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Transcriptional regulation of hematopoietic stem cell progenitors and acute myeloid leukemia by the IRE1a-XBP1 pathway [KO]

ABSTRACT: The inositol-requiring enzyme-1a (IRE1a), through its key effector transcription factor X-box binding protein-1 (XBP1), regulates cell fate and malignancy in a tissue and cell-specific manner. Here, we define the transcriptional perturbations associated with IRE1a deficiency in normal murine hematopoieitic stem cell (HSC) progenitors and HSC progenitors expressing Flt3 internal tandem duplication (FLT3-ITD) allele, a myeloid leukemia oncogne. Beyond its classical role in activating genes involved in restoring cellular proteostasis during the unfolded protein response (UPR), our transcriptome analysis reveal XBP1-dependent regulation of genes involved in diverse biological processes required for HSC homeostasis and acute myeloid leukemia (AML) development.

ORGANISM(S): Mus musculus

PROVIDER: GSE163136 | GEO | 2023/12/30

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study are to assess the transcriptional networks governed by the transcription factor XBP1 in lineage-uncommitted myeloid progenitors and in eosinophil-committed myeloid progenitors. Methods: mRNA profiles of FACS-purified granulocyte-macrophage progenitors (GMPs) from XBP1 flox/flox or XBP1 flox/flox Vav1-Cre mice were generated by sequencing, in biological triplicates, using an Illumina HiSeq2000 sequencer. The Illumina HiSeq2000 sequencer was also used to obtain mRNA profiles of FACS-purified GMPs transduced with the transcription factor GATA2, resorted 36 hours post-transduction, and cultured for 48 hours, again in biological triplicates per genotype. Sequence data from Illumina's HiSeq2000 sequencer were demuxed to generate FASTQ files for each sample using Illumina's CASAVA pipeline (version 1.8.2). The reads that passed illumina's quality/purity filter were aligned to the mouse genome (Illumina iGenomes mm9 build) using STAR aligner (version 2.3.0) with default parameters. The resulting SAM alignment files were then converted to the BAM file format, sorted and indexed using SAMtools (version 0.1.14). Mapped reads were counted with the python module HTSeq, and differential expression analyzed with the Bioconductor package DESeq. Results and conclusions: By monitoring XBP1-dependent transcriptional changes at different stages of eosinophil development, we demonstrated that classical XBP1-dependent networks such as glycosylation, chaperone production, and ERAD were downregulated in GMPs prior to eosinophil commitment, though there were no major defects in differentiation or survival. However, mRNA profiling clearly demonstrated that XBP1 deficiency causes a state of cellular stress upon eosinophil commitment. The eosinophil transcriptome was largely intact, and most dysregulated genes were associated with ER stress. However key granule protein genes required for eosinophil development such as Prg2 and Epx were selectively downregulated only after eosinophil commitment, but not in pre-committed myeloid progenitors, and this correlated with Ingenuity Pathway Analysis predictions that GATA1 function was impaired. This study documents the interplay between cellular stress and the ability to maintain key facets of cellular differentiation. Analyses of XBP1-dependent transcriptional networks at two stages of eosinophil development.

Project description:BACKGROUND AND AIM: We previously established that endoplasmic reticulum stress leads to unfolded protein response (UPR) that promotes liver fibrosis by activating hepatic stellate cell (HSC). We aimed to determine the role of X-box binding protein 1 (XBP1), one of three UPR effector pathways, in the fibrogenic HSC activation. METHODS: XBP1 expression was measured by qPCR in tunicamycin-treated human HSC lines (TWNT4 and LX2) and culture-activated human primary HSCs. XBP1 was overexpressed in primary human HSCs and the HSC lines, and fibrogenic genes (COL1A1, ACTA2, PDGFRB, MMP2, TIMP1) and encoded proteins were assessed by qPCR and Western blotting, respectively. Autophagy was inhibited by knockdown of ATG7. Genome-wide transcriptome profiling was performed to determine modulated molecular pathway by XBP1. In vivo XBP1 activation was assessed in transcriptome datasets of fibrotic mouse models and immunohistochemistry of human liver tissues. RESULTS: XBP1 overexpression accompanied both tunicamycin- and culture-based HSC activation. Ectopic overexpression of XBP1 induced fibrogenic gene expression in the HSC lines, which was inhibited by knockdown of ATG7. UPR pathway together with PDGFRB pathway, a hallmark of HSC activation, were induced in transcriptome profiles of XBP1-transduced HSC lines. Some known fibrogenic pathways such as transforming growth factor (TGF)-beta pathway were not induced, suggesting partial contribution of XBP1 to the entire fibrogenic HSC activation machinery. XBP1 target gene signature defined in our XBP1-overexpressing HSC lines was significantly induced in liver tissue transcriptome profiles of carbon tetrachloride-treated or bile duct-ligated mouse models, and XBP1 and alpha-smooth muscle actin (encoded by ACTA2, another hallmark of HSC activation) were co-localized in human fibrotic liver tissues, collectively supporting involvement of XBP1 in HSC activation and fibrogesis in vivo. CONCLUSIONS: XBP1-mediated UPR is at least partially responsible for fibrogenic HSC activation via autophagy.

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Transcriptional regulation of hematopoietic stem cell progenitors and acute myeloid leukemia by the IRE1a-XBP1 pathway [KO]

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