Several recently emerging ChIP-seq (chromatin immunoprecipitation followed by sequencing) based methods perform chemical steps on bead-bound immunoprecipitated chromatin, posing a challenge for generating similarly treated input controls required for bioinformatics and data quality analyses. Here we present a versatile method for producing technique-specific input controls for ChIP-based methods that utilize additional bead-bound processing steps. Application of this method allowed for discovery of a novel CTCF binding motif from ChIP-exo data. Overall design: HeLa cells were transfected with either a scrambled siRNA or one of two CTCF siRNAs (Thermo Fisher Scientific ? Life technologies) using Lipofectamine RNAiMAX (Thermo Fisher Scientific - Life technologies) and incubated for 24 hr.
PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond.
Cell line, Subject
View SamplesThe emerging correlation between aberrant DNA methylation patterns leading to transcriptional responses that promote and progress many cancers has prompted an interest in discerning the associated regulatory mechanisms. ZBTB33 (also known as Kaiso) is a specialized transcription factor that selectively recognizes mCpG-containing sites as well as a sequence-specific DNA target (termed the KBS) utilizing three Cys2His2 zinc fingers. Increasing reports link ZBTB33 overexpression and transcriptional activities with metastatic potential and poor prognosis, though the specific cellular consequences appear to be dependent on disease phenotype. There is currently little mechanistic insight into how various cellular phenotypes are then able to harness the transcriptional capabilities of ZBTB33 to differentially promote and progress the disease state. Here we have mechanistically interrogated the cell cycle responses mediated by the transcriptional activities of ZBTB33 in two different cell lines. Utilizing a series of ZBTB33 depletion and overexpression studies, we have determined that in HeLa cells ZBTB33 directly occupies the promoter regions of cyclin D1 and cyclin E1 in a KBS and methyl-specific manner, respectively, inducing increased proliferation by promoting RB1 hyper-phosphorylation, allowing for E2F transcriptional activity that coordinates an accelerated G1- to S-phase transition. Conversely, in HEK293 cells ZBTB33 indirectly regulates Cyclin E abundance resulting in reduced RB1 phosphorylation, decreased E2F activity and a decelerated transition through G1-phase. Thus, we have identified a novel mechanism by which ZBTB33 directly mediates the highly coordinated cyclin D1/cyclin E1/RB1/E2F signaling pathway controlling the passage through the G1-phase restriction point and accelerating cellular proliferation in a cancer cell line. Overall design: Determination of cellular and transcriptional consequences for ZBTB33 depletion in HeLa cells.
Cell-specific Kaiso (ZBTB33) Regulation of Cell Cycle through Cyclin D1 and Cyclin E1.
Cell line, Subject
View SamplesGroup 2 innate lymphoid cells (ILC2) are tissue-resident innate lymphocytes that are derived from common lymphoid progenitor (CLP). While specific progenitors and transcription factors essential for ILC2 differentiation have been well studied, external factors that regulate the commitment from CLP to ILC lineage, site that promote ILC2 terminal differentiation, and stromal cells that provide optimal microenvironment for ILC2 specific development are not fully understood. we demonstrated that the three key external factors such as concentration of IL-7 and the strength and duration of Notch signaling conditionally determined the fate of CLP toward T cell, B cell, or ILC lineages, which seems to be an important process from CLP to CHILP differentiation in the fetal liver. Furthermore, we identified ILC progenitors lacking the developmental potential to become T or B cells, and KLRG1- immature ILC2 that require STAT5 for functional maturation in the mesentery. We also identified PDGFRa+gp38+ mesenchymal cells in the mesentery that support ILC2 differentiation from ILC progenitors but not from CLP. Finally, single-cell RNA-sequencing (scRNA-seq) analysis of mesenteric cells demonstrated that PDGFRa+gp38+ cells are heterogeneous populations. Collectively, our result suggested that early differentiation of ILC2 occurs in the primary lymphoid organ with regulation of environmental factors, and final differentiation occurs in the peripheral tissues once after CHILP migrate into the periphery. Overall design: Duplicate samples (mouse 1 and mouse 2) were processed for single cell-based RNA sequencing with Illumina HiSeq 2500 with 50 paired-end reads, using barcorded RNA library.
