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SRP119557
Homo sapiens
24 Downloadable Samples
Illumina HiSeq 2000
Description
In order to identify and characterize novel human gene expression responses to glucocorticoids, we exposed the human lung adenocarcinoma cell line, A549, to the synthetic glucocorticoid dexamethasone for 1, 3, 5, 7, 9, and 11 hrs in duration as well as to a paired vehicle control, ethanol. We assayed gene expression with RNA-seq and clustered gene expression profiles using an infinite Gaussian process mixture model. Overall design: Time series treatment of human A549 cells with dexamethasone or paired vehicle control.
Publication Title
Clustering gene expression time series data using an infinite Gaussian process mixture model.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 12 Downloadable Samples
- IlluminaHiSeq2500
Description
Epigenetic modifications determine the structure and regulation of eukaryotic genomes and define key signatures of cell lineage specification. Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 (H3K27) at its target sites, leading to robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. In contrast to conventional dCas9-based activators, the acetyltransferase fusion effectively activated genes from enhancer regions and with individual guide RNAs. The core p300 domain was also portable to other programmable DNA-binding proteins. This technology enables the targeted perturbation of native epigenetic architecture and will be useful for reprogramming the epigenome for applications in genomics, genetics, disease modeling, and manipulating cell fate. Overall design: HEK293T cells were transfected in triplicate with plasmids expressing synthetic transcription factors. The synthetic TFs were either (a) dCas9-VP64 fusion protein and a targeting guide RNA (gRNA), or (b)dCas9-p300 fusion protein containing the catalytic domain of p300 and a targeting guide RNA (gRNA). As a control, cells were transfected with plasmids expressing dCas9 alone and dCas9 fused with a aceryltransferase null mutatnt form of the p300 catalytic domain (D1399Y, as in text). After transfection, RNA-seq was used to identify differential expressin at on-target and off-target sites.
Publication Title
Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 11 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Evidence for multiple roles for grainyhead-like 2 in the establishment and maintenance of human mucociliary airway epithelium.[corrected].
Sample Metadata Fields
Specimen part, Treatment
View Samples- Homo sapiens
- 8 Downloadable Samples
- Illumina HiSeq 2000
Description
The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. Here, we focus on the role of GRHL2 in primary human bronchial epithelial (HBE) cells, using either shRNA or a dominant negative protein (DN-GRHL2) to inhibit its function. We follow changes in epithelial phenotype, and in gene transcription using RNA-seq or microarray analysis, both in undifferentiated basal cells and in cells differentiating in air-liquid interface culture into a mucociliary epithelium with transepithelial electrical resistance. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2. Using ChIP-seq to map sites of GRHL2 binding in the basal cells we identify 7,687 potential primary targets, and confirm that GRHL2 binding is strongly enriched near GRHL-regulated genes. Different subsets of the large cohort of potential GRHL2 targets appear to be active in basal and differentiated cells. Taken together, the results strongly support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell adhesion, polarity and morphogenesis. Overall design: Frozen primary human bronchial epithelial (HBE) cells were obtained from three donors. Passage 2 cells at 40% confluence were infected with H2B-GFP or DN-GRHL2 lentivirus and 1 mg/ml puromycin added 48 h later. At confluence, Doxycycline 0.5 mg/ml was added for 24 h. RNA-seq was performed on all six samples, as well as samples from two donors that were not infected.
Publication Title
Evidence for multiple roles for grainyhead-like 2 in the establishment and maintenance of human mucociliary airway epithelium.[corrected].
Sample Metadata Fields
Subject
View Samples- Homo sapiens
- 5 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ progenitors. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung.
Publication Title
Evidence for multiple roles for grainyhead-like 2 in the establishment and maintenance of human mucociliary airway epithelium.[corrected].
Sample Metadata Fields
Specimen part, Treatment
View Samples- Homo sapiens
- 6 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung.
Publication Title
Evidence for multiple roles for grainyhead-like 2 in the establishment and maintenance of human mucociliary airway epithelium.[corrected].
Sample Metadata Fields
Specimen part, Treatment
View Samples- Homo sapiens
- 6 Downloadable Samples
- IlluminaHiSeq2000
Description
Synthetic transcription factors can be applied to many areas of biotechnology, medicine, and basic research. Currently, the most common method for engineering synthetic transcription factors has been based on programmable DNA-binding domains of zinc finger proteins, Transcription Activator-Like Effectors (TALEs), and most recently the CRISPR/Cas9 system. These transcription factor platforms consist of the DNA-binding domain fused to potent transcriptional activation domains, most commonly the tetramer of the minimal transactivation domain of the VP16 protein from herpes simplex virus, referred to as VP64. Although many applications are well-suited for the targeted activation of a single gene, genetic reprogramming requires the coordinated regulation of many nodes of natural gene networks as is typically performed by naturally occurring reprogramming factors. Thus we sought to combine principles from each of these approaches by attaching potent transcriptional activation domains to a natural reprogramming factor to increase the efficiency and/or rate of cell fate conversion. In this study, we evaluated the effects of fusing potent activation domains to the transcription factor MyoD, the master regulator of the skeletal myoblast lineage. In certain non-myogenic lineages, MyoD overexpression causes upregulation of the myogenic gene network and conversion to a myoblast phenotype including cell fusion into multinucleated myotubes. Compared to wild-type MyoD, the VP64-MyoD fusion protein induced greater overall reprogramming of global gene expression. This simple approach for increasing the potency of natural reprogramming factors circumvents the need for screening engineered proteins and leads to a more robust cellular reprogramming compared to treatment with the wild type transcription factor. Overall design: Human dermal fibroblasts were transduced with a single tet inducible lentivirus that expresses either WT-MyoD or VP64-MyoD in response to treatment with doxycycline. Untreated human dermal fibroblast served as the negative control. Gene expression was measured using mRNA-seq, and differential expression was calculated using DESeq. All experiments were performed in biological duplicates.
