Enhancers are cis-acting elements capable of regulating transcription in a distance and orientation-independent manner. A subset of enhancers are occupied by RNA polymerase II (RNAP II) and transcribed to produce long non-coding RNAs termed eRNAs. We thoroughly investigated the association between eRNA productivity and various chromatin marks and transcriptional regulators in mouse embryonic stem cells (ESCs) through an integrative approach. We found that eRNA-producing enhancers exhibited elevated levels of the active mark H3K27Ac, decreased DNA methylation, and enrichment for the DNA hydroxylase Tet1. Many eRNA-producing enhancers have recently been characterized as "super-enhancers," suggesting an important role in the maintenance of pluripotency. Using experimental methods, we focally investigated a well-characterized enhancer linked to the Nanog locus and confirmed its exclusive eRNA productivity in ESCs. We further demonstrate that the binding of Sall4 and Tet family proteins were required for eRNA productivity at this locus. Collectively, we demonstrate that Tet1 binding and DNA hypomethylation are hallmarks of eRNA production.

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal since this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of Polycomb Repressive Complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations.

The nephron number at birth is a quantitative trait that correlates inversely with the risk of hypertension and chronic kidney disease later in life. During kidney development, the nephron number is controlled by multiple factors including genetic, epige-netic, and environmental modifiers. Premature birth, which represents more than 12% of annual live births in the United States, has been linked to low nephron number and the development of hypertension later in life. In this report, we describe the development of a mouse model of prematurity-induced reduction of nephron number. Premature mice, delivered 1 and 2 days early, have 17.4 ± 2.3% (n = 6) and 23.6 ± 2% (n = 10) fewer nephrons, respectively, when compared with full-term animals (12,252 ± 571 nephrons/kidney, n = 10). After 5 weeks of age, the mice delivered 2 days premature show lower real-time glomerular filtration rate (GFR, 283 ± 13 vs 389 ± 26 μL/min). The premature mice also develop hypertension (mean arterial pressure (MAP), 134 ± 18 vs 120 ± 14 mm Hg) and albuminuria (286 ± 83 vs 176 ± 59 μg albumin/mg creatinine). This mouse model provides a proof of concept that prematurity leads to reduced nephron number and hypertension, and this model will be useful in studying the pathophysiology of prematurity-induced nephron number reductions and hypertension.

Cytokines and proteases are secreted by fibroblasts in response to particulate wear debris, and these proteins are felt to play an important role in the development of osteolysis and implant loosening. Although metallic and polyethlyene debris have been studied extensively, little is known about the cellular responses to hydroxyapatite, despite the wide clinical use of these materials. Therefore, the effects of hydroxyapatite (HA) and hydroxyapatitelp-tricalciumphosphate (HARCP) on cellular proliferation, cytokine gene expression and protein secretion, protease synthesis, and gelatinolytic activity were investigated in human fibroblasts.HA and HMTCP particles were synthesized, and their effects were compared to the responses elicited by titanium and cobalt chromium. Sample characterization by scanning electron microscopy and Coulter Counter demonstrated that the materials had a mean particle size of less than 10 pm, and all of the particles were compared using the same concentration ranges.Aliquots of particle suspensions were added to human fibroblasts maintained in tissue culture, and dose-response and timecourse experiments were performed. Effects of the particles on fibroblast proliferation were assessed, and alterations in cytokine levels were determined by specific enzyme linked immunosorbent assays (ELISA). Cytokines that were evaluated included interleukin-1 (IL-1 p), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a), all of which have been demonstrated to enhance bone resorption and are associated with osteolysis and implant loosening. Gene expression was determined using Northern blot analysis with cytokine-specific probes, while secretion of the proteases collagenase and stromelysin was determined by Western blot analysis.Functional gelatinolytic assay was assessed using zymogram gels.The particles were evaluated in a concentration range from 0.000021 to 0.021 ~01%. All of the particles produced increases in cellular proliferation up to 0.0021 vol%, with the largest increases being seen at 0.021 vol% with HA/TCP and titanium. At the highest concentration, both cobalt chromium and HA samples decreased cellular proliferation relative to lower doses, possibly representing cytotoxicity.Hydroxyapatite particles yielded a 30-fold increase in interleukin-6 secretion compared to unstimulated controls, which was also greater than three times the levels produced by cobalt chromium, titanium, or HAITCP. HA particles also tripled the secretion of IL-Ip at 0.00021 vol%, and doubled TNF-a secretion at 0.021 ~01%. Addition of conditioned media prepared by incubation of the particles in culture medium in the absence of cells did not alter the secretion of any of the cytokines. Northern blot analysis using IL-6 probes also demonstrated strong increases with HA compared to the other materials, suggesting that the action of the HA particles was at the level of transcription. Secretion of the protease collagenase was increased by all of the samples including HA when compared to unstimulated controls. Stro...

A previous genetic analysis comparing the Dahl salt-sensitive (S) rat to the spontaneously hypertensive rat (SHR) identified a major locus on chromosome 2 that influences proteinuria in the S rat. In the present study, blood pressure, proteinuria, and renal hemodynamics were evaluated in congenic strains with small segments of the protective SHR genome on the S background. Proteinuria and renal function were significantly improved in the congenic strains compared to the S. The causative locus interval was narrowed to <375 kb based on congenic strains, haplotype data, comparative mapping, and concordance with human genetic studies. Sequencing of the coding region of genes in this region identified 36 SNPs (13 nonsynonymous and 23 synonymous). Gene expression profiling indicated that only few genes exhibited differential expression. Arhgef11, Pear1, and Sh2d2 were identified as important candidate genes that may be linked to kidney injury in the S rat. In particular, Arhgef11 plays an important role in the activation of the Rho-ROCK signaling pathway. Inhibition of this pathway using fasudil resulted in a significant reduction of proteinuria in treated S rats (compared to untreated S). However, no difference was observed between treated or untreated SHR or congenic strains. The homologous region in humans was found to be associated with estimated glomerular filtration rate (eGFR) in the Candidate Gene Association Resource (CARe) population. In summary, these findings demonstrate that allelic variants in Arhgef11, acting through the Rho-ROCK pathway, could influence kidney injury in the S as well as provide insight into human kidney disease.