The purpose of this study was to identify putative downstream targets of the transcription factor ZIC2 in the mouse embryo. The results indicate loss of NODAL pathway expression, consistent with the observed phenotype of right isomerism in heart, lungs and viscera.
A Requirement for Zic2 in the Regulation of Nodal Expression Underlies the Establishment of Left-Sided Identity.
Specimen part
View SamplesIn order to determine the regulators of outer hair cell postnatal maturation, we utilized the RiboTag mouse model to perform a detailed transcriptomic analysis of outer hair cells at five postnatal developmental time points: P8, P14, P28, 6 weeks (6wk) and 10 weeks (10wk). This analysis resulted in consistent enrichment of outer hair cell expressed genes in the immunoprecipitated RNA compared to whole cochlear input RNA from each time point. Using transcription factor binding motif prediction on a set of defined outer hair cell enriched genes, we further use this dataset to identify the helios transcription factor as a regulator of the postnatal outer hair cell transcriptome. Overall design: Examination of the outer hair cell translatome by outer hair cell expressed HA-tagged ribosomal immunoprecipitation at 5 postnatal timepoints (P8, P14, P21, 6wk and 10wk). Immunoprecipitated samples were compared to input cochlear RNA controls in independent biological duplicates or triplicates.
Helios is a key transcriptional regulator of outer hair cell maturation.
Specimen part, Subject
View SamplesIn order to determine the transcriptional effect of Ikzf2 overexpression in mammalian auditory hair cells, mouse cochleae were transfected with either a control GFP or a Ikzf2 virus between postnatal day 1 and 3 (P1-3) and then harvested for single cell gene expression profiling at postnatal day 8 (P8) on the 10X Genomics Single Cell 3' v2 platform. Overall design: Dataset is composed of two separate single cell RNA-Seq samples captured on the 10X Genomics Chromium platform with the Single Cell 3' Solution v2 chemistry. Viral GFP- (control) or Ikzf2-transfected cochleae were harvested and single cell suspensions prepared from transgenic mice expressing tdTomato in hair cells. Hair cells expressing tdTomato were enriched by flow sorting and then captured on the Chromium system in parellel. Library preparation and sequencing was performed as defined by the 10X Genomics Single Cell 3' Solution v2 protocol.
Helios is a key transcriptional regulator of outer hair cell maturation.
Specimen part, Cell line, Treatment, Subject
View SamplesPurpose: The ability to rationally manipulate the transcriptional states of cells would be of great use in medicine and bioengineering. We have developed a novel algorithm, NetSurgeon, which utilizes genome-wide gene regulatory networks to identify interventions that force a cell toward a desired expression state. Results: We used NetSurgeon to select transcription factor deletions aimed at improving ethanol production in S. cerevisiae cultures that are catabolizing xylose. We reasoned that interventions that move the transcriptional states of cells utilizing xylose toward the fermentative state typical of cells that are producing ethanol rapidly (while utilizing glucose) might improve xylose fermentation. Some of the interventions selected by NetSurgeon successfully promoted a fermentative transcriptional state in the absence of glucose, resulting in strains with a 2.7-fold increase in xylose import rates, a 4-fold improvement in xylose integration into central carbon metabolism, or a 1.3-fold increase in ethanol production rate. Conclusions: We conclude by presenting an integrated model of transcriptional regulation and metabolic flux that will enable future metabolic engineering efforts aimed at improving xylose fermentation to prioritize functional regulators of central carbon metabolism. Overall design: Mutant and wildtype S. cerevisiae cells were put into 48 hour aerobic batch fermentations of synthetic complete medium supplmented with 2% glucose and 5% xylose and culture samples were taken at 4 hours and 24 hours for transcriptional profiling performed by RNA-Seq analysis. In addition, wildtype S. cerevisiae cells were grown in various single carbon sources for 12 hours and culture samples were taken for transcriptional profiling performed by RNA-Seq analysis.
Model-based transcriptome engineering promotes a fermentative transcriptional state in yeast.
Subject
View SamplesWe recently demonstrated mitochondrial degenerations precede muscle wasting in time course progression of CC. However, the extent of muscle perturbations prior to wasting in CC is unknown. Therefore, we performed global gene expression analysis in CC-induced muscle wasting to enhance understanding of intramuscular perturbations across the development of CC. Overall design: Lewis Lung Carcinoma (LLC) was injected into the hind-flank of C57BL6/J mice at 8 wks age with tumor allowed to develop for 1, 2, 3, or 4 wks and compared to PBS injected control. Muscle wasting was evident at 4 wks LLC. Animals were anesthetized using isoflourane and gastrocnemius muscles were collected for analysis. Conclusions: Current findings present novel evidence of transcriptomic shifts and altered cellular pathways in CC-induced muscle wasting.
Transcriptomic analysis of the development of skeletal muscle atrophy in cancer-cachexia in tumor-bearing mice.