Peripheral PDGFRα<sup>+</sup>gp38<sup>+</sup> mesenchymal cells support the differentiation of fetal liver-derived ILC2.
Sex, Cell line, Subject
View SamplesCD133 (Prominin1) is pentaspan transmembrane glycoprotein expressed in several stem cell populations and cancers. Reactivity with an antibody (AC133) to a glycoslyated form of CD133 has been widely used for the enrichment of cells with tumor initiating activity in xenograph transplantation assays. We have found by fluorescence-activated cell sorting that increased AC133 reactivity in human embryonic stem cells, colon cancer and melanoma cells is correlated with increased DNA content and reciprocally, that the least reactive cells are in the G1/G0 portion of the cell cycle. Continued cultivation of cells sorted on the basis of high and low AC133 reactivity results in a normalization of the cell reactivity profiles indicating that cells with low AC133 reactivity can generate highly reactive cells as they resume proliferation. The association of AC133 with actively cycling cells may contribute to the basis for enrichment for tumor initiating activity.
Cell cycle-dependent variation of a CD133 epitope in human embryonic stem cell, colon cancer, and melanoma cell lines.
No sample metadata fields
View SamplesGene expression profiling of the medial (MGE), lateral (LGE) and caudal (CGE) ganglionic eminence, and cerebral cortex (CTX) at various embryonic stages (E12.5, E14 and E16).
Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision.
Sex, Specimen part
View SamplesMouse erythroid progenitors (EP) in comparison to granulocyte/monocyte - macrophage progenitors (GMP) from 10 - 16 week old C57/Bl6 - S129Ola (mixed genetic background) purified by flow cytometry
Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E).
No sample metadata fields
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Nos3-/- iPSCs model concordant signatures of in utero cardiac pathogenesis.
Specimen part, Time
View SamplesThrough genome-wide transcriptional comparisons, this study interrogates the capacity of iPSCs to accurately model pathogenic signatures of structural cardiac defects. Herein, we studied the molecular etiology of structural cardiac defects in Nos3-/- mice via transcriptional analysis of stage-matched embryonic and iPSC-derived tissues. In vitro comparisons of differentiated embryoid bodies were calibrated to in utero benchmarks of health and disease. Integrated systems biology analysis of WT and Nos3-/- transcriptional profiles revealed 50% concordant expression patterns between in utero embryonic and ex vivo iPSC-derived tissue. In particular, up-regulation of glucose metabolism (p-value = 3.95x10-12) and down-regulation of fatty acid metabolism (p-value = 6.71x10-12) highlight a bioenergetic signature of early Nos3 deficiency during cardiogenesis that can be recapitulated in iPSC-derived tissues. The in vitro concordance of early Nos3-/- disease signatures supports the utility of iPSCs as a cell-autonomous model of structural heart defects. Moreover, this study supports the use of iPSCs as a platform to pinpoint initial stages of cardiac pathogenesis.
Nos3-/- iPSCs model concordant signatures of in utero cardiac pathogenesis.
Specimen part, Time
View SamplesRationale: Cardiac development is a complex process that results in the first integrated, multi-lineage embryonic tissue. Imperfect developmental progression leads to congenital heart disease, the most common birth defect with developmental corruption affecting more than 1% of all live births. Interrogation of individual genes has provided the backbone for cardiac developmental biology, yet a comprehensive transcriptome derived from natural cardiogenesis is required to establish an unbiased roadmap to gauge innate developmental milestones necessary for stem cell-based differentiation and in vitro disease modeling.
Natural cardiogenesis-based template predicts cardiogenic potential of induced pluripotent stem cell lines.
Specimen part, Cell line, Time
View SamplesWhole testes were dissected from adult males. RNA from five or six biological replicates were generated and the expression profiles were determined using Affymetrix Drosophila Genechip 1 arrays. Comparisons between the bgcn- and Os+bgcn- groups allowed for the identification of stem cell genes.
Novel regulators revealed by profiling Drosophila testis stem cells within their niche.
No sample metadata fields
View Samples