ABSTRACT: We compared transcriptional differences between Periostin siRNA treated and GFP-siRNA treated in OP9 cells using Affymetrix mouse 430_2 array.
Project description:DNA methylation is an essential epigenetic mark that is required for normal development. Knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment reveals that methylation is critical for hematopoietic differentiation. To better understand the role of DNA methylation in hematopoiesis, we characterized genome-wide DNA methylation in primary mouse hematopoietic stem cells (HSC), common myeloid progenitors (CMP), and erythroblasts (ERY). Methyl Binding Domain protein 2 (MBD) enrichment of DNA followed by massively-parallel sequencing (MBD-Seq) was used to map genome-wide DNA methylation. Globally, DNA methylation was most abundant in HSC, with a 40% reduction in CMP, and 67% reduction in ERY. Only 3% of peaks arise during differentiation demonstrating a genome-wide decline in DNA methylation during erythroid development. Analysis of genomic features revealed that 98% of promoter CpG islands are hypomethylated, while 20-25% of non-promoter CpG islands are methylated. Proximal promoter sequences of expressed genes are hypomethylated in all cell types, while gene body methylation positively correlates with gene expression in HSC and CMP. Elevated genome-wide DNA methylation in HSC and the positive association between methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSC. Utilizing de novo motif discovery we identified overrepresented transcription factor consensus binding motifs in methylated sequences. Motifs for several ETS transcription factors, including GABPalpha and ELF1 are overrepresented in methylated regions. Our genome-wide survey demonstrates that DNA methylation is markedly altered during myeloid differentiation and identifies critical regions of the genome and transcription factor programs that contribute to hematopoiesis. Examination of changes in methylation profiles during hematopoietic stem cell differentiation
Project description:Analysis of the RNA-seq data performed in IR vs NIR hematopoietic stem cells show the loss of the TNF_via_NFKB signature. We showed that the loss of this signature could be associated with H3K9me3 loss at specific retrotransposable elements . To validate this association, we tested if TNFa treatment before irradiation was able to prevent IR-effect on H3K9me3 loss at retrotransposable elements. For this purpose, we treated mice with TNFa 1h before irradiation (IR_TNF) and performed H3K9me3 cut&tag experiments on hematopoietic stem cells 1 month after irradiation and compared them to hematopoietic stem cells sorted from non irradiated mice (NIR) and from non-treated irradiated mice (IR).
Project description:Mature lymphoid cells express the transcriptional repressor Bach2, which imposes regulation on humoral and cellular immunity. Here we found critical roles for Bach2 in the development of cells of the B lineage, commencing from the common lymphoid progenitor (CLP) stage, with Bach1 as an auxiliary. Overexpression of Bach2 in pre-pro-B cells deficient in the transcription factor EBF1 and single-cell analysis of CLPs revealed that Bach2 and Bach1 repressed the expression of genes important for myeloid cells (M-bM-^@M-^Xmyeloid genesM-bM-^@M-^Y). Bach2 and Bach1 bound to presumptive regulatory regions of the myeloid genes. Bach2hi CLPs showed resistance to myeloid differentiation even when cultured under myeloid conditions. Our results suggest that Bach2 functions with Bach1 and EBF1 to promote B cell development by repressing myeloid genes in CLPs. WT and Bach1 and Bach2 double deficient (DD) CLPs. Multipotent progenitors (MPPs) infected with control-GFP and Bach2-GFP and cultured several condition. Follicular B cells (Fo B) stimulated with IgM. Three expreriments was performed in this paper.
Project description:We aim at understanding how ionizing radiations (IR) increase the risk of developing myeloid leukemia. We recently showed that IR leads to the derepression of retroelements. Retroelements are major contributors of gene regulatory networks. However, the impact of retroelements derepression on the HSC transcriptome and function remains to be addressed. We hypothesized that retroelements derepression is involved in HSC transcriptomic alterations. To answer this question, we performed RNA-seq experiments in hematopoietic stem cells sorted from mice one month after they were irradiated and compared them to controls hematopoietic stem cells sorted from non-irradiated mice.
