Project description:Supplemental data for the article: Characterization of functional reprogramming during osteoclast development using quantitative proteomics and mRNA profiling Eunkyung An, Manikandan Narayanan, and Aleksandra Nita-Lazar* *corresponding author: Cellular Networks Proteomics Unit Laboratory of Systems Biology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland, 20892, USA Tel. +1 301-451-4394 Fax: +1 301-480-5170 E-mail: nitalazarau@niaid.nih.gov This dataset includes: 1. Raw LC-MS(/MS) spectra (*.raw), and 2. The output from data analyses using IP2 (Intergrated Proteomics Application, San Diego, CA) searched against the UniProt_mouse_01-18-2011 set of protein sequences (normal + reversed). Note that the version of IP2 that was used could only analyze two SILAC channels at a time, so two analyses were performed (light-medium, light-heavy) (the current version can analyze 3-plex SILAC all together in one analysis). Also, IP2 was run using two criteria (1 or 2 peptides per protein). Lanes D and E were lanes of an SDS-PAGE gel, and each was 3-plex SILAC: Lane D: Light (Osteoclast Precursor), Medium (Mature Osteoclast), Heavy (Intermediate Osteoclast) Lane E: Light (Osteoclast Precursor), Medium (Intermediate Osteoclast), Heavy (Mature Osteoclast)
Project description:Using quantitative proteomics we identified group of synaptic genes with decreased protein synthesis during homeostatic plasticity. To obtain further information about their mRNA levels/sequences (3’UTR) we performed polyA RNAseq. At the end, small RNAseq helped us to identify miRNAs that are increased during homeostatic plasticity and might regulate downregulated genes.
Project description:Osteoclasts are multinucleated cells specialized in degrading the mineralized bone matrix. Osteoclast differentiation and function are tightly regulated, to prevent excessive or insufficient bone resorption. Several control mechanisms participate in modulating osteoclastogenesis, and an increasing number of reports describe the role of microRNAs (miRNAs) in this process. Disrupting the expression of specific miRNAs can result in alterations of osteoclast formation and bone homeostasis. We and others have previously characterized 9 miRNAs whose levels change during osteoclast differentiation, and identified some of the target genes that mediate their function. However, little is known about changes in the miRNA expression profile during osteoclastogenesis. In this study, we isolated a murine primary bone marrow population enriched for osteoclast precursors, and used the Agilent microarray platform to analyze the expression of mature miRNAs after 1, 3, and 5 days of RANKL-driven differentiation. 93 miRNAs showed greater than 2 fold-change during these early, middle, and late stages of osteoclastogenesis. Many of these miRNAs were detected for the first time in osteoclasts, and we validated the expression of selected miRNAs by quantitative RT-PCR. We identified clusters of differentially expressed miRNAs, and performed computational analyses to predict functional pathways that may be regulated by these miRNAs. Several miRNAs were predicted to regulate genes involved in cytoskeletal remodeling, a crucial mechanism for the migration of osteoclast precursors, their maturation, and bone resorbing activity. Our results suggest that clusters of miRNAs differentially expressed during the course of osteoclastogenesis converge on the regulation of several key functional pathways. Overall, this study identified miRNAs expressed during early, middle and late osteoclastogenesis, contributing to understanding the molecular mechanisms regulating this complex differentiation process. Mouse primary bone marrow cultures were enriched for osteoclast precursors by depletion of B220/CD45R+ and CD3+ cells (B and T lymphocytes, respectively). Cells were differentiated with M-CSF and RANKL, and miRNA expression was analyzed at days 1, 3, and 5. Four biological replicates for each time point were used.
Project description:Though limited proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during mammalian differentiation, however the specific genomic sites targeted for H3NT proteolysis and their functional significance of H3NT cleavage remain unknown.We used genome wide RNA-seq approaches to an established cell model of osteoclast differentiation. We discovered that H3NT proteolysis is selectively targeted near transcription start sites of a small group of genes and that most of these H3NT-cleaved genes are epigenetically regulated during osteoclastogenesis.We have identified that the principal H3NT protease of osteoclastogenesis is matrix metalloproteinase 9 (MMP-9). We next studied genomewide mRNA expression in MMP9 knockout cells and its effect in the epigenetic reprogramming of gene pathways required for proficient osteoclastogenesis.