Specimen part, Cell line, Subject
View SamplesPurpose: To compare the transcriptome profiles (RNA-seq) of cultured human epididymis cells and tissue from the caput, corpus and cauda regions of the human epididymis. Methods: Human epididymis tissue was obtained with Institutional Review Board approval from 3 patients (UC05, UC06, UC09, range: 22 - 36 years) undergoing inguinal radical orchiectomy for a clinical diagnosis of testicular cancer. None of the epididymides had extension of the testicular cancer. The three anatomical regions: caput, corpus and cauda, were separated and segments of each snap frozen. Adult human epididymis epithelial (HEE) cultures were also established from tissue. RNA was extracted from both tissue and cultured HEE cells and RNA-seq libraries prepared (TruSeq RNA Sample Preparation Kit v2, Low-Throughput protocol, Illumina). Libraries were sequenced on Illumina HiSeq2500 machines. Data were analyzed using TopHat and Cufflinks. Results: Libraries generated ~19-39 million reads per library from the cells (95-99% mapping to the human genome) and ~14-39 million reads from the tissue samples (84-99% mapped). Raw reads were aligned to the genome with Tophat and gene expression values were processed using Cufflinks as Fragments Per Kilobase per Million mapped fragments (FPKM). FPKM values were subject to principle component analysis, which revealed that though caput, corpus and cauda cell samples respectively from UC05, UC06 and UC09 clustered together. RNA-seq data from the 3 biological replicas (UC05, UC06 and UC09) of caput, corpus and cauda were pooled for further analysis. Cufflinks was used to determine differentially expressed genes (DEGs) between caput, corpus and cauda cells, combined from the 3 donors. The gene expression profiles of corpus and cauda are remarkably similar and both differ from the caput to a similar degree. We identified ~40 genes differentially expressed between corpus and cauda and more than 1600 DEGs between caput and cauda. The DEGs for each comparison (caput and corpus/cauda) were analysed using a gene ontology process enrichment analysis (DAVID, Huang et al., NAR 2009;37:1-13, Huang et al., 2009 Nat Prot 4:44-57). Conclusions: Here we describe an in depth analysis of the gene expression repertoire of primary cultures of epithelial cells and intact tissues from each region of the adult human epididymis. These data will be valuable to decipher pathways of normal epididymis function and aspects of epididymis disease that cause male infertility. Overall design: RNA-seq was performed on libraries generated from caput, corpus and cauda-derived cultured cells (passage 2 or 3) from 3 donors and on caput, corpus and cauda tissue from 2 of the same donors. Donor age range: 22 - 36 years.
Expression profiles of human epididymis epithelial cells reveal the functional diversity of caput, corpus and cauda regions.
No sample metadata fields
View SamplesWe hypothesized that broad-scale expression profiling would provide insight into the regulatory pathways that control gene expression in response to stress, and potentially identify novel heat-responsive genes. HEp2 cells were heated at 37 to 43 C for 60 min to gauge the heat shock response, using as a proxy inducible HSP-70 quantified by western blot analysis. Based on these results, microarray experiments were conducted at 37, 40, 41, 42 and 43C (3 replicates/temperature x 5 groups = 15 U95Aver2 GeneChips). Using linear modeling, we compared the sets of microarrays at 40, 41, 42 and 43C with the 37C baseline temperature and took the union of the genes exhibiting differential gene expression signal to create two sets of heat shock response genes, each set reflecting either increased or decreased RNA abundance. Leveraging human and mouse orthologous alignments, we used the two lists of co-expressed genes to predict transcription factor binding sites in silico, including those for heat shock factor 1 (HSF1) and heat shock factor 2 (HSF2) transcription factors. We discovered HSF1 and HSF2 binding sites in 15 genes not previously associated with the heat shock response. We conclude that microarray experiments coupled with upstream promoter analysis can be used to identify novel genes that respond to heat shock. Additional experiments are required to validate these putative heat shock proteins and facilitate a deeper understanding of the mechanisms involved during the stress response.
Transcriptional profiles of human epithelial cells in response to heat: computational evidence for novel heat shock proteins.
No sample metadata fields
View SamplesMYB-bHLH-TTG1 regulates Arabidopsis seed coat biosynthesis pathways directly and indirectly via multiple tiers of transcription factors
MYB-bHLH-TTG1 Regulates Arabidopsis Seed Coat Biosynthesis Pathways Directly and Indirectly via Multiple Tiers of Transcription Factors.
Specimen part
View SamplesHNF1a and HNF1ß recognize the same DNA consensus sequence in the genome, to which they bind as homodimers or heterodimers. Both factors share a high degree of homology their DNA binding and dimerization (N-terminus) regions but have a more divergent C-terminal transactivation domain. HNF1ß is essential for the generation of a functional male reproductive tract in mice and genital tract abnormalities are evident in humans with recessive mutations in HNF1ß. The functions of HNF1a and HNF1ß have been studied in epithelia from other several tissues (liver, kidney, intestine, and pancreas) but their role in the adult human epididymis epithelium (HEE) remains unexplored. We established that HNF1a/ß are expressed in caput HEE cells and are predicted to occupy cis-regulatory elements in these cells. To investigate the contribution of HNF1 in controlling gene expression in caput cells we performed siRNA-mediated depletion of HNF1a and HNF1ß together, followed by RNA-seq analysis. Three replicas of caput cells were transfected with the specific siRNAs or with a non-targeting control siRNA. RNA-seq after HNF1 depletion showed significant alterations in the expression of genes encoding ion channels and exchangers that are involved in controlling the luminal environment in the caput epididymis. Overall design: mRNA profiles from Caput HEE cells transfected with negative control (NC) or HNF1alpha and HNF1beta siRNA, in triplicate.
HNF1 regulates critical processes in the human epididymis epithelium.
No sample metadata fields
View SamplesThree groups of male +b and bb rats were obtained (ages between 6 and 14 months) and intestinal scrapes were taken. Tissues was combined from 3 rats per group and processed for gene chip analysis.
Induction of arachidonate 12-lipoxygenase (Alox15) in intestine of iron-deficient rats correlates with the production of biologically active lipid mediators.
No sample metadata fields
View Samples