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SRP056859
Mus musculus
44 Downloadable Samples
Illumina HiSeq 2000
Description
The DNA-binding protein, Ikaros, functions as a potent tumor suppressor and hematopoietic regulator. However, the mechanisms by which Ikaros functions in the nucleus remain largely undefined, due in part to its atypical DNA-binding properties and partnership with the poorly understood Mi-2/NuRD complex. In this study, we extended our analysis of thymocyte development and lymphomagenesis in a mouse strain containing a specific deletion of Ikaros zinc finger 4, which exhibits a select subset of abnormalities observed in Ikaros null mice. By examining thymopoiesis in vivo and in vitro, numerous abnormalities were observed. RNA-sequencing revealed that each developmental stage is characterized by mis-regulation of a limited number of genes, with a strong preference for genes modulated in a stage-specific manner. Strikingly, individual genes and pathways rarely exhibited Ikaros-dependence at all developmental stages. Instead, the most consistent feature of aberrantly expressed genes was a reduced magnitude of expression level change during a developmental transition. These results and others suggest that Ikaros may not be a dedicated and consistent activator or repressor of a defined set of genes. Instead, its primary function may be to support the dynamic range of gene expression changes during developmental transitions via atypical molecular mechanisms that remain undefined. Overall design: RNA-Seq of T cells at varying developmental stages and T cells expressing activated Notch in WT and Ikzf1-dF4/dF4 mutant backgrounds
Publication Title
Regulation of gene expression dynamics during developmental transitions by the Ikaros transcription factor.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 55 Downloadable Samples
- IlluminaHiSeq2000
Description
Human neonates and older adults frequently exhibit a reduced capacity to control microbial infections. A variety of mechanisms involving both the innate and adaptive immune systems have been proposed to contribute to these deficiencies. The emergence of RNA sequencing (RNA-seq) as an accurate and quantitative method for examining mRNA levels provides an opportunity to compare transcriptional responses to a stimulus at a global scale in neonates, adults, and older adults. An examination of ex vivo monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection (with cord blood monocytes representing neonatal monocytes) revealed extensive similarities between all three age groups, with only a small number of genes exhibiting statistically significant differences. Using transcription factor motif analyses and RNA-seq data sets from a variety of mouse mutants, the most significant neonatal deficiencies corresponded to genes that require interferon response factor-3 or type 1 interferon signaling for their activation. In older adults, the most striking difference was broad, low-level activation of inflammatory genes prior to stimulation, consistent with prior evidence of a chronic inflammatory state in older adults. These results demonstrate the value of quantitative RNA-seq analyses and the feasibility of cross-species comparisons between well-defined mouse networks and human data sets. Overall design: RNA-seq of primary cells from three independent donors in three different age-groups across 3 time-points stimulated with either LPS or Listeria monocytogenes.
Publication Title
Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 228 Downloadable Samples
- Illumina HiSeq 2000
Description
Bone marrow-derived macrophages derived from C57Bl/6, Myd88-/- and Trif-/-, Ifnar-/-, Atm-/-, Sting-/-, Scid, Irf3-/-, Irf1-/-, p53-/-, Nrf2-/-mice were irradiated with 6Gray ioninzing radiation; C57Bl/6 macrophages were Irradiated in the presence of MAPK inhibitors or Reactive Oxygen Species Scavenger (N-Acetyl Cysteine), Two biological replicates were generated for each time point. RNA samples were collected at 0 (unirradiated), 0.5, 1, 2, 6, and 24h post irradiation except where ever mentioned. Overall design: Bone marrow-derived macrophages derived from C57Bl/6, Myd88-/- and Trif-/-, Ifnar-/-, Atm-/-, Sting-/-, Scid, Irf3-/-, Irf1-/-, p53-/-, Nrf2-/-mice were irradiated with 6Gray ioninzing radiation; C57Bl/6 macrophages were Irradiated in the presence of MAPK inhibitors or Reactive Oxygen Species Scavenger (N-Acetyl Cysteine), Two biological replicates were generated for each time point. RNA samples were collected at 0 (unirradiated), 0.5, 1, 2, 6, and 24h post irradiation except where ever mentioned.
Publication Title
Defined Sensing Mechanisms and Signaling Pathways Contribute to the Global Inflammatory Gene Expression Output Elicited by Ionizing Radiation.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 120 Downloadable Samples
- IlluminaHiSeq2000
Description
Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput data sets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif data sets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well- defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks. Overall design: Bone marrow-derived macrophages derived from C57Bl/6, Myd88-/-, Trif-/-, Irf3-/-, Ifnar-/-, and RelA-/- mice were stimulated with lipid A; C57Bl/6 macrophages were stimulated with lipid A in the presence of MAPK inhibitors or cycloheximide, or stimulated with PAM3CSK4 for 0, 15, 30, 60, and 120 minutes, or stimulated with lipid A for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 minutes. Two biological replicates were generated for each time point for each treatment type.
