YY1 Overexpression Negatively Impacts B Cells Figure 2. High levels of YY1 do not affect myeloid lineage development. Chimeric mice were prepared as described in Fig. 1. Recipient mice were subject to analysis at 10 weeks, 14 weeks, 18 weeks or 26 weeks post transplantation. (A) The top panel shows the cell gating strategy. Bone marrow cells were evaluated for the presence of CMP (lin2 c-kit hi Sca2 CD16/32lo CD34+), GMP (lin2 c-kit hi Sca2 CD16/32hi CD34+), and myeloid (Gr- 1 + CD11b+) cells. GFP+ percentages are listed in each cell compartment in MigR1 or MigR1-FlagYY1 reconstituted mice 10 weeks (left panel) or 26 weeks (right panel) post reconstitution. (B) GFP+ percentages of MigR1-FlagYY1 reconstituted mice are the same as MigR1 control mice in myeloid lineage cells. GFP+ percentages are shown for individual mice in the LSK, CMP, GM and myeloid cells in reconstituted mice 14 or 18 weeks post reconstitution. Each data point represents a GFP+ percentage of an individual mouse. (C) GFP MFI remains stable throughout myeloid cell development in MigR1-FlagYY1 reconstituted mice. GFP MFI of bone marrow LSK, CMP, GMP and myeloid cells in MigR1-FlagYY1 and MigR1 reconstituted mice are normalized to GFP MFI in the LSK population of each group (MigR1 versus MigR1-FlagYY1). Each dot represents the MFI of an individual mouse 14 or 18 weeks post reconstitution. (D) Cells expressing high levels of YY1 experience negative selection in the B cell lineage but not the myeloid lineage. GFP+ percentages of CMP, GMP, myeloid, pro-B, pre-B, immature B and re-circulating B cells are normalized to the GFP+ percentage of bone marrow LSK cells. Mean and standard error of the mean are shown. In B, C, and D, cell fractions marked with one or two asterisks show significant differences between MigRI and MigR1-FlagYY1 populations at P,0.05 and 0.01, respectively. doi:10.1371/journal.pone.0030656.g002 YY1 can induce apoptosis in B cells To determine the mechanism of YY1-induced selective disadvantage in B cells, we assessed whether elevated YY1 expression affected cell growth parameters. MigR1 and MigR1- YY1 transduced 38B9 cells were evaluated for DNA content by PI staining. Comparing MigR1-FlagYY1 to empty vector there was little difference in G1/S, G2, or M stage profiles (Fig. 5A). Similarly, there was no difference in Budr incorporation comparing GFP+ and GFP2 38B9 cells transduced with MigRI-YY1 (Fig. 5B). However, we observed a 7 fold increase in the sub-G1 phase cell population consistent with a substantial increase in apoptotic cells (Fig. 5A). MigR1-FlagYY1 and MigR1 transduced 38B9 pro-B cells were stained with the dead cell exclusion dye 7-AAD and the apoptosis indicator AnnexinV. Interestingly, we observed a substantial increase of early apoptotic cells (AnnexinV+, 7AAD2) in MigR1-FlagYY1 transduced cells as compared to those transduced with MigR1 empty vector (Fig. 5C). We also tested the impact of elevated YY1 on the apoptosis marker protein, cleaved caspase-3. In MigR1-FlagYY1 transduced 38B9 pro-B cells, increased YY1 expression led to a 6.6 fold elevation of cleaved caspase-3 (Fig. 5D). As a negative control, we transduced the murine myeloid cell line 32D with MigR1- FlagYY1 or MigR1 and GFP-positive cells were sort-purified. Increased YY1 expression did not change the level of cleaved caspase-3 when comparing MigR1-FlagYY1 expressing cells to empty vector MigR1 expressing cells (Fig. 5E). Thus, consistent with our in vivo results, forced expression of YY1 did not cause apoptosis in myeloid cells. To gain further insight into YY1 induced B cell-specific apoptosis, we used a genome-wide microarray approach to identify YY1 target genes in murine pro-B cells. We performed microarray analyses on 38B9 pro-B cells transduced either with MigR1-FlagYY1 or MigR1 vector control. This comparison enabled us to identify approximately 700 genes whose expression was affected by YY1. RT-PCR analyses