Project description:Systems vaccinology has emerged as an interdisciplinary field that combines systems wide measurements and network and predictive modeling applied to vaccinology. Here we used the systems vaccinology approach to study the molecular mechanisms underlying the innate responses to the trivalent inactivated influenza (TIV) and live attenuated influenza (LAIV) vaccination in humans, and to identify early gene signatures that predict the magnitude of the antibody responses to influenza vaccination. During the 2008 influenza season, healthy adults were vaccinated with TIV (6 vaccinees) or LAIV (6 vaccinees), and blood samples isolated at day 0 and at day 7 post-vaccination. Cell subsets (B cells, Monocytes, mDCs and pDCs) were FACS-sorted from frozen PBMCs. Microarrays were performed using amplified total RNA.
Project description:Viral dsRNA binds to Retinoic acid Inducible Gene I (RIG-I) Like Receptors (RLRs), promoting the production of Interferon (IFN). Interferon then stimulates the innate and adaptive immune system in an autocrine and paracrine manner. Outside of conical pathways, regulators of the interferon (IFN) activation/response system are poorly characterized. In this study, we used a discovery-biased approach to identify Kinases that are part of the interferon system. Differential changes in phosphorylation sites, in the context of dsRNA RIG-I stimulation, were identified with unbiased mass-spec biased phospho-proteomics. We then computationally identified several Kinases upregulated after RIG-I stimulation from phospho-proteomics data. A Chemoproteomics screen was then used to characterize the altered interferon response in the presence of Kinases inhibitors for the upregulated kinases. Combining unbiased phosphoproteomics with a chemoproteomics screen, we identified several potentially novel regulators of the Interferon system whose inhibition blocked the production of Interferon Stimulated Genes.
Project description:Aberrant expression of the homeodomain transcription factor CDX2 occurs in most cases of acute myeloid leukemia (AML) and promotes leukemogenesis, making CDX2, in principle, an attractive therapeutic target. Conversely, CDX2 acts as a tumor suppressor in colonic epithelium. The effectors mediating the leukemogenic activity of CDX2 and the mechanism underlying its context-dependent properties are poorly characterized, and strategies for interfering with CDX2 function in AML remain elusive. We report data implicating repression of the transcription factor KLF4 as important for the oncogenic activity of CDX2, and demonstrate that CDX2 differentially regulates KLF4 in AML versus colon cancer cells through a mechanism that involves tissue-specific patterns of promoter binding and epigenetic modifications. Furthermore, we identified deregulation of the PPARγ signaling pathway as a feature of AML expressing CDX2, and observed that PPARγ agonists derepress KLF4 and are preferentially toxic to CDX2-positive leukemic cells. These data delineate transcriptional programs associated with CDX2 expression in hematopoietic cells; provide insight into the antagonistic duality of CDX2 function in AML versus colon cancer; and suggest reactivation of KLF4 expression, through modulation of PPARγ signaling, as a new therapeutic modality in a large proportion of AML patients. Experiment 1 (Samples 1-6): The transcriptional changes induced by Cdx2 were assessed ex vivo in primary murine hematopoietic stem and progenitor cells. Bone marrow cells were harvested from 3x15 C57BL/6 mice, and differentiated cells were removed by incubation with rat antibodies against lineage antigens (CD3, CD4, CD8, Gr-1, B220, CD19, IL-7R, Ter119) followed by depletion with magnetic beads (Dynabeads, Invitrogen). Lineage-depleted cells were stained with APC-conjugated anti-c-Kit and PE-Cy5-conjugated goat anti-rat antibodies, and c-Kit+ Lin– cells were sorted using a BD FACSAria cell sorter (BD Biosciences). Cells were plated in RetroNectin-coated tissue culture dishes (Takara Bio) and transduced twice with pMSCV-Cdx2-IRES-GFP or pMSCV-IRES-GFP retroviral constructs. After 48 hours, GFP-positive cells were sorted using a BD FACSAria cell sorter. Experiment 2 (Samples 7-10): The transcriptional changes induced by Cdx2 were assessed in vivo in Cdx2-initiated murine leukemias and compared with those of leukemias initiated by 5 different MLL fusion oncogenes (previously published data, available at GSE13690). Bone marrow cells isolated from C57BL/6 mice were transduced with pMSCV-Cdx2-IRES-GFP, and 8x10e5 GFP-positive cells were injected into lethally irradiated syngeneic recipient mice after 48 hours, followed by injection of 1x10e6 spleen cells from primary leukemic animals into sublethally irradiated secondary recipients. Spleen cells from secondary leukemic animals were harvested.
