Analysis of wildtype and Xbp1-deficient hematopoietic progenitor cell transcriptomes

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.研究目的：本研究旨在解析转录因子X盒结合蛋白1（XBP1）在谱系未定向髓系祖细胞及嗜酸性粒细胞定向髓系祖细胞中调控的转录网络。 实验方法：采用Illumina HiSeq2000测序仪，对来自XBP1 flox/flox或XBP1 flox/flox Vav1-Cre小鼠的、经荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）纯化的粒细胞-巨噬细胞祖细胞（GMPs）进行测序，以生成其mRNA表达谱，每组设置3次生物学重复。同时，使用Illumina HiSeq2000测序仪获取另一组样本的mRNA表达谱：该组样本为经转录因子GATA结合蛋白2（GATA2）转导、转导后36小时重分选且培养48小时的FACS纯化GMPs，每组基因型同样设置3次生物学重复。Illumina HiSeq2000测序仪产生的测序数据通过Illumina CASAVA分析流程（版本1.8.2）进行解复用，为每个样本生成FASTQ文件。通过Illumina质量/纯度过滤的测序读段，使用STAR比对软件（STAR aligner）并以默认参数比对至小鼠基因组（Illumina iGenomes mm9版本）。生成的SAM比对文件随后通过SAMtools（版本0.1.14）转换为BAM文件格式，并进行排序与索引。使用Python模块HTSeq对比对上的读段进行计数，并通过Bioconductor工具包（Bioconductor）DESeq进行差异表达分析。 研究结果与结论：通过监测嗜酸性粒细胞发育不同阶段中XBP1依赖的转录变化，本研究发现，在嗜酸性粒细胞定向分化前的粒细胞-巨噬细胞祖细胞中，经典的XBP1依赖调控网络（如糖基化、分子伴侣生成及内质网相关降解（ERAD））均被下调，尽管此时细胞的分化与存活未出现明显异常。然而，mRNA表达谱分析明确显示，XBP1缺失会在嗜酸性粒细胞定向分化阶段引发细胞应激状态。嗜酸性粒细胞的转录组整体保持完整，多数表达失调的基因均与内质网应激相关。但嗜酸性粒细胞发育所需的关键颗粒蛋白基因（如Prg2与Epx）仅在嗜酸性粒细胞定向分化后才被选择性下调，而在未定向髓系祖细胞中则无此现象；该现象与Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）预测的转录因子GATA结合蛋白1（GATA1）功能受损结果一致。本研究阐明了细胞应激与维持细胞分化关键特征的能力之间的相互作用关系。本研究针对嗜酸性粒细胞发育两个阶段的XBP1依赖转录调控网络进行了分析。