Publication Title
A Stringent Systems Approach Uncovers Gene-Specific Mechanisms Regulating Inflammation.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 68 Downloadable Samples
- Illumina HiSeq 2000
Description
Successful host defense against pathogens requires innate immune recognition of the correct pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs) to trigger the appropriate gene program tailored to the pathogen. While many PRR pathways have been shown to contribute to the innate immune response to specific pathogens, the relative importance of each pathway for the complete transcriptional program elicited has not been examined in detail. Herein, we used RNA-sequencing with wildtype and mutant macrophages to delineate the innate immune pathways responsible for the early transcriptional response to Staphylococcus aureus, a ubiquitous microorganism that can activate a wide variety of PRRs. Unexpectedly, only two PRR pathways – the Toll-like receptor (TLR) and Stimulator of Interferon Gene (STING) pathways - were identified as dominant regulators of approximately 95% of the genes that were potently induced within the first four hours of macrophage infection with live S. aureus. TLR signaling predominantly activated an inflammatory program, STING signaling activated an antiviral/type I interferon response, and both pathways contributed to a program linking innate and adaptive immunity. Only a small number of genes were induced in the absence of TLR or STING signaling, and these genes possessed a strong hypoxia signature. STING pathway activation required live S. aureus and was largely dependent on the DNA sensor cyclic guanosine-adenosine synthase (cGAS) recognition of S. aureus DNA. Interestingly, using a cutaneous infection model, we found that the TLR and STING pathways played opposite roles in host defense to S. aureus, with TLR signaling being required for protective interleukin (IL)-1? and neutrophil recruitment and STING signaling having an opposite effect. These results provide novel insights into the complex interplay of innate immune signaling pathways triggered byS. aureus and uncover opposing roles of TLR and STING in cutaneous host defense to S. aureus. Overall design: Files are labeled according to the figures in which they were used. Note, that many data files were used in multiple figures or figure panels. Files are labeled by genotype of macrophages (WT=wildtype; KO= StingGt/Gt; DKO=MyD88-/-TRIF-/-) and whether the macrophages were treated with live (Live) or heat killed (HK) or uninfected (zero hour). Labeling of time points is in the order of "minutes_replicate #." For example, "WT_HK_30_2" indicates that this is wild type mouse macrophages stimulated with heat killed bacteria at the 30-minute time point and is replicate number 2. Reads were converted into RPKM, and the RPKM for all replicates listed for a given time point were averaged to obtain the average RPKM that was used for figures and analyses. For samples listed as contributing to either figure 3 or supplemental figure 2, the replicates that do NOT end in either KO_analysis nor DKO analysis were used to determine induced genes in wild type macrophages. In contrast, the replicates that end in KO_analysis or DKO_analysis were used to determine dependence on either STING signaling or MyD88/TRIF signaling, respectively. If a replicate was used in the STING or MyD88/TRIF dependence analysis for both live and heat-killed S. aureus, "live_and_hk" was added after the dependence analysis it contributed to. Some 0h samples were used in both live and heat-killed analyses.
Publication Title
Opposing roles of Toll-like receptor and cytosolic DNA-STING signaling pathways for Staphylococcus aureus cutaneous host defense.
Sample Metadata Fields
Sex, Specimen part, Cell line, Subject
View Samples- Mus musculus
- 6 Downloadable Samples
- IlluminaHiSeq2000
Description
The physiological responses to B cell receptors (BCR) and Toll-like receptors (TLRs) so vital to immunity are well known but the transcriptional signatures and regulatory mechanisms that initiate activation and release cells from quiescence remain unclear. Here, we show that BCR- or TLR-mediated activation of B cells involves a large shared transcriptional signature and a smaller subset of distinct signal-specific transcriptional responses. Signal-specific transcription is observable within 2 hours of ligand exposure; suggesting different modes of activation begin soon after ligand binding and long before the well-documented BCR and TLR-dependent physiological responses occur. Ligand-specific differences in regulatory mechanisms including RNA Pol II recruitment, activating (H3K4me3) and repressing (H3K27me3) histone marks, transcription factor binding sites in responsive gene promoters, and miRNA expression were observed. These results begin to define the transcriptional landscape of early B cell activation revealing more ligand-specific regulation and character than occurs much earlier than previously expected. Overall design: CD43- mouse resting B cells were stimulated with ligands against the B cell receptor and TLR4 (LPS). RNA-sequencing was performed to describe differential transcription and ChIP-sequencing was performed to describe regulatory mechanism responses.