Publication Title
Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 13 Downloadable Samples
- IlluminaHiSeq2000
Description
Synthetic DNA-binding proteins have found broad application in gene therapies and as tools for interrogating biology. Engineered proteins based on the CRISPR/Cas9 and TALE systems have been used to alter genomic DNA sequences, control transcription of endogenous genes, and modify epigenetic states. Although the activity of these proteins at their intended genomic target sites have been assessed, the genome-wide effects of their action have not been extensively characterized. Additionally, the role of chromatin structure in determining the binding of CRISPR/Cas9 and TALE proteins to their target sites and the regulation of nearby genes is poorly understood. Characterization of the activity these proteins using modern high-throughput genomic methods would provide valuable insight into the specificity and off-target effects of CRISPR- and TALE-based genome engineering tools. We have analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators targeted to the promoters of two different endogenous human genes in HEK293T cells using a variety of high-throughput DNA sequencing methods. In particular, we assayed the DNA-binding specificity of these proteins and their effects on the epigenome. DNA-binding specificity was evaluated by ChIP-seq and RNA-seq was used to measure the specificity of these activators in perturbing the transcriptome. Additionally, DNase-seq was used to identify the chromatin state at target sites of the synthetic transcriptional activators and the genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these genome engineering technologies are highly specific in both binding to their promoter target sites and inducing expression of downstream genes when multiple activators bind to a single promoter. Moreover, we show that these synthetic activators are able to induce the expression of silent genes in heterochromatic regions of the genome by opening regions of closed chromatin and decreasing DNA methylation. Interestingly, the transcriptional activation domain was not necessary for DNA-binding or chromatin remodeling in these regions, but was critical to inducing gene expression. This study shows that these CRISPR- and TALE-based transcriptional activators are exceptionally specific. Although we detected limited binding of off-target sites in the genome and changes to genome structure, these off-target event did not lead to any detectable changes in gene regulation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. Overall design: HEK293T cells were transfected in triplicate with plasmids expressing synthetic transcription factors. The synthetic TFs were either (a) dCas9-VP64 fusion protein and a targeting guide RNA (gRNA), or (b) a TALE-VP64 fusion protein engineered to bind to a specific target site in the genome. As a control, cells were transfected with plasmids expressing GFP. After transfection, RNA-seq was used to identify both on-target and off-target binding sites for the synthetic TFs. The data in this submission were generated using the TALE transfection experiments.
Publication Title
Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 26 Downloadable Samples
- Illumina HiSeq 2000
Description
Synthetic transcription factors can be applied in many areas of biotechnology, medicine, and basic research. In contrast to current methods based on engineering new DNA-binding proteins, we show that Cas9 fused to a transcriptional activation domain can be targeted by combinations of guide RNA molecules to induce the expression of endogenous human genes. This simple approach for targeted gene activation circumvents the need for engineering new proteins and will enable widespread synthetic gene regulation. Overall design: HEK293T cells were transfected with plasmid expressing Cas9-VP64 fusion protein and a guide RNA. As a control, empty guide RNA was transfected. Gene expression was then measured using mRNA-seq, and differential expression calculated using DESeq. All experiments were performed in biological duplicates or triplicates.
Publication Title
RNA-guided gene activation by CRISPR-Cas9-based transcription factors.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 8 Downloadable Samples
- Affymetrix Human Exon 1.0 ST Array [transcript (gene) version (huex10st)
Description
Epstein-Barr virus (EBV) is an oncogenic virus that is associated with the pathogenesis of several human lymphoid malignancies, including Hodgkin's lymphoma. Infection of normal resting B cells with EBV results in activation to lymphoblasts that are phenotypically similar to those generated by physiological stimulation with CD40L plus IL-4. One important difference is that infection leads to the establishment of permanently growing lymphoblastoid cell lines, whereas CD40L/IL-4 blasts have finite proliferation life-spans. To identify early events which might later determine why EBV infected blasts go on to establish transformed cell lines, we performed global transcriptome analyses on resting B cells and on EBV and CD40L/IL-4 blasts after 7 days culture. As anticipated, there was considerable overlap in the transcriptomes of the two types of lymphoblasts when compared to the original resting B cells, reflecting common changes associated with lymphocyte activation and proliferation.
Publication Title
Induction of interferon-stimulated genes on the IL-4 response axis by Epstein-Barr virus infected human b cells; relevance to cellular transformation.
Sample Metadata Fields
No sample metadata fields
View Samples