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...

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)

The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the ‘rules’ for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25–45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells.