CCAAT/enhancer binding protein (C/EBP) ɛ is a recently cloned member of the C/EBP family of transcription factors and is expressed exclusively in cells of hematopoietic origin. The human C/EBPɛ gene is transcribed by two alternative promoters, Pα and Pβ. A combination of differential splicing and alternative use of promoters generates four mRNA isoforms, of 2.6 kb and 1.3–1.5 kb in size. These transcripts can encode three proteins of calculated molecular mass 32.2 kDa, 27.8 kDa, and 14.3 kDa. Accordingly, Western blots with antibodies specific for the DNA-binding domain, that is common to all forms, identify multiple proteins. C/EBPɛ mRNA was greatly induced during in vitro granulocytic differentiation of human primary CD34 ⁺ cells. Retinoic acid treatment of HL60 promyelocytic leukemia cells for 24 hr induced C/EBPɛ mRNA levels by 4-fold, while prolonged treatment gradually reduced mRNA expression to pretreatment levels. Transient transfection experiments with expression vectors for two of the isoforms demonstrated that the 32.2-kDa protein is an activator of transcription of granulocyte colony-stimulating factor receptor promoter, while the 14.3-kDa protein is not. Thus, C/EBPɛ is regulated in a complex fashion and may play a role in the regulation of genes involved in myeloid differentiation.

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 Foxp3^neg 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 Foxp3^neg 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 bone-sparing 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.

The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in the fusion of the SYT gene on chromosome 18 to either of two closely related genes SSX1 and SSX2 on chromosome X. The resulting chimaeric genes express SYT-SSX1 or SYT-SSX2 fusion proteins in which the C-terminal amino acids of SYT are replaced by amino acids from the C-terminus of the SSX proteins. Using green fluorescent protein fusions we demonstrate that the SYT, SSX and the SYT-SSX proteins are nuclear proteins.We demonstrate that whilst the SSX1 protein has a uniform nuclear distribution the SYT protein has a speckled distribution in the cell nucleus, and this distribution is retained with the SYT-SSX2 fusion protein. Since the SYT speckles do not co-localise with PML-containing bodies (PODs) or spliceosomes it is possible that they represent a novel nuclear structure. Transfection of constructs expressing GAL4 fusion proteins demonstrate that the SYT domains present in the SYT-SSX fusion proteins can activate transcription of a luciferase reporter. It is proposed that the t(X;18) translocation results in the generation of an SYT-SSX transcriptional co-activator in which the addition of the C-terminal SSX domain to SYT provides a new interacting domain that redirects the SYT activation domain to different target promoters.

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.