(Fig. 4C) and ara-C sensitivity (Fig. 4D) compared to the CMK- neg cells. Identification of GATA1s Responsive Genes in CMK cells To further investigate the molecular mechanisms that underlie the effects of GATA1s on DS AMkL cell proliferation, differentiation, and sensitivitiy to ara-C, we performed oligonuc- teotide microarray analyses on RNAs from the CMK-5a or CMK- 5b, and CMK-neg cells. Average log ratios representing the differences in expression between the GATA1 shRNA clone (CMK-5a or -5b) and the CMK-neg samples were derived for each array feature by combining replicate array data, using the error-weighted algorithm of Rosetta ResolverH.[21] Differentially expressed genes were identified by their p-values, calculated with the Resolver error-model and the replicate data. Using a p- value#0.001 and a minimum 6 2.0 fold change as cutoffs, 1782 differentially expressed features (probes) were identified (Table S2), with a false discovery rate ,1%. These probe IDs were then cross referenced to Entrez Gene IDs. That provided 1111 Entrez IDs. When these Entrez IDs were compared to those from our previously reported microarray gene set derived from a compar- ison between DS and non-DS AMkL patient samples, 91 Entrez IDs were found to be common in both gene sets (Figure 5). These results strongly suggest that GATA1s may contribute to the DS AMkL phenotype through regulating its responsive genes. Interestingly, a large number of these 1782 differentially expressed probes are involved with cell differentiation (Table S3), proliferation (Table S4), and death (Table S5). Among the genes relevant to cell differentiation and proliferation, IL1A (interleukin 1 alpha), PF4 (platelet factor 4), and TUBB1 (tubulin beta 1), were of particular interest. IL1A encodes a cytokine which enhances proliferation of megakaryocytic progenitors.[25] PF4 and TUBB1 encode proplatelet formation proteins and increased expression of these genes would suggest differentiation toward the megakaryocytic lineage. Oligonucleotide microarray analysis Figure 5. Identification of overlapping genes between the CMK microarray gene set and the previously reported microarray gene set derived from a comparison between DS and non-DS AMkL patient samples. For each gene set, differentially expressed probes were cross referenced to Entrez Gene IDs. That provided 1111 and 445 Entrex IDs for the CMK gene set and the DS vs non-DS AMkL gene set, respectively. The common set between the 2 groups was found and shown in a Venn representation. doi:10.1371/journal.pone.0027486.g005 The Role of GATA1s in DS AMkL revealed 11.0- and 2.4-fold increased expression of PF4 and TUBB1, respectively, and a 2.1-fold decreased expression of IL1A in the CMK-5a and -5b clones compared to the CMK-neg cells. Real-time RT-PCR confirmed a nearly complete lack of IL1A expression and substantially increased expression of PF4 in the CMK-5a and -5b clones compared to the CMK-neg cells (Fig. 6A&C). Substantially increased expression of TUBB1 was also detected by real-time RT-PCR in the CMK-5a but not in the CMK-5b clone, compared to the CMK-neg cells (Fig. 6B). As expected, cell proliferation was significantly increased when CMK-5a cells were treated with 40 ng/mL recombinant IL1a (Fig. 6D). Thus, GATA1s may impact proliferation and mega- karyocytic differentiation of CMK cells by regulating the expression of relevant genes. IL1A is a Direct GATA1s Target Gene It was of interest to establish whether IL1A is a direct GATA1s target gene. Three putative GATA1 binding sites (GATA) located at positions 2993 to 2990, 2909 to 2906, and 2869 to 2866 (relative to the transcription start site, based on IL1A mRNA sequence NM_000575) are present in the IL1A promoter region (Fig. 7A). In CMK cells, GATA1s directly bound to the GATA1 binding sites of IL1A in vivo when assayed by ChIP with either regular PCR (Fig. 7B) or real-time PCR (Fig. 7C). These results demonstrated that IL1A is a bona fide GATA1s target in CMK cells. Thus, GATA1s likely exerts its functions by directly regulating expression of genes important for cell proliferation, survival, and megakaryocytic differentiation. Discussion The GATA1 gene encodes a zinc finger transcription factor that is essential for normal erythroid and megakaryocytic differentia- tion.[13,26–29] Acquired somatic mutations in exon 2 of GATA1 gene have been consistently detected in nearly all DS TMD and AMkL cases, whereas none were detected in non-DS AML and non-AMkL DS leukemia except for rare cases,[10–12,30–32] highlighting their critical roles in leukemogenesis in DS. The net effect of these mutations is the introduction of stop codons and synthesis of GATA1s (40 kDa), initiated from a downstream initiation site and distinguishable from GATA1 (50 kDa).[12] Both GATA1s and GATA1 show similar DNA binding abilities and interact with partner proteins, such as FOG1, although GATA1s exhibits altered transactivation capacity due to the loss of the N- terminal activation domain.[12] Lineage selective knockdown of GATA1 in megakaryocytes has been reported to cause increased megakaryocyte proliferation coupled with impaired maturation in mice.[27] It is believed that the presence of constitutional chromosome 21 disturbs fetal hematopoiesis promoting the acquisition of GATA1 mutations.[33,34] As a consequence, GATA1 mutations would promote accumulation of poorly differentiated megakaryocytic precursors and would represent initiating or early ‘‘genetic hits’’ in a multi-step process of leukemogenesis initiated prenatally.[35] One mechanism by which those effects can be explained is that GATA1s fails to repress certain genes normally regulated by GATA1.[36] However, the possibility that expression of GATA1s in DS AMkL may itself be causal, due to its unique function in regulating specific genes, cannot be excluded. To date, the possibly unique role of GATA1s in promoting leukemogenesis and chemotherapy sensitivity in a human DS AMkL model has not been considered. In this report, we established that GATA1s has novel biological functions in relation to DS AMkL leukemogenesis and responses PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27486