Hematopoietic stem cells have the ability to promote continuous self-renewal by controlled proliferation and expansive differentiation into all cells of hematopoietic lineages. Regulation of the molecular mechanisms that direct cellular proliferation and differentiation during hematopoiesis depends, partly, on the action of certain transcription factors (1-3).Several members of the basic region-leucine zipper (bZIP) class of transcription factors, including the CCAAT͞enhancer binding protein (C͞EBP) family have been implicated in the differentiation of hematopoietic cells (1-5). C͞EBP proteins have highly conserved bZIP C-terminal regions, but differ in their N-terminal regions (6, 7). A consensus site,

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4+ T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3neg CD4+ T cells that displays regulatory activity unlike other IL-10-producing CD4+ T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4+ T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3neg CD4+ T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.

The bone-sparing effect of estrogen in both males and females is primarily mediated via estrogen receptor-α (ERα), encoded by the Esr1 gene. ERα in osteoclasts is crucial for the trabecular bonesparing effect of estrogen in females, but it is dispensable for trabecular bone in male mice and for cortical bone in both genders. We hypothesized that ERα in osteocytes is important for trabecular bone in male mice and for cortical bone in both males and females. Dmp1-Cre mice were crossed with ERα flox/flox mice to generate mice lacking ERα protein expression specifically in osteocytes (Dmp1-ERα −/− ). Male Dmp1-ERα −/− mice displayed a substantial reduction in trabecular bone volume (−20%, P < 0.01) compared with controls. Dynamic histomorphometry revealed reduced bone formation rate (−45%, P < 0.01) but the number of osteoclasts per bone surface was unaffected in the male Dmp1-ERα −/− mice. The male Dmp1-ERα −/− mice had reduced expression of several osteoblast/osteocyte markers in bone, including Runx2, Sp7, and Dmp1 (P < 0.05). Gonadal intact Dmp1-ERα −/− female mice had no significant reduction in trabecular bone volume but ovariectomized Dmp1-ERα −/− female mice displayed an attenuated trabecular bone response to supraphysiological E2 treatment. Dmp1-ERα −/− mice of both genders had unaffected cortical bone. In conclusion, ERα in osteocytes regulates trabecular bone formation and thereby trabecular bone volume in male mice but it is dispensable for the trabecular bone in female mice and the cortical bone in both genders. We propose that the physiological trabecular bone-sparing effect of estrogen is mediated via ERα in osteocytes in males, but via ERα in osteoclasts in females.

Ligand-induced transcriptional activation of gene expression by nuclear receptors is dependent on recruitment of coactivators as intermediary factors. The present work describes the cloning and characterization of RAP250, a novel human nuclear receptor coactivator. The results of in vitro and in vivo experiments indicate that the interaction of RAP250 with nuclear receptors is ligand-dependent or ligand-enhanced depending on the nuclear receptor and involves only one short LXXLL motif called nuclear receptor box. Transient transfection assays further demonstrate that RAP250 has a large intrinsic glutamine-rich activation domain and can significantly enhance the transcriptional activity of several nuclear receptors, acting as a coactivator. Interestingly, Northern blot and in situ hybridization analyses reveal that RAP250 is widely expressed with the highest expression in reproductive organs (testis, prostate and ovary) and brain. Together, our data suggest that RAP250 may play an important role in mammalian gene expression mediated by nuclear receptor.

The promoter region of the mouse CCAAT-Enhancer Binding Protein (C/EBP alpha) gene is capable of directing high levels of expression of reporter constructs in various cell lines, albeit even in cells that do not express their endogenous C/EBP alpha gene. To understand the molecular mechanisms underlying this ubiquitous expression, we have characterized the promoter region of the mouse C/EBP alpha gene by a variety of in vitro and in vivo methods. We show that three sites related in sequence to USF, BTE and C/EBP binding sites and present in promoter region -350/+3, are recognized by proteins from rat liver nuclear extracts. The sequence of the C/EBP alpha promoter that includes the USF binding site is also capable of forming stable complexes with purified Myc+Max heterodimers and mutation of this site drastically reduces transcription of C/EBP alpha promoter luciferase constructs both in liver and non liver cell lines. In addition, we identify three novel protein-binding sites two of which display similarity to NF-1 and a NF kappa B binding sites. The region located between nucleotides -197 and -178 forms several heat-stable complexes with liver nuclear proteins in vitro which are recognized mainly by antibodies specific for C/EBP alpha. Furthermore, transient expression of C/EBP alpha and to a lesser extent C/EBP beta expression vectors, results in transactivation of a cotransfected C/EBP alpha promoter-luciferase reporter construct. These experiments support the notion that the C/EBP alpha gene is regulated by C/EBP alpha but other C/EBP-related proteins may also be involved.

Coactivators constitute a diverse group of proteins that are essential for optimal transcriptional activity of nuclear receptors. In the past few years many coactivators have been identified but it is still unclear whether these proteins interact indiscriminately with all nuclear receptors and whether there is some redundancy in their functions. We have previously cloned and characterized RAP250 (ASC-2/PRIP/TRBP/NRC), an LXXLLcontaining coactivator for nuclear receptors. In order to study its biological role, Rap250 null mice were generated by gene targeting. Here we show that genetic disruption of Rap250 results in embryonic lethality at embryonic day (E) 13.5. Histological examination of placentas revealed a dramatically reduced spongiotrophoblast layer, a collapse of blood vessels in the region bordering the spongiotrophoblast, and labyrinthine layers in placentas from Rap250 ؊/؊ embryos. These findings suggest that the lethality of Rap250 ؊/؊ embryos is the result of obstructed placental blood circulation. Moreover, the transcriptional activity of PPAR␥ is reduced in fibroblasts derived from Rap250 ؊/؊ embryos, suggesting that RAP250 is an essential coactivator for this nuclear receptor in the placenta. Our results demonstrate that RAP250 is necessary for placental development and thus essential for embryonic development.Nuclear receptors comprise a family of transcription factors that regulate gene expression in a ligand-dependent manner. Upon ligand binding these proteins activate transcription of specific genes involved in the control of diverse physiological processes, such as cellular growth, development, differentiation, and homeostasis (20). Binding of ligands to the ligandbinding domain of nuclear receptors causes conformational changes of the receptor, enabling the recruitment of coactivators harboring LXXLL motifs (see below). A number of coactivators for nuclear receptors have been identified (for a review, see reference 6). Among the most studied is the steroid receptor coactivator 1 (SRC-1) family, which contains three related coactivators, referred to as SRC-1, SRC-2/GRIP1/ TIF2, and SRC-3/p/CIP/RAC3/ACTR/AIB1/TRAM1 (1,4,7,17,23,28,30,33). These factors, together with CBP/p300 and pCAF, bind nuclear receptors and gain access to target promoter regions through histone acetyltransferase-mediated nucleosome remodeling. Another class of coactivators is represented by the TRAP/DRIP/ARC multiprotein complex (5, 22, 26), with one major nuclear receptor interacting subunit, PBP/ TRAP220/DRIP205 (25,31,41,45), that appears to act independently of histone acetyltransferase activity.Nuclear receptor-activating protein 250 (RAP250) (3), also called ASC-2, PRIP, TRBP, and NRC (13,15,19,42), was recently cloned and described as a novel nuclear receptor coactivator which interacts with the ligand-binding domain of ligand-bound nuclear receptors. The interaction is mediated through a short hydrophobic motif called the LXXLL motif (or NR box) which is found in most nuclear receptor coactivators. In contrast to ...

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