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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu) A transcriptome-wide approach reveals the key contribution of NFI-A in promoting erythroid differentiation of human CD34 þ progenitors and CML cells Leukemia (2010) 24, 1220–1223; doi:10.1038/leu.2010.78; NFI-A or a control empty Vector as previously described.2 published online 29 April 2010 Forty-eight hours after transduction, GFP þ cells were sorted using FACSAria instrument (BD Biosciences, San Jose`, CA, USA), subjected to total RNA extraction and hybridized on Agilent whole human genome oligo microarray (No. G4112F; Nuclear factor I-A (NFI-A) is a member of the NFI family Agilent Technologies, Palo Alto, CA, USA; detailed protocols of transcription factors, composed of four separate genes are summarized in Supplementary Information). (Nfia, Nfib, Nfic, Nfix) with distinct functions depending on To quantify the resulting amount of NFI-A in both the Vector the cell type and target promoter context.1,2 Recent studies and NFI-A samples, we performed a real-time PCR using primers showed a novel and considerable role for NFI-A in the specific for NFI-A (primers used are listed in Supplementary specification of hematopoietic lineages from CD34 þ human Table S2). As shown in Figure 1a, the relative amount of hematopoietic progenitor cells (HPCs). In particular, NFI-A was NFI-A is greatly increased in the NFI-A-transduced compared to shown to promote the erythroid lineage at the expense of the Vector-infected cells. Microarray results were then analyzed by granulocytic lineage, by direct repression of the G-CSF receptor using the GeneSpring GX 10 software (Agilent Technologies). (G-CSFR) gene, and to activate b-globin transcription in normal Data transformation was applied to set all the negative raw erythroblasts and remarkably in K562 chronic myeloid leukemia values at 1.0, followed by a Quantile normalization. A filter on (CML) cells, which do not express endogenous b-globin.2 low gene expression was used to keep only the probes expressed Notably, NFI-A expression is shut off to allow terminal in at least one sample (flagged as Marginal or Present). Expressed granulocytic or monocytic differentiation by the activity of genes having a fold change of 41.5 between the Vector and microRNA-223 and microRNA-424, respectively,3,4 further NFI-A samples were selected. Interestingly, numerous genes indicating a common negative regulatory role for NFI-A in the were upregulated in the exogenous NFI-A-expressing myeloid lineage. sample, whereas relatively less genes were downregulated (for In line with these findings, we recently reported that the complete raw and normalized data see Supplemental exogenous NFI-A expression in K562 CML blast crisis cells Table S1), suggesting that NFI-A per se behaves both as a resulted in a dramatic restoration of the normal erythroid transcriptional activator and repressor in undifferentiated program and increased differentiation responsiveness to the CD34 þ cells. anti-metabolite cytosine arabinoside (Ara-C).2 Notably, during On careful analysis and classification of the data, we noted Ara-C-induced differentiation of K562 wild-type cells, no NFI-A among the modulated genes a consistent number of erythroid protein upregulation was observed compared to the physio- genes. Many of these genes reflected an intermediate-to-mature logical erythroid program of human HPCs, which is normally erythroid developmental stage, and were in general accordance accompanied by increased NFI-A accumulation.2 Altogether with recent genome-wide analyses performed on a variety of this evidence suggests a defective tumor suppressor-like func- in vitro erythroid differentiation systems.6–8 Out of this extensive tion of NFI-A in K562 CML cells. In fact, a recent study number of potential target genes, a list of 27 genes correspond- performed using array-comparative genomic hybridization and ing to well-characterized erythroid-affiliated genes was used for DNA sequencing reported structural alterations of the NFI-A cluster analysis of samples using the Euclidean distance as a gene associated with different chronic malignant myeloid measure of similarity (Figure 1b). As shown in Figure 1b, the diseases.5 NFI-A cells displayed considerable induction of erythroid cell In this study, to adequately dissect the molecular network membrane molecules, including CDH1, the Rh antigen family downstream of NFI-A, we performed