Publication Title
Divergence of transcriptional landscape occurs early in B cell activation.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
The C2H2 zinc finger is the most prevalent DNA-binding motif in the mammalian proteome, with DNA-binding domains usually containing more tandem fingers than are needed for stable sequence-specific DNA recognition. To examine the reason for the frequent presence of multiple zinc fingers, we generated mice lacking finger 1 or finger 4 of the 4-finger DNA-binding domain of Ikaros, a critical regulator of lymphopoiesis and leukemogenesis. Each mutant strain exhibited a specific subset of the phenotypes observed with Ikaros null mice. Of particular relevance, fingers 1 and 4 contributed to distinct stages of B- and T-cell development and finger 4 was selectively required for tumor suppression in thymocytes and in a new model of BCR-ABL+ acute lymphoblastic leukemia. These results, combined with transcriptome profiling (this GEO submission: RNA-Seg of whole thymus from wt and the two ZnF mutants), reveal that different subsets of fingers within multi-finger transcription factors can regulate distinct target genes and biological functions, and they demonstrate that selective mutagenesis can facilitate efforts to elucidate the functions and mechanisms of action of this prevalent class of factors. Overall design: Ikaros RNA-Seq from double positive thymocytes comparing wt (n=2), Ikaros-ZnF1-/- mutant (n=2) and Ikaros-ZnF4-/- mutant (n=2)
Publication Title
Selective regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros.
Sample Metadata Fields
Sex, Age, Specimen part, Cell line, Subject
View Samples- Mus musculus
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
The C2H2 zinc finger is the most prevalent DNA-binding motif in the mammalian proteome, with DNA-binding domains usually containing more tandem fingers than are needed for stable sequence-specific DNA recognition. To examine the reason for the frequent presence of multiple zinc fingers, we generated mice lacking finger 1 or finger 4 of the 4-finger DNA-binding domain of Ikaros, a critical regulator of lymphopoiesis and leukemogenesis. Each mutant strain exhibited a specific subset of the phenotypes observed with Ikaros null mice. Of particular relevance, fingers 1 and 4 contributed to distinct stages of B- and T-cell development and finger 4 was selectively required for tumor suppression in thymocytes and in a new model of BCR-ABL+ acute lymphoblastic leukemia. These results, combined with transcriptome profiling (this GEO submission: RNA-Seg of whole thymus from wt and the two ZnF mutants), reveal that different subsets of fingers within multi-finger transcription factors can regulate distinct target genes and biological functions, and they demonstrate that selective mutagenesis can facilitate efforts to elucidate the functions and mechanisms of action of this prevalent class of factors. Overall design: RNA-Seq from sorted primary proB cell Hardy Fractions B and C+C'', comparing wt, Ikaros-ZnF1-/- mutant and Ikaros-ZnF4-/- mutant.
Publication Title
Selective regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 16 Downloadable Samples
Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The role of estrogen and testosterone in the regulation of gene expression in the proximal reproductive tract is not completely understood. To address this question, mice were treated with testosterone or estradiol and RNA from the efferent ducts and caput epididymis was processed and hybridized to Affymetrix MOE 430 2.0 microarrays. Analysis of array output identified probe sets in each tissue with altered levels in hormone treated versus control animals. Hormone treatment efficacy was confirmed by determination of serum hormone levels pre- and post-treatment and observed changes in transcript levels of previously reported hormone-responsive genes. Tissue-specific hormone sensitivity was observed with 2867 and 3197 probe sets changing significantly in the efferent ducts after estrogen and testosterone treatment, respectively. In the caput epididymis, 117 and 268 probe sets changed after estrogen and testosterone treatment, respectively, demonstrating a greater response to hormone in the efferent ducts than the caput epididymis. Transcripts sharing similar profiles in the intact and hormone-treated animals compared with castrated controls were also identified. Ontological analysis of probe sets revealed a significant number of hormone-regulated transcripts encode proteins associated with lipid metabolism, transcription and steroid metabolism in both tissues. Real-time RT-PCR was employed to confirm array data and investigate other potential hormone-responsive regulators of proximal reproductive tract function. The results of this work reveal previously unknown responses to estrogen in the caput epididymis and to testosterone in the efferent ducts as well as tissue specific hormone sensitivity in the proximal reproductive tract.
Publication Title
Regulation of gene expression by estrogen and testosterone in the proximal mouse reproductive tract.
Sample Metadata Fields
Sex, Specimen part, Treatment
View Samples- Danio rerio
- 12 Downloadable Samples
- Affymetrix Zebrafish Genome Array (zebrafish)
Description
A microarray study of sex- and gonad-biased gene expression was conducted to determine whether zebrafish demonstrate male-specific patterns consistent with those observed in other animals. We identified a large number of genes (5899) demonstrating statistical differences in transcript abundance between male and female Danio rerio. All sex-biases in gene expression were due to differences between testis and ovary, although differences between male and female body likely went undetected due to constraints imposed by study design and statistical criteria. Male-enriched genes were more abundant than female-enriched genes, and the magnitude of expression bias for male-enriched genes was greater than that for female-enriched genes. We also identified a large number of candidate reproductive genes based on elevated transcript abundance in testes and ovaries, relative to male body and female body, respectively. Gene expression patterns in adult zebrafish from this study are consistent with the male-biased patterns typical of most animal taxa studied to date. Recent zebrafish studies designed to address more specific questions have not reported the same findings, but major methodological and analytical differences across these studies could explain discrepancies.
Publication Title
A microarray analysis of sex- and gonad-biased gene expression in the zebrafish: evidence for masculinization of the transcriptome.
Sample Metadata Fields
Sex
View Samples