Allele-specific DNA methylation in mouse strains is mainly determined by cis-acting sequences.

DNA methylation is a vital epigenetic mark that participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. Differences in epigenetic patterns between individuals may contribute to phenotypic variation and disease susceptibility; however, little is known about the extent of such variation or how different epigenetic patterns are established. Here we have compared DNA methylation profiles of macrophages from two inbred mouse strains (C57BL/6 and BALB/c) at 181 large genomic intervals that were selected based on differential gene expression patterns. Using a DNA methylation-dependent fractionation approach based on a combination of methyl-CpG immunoprecipitation and locus-wide tiling arrays, we identified several hundred differentially methylated regions, and simultaneously uncovered previously unrecognized genetic variability between both mouse strains at the studied loci. DNA sequence and methylation differences were validated by DNA sequencing and mass spectrometry analysis of bisulfite-treated DNA for a subset of regions. Importantly, we show that in F1 hybrids, the majority of strain-specific methylation patterns in somatic cells were maintained on the parental allele, regardless of their status in the male germ line. The common association of differentially methylated regions with sequence polymorphisms suggests that the genomic context determines the developmentally regulated epigenetic status at most nonimprinted regions of mammalian genomes.

Comprehensive analysis of TLR4-induced transcriptional responses in interleukin 4-primed mouse macrophages.

Interferon (IFN)gamma and interleukin (IL)-4 are central regulators of T helper 1 (Th1) and T helper 2 (Th2) immune responses, respectively. Both cytokines have a major impact on macrophage phenotypes: IFNgamma-priming and subsequent TLR4 activation induces so-called "classically activated" macrophages that are characterized by pronounced pro-inflammatory responses, whereas IL-4-treated macrophages, commonly called "alternatively activated", are known to develop enhanced capacity for endocytosis, antigen presentation and tissue repair and are generally considered anti-inflammatory. Considering IL-4 as priming rather than activating stimulus, we now compared the TLR4-dependent global gene activation program in IFNgamma- versus IL-4-pretreated mouse macrophages, which has rarely been studied so far. Although both cytokines frequently induced opposing effects on gene transcription, the subsequent activation of bone marrow-derived macrophages by lipopolysaccharide (LPS) produced a strong, priming-dependent pro-inflammatory response in both macrophage types. For example, the production of key pro-inflammatory cytokines IL-6 and IL-12 was significantly higher in IL-4- versus IFNgamma-primed macrophages and several cytokine genes, including Il19, Ccl17, Ccl22, Ccl24 and Cxcl5, were preferentially induced in "alternatively" primed and LPS activated mouse macrophages. In a subset of genes, including IL12a, IFNgamma-priming was actually found to suppress LPS-induced gene expression in a Stat1-dependent manner. Our data suggest that IL-4-priming is not per se anti-inflammatory but generates a macrophage that is "tissue protective" but still capable of mounting a strong inflammatory response after TLR4-dependent activation.

Interleukin-4 induced interferon regulatory factor (Irf) 4 participates in the regulation of alternative macrophage priming.

Interleukin (IL)-4 is a central regulator of T helper 2 (Th2) immune responses, and also has a major impact on innate immune cells. This cytokine primes macrophages for immune responses to parasites and induces a distinct macrophage phenotype that may also promote tissue repair. IL-4 signaling in macrophages is primarily mediated by the transcription factor signal transducer and activator of transcription 6 (Stat6), which in turn regulates a number of secondary DNA binding proteins that may participate in shaping the resulting phenotype. The impact of secondary transcription factors on IL-4-treated macrophages, however, is largely unknown. Here we show that interferon regulatory factor 4 (Irf4) is strongly induced on RNA and protein level in bone marrow-derived macrophages upon priming with IL-4. Using microarray-based whole genome expression analysis, we also demonstrate that a subset of IL-4 regulated genes, including several MHC-II genes, Ciita, Cyp1b1, and Il1rn, are dysregulated in Irf4-deficient macrophages. The presented data suggests a non-redundant role for Irf4 in shaping the phenotype of alternatively primed macrophages.

Transcriptional effects of colony-stimulating factor-1 in mouse macrophages.

Colony-stimulating factor-1 (CSF-1) is a major regulator of macrophage development. CSF-1-dependent signalling has been implicated in proliferation, survival, and differentiation of mononuclear phagocytes, however, relatively little is known about the effects of CSF-1 on macrophage gene transcription and on CSF-1-responsive gene promoters. We used a combination of transcription profiling and in silico motif search to characterize genes that are regulated in mature bone marrow-derived macrophages cultured in the presence or absence of CSF-1. The expression of many known differentiation-associated macrophage markers was not significantly affected in the absence of CSF-1. Genes repressed by CSF-1 comprised a considerable number of granulocyte-specific genes. The respective gene promoters; however, were not significantly enriched for specific DNA patterns, suggesting that these genes are regulated by promoter-distal elements or at a post-transcriptional level. Genes downregulated upon CSF-1 deprivation showed a highly significant association with cell division which is in line with the known role of CSF-1 as a proliferation stimulus for mouse macrophages. Interestingly, three DNA patterns were significantly co-enriched in CSF-1-dependent gene promoters, including motifs related to NFY, CHR, and E2F sites. These motifs showed a strong positional preference on target promoters at -60, -30 and 0 bp upstream of the transcription start site, and define the common promoter structure of CSF-1-responsive genes.

CCAAT enhancer-binding protein beta regulates constitutive gene expression during late stages of monocyte to macrophage differentiation.

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/EBPbeta and PU.1 at the proximal promoter in parallel with CHIT1 mRNA induction. Notably, the induction of C/EBPbeta promoter binding strongly correlated with increased nuclear levels of Thr-235-phosphorylated C/EBPbeta protein during the differentiation process, whereas C/EBPbeta mRNA and total protein expression remained relatively stable. Our data suggest an important constitutive gene regulatory function for C/EBPbeta in differentiated macrophages but not in human blood monocytes.

Transcription factor Tfec contributes to the IL-4-inducible expression of a small group of genes in mouse macrophages including the granulocyte colony-stimulating factor receptor.

Expression of the mouse transcription factor EC (Tfec) is restricted to the myeloid compartment, suggesting a function for Tfec in the development or function of these cells. However, mice lacking Tfec develop normally, indicating a redundant role for Tfec in myeloid cell development. We now report that Tfec is specifically induced in bone marrow-derived macrophages upon stimulation with the Th2 cytokines, IL-4 and IL-13, or LPS. LPS induced a rapid and transient up-regulation of Tfec mRNA expression and promoter activity, which was dependent on a functional NF-kappaB site. IL-4, however, induced a rapid, but long-lasting, increase in Tfec mRNA, which, in contrast to LPS stimulation, also resulted in detectable levels of Tfec protein. IL-4-induced transcription of Tfec was absent in macrophages lacking Stat6, and its promoter depended on two functional Stat6-binding sites. A global comparison of IL-4-induced genes in both wild-type and Tfec mutant macrophages revealed a surprisingly mild phenotype with only a few genes affected by Tfec deficiency. These included the G-CSFR (Csf3r) gene that was strongly up-regulated by IL-4 in wild-type macrophages and, to a lesser extent, in Tfec mutant macrophages. Our study also provides a general definition of the transcriptome in alternatively activated mouse macrophages and identifies a large number of novel genes characterizing this cell type.