Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis

This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs) are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis). Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf) described in the research article. Also included are 5′ untranslated sequences (UTR) for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT)18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf.


a b s t r a c t
This paper describes data related to a research article titled, "Fasantisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fasmediated cell death" [1]. Long noncoding RNAs (lncRNAs) are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis). Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf) described in the research article. Also included are 5 0 untranslated sequences (UTR) for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing

Value of the data
Expression data for 82 long noncoding RNAs at early and late stages of human erythroid maturation are provided.
Methods to determine poly-adenylation status of lncRNAs using end point and quantitative RT-PCR analysis of cDNA.
Methods to transduce human CD34 þ cells with lentiviral vectors encoding for expression of a functional lncRNA and subsequently monitor Fas receptor levels by flow cytometry.

Data
Here we provide expression data for a focused set of lncRNAs during culture-induced differentiation of human CD34 þ cells into erythroblasts (Table 1). We provide sequences for the cloned 5 0 untranslated region (UTR) of Saf highlighting transcription factor binding sites including GATA-1, KLF1, and NF-κB, which were studied in detail in the research article ( Fig. 1). We include relative transcript levels of GATA-1 and KLF1 in K562 human erythroleukemia cells and maturing human erythroblasts (Figs. 2 and 3). We share end point and quantitative RT-PCR analysis of cDNA prepared from total RNA isolated from human erythroblasts using random hexamers versus oligo(dT) 18 (Fig. 4). Finally, we include flow cytometry histograms demonstrating Fas surface levels on maturing erythroblasts derived from human CD34 þ cells transduced using mock conditions or Saf-encoding lentivirus particles (Fig. 5). CD34 þ cells isolated from fetal liver (FL), umbilical cord blood (CB), or adult bone marrow (BM) were purchased commercially (Lonza; Walkersville, MD) and used in accordance with protocols approved by the Springfield Committee for Research Involving Human Subjects at Southern Illinois University School of Medicine. Samples for FL and BM were from two unrelated donors. CD34 þ cells were cultured under conditions that promote erythroid differentiation as described [2]. Erythrocytes collected at early, intermediate and late stages of maturation were extracted of total RNA using spin columns (Ambion) with on-column DNase treatment (Promega). RNA was quantified by a Nanodrop 2000 (Life Technologies) and quality assessed by visualizing 18S and 28S ribosomal RNA bands separated through 1% agarose and stained with ethidium bromide. RNA (300 ng) was reverse transcribed into cDNA using SuperScript VILO Master Mix (Life Technologies) and conditions: 25°C-10 min, 42°C-60 min, 85°C-5 min, 4°C-hold. cDNA (40 ng/reaction) was used as template for a lncRNA PCR array (LncRNA Profiler™ qPCR Array, System Biosciences) that included primer sequences for 82 unique lncRNAs and 14 controls optimized for use with SYBR Green as the fluorophore. A version of this array has previously been validated [3]. Quantitative PCR reactions were performed with a StepOnePlus thermocycler (Applied Biosystems) using SYBR Green settings that included a melt curve. Changes in transcript levels were assessed by the delta-delta C T formula [4] with normalization to internal controls.

Relative expression of GATA-1 and KLF1
RNA, extracted from K562 human erythroleukemia cells or culture-differentiated human erythroblasts collected at early, intermediate and late stages of maturation, was reverse transcribed into cDNA as described in Section 2.1. Quantitative PCR was performed on a StepOnePlus thermocycler (Applied Biosystems) using cDNA (100 ng) and TaqMan primer:probe sets specific to GATA-1 (Hs01085823_m1; FAM), KLF1 (Hs00610592_m1; FAM), and RNaseP (4316844; VIC-TAMRA) all from Life Technologies. Changes in transcript levels were assessed by the delta-delta C T formula with normalization to RNAseP.

Saf polyadenylation status
RNA was extracted from CD34 þ -derived erythroblasts on day 8 in culture and subjected to first strand cDNA synthesis with either random hexamers or oligo(dT) 18   an internal control. End point PCR products were resolved on 2% agarose gels containing ethidium bromide and photographed. Erythroid cells derived from fetal liver or adult bone marrow CD34 þ cells exposed to identical conditions were collected during early, intermediate (mid), or late stages of differentiation. Cells were isolated of total RNA and levels of GATA-1 and KLF1 transcripts determined by quantitative RT-PCR. Data were normalized to RNAseP, set relative to fetal liver, and plotted as mean þSD; n¼2 independent donors performed in duplicate.

Flow cytometry analysis of Fas on maturing erythroblasts
Flow cytometry studies were performed on a FACSAriaII (BD Biosciences) using FlowJo v10.0 analysis software. Human CD34 þ cells transduced using mock conditions or with lentiviral vector particles encoding for Saf were collected on days 6 and 10 post-transduction, times corresponding to  proliferation and differentiation stages of culture. Erythroblasts (1 Â 10 5 ) were washed with PBS, suspended in staining medium (PBSþ 0.5% BSA) and reacted with allophycocyanin (APC)-conjugated mouse anti-human CD95 (DX2, BD Biosciences, 1:50 dilution); mouse anti-human IgG-APC was used as an isotype control.

Statistical analysis
Microsoft Excel or Prism 5 (GraphPad) were used to determine descriptive statistics (mean þSD).