Genome-wide analysis of gene expression in human iPSCs and NSCs engineered by safe-harbor TALEN-mediated gene targeting
ABSTRACT: Human iPSCs and NSCs were engineered by AAVS1 and/or C13 safe-harbor TALENs which mediated targeted integration of various reporter genes at single or dual safe-harbor loci. Multiple clones of targeted human iPSCs were used to compare with parental untargeted NCRM5 iPSCs. Polyclonal targeted human NSCs were used to compare with their parental untargeted NCRM1NSCs or H9NSCs. Total RNA obtained from targeted human iPSCs or NSCs compared to untargeted control iPSCs or NSCs.
Project description:PDX1 binds and regulates numerous genes involved in human pancreatic development but also binds hepatic genes Total RNA was extracted from day 0, day 10 and day17 of differentiated HES3 cells. Comparisons were made for day 10 and day 17 to day 0.
Project description:Recent data demonstrate that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways. This network model suggests why inhibition of a single component of a canonical pathway, even when targeting a mutationally activated driver of cancer, has insufficiently dramatic effects on the treatment of cancer. The biological outcome of signals propagated through a network is inherently more robust and resistant to inhibition of a single network component due to compensatory and redundant signaling events. In this study, we performed a functional chemical genetic screen analogous to synthetic lethal screening in yeast genetics to identify novel interactions between signaling inhibitors that would not be predicted based on our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. Among the most robust and surprising results was synergy between sorafenib, a multi-kinase inhibitor with activity against Raf, and diclofenac, a non-steroidal anti-inflammatory drug (NSAID). This synergy did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. The NSAIDs celecoxib and ibuprofen could qualitatively substitute for diclofenac. Similarly, the MEK inhibitor PD325901 and the Raf inhibitor RAF265 could qualitatively substitute for sorafenib. These drug substitution experiments suggest that inhibition of cyclo-oxygenase and MAP kinase signaling are components of the observed synergistic cytotoxicity. Genome-wide expression profiling demonstrates synergy-specific down-regulation of survival-related genes. This study provides proof of principle that synthetic lethal screening can uncover novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially depending on unexplored components of the cell genotype. RNA from VMM39, DM331, and SLM2 cells with/without mutations in Ras and/or Braf, treated with Sorafenib and/or Diclofenac.
Project description:Molecular subtypes of breast cancer are characterized by patterns of gene expression, which can be used to predict response to therapy and overall clinical outcome. The luminal breast cancer subtypes are defined by the expression of ER-alpha (ERa) and a set of ERa-associated genes. The transcription factor activator protein 2C (TFAP2C, AP-2C, AP-2g) transcription factor plays a critical role in regulating cell growth and differentiation during ectodermal development and has been implicated in the regulation of ERa and other luminal-associated genes in breast cancer. While TFAP2C has been established as a prognostic factor in human breast cancer, the role of TFAP2C in development of the luminal epithelial cells in the normal mammary gland and in breast cancer have remained elusive. Herein, we demonstrate a critical role of TFAP2C in maintaining the luminal differentiation phenotype during normal mammary development and in luminal breast carcinoma cell lines. Total RNA from MCF7 cells with and without knockdown of TFAP2c. 3 biological replicates, with 2 technical replicates each, were performed for each sample type.
Project description:The root cap surrounds the root meristem, protecting it from harsh environments and facilitating root movement through soil. In Arabidopsis, new root cap cells produced in the meristem displace older cells toward the root periphery, where they undergo programmed cell death and are released from the root. This rapid cell turnover is a unique feature of the root cap and is modulated in part by the combined action of four NAC transcription factors, as well as cell wall modification enzymes. Here we show that the transcription factor NIN-LIKE PROTEIN 7 (NLP7) regulates root cap maturation in Arabidopsis by modulating expression of each of the NAC TFs as well as several cell wall modifying enzymes. NLP7 is a homolog of NIN, a transcription factor required for nodulation in Lotus japonicas, and is highly expressed in the columella root cap. Mutants have altered columella root cap development and decreased levels of homogalacturonan, a major component of pectin. Reverse genetic analysis of genes differentially expressed in nlp7 roots showed that two cell wall modifying enzymes, CELLULASE5 and XTH5, modulate root cap maturation likely downstream of NLP7. Plant cell wall modification is critical for nodulation, and we propose that this characteristic is also present in NLPs. We used microarrays to identify the differences in gene expression between whole roots of WT and nlp7-1 Arabidopsis plants Roots were cut at the root/hypocotyl junction and collected into RLT buffer in the RNeasy plant mini kit. Approximately 60 roots were collected per replicate, with two biological replicates. RNA was extracted using the RNeasy Micro Kit. Probes for array analysis were preparedfrom 1μg total RNA with the one-cycle amplification protocol by Affymetrix according to the manufacturer's instructions. Samples were submitted to Expression Analysis Inc. (Durham, NC) for hybridization to Arabidopsis Whole Genome ATH1 Affymetrix GeneChips.