for a subset of transcripts confirmed our microarray results as over 80% of transcripts matched closely in fold increase or decrease in the presence of overexpressed YY1 (Fig. S3; see also Fig. 6A). Evaluation of identified transcripts using David Bioinformatics Resources (NIAID) and the KEGG Functional Annotation Chart revealed that genes involved in cell cycle control and BCR-ABL signaling were down-regulated by YY1. The complete list of YY1- responsive genes is provided in Table S3. Consistent with the YY1-induced apoptotic effect we observed in B cells, several anti- apoptotic related genes (NFkB2 and Bcl-xl) were deregulated by elevated YY1 expression. We found 4 fold decreases in NFkB2 and Bcl-xl transcripts in MigR1-FlagYY1 transduced cells compared to MigR1 vector alone (Fig. 6A) and a corresponding 2.4 fold decrease in the levels of NFkB2 and 3.4 fold decrease in Bcl-xl proteins (Fig. 6B). On the contrary, in MigR1-FlagYY1 transduced 32D myeloid cells we observed the opposite results. In contrast to YY1-induced down regulation of Bcl-xl and NFkB2 in B cells, we observed a 4.5-fold increase in Bcl-xl transcripts and 2.6-fold increase in Bcl-xl protein in 32D myeloid cells. Similarly, we observed a 1.5-fold increase of NFkB2 transcripts but no change of NFkB2 protein in 32D myeloid cells (Fig. 6C and D). Thus, elevated YY1 results in down regulation of anti-apoptotic genes in B cells but not in myeloid cells. The mechanism of down regulation of NFkB2 and Bcl-xl by YY1 in B cells could be either direct or indirect. ChIP assays were performed to determine if YY1 bound directly to the Bcl-xl and NFkB2 promoters. Multiple primer sets were designed covering approximately 1 kb of the Bcl-xl and NFkB2 promoters, respectively. Whereas YY1 was clearly enriched at the positive control rpL30 promoter in 38B9 pro-B cells, we did not detect obvious enrichment of YY1 at either Bcl-xl or NFkB2 promoters above that observed at the negative control b-actin promoter (Figure S4). Thus, YY1 may influence these two anti-apoptotic genes indirectly, or YY1 might bind to other DNA sequences that regulate these two genes. Consistent with the former option, overexpression of NFkB2 and Bcl-xl either separately or together failed to reverse the pro-apoptotic effect of YY1 overexpression (data not shown). Thus, reduced NFkB2 and Bcl-xl proteins in YY1 overexpressing B cells appears to be a consequence of YY1 apoptotic induction rather than due to direct regulation of the NFkB2 and Bcl-xl genes. None-the-less, reduced levels of these proteins confirm that YY1 overexpression induces apoptosis in B lineage cells. Discussion The functional consequences of YY1 regulation in hematopoi- etic cell development are poorly understood. Conditional knock- out of YY1 in the B cell lineage by action of mb1-driven CRE results in arrest at the pro-B cell stage and impairment of immunoglobulin distal V gene rearrangement [31]. Loss of YY1 may have an impact on both immunoglobulin locus contraction needed for rearrangement of the most distal V genes, as well on cell proliferation [31,32]. Other investigators have shown that YY1 can inhibit neutrophil differentiation both in vivo and in vitro [35]. In this study, we used bone marrow transduction and reconstitution to investigate the impact of elevated YY1 expression on the B cell and myeloid hematopoietic compart- ments in vivo. Significantly, we found that forced expression of YY1 resulted in selective disadvantage as cells developed within the B cell lineage. We observed a progressive decline in the percentage of YY1-transduced (GFP positive) cells at each successive B cell stage, and the GFP MFI signal progressively decreased indicating selection against cells expressing high levels of YY1 (Figs. 1 and 2). The inhibitory effect of YY1 on B cell development was not caused by a non-specific toxic effect of PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30656