RNA-seq of control and CHH (cartilage-hair hypoplasia) fibroblasts transdifferentiated into chondrocytes-like cells
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ABSTRACT: We used primary human CHH (cartilage-hair hypoplasia) and control fibroblasts in a chondrogenic transdifferentiation model (FDC; fibroblast-derived chondrocytes) to determine the chondrogenic capacity and differential pathway regulation of CHH cells. For the sequencing experiment, dermal fibroblasts from control donors (n=4) and CHH patients (n=4) were isolated from skin biopsies, plated at high density into wells coated with aggrecan, and cultured for three days in an FDC transdifferentiation medium. Total RNA was isolated at three different time points (Day 0, 1, and 3; in total 24 RNA samples), and sequenced using the NextSeq platform (Illumina).
Project description:Nuclear lamins contact the genome at the nuclear periphery through large domains and are involved in chromatin organization. Among broad peak calling algorithms available to date, none are suited for mapping lamin-genome interactions genome-wide. We disclose a novel algorithm, Enriched Domain Detector (EDD), for analysis of broad enrichment domains from ChIP-seq data. EDD enables discovery of genomic domains interacting with broadly distributed chromatin-associated proteins such as lamins. The main advantage of EDD over existing broad peak callers is sensitivity to domain width rather than enrichment strength at a particular site, and robustness against local variations. EDD is downloadable from http://github.com/eivindgl/edd. RNA-seq experiments in human normal dermal fibroblasts (Lonza CC-2511; LDFs) and human normal primary dermal fibroblasts (Norwegian Stem Cell Center AD04DFs).
Project description:Follicular dendritic cells (FDC) are important stromal cells within the B cell follicles and germinal centres (GC) of secondary lymphoid tissues. FDC trap and retain native antigens on their surfaces in the form of immune complexes which they display to B cells, in order to select those cells with the highest antigen affinity. MicroRNAs are short, non-coding RNAs of approximately 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level by repressing the translation of target genes. In the current study in vivo and in vitro systems were used to identify microRNAs that are differentially expressed as a result of FDC depletion. Constitutive lymphotoxin-β receptor (LTβR) stimulation is required to maintain FDC in their differentiated state. We show that the rapid de-differentiation of spleen FDC that followed LTβR-blockade, coincided with a significant decrease in the expression of mmu-miR-100-5p, mmu-miR-138-5p and mmu-miR-2137. These microRNAs were shown to be expressed in the FDC-like cell line, FL-YB, and specific inhibition of mmu-miR-100-5p significantly enhanced expression of Il6, Ptgs1/2 and Tlr4 in this cell line. The expression of each of these genes by FDC plays an important role in regulating GC size and promoting high-affinity antibody responses, suggesting that mmu-miR-100-5p may help regulate their expression during GC reactions. C57BL/6 mice were given a single intravenous injection of 100 µg of LTβR to temporarily deplete their FDC. At intervals after treatment 4 spleens from each group were harvested and RNA prepared. For each group samples were pooled into 2 groups of 2 and microRNA expression levels compared. Spleens from LTb-/- mice were also analysed.
Project description:We observed that follicular dendritic cell line induced a new type of CD11b+ myeloid cells (FDMCs) when cultured with a lineage-negative c-kit+ population from mouse spleen cells. Thus, we used microarrays to characterize gene expression of FDMCs. FDMCs were induced on FDC line for RNA extraction and hybridization on Affymetrix microarrays. Microarray data of "FDC-induced myeloid cells (FDMCs)" sample were combined with a large collection of other cell- and tissue-specific gene expression data sets (total: 205 individual data sets). Raw data (.cel) files were normalized using Robust Multichip Analysis (RMA Express; http://rmaexpress.bmbolstad.com/), annotated using the latest libraries available from Affymetrix, and arranged according to cell-type grouping.
Project description:Follicular dendritic cells (FDC) are important stromal cells within the B cell follicles and germinal centres (GC) of secondary lymphoid tissues. FDC trap and retain native antigens on their surfaces in the form of immune complexes which they display to B cells, in order to select those cells with the highest antigen affinity. MicroRNAs are short, non-coding RNAs of approximately 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level by repressing the translation of target genes. In the current study in vivo and in vitro systems were used to identify microRNAs that are differentially expressed as a result of FDC depletion. Constitutive lymphotoxin-M-NM-2 receptor (LTM-NM-2R) stimulation is required to maintain FDC in their differentiated state. We show that the rapid de-differentiation of spleen FDC that followed LTM-NM-2R-blockade, coincided with a significant decrease in the expression of mmu-miR-100-5p, mmu-miR-138-5p and mmu-miR-2137. These microRNAs were shown to be expressed in the FDC-like cell line, FL-YB, and specific inhibition of mmu-miR-100-5p significantly enhanced expression of Il6, Ptgs1/2 and Tlr4 in this cell line. The expression of each of these genes by FDC plays an important role in regulating GC size and promoting high-affinity antibody responses, suggesting that mmu-miR-100-5p may help regulate their expression during GC reactions. C57BL/6 mice were given a single intravenous injection of 100 M-BM-5g of LTM-NM-2R to temporarily deplete their FDC. At intervals after treatment 4 spleens from each group were harvested and RNA prepared. For each group samples were pooled into 2 groups of 2 and microRNA expression levels compared. Spleens from LTb-/- mice were also analysed. One channel was used for the actual sample, the second channel was used for internal QC reference.
