Summary How the glucocorticoid receptor (GR) activates some genes while potently repressing others remains an open question. There are three current models for suppression: trans-repression via GR ‘tethering’ to AP-1/NF-κB sites, direct GR association with inhibitory elements (nGREs), and GR recruitment of the corepressor GRIP1. To gain insights into GR suppression, we used genomic analyses and genome-wide profiling of GR, p65, and c-Jun in LPS-stimulated macrophages. We show that GR mediates both activation and repression at tethered sites, GREs, and GRIP1-bound elements, indicating that motif classification is insufficient to predict regulatory polarity of GR binding. Interestingly, sites of GR repression utilize GRIP1’s corepressor function and display reduced histone acetylation. Together, these findings suggest that while GR occupancy confers hormone responsiveness, the receptor itself may not participate in the regulatory effects. Furthermore, transcriptional outcome is not established by sequence, but is influenced by epigenetic regulators, context, and other unrecognized regulatory determinants.

In the macrophage, toll-like receptors (TLRs) are key sensors that trigger signaling cascades to activate inflammatory programs via the NF-kB gene network. However, the genomic network targeted by TLR/NF-kB activation and the molecular basis by which it is restrained to terminate activation and re-establish quiescence is poorly understood. Here, using chromatin immunoprecipitation sequencing (ChIP-seq), we define the NF-kB cistrome, which is comprised of 31,070 cis-acting binding sites responsive to lipopolysaccharide (LPS)-induced signaling. In addition, we demonstrate that the transcriptional repressor B-cell lymphoma 6 (Bcl-6) regulates nearly a third of the Tlr4-regulated transcriptome, and that 90% of the Bcl-6 cistrome is collapsed following Tlr4 activation. Bcl-6-deficient macrophages are acutely hypersensitive to LPS and, using comparative ChIP-seq analyses, we found that the Bcl-6 and NF-kB cistromes intersect, within nucleosomal distance, at nearly half of Bcl-6-binding sites in stimulated macrophages to promote opposing epigenetic modifications of the local chromatin. These results reveal a genomic strategy for controlling the innate immune response in which repressive and inductive cistromes establish a dynamic balance between macrophage quiescence and activation via epigenetically marked cis-regulatory elements.

How type I skeletal muscle inherently maintains high oxidative and vascular capacity in absence of exercise in unclear. We show that nuclear receptor ERRγ is highly expressed in type I muscle and when transgenically expressed in anaerobic type II muscles (ERRGO mice), dually induces metabolic and vascular transformation in absence of exercise. ERRGO mice show increased expression of genes promoting fat metabolism, mitochondrial respiration and type I fiber specification. Muscles in ERRGO mice also display an activated angiogenic program marked by myofibrillar induction and secretion of pro-angiogenic factors, neo-vascularization and a 100% increase in running endurance. Surprisingly, the induction of type I muscle properties by ERRγ does not involve PGC1α. Instead, ERRγ genetically activates the energy sensor AMPK, in mediating the metabo-vascular changes in the ERRGO mice. Therefore, ERRγ represents a previously unrecognized determinant that specifies intrinsic vascular and oxidative metabolic features that distinguish type I from type II muscle.

Mitsugumin 53 (MG53) negatively regulates skeletal myogenesis by targeting insulin receptor substrate 1 (IRS-1). Here, we show that MG53 is a ubiquitin E3 ligase that induces IRS-1 ubiquitination with the help of an E2-conjugating enzyme UBE2H. Molecular manipulations that disrupt the E3 ligase function of MG53 abolishes IRS-1 ubiquitination and enhances skeletal myogenesis. Skeletal muscles derived from the MG53−/− mice show an elevated IRS-1 level with enhanced insulin signaling, which protects the MG53−/− mice from developing insulin resistance when challenged with a high fat/high sucrose diet. Muscle samples derived from human diabetic patients and mice with insulin resistance show normal expression of MG53, indicating that altered MG53 expression does not serve as a causative factor for the development of metabolic disorders. Thus, therapeutic interventions that target the interaction between MG53 and IRS-1 may be a novel approach for the treatment of metabolic diseases that are associated with insulin resistance.

SUMMARY Chronic inflammation is a hallmark of atherosclerosis, but its transcriptional underpinnings are poorly understood. We show that the transcriptional repressor Bcl6 is an anti-inflammatory regulator whose loss in bone marrow of Ldlr−/− mice results in severe atherosclerosis and xanthomatous tendonitis, a virtually pathognomonic complication in patients with familial hypercholesterolemia. Disruption of the interaction between Bcl6 and SMRT or NCoR with a peptide inhibitor in vitro recapitulated atherogenic gene changes in mice transplanted with Bcl6-deficient bone marrow, pointing to these cofactors as key mediators of Bcl6 inflammatory suppression. Using ChIP-seq, we reveal the SMRT and NCoR co-repressor cistromes, each consisting of over 30,000 binding sites with a nearly 50% overlap. While the complete cistromes identify a diversity of signaling pathways, the Bcl6-bound sub-cistromes for each co-repressor are highly enriched for NF-κB-driven inflammatory and tissue remodeling genes. These results reveal that Bcl6-SMRT/NCoR complexes constrain immune responses and contribute to the prevention of atherosclerosis.

Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.

MicroRNAs (miRNAs) have been extensively reported to be associated with hematological malignancies. The loss of miR-15a/16-1 at chromosome 13q14 is a hallmark of most of human chronic lymphocytic leukemia (CLL). Deletion of murine miR-15a/16-1 and miR-15b/ 16-2 has been demonstrated to promote B cell malignancies. Here, we evaluate the biological role of miR-15/16 clusters, crossbreeding miR-15a/16-1 and miR-15b/16-2 knockout mice. Unexpectedly, the complete deletion of both clusters promoted myeloproliferative disorders in the majority of the mice by the age of 5 months with a penetrance of 70%. These mice showed a significant enlargement of spleen and abnormal swelling of lymph nodes. Flow cytometry characterization demonstrated an expanded CD11b/Gr-1 double-positive myeloid population both in spleen and in bone marrow. The transplantation of splenocytes harvested from double-KO mice into wildtype recipient mice resulted in the development of myeloproliferative disorders, as observed in the donors. In vivo, miR-15/16 cluster deletion up-regulated the expression of Cyclin D1, Cyclin D2, and Bcl-2. Taken together, our findings identify a driver oncogenic role for miR-15/16 cluster deletion in different leukocytic cell lineages. miR-15/16 cluster | acute myeloid leukemia | mouse model T he loss of miR-15a/16-1 at chromosome 13q14, encoding two miRNAs residing in the same polycistrocnic RNA, is a hallmark of most of human chronic lymphocytic leukemias (CLL) (1). This dysregulation has been proved as the first evidence that genetic alteration in noncoding genes can cause malignancy (1).Most of CLLs are indolent but may progress to an aggressive disease. Analysis for mutations in many CLL showed that germline mutations just seven nucleotides 3′ of miR-16-1 are present in familial CLL (2), and this region was found to be necessary for processing of the miRNA precursor (3). Furthermore, the mouse strain NZB that develops an indolent CLL late in life carries a mutation six nucleotides 3′ of miR-16-1 affecting miR-15/16 processing, indicating that loss of function of miR-15a/16-1 leads to the development of indolent CLL (4). The frequency of loss of one or both miR-15a/16-1 clusters on chromosome 13q14 occurs in over 70% of the CLL patients and is the most common genetic alteration in CLL (1). Klein et al. (5) knocked out the miR-15a/16-1 locus in the mouse, and the knockout (KO) mice developed late in life the indolent form of CLL, as in human patients (6). Thus, loss of miR-15a/16-1 leads to the development of indolent CLL both in humans and mice. Since an additional locus of miR-15/16 exists on chromosome 3 (3q25), we have knocked out this second miR-15b/16-2 locus in the mouse (7). These mice developed mainly CLL and few diffuse large B cell lymphoma at higher penetrance (60%) and earlier age at 15-18 months (7) than the miR-15a/16-1 KO mice described by Klein et al. (5). Here, we evaluated the biological role of two different miR-15/16 clusters, crossbreeding miR-15a/16-1 and miR-15b/16-2 knockout (KO) mice. Unexpec...

Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML. We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls. Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT. EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells. Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML. EGFL7 | acute myeloid leukemia | clinical outcomeA cute myeloid leukemia (AML) is a clonal hematopoietic disease characterized by the proliferation of immature blasts in the bone marrow (BM) and blood (1). Genetic alterations, including chromosomal translocations and deletions and gene mutations leading to aberrant downstream target gene expression, contribute to AML initiation and maintenance. Previously, our group demonstrated that increased miRNA-126-3p (miR-126) expression in patients with cytogenetically normal AML (CN-AML) correlated with shorter overall survival (OS). Furthermore, we found miR-126 to be essential for leukemia stem cell (LSC) homeostasis, and in vivo targeting of miR-126 in a patientderived xenograft model resulted in prolonged survival in secondary bone marrow transplant (BMT) recipients (2). miR-126 is located within intron 7 of a protein-coding gene known as Epithelial growth factor-like 7 (EGFL7) (3). Although we and others (2, 4, 5) have shown an important role for miR-126 in AML biology, we know of no studies that have been performed to understand the prognostic and functional implications of expression of its host gene, EGFL7, in AML.EGFL7 is a secreted protein of ∼30 kDa and plays an important physiological role in angiogenesis (6-8). Unlike other angiogenic factors (e.g., VEGF), physiological EGFL7 expression and function has been restricted mainly to the endothelial cells where it regulates survival, migration, and differentiation (6). Aberrant expression of EGFL7 has been shown to be involved in tumor growth and disease progression of several solid tumors, including hepatocellular carcinoma, malignant glioma, and breast, lung, and pancreatic cancers (9), but its role in hematopoietic malignancies is currently unknown. Therefore, we investigated the prognostic and biological function of EGFL7 expression in A...