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Regulation of Macrophage IFNγ-Stimulated Gene Expression by the 1 Transcriptional Coregulator CITED1

ABSTRACT: Macrophages are highly dynamic innate immune cells that adopt temporary phenotypes, known as polarization states, in response to changing environmental signals to combat microbial infection and contribute to the maintenance of tissue homeostasis. During the early stages of an infection, exposure to interferon-gamma (IFNγ) and microbial ligands induce M1 polarization, a heightened proinflammatory anti-microbial state, by regulating the expression of interferon stimulated genes (ISGs). While this response must be sufficiently vigorous to ensure the successful clearance of invading pathogens, it must also be spatially and temporally restricted to prevent uncontrolled inflammation that could result in tissue damage and disease. This is controlled by a variety of cell-extrinsic and -intrinsic mechanisms, including the expression of CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2), a transcriptional coregulator that limits IFNγ-stimulated proinflammatory gene expression by inhibiting STAT1- and IRF1-regulated ISGs. In this study, we show that CITED1, another member of the CITED family of proteins, is itself an ISG and is expressed in a STAT1-dependent manner, and that IFNγ stimulates the nuclear accumulation of fluorescently tagged CITED1 proteins. In contrast to CITED2, ectopic expression of CITED1 enhanced the expression of a subset of ISGs, including Ccl2, Ifit3b, Isg15, and Oas2. This effect was reversed in a Cited1 null cell line produced by CRISPR-based genomic editing. Collectively, these data show that CITED1 helps to maintain proinflammatory gene expression during periods of prolonged IFNγ exposure and suggests a distinct and antagonistic relationship between CITED proteins in the regulation of macrophage inflammatory function.

ORGANISM(S): Mus musculus

PROVIDER: GSE217961 | GEO | 2023/01/01

REPOSITORIES: GEO

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Project description:Purpose: Bulk transcriptomics analysis of Wilms tumor SIX2+CITED1+ cells to compare and identify unique nephron progenitor transcriptome profiling (RNA-seq) signature between unfavorable and favorable Wilms Tumors and against human fetal kidney Methods: Wilms tumor samples were collected and transported on ice at 4°C in RPMI-1640 and a single cell suspensions were prepared following mechanical and enzymatic dissociation as previously publication. Enzymatic dissociation was performed with 125 U/ml collagenase I in RPMI-1640 at 37 °C for 35 min. The digested cells were then passed through a 100-μm cell strainer and a 40-μm cell strainer with washes of 1x PBS. The cell suspension was than centrifuged at 1500 rpm for 5 min and erythrocytes were eliminated using a red blood cell lysis kit. WT SIX2+CITED1+ cells were isolated using SIX2-Cy5 and CITED1-Cy3 Smartflare RNA probes following manufacturer’s instructions and previous publication. Briefly, cells were incubated over night at 37 °C with both RNA probes diluted at 1:20 in PBS and 25ul/ml in RPMI-1640 supplemented with 5% FBS, and 0.2% antimicrobial agent Primocin. After 16-18 h, cells were dissociation using TrypLE 1x for 5 min, cells were centrifuged at 1500 rpm for 5 min and prepared for FACS. RNA extraction was performed immediately after FACS using the RNeasy Micro Kit following manufacturer’s recommendations. After cDNA production and construction of DNA libraries, the samples were run on an Illumina NextSep500 (Illumina). Differential gene expression was analyzed using ERCC ExFold probes with the Remove Unwanted Variation R/Bioconductor software package combined with edgeR Results: Transcriptomics analysis of Wilms Tumor SIX2+CITED1+ cells in comparison to different tumor types and human fetal kidneys confirmed the nephrogenic signature of hFK-SIX2+CITED1+ and WT-SIX2+CITED1+ cells but highlighted differences in expression of pluripotency and self renewal-related genes like OCT4, FOXO1, SALL, NANOG along with a lower expression of β-catenin, TCF, and other growth factors known to promote differentiation (BMPs, FGFs). Hierarchical clustering of gene expression showed shared similarities between Wilms tumor samples against human fetal kidney samples however, with major differences in gene expression between tumor-to-tumor type was also present. Conclusion: Our study represents the first transcriptomic characterization of Wilms Tumor cancer stem cells (SIX2+CITED1+) against human fetal kidney SIX2+CITED1+ cells. Identifying specify gene expression and signaling pathway profiles across different subtypes of Wilms Tumor and against human fetal kidney SIX2+CITED1+ cells.

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Regulation of Macrophage IFNγ-Stimulated Gene Expression by the 1 Transcriptional Coregulator CITED1

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