Project description:Dendritic cells (DC) arise from a diverse group of hematopoietic progenitors and have marked phenotypic and functional heterogeneity. We have found previously that activation of protein kinase C beta 2 (PRKCB2) by cytokines or phorbol esters drives normal human CD34(+) hematopoietic progenitors and myeloid leukemic blasts (KG1, K562 cell lines, and primary patient blasts) to differentiate into DC, but the genetic program triggered by PRKCB2 activation that results in DC differentiation is only beginning to be characterized. Of the cPKC isoforms, only PRKCB2 was consistently activated by DC differentiation-inducing stimuli in normal and leukemic progenitors. To examine early changes in gene expression following PRKCB2 activation, we employed the following cell lines: (1) the CD34(+) human acute myeloid leukemia derived cell line KG1, which undergoes DC differentiation following phorbol ester treatment; (2) KG1a, a spontaneously arising differentiation-resistant daughter cell line of KG1 that has lost PRKCB2 expression; (3) clones established from KG1a that stably express exogenous PRKCB2-GFP fusion proteins and are once again able to undergo DC differentiation (KG1a-PRKCB2-GFP Clone E9 and Clone E11). We examined changes in gene expression in these cells following treatment with the phorbol ester PMA (phorbol 12-myristate 13-acetate) for 2 hours. Since KG1 and KG1a differ in PRKCB2 expression but have similar expression of the other protein kinase C isoforms, this protocol will allow for the identification of genes regulated by PRKCB2 activation. KG1, KG1a, E9, and E11 were cultured for 2 hours in normal media +/- PMA (10 ng/ml). RNA was then isolated using RNeasy mini-columns (Qiagen) following the standard protocol. Samples were then sent to Expression Analysis, who carried out quality control, sample processing/labeling, hybridization to Affymetrix GeneChip arrays (Human U133 2.0 Plus), imaging, and analysis according to their standard protocols. Gene expression profiles were generated for each cell line in the presence and absence of phorbol ester stimulation, for a total of 12 samples (E9 +/- PMA and E11 +/- PMA were run in duplicate).
Project description:Mechanisms of neuroendocrine tumor (NET) proliferation are poorly understood and therapies that effectively control NET progression and metastatic disease are limited. We found amplification of a putative oncogene, RABL6A, in primary human pancreatic NETs (PNETs) that correlated with high level RABL6A protein expression. Consistent with those results, stable silencing of RABL6A in cultured BON-1 PNET cells revealed that it is essential for their proliferation and survival. Cells lacking RABL6A predominantly arrested in G1 phase with a moderate mitotic block. Pathway analysis of microarray data suggested activation of the p53 and retinoblastoma (Rb1) tumor suppressor pathways in the arrested cells. Specific inactivation of p53 had no effect on the RABL6A knockdown phenotype, indicating RABL6A functions independent of p53 in this setting. By comparison, Rb1 inactivation restored G1 to S phase progression in RABL6A knockdown cells although it was insufficient to override the mitotic arrest and cell death caused by RABL6A loss. Thus, RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET proliferation and development. This work identifies RABL6A as a new negative regulator of Rb1 that is essential for PNET proliferation and survival. We suggest RABL6A is a new potential biomarker and target for anticancer therapy in PNET patients. Total RNA obtained from human BON-1 PNET cells with RABL6A shRNA knockdown compared to BON-1 cells expressing control vector.
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: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