Project description:Derivation of human naïve cells in the ground state of pluripotency provides promising avenues for developmental studies and therapeutic manipulations. However, the molecular mechanisms involved in establishment and maintenance of human naïve pluripotency remain poorly understood. Using the human inducible reprogramming system together with 5iLAF naïve induction strategy, we performed integrative analysis across the timeline from human fibroblasts to naïve iPSCs, which reveals ordered expression waves of gene networks sharing signatures with embryonic development process from post-implantation to pre-implantation stages. We also observed a significant transient re-activation of transcripts with 8-cell-stage-like characteristics in late naïve reprogramming stages. Moreover, combined quantitative proteomics analysis with transcriptional dynamics during naïve pluripotency induction, we identified ALPPL2 as the specific surface marker for human naïve pluripotency. Altogether, our study deepens the understanding of molecular mechanisms of human naïve pluripotency.
Project description:Derivation of human naïve cells in the ground state of pluripotency provides promising avenues for developmental studies and therapeutic manipulations. However, the molecular mechanisms involved in establishment and maintenance of human naïve pluripotency remain poorly understood. Using the human inducible reprogramming system together with 5iLAF naïve induction strategy, we performed integrative analysis across the timeline from human fibroblasts to naïve iPSCs, which reveals ordered expression waves of gene networks sharing signatures with embryonic development process from post-implantation to pre-implantation stages. We also observed a significant transient re-activation of transcripts with 8-cell-stage-like characteristics in late naïve reprogramming stages. Moreover, combined quantitative proteomics analysis with transcriptional dynamics during naïve pluripotency induction, we identified ALPPL2 as the specific surface marker for human naïve pluripotency. Altogether, our study deepens the understanding of molecular mechanisms of human naïve pluripotency.
Project description:Derivation of human naïve cells in the ground state of pluripotency provides promising avenues for developmental studies and therapeutic manipulations. However, the molecular mechanisms involved in establishment and maintenance of human naïve pluripotency remain poorly understood. Using the human inducible reprogramming system together with 5iLAF naïve induction strategy, we performed integrative analysis across the timeline from human fibroblasts to naïve iPSCs, which reveals ordered expression waves of gene networks sharing signatures with embryonic development process from post-implantation to pre-implantation stages. We also observed a significant transient re-activation of transcripts with 8-cell-stage-like characteristics in late naïve reprogramming stages. Moreover, combined quantitative proteomics analysis with transcriptional dynamics during naïve pluripotency induction, we identified ALPPL2 as the specific surface marker for human naïve pluripotency. Altogether, our study deepens the understanding of molecular mechanisms of human naïve pluripotency.
Project description:Enhanced osteoclastogenesis and osteoclast activity contribute to the development of osteoporosis, which is characterized by increased bone resorption and inadequate bone formation. As novel anti-osteoporotic therapeutics are needed, understanding the genetic regulation of human osteoclastogenesis could help identify potential treatment targets. This study aimed to provide an overview of the transcriptional reprogramming during human osteoclast differentiation. Osteoclasts were differentiated from CD14+-monocytes from eight female donors. RNA-sequencing during differentiation demonstrated 8980 differentially expressed genes grouped into eight temporal patterns conserved across donors. These patterns showed distinct molecular functions, associated with postmenopausal osteoporosis susceptibility genes based on RNA from iliac crest biopsies, and bone mineral density SNPs. Network analyses showed mutual dependencies between the temporal expression patterns and provides insight into subtype-specific transcriptional networks. Donor specific expression patterns identified genes at monocyte stage, such as filamin B (FLNB) and oxidized low density lipoprotein receptor 1 (OLR1, encoding LOX-1), that are predictive for the resorptive activity of mature osteoclasts. Differentially expressed G-protein coupled receptors showed strong expression during osteoclast differentiation and associated with bone mineral density SNPs, implying a pivotal role in osteoclast differentiation and activity. The regulatory effects of three differentially expressed G-protein coupled receptors were exemplified by in vitro pharmacological modulation of complement 5A receptor 1 (C5AR1), somatostatin receptor 2 (SSTR2), and free fatty acid receptor 4 (FFAR4/GPR120). Activating C5AR1 enhanced osteoclast formation, while activating SSTR2 decreased resorptive activity of mature osteoclasts, and activating FFAR4 decreased both number and resorptive activity of mature osteoclasts. In conclusion, we report the transcriptional reprogramming during human osteoclast differentiation and identified SSTR2 and FFAR4 as anti-resorptive G-protein coupled receptors as well as FLNB and LOX-1 as potential molecular markers of osteoclast activity. These data can help future investigations to identify molecular regulators of osteoclast differentiation and activity and provide the basis for novel anti-osteoporotic targets.
Project description:Expressions of mRNA in osteoclast precursors treated with RANKL and M-CSF for 0, 24, and 72 h. We used DNA microarray to analyze the changes in mRNA expressions during osteoclastogegesis. Experiment Overall Design: Osteoclast precursors were induced from mouse bone marrow cells by M-CSF for 3 days and treated with RANKL. WE extracted total RNA and analyzed by DNA microarray.