylation at promoter-distal sites. Here, we present a comprehensive analysis of differential DNA methylation in human conventional CD4(+) T cells (Tconv) and CD4(+)CD25(+) regulatory T cells (Treg), cell types whose differentiation and function are known to be controlled by epigenetic mechanisms. Using a novel approach that is based on the separation of a genome into methylated and unmethylated fractions, we examined the extent of lineage-specific DNA methylation across whole gene loci. More than 100 differentially methylated regions (DMRs) were identified that are present mainly in cell type-specific genes (e.g., FOXP3, IL2RA, CTLA4, CD40LG, and IFNG) and show differential patterns of histone H3 lysine 4 methylation. Interestingly, the majority of DMRs were located at promoter-distal sites, and many of these areas harbor DNA methylation-dependent enhancer activity in reporter gene assays. Thus, our study provides a comprehensive, locus-wide analysis of lineage-specific methylation patterns in Treg and Tconv cells, links cell type-specific DNA methylation with histone methylation and regulatory function, and identifies a number of cell type-specific, CpG methylation-sensitive enhancers in immunologically relevant genes.

Gateways to the FANTOM5 promoter level mammalian expression atlas

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.

Genome-Wide Profiling of CpG Methylation Identifies Novel Targets of Aberrant Hypermethylation in Myeloid Leukemia

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.

Differential screening identifies genetic markers of monocyte to macrophage maturation.

Maturation of cells of the mononuclear phagocyte lineage from bone marrow precursors to tissue macrophages (MAC) via circulating blood monocytes (MO) is a multistep process only partially understood. Similarly, MAC differentiation can be observed if MO are cultured in vitro. In an attempt to further characterize molecular changes occurring during this process we carried out differential screening of a MO‐derived MAC cDNA library using MO and MAC cDNA. After subcloning and confirmation by a second round of screening, partial sequencing of 41 cDNA clones was performed. In 33 clones the sequences of 7 different previously identified cDNAs were found. The mRNA expression of two of the corresponding genes (apolipoprotein E, ferritin light chain) is already known to be up‐regulated during MAC maturation. For one gene (cathepsin B), a specific up‐regulation of mRNA expression could be shown corresponding to previous protein data. For four genes [human cartilage glycoprotein (HC‐gp39), osteopontin, type IV collagenase, and tryptophanyl‐tRNA synthetase] the specific expression in MAC versus MO was previously unknown but could be confirmed by the use of Northern blot analysis. Of these genes, HC‐gp39 is especially interesting because it is only expressed during the late stages of MAC differentiation. J. Leukoc. Biol. 60: 540–545; 1996.

Transcriptional regulation of CHI3L1, a marker gene for late stages of macrophage differentiation.

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 ∼300 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.

Transcriptional Regulation of the Human Toll-Like Receptor 2 Gene in Monocytes and Macrophages

This report investigates the molecular basis for tissue-restricted and regulated expression of the pattern recognition receptor Toll-like receptor (TLR)2 in human monocytes and macrophages. To define the proximal promoter, the full 5′-sequence and transcriptional start sites of TLR2 mRNA were determined. The human TLR2 gene was found to consist of two 5′ noncoding exons followed by a third coding exon. Alternative splicing of exon II was detected primarily in human blood monocytes. The proximal promoter, exon I, and part of intron I were found to be located in a CpG island. Although CpG methylation of the proximal human TLR2 promoter in cell lines correlated with TLR2 repression, the promoter was unmethylated in primary cells, indicating that CpG methylation does not contribute to the cell-type specific expression of human TLR2 in normal tissues. The promoter sequence contains putative binding sites for several transcription factors, including Sp1 and Ets family members. Reporter gene analysis revealed a minimal promoter of 220 bp that was found to be regulated by Sp1, Sp3, and possibly PU.1. Interestingly, no sequence homology was detected between human and murine TLR2 promoter regions. In contrast to murine TLR2, expression of human TLR2 in monocytes/macrophages is not induced by the proinflammatory stimuli LPS or macrophage-activating lipopeptide-2, and reporter activity of the promoter was not enhanced by stimuli-induced NF-κB activation in THP-1 or MonoMac-6 cells. Our findings provide an initial definition of the human TLR2 promoter and reveal profound differences in the regulation of an important pattern recognition molecule in humans and mice.

Evolution of the TIR, Tolls and TLRs: Functional Inferences from Computational Biology

The mammalian toll-like receptors (TLRs) are products of an evolutionary process that began prior to the separation of plants and animals. The most conserved protein motif within the TLRs is the TIR, which denotes Toll, the Interleukin-1 receptor, and plant disease Resistance genes. To trace the ancestry of the TLRs, it is desirable to draw upon the sequences of TIR domains from TLRs of diverse vertebrate species, including species with known dates of divergence (i.e., representatives of Mammalia and Aves) in order to establish a relationship between time and genetic divergence. It appears that a gene ancestral to modern TLRs 1 and 6 duplicated approximately 130 million years ago, only shortly before the speciation event that led to humans and mice. Though it is not represented in mice, TLR10 split from the TLR[1/6] precursor about 300 million years ago. The origins of other TLRs are more ancient, dating to the origins of vertebrate life, and some present-day vertebrate species appear to have many more TLRs than others. Moreover, the patterns of TLR expression are quite variable at the level of tissues, even among closely related species. A given TLR in species that are related by descent from a common ancestor may acquire different duties within each descendant line, so that some microbial inducers are avidly recognized in one species but not in others; likewise the intensity and the antomic location of an innate immune response may vary considerably. In this review, we discuss the computational methods used to analyze divergence of the TIR, and the conclusions that may be safely drawn.