Project description:Previous research has reported that FDC-SP had similar molecular properties to statherin, a protein exists in saliva which plays important roles in preventing Ca precipitation. Further biomolecular study has suggested that the expression of FDC-SP may be associated with periodontal ligament (PDL) phenotype expression. Therefore, we hypothesized that FDC-SP may play specific roles in the inhibition of calcium precipitation during periodontal regeneration, as well as affect phenotype expression of periodontal ligament cells (PDLCs) during the differentiation process. To investigate this, we applied microarray technology to identify gene expression changes in hPDLCs transfected with FDC-SP and then clustered them according to their biological functions. We firstly established a recombinant lentiviral vector containing FDC-SP and obtained safe and efficient FDC-SP overexpression in human periodontal ligament cells (hPDLCs). After that, we applied Agilent Whole Human Genome Oligo Microarray (4M-CM-^W44K) to identify differentially expressed genes between empty vector-transfected hPDLCs and FDC-SP -transfected ones and then clustered them according to their biological functions. 3 independent experiments were performed and the empty vector-transfected hPDLCs were used as control.
Project description:To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. EMSA confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50X methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. mRNA-seq of primary female mouse dermal fibroblasts with and without thapsigargin identified differentially expressed genes. Genes that are commonly bound by CEBPB and ATF4 to TGAT|GCAA (the best-bound 8-mer in the array) at the promoters were highly expressed and up-regulated or remained unchanged in the thapsigargin treated primary female mouse dermal fibroblasts. RNA-Seq: Examination of whole genome transcriptome profiles (RNA-seq) of primary mouse dermal fibroblasts with and without Thapsigargin treatment ChIP-Seq: Examination of transcription factor binding in dermal fibroblasts with and without Thapsigargin teratment BS-Seq: Determination of whole genome DNA methylation profiles (BS-seq) of primary mouse dermal fibroblasts
Project description:The goal of this study was to determine the similarity between human dermal microvascular endothelial cells, induced endothelial cells from fibroblasts, and fibroblasts through RNA-seq expression analysis. RNA samples from independently induced cultures, plus fibroblast and human dermal microvascular endothelial cultures were converted into individual cDNA libraries using Illumina TruSeq methods and subjected to single-end 50 base-sequence analysis at 20-30 million read depths. Examination of one fibroblast culture, one human dermal mibrovascular endothelial cell culture, and two induced endothelial cell cultures.
Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies. Duplicate cDNA libraries of two IVF-ESCs, three sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, and the parental fibroblast line were sequenced using Illumina HiSeq 2000. The sequence reads were mapped to hg19 reference genome and hits that passed quality filters were analyzed for differential expression.
Project description:Follicular dendritic cell (FDC) sarcomas are rare mesenchymal tumours (MTs) with variable clinical, morphologic and phenotypic characteristics. We investigated the transcriptome of 29 FDC sarcomas and compared it with that of other MTs, microdissected Castleman FDCs, and normal fibroblasts. On unsupervised analysis, FDC sarcoma clustered with microdissected FDCs, distinct from other MTs and fibroblasts. The specific endowment of FDC-related programs in FDC sarcomas emerged by applying a gene signature consisting of 1,289 genes differentially expressed between microdissected FDCs and fibroblasts. Supervised analysis comparing FDC sarcomas with microdissected FDCs and other MTs identified 370 and 2,927 differentially expressed transcripts, respectively, which pathway enrichment analysis mainly ascribed to signal transduction, chromatin organization, and extracellular matrix organization programs. Since the transcriptome of FDC sarcomas retained similarity with that of FDCs, we investigated the immune landscape of FDC sarcoma by applying the CIBERSORT analysis to FDC sarcomas and non-FDC MTs, and demonstrated that FDC sarcomas were enriched in TFH and TREG populations, as confirmed in situ by immunohistochemistry. The enrichment in specific T-cell subsets prompted investigating the mRNA expression of the inhibitory immune receptor PD-1 and of its ligands PD-L1 and PD-L2, which were found to be significantly more expressed in FDC sarcomas as compared with other MTs, a finding also confirmed in situ. We demonstrated for the first time the transcriptional relationship of FDC sarcomas with non-malignant FDCs and their distinction from other MTs. Furthermore, we provided evidence of a peculiar immunological microenvironment associated with FDC sarcomas.