Long noncoding RNA expression in several hematopoietic progenitor and differentiated cell populations using a custom Agilent microarray

Mammals express thousands of long noncoding (lnc) RNAs, a few of which are shown to function in tissue development. However, the entire repertoire of lncRNAs and the extent to which they regulate biological processes in different tissues and species are not defined. Indeed, most lncRNAs are not conserved between species, raising questions about function. We used RNA-Seq to identify lncRNAs in primary murine fetal liver erythroblasts expressing the lineage marker TER119, megakaryocytes (CD41+) cultured from embryonic day (E) 14.5 murine fetal liver and megakaryocyte erythroid progenitors (MEPs) isolated from mouse bone marrow. We identified 683 and 594 polyadenylated lncRNAs expressed in red blood cell (erythroid) precursors of mice and humans, respectively. More than one half of erythroid lncRNAs are un-annotated, emphasizing the opportunity for new discovery through studies of specialized cell types. We analyzed the expression of these identified lncRNAs in several hematopoietic compartments using a custom microarray to identify erythroid-specific lncRNAs that were robustly expressed in both fetal liver and adult erythroid cells as targets for knockdown. Over 90% of fetal liver erythroid lncRNAs detected using RNA-seq were expressed in adult erythroblasts measured on the microarray. Analysis of the murine erythroid lncRNA transcriptome indicates that ~75% arise from promoters and 25% from enhancers, many of which are regulated by the key erythroid transcription factors GATA1 and SCL/TAL1. Erythroid lncRNA expression is largely conserved among 8 different mouse strains, yet only 15% of mouse lncRNAs are expressed in humans and vice versa, reflecting dramatically greater species-specificity than coding genes. We investigated potential functions of 21 relatively abundant erythroid-specific murine lncRNAs (both conserved and non-conserved) by RNA interference in primary mouse erythroid precursors, and identified 7 whose knockdown inhibited features of terminal erythroid maturation including cell size reduction and enucleation. Strikingly, at least 6 of the 7 lncRNAs have no detectable expression in human erythroblasts, demonstrating that lack of conservation between mammalian species does not predict lack of function. These results reflect marked evolutionary differences between protein-coding genes and lncRNAs and indicate that the latter exert tissue- and species-specific roles in development. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf A custom Agilent microarray was designed to interrogate expression levels of long noncoding RNAs identified using RNA-seq in several different hematopoietic progenitor and differentiated cell populations. LncRNA gene definitions and RNA-seq data used to identify long noncoding RNAs are deposited in GEO with the accession numbers GSE51667 and GSE40522 respectively. The Agilent eArray platform was used to customize the SurePrint G3 Mouse GE 8x60K microarray to add 3 custom-designed probes (60nt length) each against several categories of genes, namely pseudogene, genes with small RNA overlap, low stringency lncRNAs and high stringency lncRNAs . We interrogated 7 types of hematopoietic cells, HSC, CMP, MEP, GMP, early and late erythroblasts and granulocytes. Expression measurements were determined at least in duplicate for all samples and in triplicate for most samples.哺乳动物可表达数千种长链非编码RNA（long noncoding RNA，lncRNA），其中少数已被证实参与组织发育过程。然而，长链非编码RNA的完整全集，以及它们在不同组织和物种中调控生物学过程的程度，仍未得到明确界定。事实上，大多数长链非编码RNA在物种间并不保守，这引发了关于其功能的诸多疑问。 我们通过RNA测序（RNA-Seq）在表达谱系标志物TER119的原代小鼠胎肝成红细胞、从胚胎14.5天（E14.5）小鼠胎肝中培养的巨核细胞（CD41+），以及从小鼠骨髓分离的巨核红细胞祖细胞（megakaryocyte erythroid progenitors，MEPs）中鉴定长链非编码RNA。我们分别在小鼠和人类的红细胞（红系）前体细胞中鉴定出683种和594种聚腺苷酸化（polyadenylated）长链非编码RNA。超过半数的红系长链非编码RNA尚未被注释，这凸显了通过对特化细胞类型的研究开展新发现的潜力。 我们通过定制微阵列（microarray）分析了这些已鉴定长链非编码RNA在多个造血隔室中的表达情况，以筛选出在胎肝和成体红系细胞中均稳定表达的红系特异性长链非编码RNA，作为敲降（knockdown）实验的靶标。经微阵列检测的成体红系细胞中，超过90%的RNA测序检出的胎肝红系长链非编码RNA均有表达。对小鼠红系长链非编码RNA转录组（transcriptome）的分析显示，约75%的长链非编码RNA来源于启动子（promoter）区域，25%来源于增强子（enhancer）区域，其中多数受关键红系转录因子（transcription factor）GATA1和SCL/TAL1调控。红系长链非编码RNA的表达在8种不同小鼠品系中基本保守，但仅15%的小鼠长链非编码RNA在人类红系细胞中表达，反之亦然，这反映出长链非编码RNA的物种特异性远高于编码基因。 我们通过RNA干扰（RNA interference）技术对21种丰度较高的小鼠红系特异性长链非编码RNA（包括保守和非保守类型）进行了功能探究，这些RNA来自原代小鼠红系前体细胞；最终鉴定出7种长链非编码RNA，其敲降会抑制终末红系成熟的相关特征，包括细胞体积缩小和脱核（enucleation）。值得注意的是，7种长链非编码RNA中至少有6种在人类红系细胞中无法检测到表达，这表明哺乳动物物种间的序列保守性并不等同于功能保守性。上述结果反映了蛋白质编码基因与长链非编码RNA之间显著的进化差异，并表明长链非编码RNA在发育过程中发挥组织和物种特异性的功能。 有关数据使用的条款与条件，请参阅http://www.genome.gov/27528022及http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf。 我们设计了定制安捷伦（Agilent）微阵列，用于检测通过RNA测序鉴定的多种造血祖细胞和分化细胞群体中的长链非编码RNA表达水平。用于鉴定长链非编码RNA的长链非编码RNA基因定义及RNA测序数据已分别保存在基因表达综合数据库（Gene Expression Omnibus，GEO），登录号分别为GSE51667和GSE40522。我们使用安捷伦eArray平台定制了SurePrint G3小鼠全基因8x60K微阵列，为假基因、与小RNA存在重叠的基因、低严谨性长链非编码RNA及高严谨性长链非编码RNA这几类基因分别添加了3条定制设计的探针（长度为60nt）。 我们对7种造血细胞进行了检测，分别为造血干细胞（hematopoietic stem cell，HSC）、共同髓系祖细胞（common myeloid progenitor，CMP）、巨核红细胞祖细胞（MEP）、粒细胞-单核细胞祖细胞（granulocyte-monocyte progenitor，GMP）、早幼和成红细胞以及粒细胞。所有样本的表达检测至少设置2次生物学重复，多数样本设置3次重复。