Project description:In order to investigate how transcription factor dose impacts B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. While combined reduction of Pax5 and Ebf1 dose had minimal impact on the development of the earliest CD19+ progenitors, these cells displayed an increased T-cell potential in vivo and in vitro. Alteration in lineage fate depended on a Notch1 mediated conversion process while no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5+/-Ebf1+/- pro-B cells was reflected in the transcriptional response because even though cells of both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, only the Pax5+/-Ebf1+/- pro-B cells down-regulated genes central for the preservation of stable B-cell identity. This report stresses the importance of transcription factor dose in lymphocyte development and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling after the generation of activated intracellular Notch1. This provides an insight to how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation. EBF-1 ChIP-seq: Cultivated CD43+IgM- cells (ProB) cells from Wt, EBF-1 +/-, PAX-5 +/- and EBF-1 +/- PAX-5 +/- (TH) were assessed for EBF-1 binding by ChIP-seq. Replicate Ebf1 ChIP-seq runs on each genotype (Wt, TH, Ebf1+/- and Pax5+/-) and corresponding inputs were pooled into one dataset and analyzed as one combined sample per genotype. RNA-seq no treatment: Briefly, ProB-cells from C57BL/6J Ebf1+/-Pax5+/- (n=4), WT (n=4), Ebf1+/- (n=2) and Pax5+/- (n=2) were sorted and RNA extracted with Qiagen RNeasy Micro Kit. RNA was sent to UCLA Microarray Core for library preparation and were subsequently for 50 cycles of HiSeq 2000 SBS sequencing generating 20-30 million reads/sample. Data analysis was performed with Arraystar® (DNASTAR)). RNA-seq 1 day on OP9DL1 and OP9: In short, in vitro expanded ProB-cells from C57BL/6J Ebf1+/-Pax5+/- (n=4) and WT (n=4) were exposed either on OP9 or OP9-DL1 stromal cells for 24 hours and RNA extracted with Qiagen RNeasy Micro Kit. Due to low reads, two Wt and Ebf1+/-Pax5+/- were sequenced twice. Libraries were constructed using Nugen's Ovation Ultralow Library systems and were subsequently for 50 cycles of NextSeq500 sequencing generating 20-30 million reads/sample. Data analysis was performed with Arraystar® (DNASTAR)).
Project description:HSC (Sca+ SP) were isolated from 8-12 week C57B6 mice at various time points after treatment with 5-Fluorouracil. RNA was isolated from 50,000-100,000 FACS sorted cells and subjected to two rounds of T7 based linear amplification using Ambion's Message Amp kit. Two replicates from each time point were analyzed. http://www.plosbiology.org/plosonline/?request=get-document&doi=10.1371%2Fjournal.pbio.0020301
Project description:This SuperSeries is composed of the following subset Series: GSE29614: Time Course of Young Adults Vaccinated with Influenza TIV Vaccine during 2007/08 Flu Season GSE29615: Time Course of Young Adults Vaccinated with Influenza LAIV Vaccine during 2008/09 Flu Season GSE29617: Time Course of Young Adults Vaccinated with Influenza TIV Vaccine during 2008/09 Flu Season GSE29618: FACS-sorted cells from Young Adults Vaccinated with Influenza TIV or LAIV Vaccines during 2008/09 Flu Season Refer to individual Series