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.

Human monocyte to macrophage differentiation is accompanied by pronounced phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are poorly understood. Here, we studied the regulation of the macrophage-specific chitotriosidase (CHIT1) gene promoter to gain insights into the mechanisms of transcriptional control during the differentiation of human blood monocytes into macrophages. We used transient transfections to define a cell type-specific minimal promoter that was mainly dependent on a proximal C/EBP motif that bound multiple C/EBP factors in gel shift assays. In depth analysis of occupied promoter elements using in vivo footprinting and chromatin immunoprecipitation analyses demonstrated the differentiation-associated recruitment of C/EBP␤ and PU.1 at the proximal promoter in parallel with CHIT1 mRNA induction. Notably, the induction of C/EBP␤ promoter binding strongly correlated with increased nuclear levels of Thr-235-phosphorylated C/EBP␤ protein during the differentiation process, whereas C/EBP␤ mRNA and total protein expression remained relatively stable. Our data suggest an important constitutive gene regulatory function for C/EBP␤ in differentiated macrophages but not in human blood monocytes.Peripheral blood monocytes are able to differentiate into morphologically and functionally divergent effector cells, including macrophages, myeloid dendritic cells, and osteoclasts (1). These cell types together with their bone marrow progenitors constitute the mononuclear phagocyte system, a widely used model to study cellular differentiation, lineage commitment, and mechanisms of cell type-specific gene regulation. Extensive research during the last two decades revealed several transcription factors that are important for lineage commitment and subsequent differentiation of myeloid progenitor cells as well as myeloid-specific gene regulation. These include members of the Ets-, CCAAT-enhancer-binding protein (C/EBP) 2 , and core binding factor (CBF) families and several other transcription factors (2, 3). In particular the Ets family transcription factor PU.1 and C/EBP family members C/EBP␣ and C/EBP␤ represent master regulators of the myeloid lineage. PU.1 is known to be essential for normal monocyte/macrophage differentiation and cell type-specific gene expression (4 -6), and both C/EBP family members are implicated in myeloid development and myeloid gene expression and are capable of reprogramming committed B-or T-cell progenitors to macrophages (7-9). In humans, the differentiation process of progenitor cells into monocyte/macrophage-like cells has been studied mainly using myeloid progenitor cell lines like HL-60, U937, and THP-1, which acquire monocyte/macrophage-like phenotypes upon vitamin D3 or phorbol ester treatment (10). However, the above cell lines, treated or untreated, differ from primary macrophages (11) and are not necessarily well suited to investigate the transition of primary monocytes into macrophages, which generally proceed...

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3875 The differentiation of human macrophages is accompanied by distinctive phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are still poorly understood. Using ChIP-Seq technology we studied epigenetic changes as well as alterations in transcription factor occupancy during human monocyte differentiation and correlated these events with gene expression levels in hematopoietic cell types. We show that putative enhancer regions marked by histone H3 lysine4 monomethylation (H3K4me1) at different developmental stages (human progenitor cells, peripheral blood monocytes and in vitro differentiated macrophages) are enriched in distinct sets of transcription factor motifs corresponding to lineage-determining factors. Cell stage-specific histone methylation at promoter-distal sites corresponds with increased mRNA expression levels of neighboring genes. We generated global DNA-binding maps in monocytes and macrophages for two transcription factors (PU.1 and C/EBPβ) with a well established role in monocyte/macrophage differentiation. Comparison of human binding sites with corresponding mouse data revealed a surprisingly low level of conservation (∼10-15%) of PU.1-or C/EBPβ -bound sites between man and mouse, despite a highly conserved binding preference for both transcription factors. During monocytic differentiation, human macrophages primarily gained additional binding sites for both transcription factors (as well as promoter-distal H3K4me1). Interestingly, only neighboring genes with multiple binding events showed significantly increased, macrophage-specific mRNA expression as compared to monocytic as well as lymphocytic cell types. Human macrophage-specific H3K4me1-marked regions as well as macrophage-specific PU.1- and C/EBP-bound sites were characterized by overlapping sets of novel sequence motifs, suggesting that the combinatorial interaction of corresponding DNA-binding factors with PU.1 and C/EBPβ may be required for the establishment of human macrophage-specific enhancers. These data provide novel insights into PU.1 and C/EBPβ mediated gene regulation during human macrophage differentiation. Disclosures: No relevant conflicts of interest to declare.