Project description:Genome-wide profiling of PPAR?:RXR and RNA polymerase II reveals temporal activation of distinct metabolic pathways in RXR dimer composition during adipogenesis. Chromatin immunoprecipitation combined with deep sequencing was performed to generate genome-wide maps of peroxisome prolifelator-activated receptor gamma (PPARg) and retinoid X receptor (RXR) binding sites, and RNA polymerase II (RNAPII) occupancy at high resolution throughout adipocyte differentiation of 3T3-L1 cells. The data provides the first positional and temporal map PPAR? and RXR occupancy during adipocyte differentiation at a global scale. The number of PPAR?:RXR shared binding sites is steadily increasing from D0 to D6. At Day6 there are over 5000 high confidence shared PPARy:RXR binding sites. We show that at the early days of differentiation several of these sites bind not only PPAR?:RXR but also other RXR dimers. The data also provides a comprehensive temporal map of RNAPII occupancy at genes throughout 3T3-L1 adipogenesis thereby uncovering groups of similarly regulated genes belonging to glucose and lipid metabolic pathways. The majority of the upregulated but very few downregulated genes have assigned PPAR?:RXR target sites, thereby underscoring the importance of PPAR?:RXR in gene activation during adipogenesis and indicating that a hitherto unrecognized high number of adipocyte genes are directly activated by PPAR?:RXR Examination of PPARg and RXR bindingsites during adipocyte differentiation (day 0 to 6) and association with transcription via RNAPII occupancy.
Project description:Data from nuclear receptors RXR and PPARg binding to the genome was visualized using Genome Browser (UCSC) in order to test their role in Mafb transcriptional regulation RXR and PPARg ChIP was performed in thioglycollate-elicited peritoneal macrophages in basal conditions
Project description:Osteoclasts are absorptive cells and play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role for epigenetic regulation of osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation during osteoclast formation. DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. DOT1L inhibition also increased osteoclast area and accelerated bone mass reduction in a mouse ovariectomy (OVX) model of osteoporosis. DOT1L inhibitors did not alter osteoblast differentiation in vitro and in vivo. Proteomics data, together with bioinformatics analysis, revealed that DOT1L inhibition altered reactive oxygen species (ROS) generation, autophagy activation, and cell fusion-related protein expression. ROS generation increased, and autophagy activation and cell migration ability enhancement were verified subsequently by flow cytometry and transwell migration assays. DOT1L inhibition increased NFATc1 nuclear translocation and NF-κB activation and strengthend osteoclast fusion and expression of resorption-related protein CD9, and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-mediated H3K79me2 epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.
Project description:Tight control of gene expression networks involved in adipose tissue formation and plasticity is required to adapt to energy needs and environmental cues. However, little is known about the mechanisms that orchestrate the dramatic transcriptional changes leading to adipocyte differentiation. We investigated the regulation of nascent transcription by SUMO during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that SUMO has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing SUMOylome dynamics in differentiating adipocytes by mass spectrometry, we found that SUMOylation of specific transcription factors like PPARG/RXR and chromatin modifiers promotes the transcription of adipogenic genes. Our data demonstrate that the sumoylation pathway helps coordinates the rewiring of transcriptional networks required for formation of functional adipocytes.
Project description:Genome-wide profiling of PPARγ:RXR and RNA polymerase II reveals temporal activation of distinct metabolic pathways in RXR dimer composition during adipogenesis. Chromatin immunoprecipitation combined with deep sequencing was performed to generate genome-wide maps of peroxisome prolifelator-activated receptor gamma (PPARg) and retinoid X receptor (RXR) binding sites, and RNA polymerase II (RNAPII) occupancy at high resolution throughout adipocyte differentiation of 3T3-L1 cells. The data provides the first positional and temporal map PPARγ and RXR occupancy during adipocyte differentiation at a global scale. The number of PPARγ:RXR shared binding sites is steadily increasing from D0 to D6. At Day6 there are over 5000 high confidence shared PPARy:RXR binding sites. We show that at the early days of differentiation several of these sites bind not only PPARγ:RXR but also other RXR dimers. The data also provides a comprehensive temporal map of RNAPII occupancy at genes throughout 3T3-L1 adipogenesis thereby uncovering groups of similarly regulated genes belonging to glucose and lipid metabolic pathways. The majority of the upregulated but very few downregulated genes have assigned PPARγ:RXR target sites, thereby underscoring the importance of PPARγ:RXR in gene activation during adipogenesis and indicating that a hitherto unrecognized high number of adipocyte genes are directly activated by PPARγ:RXR
Project description:Def6 suppresses osteoblast differentiation and mineralization both in vitro and in vivo. Def6 knockout (KO) mice exhibit osteoporotic phenotype with enhanced osteoclast formation. Osteoblast differentiation and bone formation are elevated as well in Def6 KO mice, indicating a high bone turnover rate that leads to bone loss in Def6 KO mice. Thus, lack of Def6 leads towards unbalanced activities between osteoclastic resorption and osteoblast mediated bone formation and disrupts normal bone remodeling. Def6 suppresses osteoblast differentiation via endogenous type I IFN-mediated feedback inhibition. These findings reveal that Def6 is a novel bone remodeling regulator that controls both osteoclast and osteoblast differentiation to maintain bone remodeling.
Project description:Exosomes are secreted into the blood by various types of cells. These small vesicles are involved in bone remodeling by mediation of intercellular communication in osteoblasts, osteoclasts or their precursors. Alterations in exosomal proteins are related to the failure of bone remodeling, which results in progressive loss of bone mass and poor quality of bone structure. However, the molecular changes in serum-derived exosomes (SDEs) from patients with low bone density and their functions in bone remodeling remain to be fully elucidated. We present a quantitative proteomics analysis of exosomes purified from the serum of patients with osteoporosis/osteopenia and normal volunteers; these data are available via Proteome Xchange with the identifier PXDXXXXX. Overall, 1,371 proteins were identified with an overlap of 1,160 Gene IDs among the ExoCarta proteins. In vitro studies and bioinformatics analysis revealed that the main changes in the SDEs of osteoporosis patients were proteins playing critical roles in integrins-mediated mechanosensation and signaling cascades, which are implicated in aggravating the failure of bone remodeling, including the enhancement of osteoclast differentiation and inhibition of bone mineralization. In contrast, the main changes in SDEs of osteopenia patients were proteins known to facilitate both osteoclast differentiation and osteoblastic bone formation, which resulted in a compensatory elevation of bone remodeling. In addition, bioinformatics analysis indicated that SDEs from elderly volunteers mediate negative regulation of bone remodeling via selenium deficiency-associated oxidative stress. This information will be helpful in elucidating the pathophysiological functions of SDEs and aid in the development of osteoporosis diagnostics and therapeutics.
Project description:Data from nuclear receptors RXR and PPARg binding to the genome was visualized using Genome Browser (UCSC) in order to test their role in Mafb transcriptional regulation