The Yin and Yang of microRNAs: leukemia and immunity

Yin and Yang are two complementary forces that together describe the nature of real-world elements. Yin is the dark side; Yang is the light side. We describe microRNAs having both Yin and Yang characteristics because they can contribute to normal function (Yang) but also to autoimmunity, myeloproliferation, and cancer (Yin). We have been working on a number of microRNAs that have these dual characteristics and here we focus on two, miR-125b and miR-146a. We have concentrated on these two RNAs because we have very extensive knowledge of them, much of it from our laboratory, and also because they provide a strong contrast: the effects of overexpression of miR-125b are rapid, suggesting that it acts directly, whereas the effects of miR-146a are slow to develop, suggesting that they arise from chronic alterations in cellular behavior

Determinants of Cell- and Gene-Specific Transcriptional Regulation by the Glucocorticoid Receptor

The glucocorticoid receptor (GR) associates with glucocorticoid response elements (GREs) and regulates selective gene transcription in a cell-specific manner. Native GREs are typically thought to be composite elements that recruit GR as well as other regulatory factors into functional complexes. We assessed whether GR occupancy is commonly a limiting determinant of GRE function as well as the extent to which core GR binding sequences and GRE architecture are conserved at functional loci. We surveyed 100-kb regions surrounding each of 548 known or potentially glucocorticoid-responsive genes in A549 human lung cells for GR-occupied GREs. We found that GR was bound in A549 cells predominately near genes responsive to glucocorticoids in those cells and not at genes regulated by GR in other cells. The GREs were positionally conserved at each responsive gene but across the set of responsive genes were distributed equally upstream and downstream of the transcription start sites, with 63% of them >10 kb from those sites. Strikingly, although the core GR binding sequences across the set of GREs varied extensively around a consensus, the precise sequence at an individual GRE was conserved across four mammalian species. Similarly, sequences flanking the core GR binding sites also varied among GREs but were conserved at individual GREs. We conclude that GR occupancy is a primary determinant of glucocorticoid responsiveness in A549 cells and that core GR binding sequences as well as GRE architecture likely harbor gene-specific regulatory information

Regulation of APC development, immune response, and autoimmunity by Bach1/HO-1 pathway in mice

APCs are essential for innate and adaptive immunity as well as self-immune tolerance. Here, we show that the Cap’n’collar member Bach1 regulates the generation of APCs, specifically macrophages and dendritic cells, in mice. The impaired APC development in Bach1^(-/-) mice was accompanied by defects in downstream T-cell responses and partial protection from experimental autoimmune encephalomyelitis. Genomewide analyses identified a panel of Bach1 target genes and ablation of the direct Bach1 target gene HO-1 exacerbated the impaired APC development observed in Bach1^(-/-) mice. This was attributed to the impaired ability of HO-1^(-/-)Bach1^(-/-) double mutants to produce upstreamAPC progenitor cells, including common myeloid progenitor (CMP)–Flk2^+. By contrast, we observed an increase in hematopoietic stem-progenitor cells (HSPCs) in these mice, suggesting a developmental block in the progression of HSPCs to CMP-Flk2^+ and subsequently APCs

MicroRNA-125b Potentiates Macrophage Activation

MicroRNA (miR)-125b expression is modulated in macrophages in response to stimulatory cues. In this study, we report a functional role of miR-125b in macrophages. We found that miR-125b is enriched in macrophages compared with lymphoid cells and whole immune tissues. Enforced expression of miR-125b drives macrophages to adapt an activated morphology that is accompanied by increased costimulatory factor expression and elevated responsiveness to IFN-y, whereas anti–miR-125b treatment decreases CD80 surface expression. To determine whether these alterations in cell signaling, gene expression, and morphology have functional consequences, we examined the ability of macrophages with enhanced miR-125b expression to present Ags and found that they better stimulate T cell activation than control macrophages. Further indicating increased function, these macrophages were more effective at killing EL4 tumor cells in vitro and in vivo. Moreover, miR-125b repressed IFN regulatory factor 4 (IRF4), and IRF4 knockdown in macrophages mimicked the miR-125b overexpression phenotype. In summary, our evidence suggests that miR-125b is at least partly responsible for generating the activated nature of macrophages, at least partially by reducing IRF4 levels, and potentiates the functional role of macrophages in inducing immune responses

Dual mechanisms by which MiR-125b represses IRF4 to induce myeloid and B cell leukemias

The oncomir microRNA-125b (miR-125b) is up-regulated in a variety of human neoplastic blood disorders and constitutive up-regulation of miR-125b in mice can promote myeloid and B cell leukemia. We found that miR-125b promotes myeloid and B cell neoplasm by inducing tumorigenesis in hematopoietic progenitor cells. Our study demonstrates that miR-125b induces myeloid leukemia by enhancing myeloid progenitor output from stem cells as well as inducing immortality, self-renewal, and tumorigenesis in myeloid progenitors. Through functional and genetic analyses, we demonstrated that miR-125b induces myeloid and B cell leukemia by inhibiting IRF4 but through distinct mechanisms; it induces myeloid leukemia through repressing IRF4 at the mRNA level without altering the genomic DNA and induces B cell leukemia via genetic deletion of the gene encoding IRF4

Heme-Mediated SPI-C Induction Promotes Monocyte Differentiation into Iron-Recycling Macrophages

Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80^+VCAM1^+ bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis

Determinants of Glucocorticoid Receptor (GR) Transcriptional Specificity and Genomic Occupancy

The glucocorticoid receptor (GR) associates with glucocorticoid response elements (GREs) and regulates selective gene transcription in a cell-specific manner. GREs are typically thought to be composite elements that recruit GR as well as other factors into functional complexes. We assessed whether GR occupancy is commonly a limiting determinant of GRE function as well as the extent to which core GR binding sequences (GBSs) and GRE architecture are conserved at functional loci. We found that GR was bound in A549 cells predominately near genes responsive to glucocorticoids in those cells and not at genes regulated by GR in other cells. The GREs were distributed equally upstream and downstream of the transcription start sites, with 63% of them >10kb from those sites. Strikingly, sequences flanking the GBSs varied among GREs but were conserved at individual GREs. Similarly, although the GBSs across the set of GREs varied extensively around a consensus, the precise sequence at an individual GRE was conserved across species. Thus, we further examined whether sequence conservation of sites resembling GBSs is sufficient to predict GR-occupancy of GREs at genes responsive to glucocorticoids. Indeed, we found that the level of conservation of GBSs at genes up-regulated by glucocorticoids in mouse C3H10T1/2 mesenchymal stem-like cells correlated directly with the extent of occupancy by GR. We conclude that GR occupancy is a primary determinant of glucocorticoid responsiveness and that sequence of GBSs as well as GRE architecture likely harbor gene-specific regulatory information. Moreover, GBS conservation alone is sufficient to predict GR occupancy at induced genes. In this study, we found that genes important for regulating circadian rhythm were responsive to glucocorticoids in mesenchymal stem cells (MSCs). Thus, we confirmed that GR stimulated rhythmicity in these cells and identified primary GREs at Per1, Per2, and E4bp4 genes. We investigated whether the circadian clock in MSCs became autonomous of GR function after glucocorticoids have initiated it and found that continual GR activity is required for maintaining rhythmicity. Thus, we conclude that GR initiates circadian rhythm through directly activating Per1, Per2, and E4bp4 genes, and that GR function is essential for maintenance of rhythmicity