Project description:Breast and ovarian cancer susceptibility genes, BRCA1 and PALB2 have enigmatic roles in cellular growth and mammalian development. While these genes are essential for growth during early developmental programs, inactivation later in adulthood leads to increased growth and formation of tumors, leading to their designation as tumor suppressors. We performed genome-wide analysis assessing their chromatin residence and gene expression responsiveness using high throughput sequencing in breast epithelial cells. These experiments revealed a critical role for BRCA1 and PALB2 in transcriptional responsiveness to NF-kB, a crucial mediator of growth and inflammatory response during development and cancer. Importantly, we also uncovered a vital role for these proteins in response to retinoic acid (RA), a growth inhibitory signal in breast cancer cells, which may constitute the basis for their tumor suppressor activity. MCF-7 cells were infected with PALB2, BRCA1, p65/RelA and non targeting (SCR) shRNAs. After puromycin selection, cells were stimulated with 10ng/ml TNF-alpha 1 hour
Project description:Breast and ovarian cancer susceptibility genes, BRCA1 and PALB2 have enigmatic roles in cellular growth and mammalian development. While these genes are essential for growth during early developmental programs, inactivation later in adulthood leads to increased growth and formation of tumors, leading to their designation as tumor suppressors. We performed genome-wide analysis assessing their chromatin residence and gene expression responsiveness using high throughput sequencing in breast epithelial cells. These experiments revealed a critical role for BRCA1 and PALB2 in transcriptional responsiveness to NF-kB, a crucial mediator of growth and inflammatory response during development and cancer. Importantly, we also uncovered a vital role for these proteins in response to retinoic acid (RA), a growth inhibitory signal in breast cancer cells, which may constitute the basis for their tumor suppressor activity. MCF-7 cells were infected with PALB2, BRCA1 and non targeting (SCR) shRNAs. After puromycin selection, cells were stimulated with 10microM Retinoic Acid (RA) for 24 hours
Project description:We investigated the genomic occupancy of INTS11, in normal condition and after stimulation of EGF. Total RNAPII was profiled in the presence or absence of INTS11, along with the Super Elongation Complex proteins AFF4 and ELL2. Additionally, we extensively examined the transcriptional response to EGF, before and after depletion of INTS11, using RNA-seq on ribosome-depleted total RNA and Global Run-on sequencing (GRO-seq). EGF stimulation of Hela cells, trasnfected with a control shRNA, INTS1 shRNA or INTS11 shRNA
Project description:The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is a basal transcription factor that mediates the transcriptional activation of snRNAs. Here, we describe the genome-wide occupancy of the SNAPC1_and SNAPC4 subunits of SNAPc. While the SNAPC4 occupancy was in accord with the role for SNAPc in snRNA transcription, SNAPC1_displayed a broader genomic profile mirroring that of RNA polymerase II at highly active protein-coding genes. Our functional analysis revealed a role for SNAPC1_in regulation of both basal and activator-induced transcription of protein-coding genes. These studies expand the role for SNAPC1_beyond its regulation of snRNA transcription. EGF stimulation of Hela cells, transfected with a control sh (scr) or a SNAPC1_sh
Project description:This SuperSeries is composed of the following subset Series: GSE37403: Genome-wide analysis of the SNAPc complex [ChIP-Seq] GSE41528: Genome-wide analysis of the SNAPc complex [Illumina array] Refer to individual Series
Project description:Mesenchymal stromal cells (MSCs) are of high relevance for the regeneration of mesenchymal tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age, their limited availability in human tissues and the need of in vitro expansion prior to treatment. We therefore aimed to determine to which degree in vitro aging and chronological aging may be similar processes or if in vitro culture-related changes at the cellular and molecular level are at least altered as a function of donor age. For that purpose we established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Transcriptome analysis revealed that long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function. Accordingly, functional analysis demonstrated altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging. Based on specific differences observed between the impact of chronological and in vitro MSC aging we conclude that both are distinct processes. Total mRNA obtained from MSCs from 12 month and three week old Sprague Dawley rats of passage 2, 30 and 100. Total RNA was isolated using Trizol® (Invitrogen) reagent as describes previously and purified using Qiagen RNeasy® mini kit (Qiagen, Germany, www.qiagen.com) according to manufacturers instruction. Illumina® BeadChip hybridization: Biotinylated cRNA was produced from 500ng total RNA using llumina® TotalPrep™ RNA amplification kit (Invitrogen). Illumina® RatRef-12 Expression BeadChips hybridization, washing, Cy3 streptavidin staining, and scanning were performed using Illumina® BeadStation 500 platform.
Project description:Human induced pluripotent stem (iPS) cells are capable of differentiating into derivatives of the three embryonic germ layers both in vitro and in vivo. To date the the molecular differences between teratoma-forming cells and non-teratoma-forming cells has not been analysed. A cell line, B1, bears typical ES cell-like morphology, expression of pluripotency-associated genes, and in vitro pluripotency capacity, but fails to form teratomas after subcutaneously injected into immune-deficient mice based on histological analysis. Besides histological analysis, we characterized the tumors derived from line B1, and teratomas derived from bona fida iPS and ES (line H1) cells respectively, using microarray-based gene expression analysis. The expression levels of pluripotency-associated markers in B1 cells were comparable to that in iPS and ES cells, while the complexity of tissue expression commitment was decreased upon spontaneous differentiation of B1 cells as compared to iPS and ES cells. Total RNA obtained from HFF1 (human foreskin fibroblast) cells, line B1, iPS-A4, iPS-B4 and ES (line H1) cells, and their derived tumors in immune-deficient mice.
Project description:Microarray analysis of gene expression in 2-cell embryos obtained from developmentally competent MII oocytes or developmentally incompetent MII (NSN) oocytes. In this study we have compared the expression profile of 2-cell embryos obtained after following in vitro fertilisation of developmentally competent (control) or incompetent (NSN) MII oocytes with the aim of identifying the gene expression networks that operate at this specific stage of development.
Project description:Human somatic fibroblasts can be reprogrammed to induced pluripotent stem (iPS) cells by exogenic expression of the Yamanaka factors (OCT4, SOX2, KLF4 and MYC) after about 1 month. To gain some insight into the early processes operative in fibroblast reprogramming, we profiled genome-wide transcription levels using Illumina microarrays in the starting donor cells-human foreskin fibroblast (HFF1) cells and at three time points after OSKM transduction (24 h, 48 h, 72 h), as well as two iPS cell lines (iPS2, iPS4) and hES cell lines (H1, H9). We show that within the context of the viral transduction reprogramming protocol, the donor cell response to viral transfection perturbs redox homeostasis, which induces oxidative damage on the donor cells' protein and DNA. This leads to activation of p53, senescence, and apoptosis, greatly reducing the efficiency of reprogramming. Total RNA obtained from HFF1 (human foreskin fibroblast) cells, OSKM-transduced HFF1 cells after 24h, 48h, 72h, undifferentiated hESCs, iPSCs.