gene expression profiling (RHAG; RHC/RHD), SLC4A1, AQP1, ADD3 and SPTB; mole- of normal human uncommitted CD34 þ HPCs efficiently cules involved in the hemoglobin biosynthetic pathway, expressing exogenous NFI-A at high level. CD34 þ HPCs were including ALAS2 and globin chains HBB, HBA and HBD; purified from fresh cord blood obtained from healthy donors, growth factors and growth factor receptors, including INHBA, pre-stimulated in serum-free medium supplemented with SCF EFBN2 and IGF2; signal transduction molecules, including 50 ng/ml, Flt3 ligand 50 ng/ml, IL-3 20 ng/ml and TPO 20 ng/ml JAK2, AKT2 and GAB1; transcription factors or DNA-binding overnight and transduced with a lentiviral vector encoding proteins, including JUNB, HOXB8, KLF2, SSBP3 and TRIM10; Leukemia Letters to the Editor 1221 CD34+ HPCs CD34+ HPCs 50 Vector Vector NFI-A NFI-A 40 TRIM10 30 AQP1 20 NFI-A mRNA 10 KLF2 0 ALAS2 Vector NFI-A GAPDH Fold difference Gene Symbol Gene Description (NFI-A/Vector) JAK2 Janus kinase 2 1.9 CDH1 Cadherin 1 2.0 JUNB JunB proto-oncogene 1.9 INHBA Inhibin, beta A 2.4 ADD3 Adducin 3 2.2 HBD Hemoglobin, delta 2.2 SPTB Spectrin beta, erythrocytic 2.2 EFNB2 Ephrin-B2 2.3 AKT2 v-akt homolog 2 1.5 RHAG Rh-associated glycoprotein 1.7 GAB1 GRB2-associated binding protein 1 2.1 HBB Hemoglobin, beta 3.8 HBA2 Hemoglobin, alpha2 5.3 ALAS2 Aminolevulinic acid synthase 2 2.9 RHCE Rh blood group, CcEe antigens 2.7 RHD Rh blood group, D antigen 5.4 HOXB8 Homeobox B8 5.5 KLF2 Kruppel-like factor 2 4.9 HBA1 Hemoglobin, alpha1 5.2 SELENBP1 Selenium binding protein 1 6.6 IGF2 Insulin-like growth factor 2 8.1 SLC4A1 Erythrocyte membrane protein band 3 10.5 CSF2RB GM-CSF receptor, beta, low-affinity -2.0 ETS1 v-ets homolog 1 (avian) -1.9 AQP1 Aquaporin 1 29.5 SSBP3 Single stranded DNA binding protein 3 40.7 TRIM10 Tripartite motif-containing 10 16.3 Vector NFI-A Figure 1 NFI-A regulates several erythroid-affiliated genes. (a) Real-time PCR of NFI-A expression in Vector- and NFI-A-transduced GFP þ sorted CD34 þ HPCs 48 h after transduction. Mean±s.e.m. (n ¼ 3). (b) Hierarchical clustering of selected erythroid genes in the Vector- and NFI-A expressing CD34 þ cells. Relative levels of gene expression are depicted with a color scale, red being the highest levels and green the lowest levels of expression. (c) Semiquantitative RT–PCR of transcripts from Vector- or NFI-A-transduced CD34 þ cells using GAPDH as a normalizer. finally, the intracellular transporter SELENBP1 as reported by erythroid-like gene expression signature as observed for CD34 þ Komor et al.6 and Macaulay et al.7 and reported in Supplemen- HPCs (RT–PCR primers are listed in Supplementary Table S2), tary References 1À27. Collectively, we can postulate that the thus suggesting a defective pro-differentiating or tumor suppres- gene expression profile analysis uncovered an extensive net- sor-like role of NFI-A in these cells. In fact, the kinetics of the work of erythroid genes regulated after NFI-A expression, tested mRNA accurately reflected the transcript profiles that particularly erythroid membrane skeleton proteins and hemo- emerged from the microarray performed in primary cells. globin biosynthesis effectors. To validate the microarray data, The RT–PCR screening indicated several relevant NFI-A- we performed semiquantitative reverse transcription (RT)–PCR regulated genes. However, gene expression alone cannot analysis of a few selected genes in CD34 þ HPCs transduced establish whether a transcription factor directly or indirectly with Vector or NFI-A. As shown in Figure 1c, enforced binds to its target genes. To address this question, we focused on expression of NFI-A parallels a vigorous accumulation of the two genes that have been characterized for their essential selected mRNAs, confirming the gene expression profiling function in erythroid cells: SLC4A1 and ALAS2. The SLC4A1 observations. gene encodes the major anion exchanger of the red cell To extend the significance of the microarray data in a CML (chloride-bicarbonate) also called band 3, or anion exchanger setting, we performed semiquantitative RT–PCR analysis of 10 1 (AE1), (an integral component of the cytoskeletal network modulated genes, selected for their biological relevance, in responsible for the unique functional properties of the erythro- K562 CML blast crisis cells transduced with the control Vector cyte membrane) whose alteration has been linked to the or NFI-A with or without Ara-C treatment (Figure 2a).