YY1 Overexpression Negatively Impacts B Cells panel) or 26 weeks (right panel) post reconstitution. (B) MigR1-FlagYY1 reconstituted mice have lower GFP+ percentages compared with MigR1 control mice. GFP+ percentages shown for individual mice in the LSK, pro-B, pre-B, immature B and re-circulating B cell compartments at 14 or 18 weeks post reconstitution. Each data point represents a GFP+ percentage of an individual mouse. (C) GFP MFI decreases throughout B cell development in MigR1-FlagYY1 reconstituted mice. GFP MFI of bone marrow pro-B, pre-B, immature B and re-circulating B 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. In (B) and (C), 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.g001 MigR1 reconstituted mice (Fig. 1B). In addition, YY1 reconstitut- ed mice had a substantially lower GFP mean fluorescence intensity (MFI) among GFP+ bone marrow pro-B, pre-B, immature B and re-circulating B lineages as compared with MigR1 reconstituted mice (Fig. 1C). This provided quantitative evidence for the loss of high YY1-expressing cells in these cell compartments. Thus high levels of YY1 expression appear to be detrimental to developing bone marrow B cell populations. The YY1 inhibitory effect observed during B cell development was not observed in bone marrow myeloid lineages. At 10 weeks post reconstitution, the percentage of GFP positive cells remained relatively stable in the myeloid lineage from 75% in the LSK (Lin2Scahic-Kit hi) pool (Fig. 1A) to 71% in bone marrow CMP (Lin2 Sca2 c-Kit hi CD16/32lo CD34+), 73% in bone marrow GMP (lin2 c-kit hi Sca2 CD16/32hi CD34+), and 82% of bone marrow myeloid cells (Gr-1+CD11b+) (Fig. 2A, left panel). GFP expression in splenic Gr-1+CD11b+ cells also remained relatively high at 63% (Fig. 2A, left panel). Similarly, at 26 weeks post reconstitution, we did not observe any detrimental effects of high levels of YY1 on bone marrow myeloid lineages (Fig. 2A, right panel). Compared to 10 weeks post-reconstitution, bone marrow CMP GFP positive cells increased from 71% to 82%, GMP increased from 73 to 84%, and Gr-1+CD11b+ cells increased from 82% to 91% (Fig. 2A). MigR1-Flag YY1 and MigR1 vector reconstituted mice had similar percentages of GFP positive cells in CMP, GMP and myeloid compartments (Fig. 2B), and MigR1- FlagYY1 and MigR1 vector reconstituted LSK, GMP and myeloid cells showed similar MFI among GFP+ cells, although there was a slight positive effect of YY1 on MFI in CMPs (Fig. 2C). The distinct effects of YY1 expression on the percentage of GFP+ cells among B cell and myeloid cell populations is summarized in Fig. 2D. Cells expressing elevated YY1 are enriched in LT-HSC cells PcG proteins are involved in hematopoietic development, stem cell self-renewal and cell proliferation. As YY1 is a PcG protein [17], we hypothesized that YY1 over-expression might also influence HSC biology. Consistent with this hypothesis, we observed an enrichment of MigRI-FlagYY1 transduced cells in the LSK fraction containing HSCs (Figure 1B). At 10 weeks post reconstitution, 75% of cells in the LSK pool were GFP positive and this increased to 90% by 26 weeks, indicating enrichment of cells expressing high levels of YY1 in this progenitor compartment (Fig. 1A). Within the LSK pool we further examined the phenotypically defined LT-HSC population by analysis of CD48 and CD150 cell surface markers [33], By comparing the percentages of LT-HSCs among GFP+ versus GFP2 LSK cells, we observed a higher percentage of LT-HSCs within the GFP+ LSK population of YY1 reconstituted mice as compared to the GFP2 population (Fig. 3A). Conversely, we examined the percentage of GFP expression in phenotypically defined LT- HSCs, short-term HSCs, common myeloid progenitors (CMPs), and granulocyte-macrophage progenitors (GMPs). An enrichment for GFP+ cells was observed specifically in the LT-HSC compartment of MigR1-FlagYY1 reconstituted mice but there was no difference in CMP and GMP populations (Fig. 3B). These data indicate that cells expressing high levels of YY1 are enriched in the bone marrow LT-HSC population of MigR1-FlagYY1 reconstituted mice. YY1 inhibits B cell growth in vitro To further explore the inhibitory activity of YY1 expression in the B cell lineage, we tested the effect of YY1 expression on the growth of various B cell lines. We reasoned that if YY1 inhibited B cell growth, cells transduced with MigR1-FlagYY1 should be at a selective growth disadvantage in culture and should progres- sively decrease in relative percentage compared to untransduced cells and vector control transduced cells. We retrovirally transduced murine pro-B cell line 38B9, murine pre-B cell line 3-1, murine plasmacytoma cell line S194, and Il-7 cultured primary bone marrow B cells with MigR1-FlagYY1 or empty MigR1 vector. MigR1 vector transduced GFP-positive cells proliferated at a rate similar to the untransduced GFP-negative cells as indicated by the stable percentage of GFP-positive cells at different time points after transduction (Fig. 4A). In contrast, the percentage of MigR1-YY1 transduced GFP positive cells declined dramatically in murine pro-B, pre-B, plasmacytoma, and Il-7 cultured bone marrow B cells, indicating growth arrest and/or cell death (Fig. 4A). Since our in vivo data showed that YY1 overexpression was detrimental to B cells but not myeloid cells (Figs. 1 and 2), we transduced murine myeloid cell line 32D with MigR1-YY1 or MigR1 empty vector. Consistent with our in vivo data, there was no decline of GFP-positive percentages in YY1 expressing myeloid cells over time (Fig. 4B). Thus, similar to the case in vivo, we observed no negative impact of elevated YY1 levels on myeloid cell growth. To test the impact of elevated YY1 on B-lymphomagenesis in vivo, the Myc-over-expressing B-lymphoma Myc3 [34] was infected with either MigR1-YY1 or MigR1 vector. When evaluated in vitro, YY1 transduced GFP positive cells declined dramatically over time (Fig. 4C). To test YY1 inhibitory effects in vivo, the mixture of transduced and uninfected cells was injected subcutaneously in syngeneic animals to produce tumors. Two weeks post-injection, the resulting tumors were analyzed by flow cytometry and percentages of GFP-positive cells were determined again. For MigR1 infected cells, the percentage of GFP-positive cells remained unchanged (Fig. 4D). However, there was a 3-fold decrease in the percentage of GFP-positive cells in MigR1- FlagYY1 transduced Myc3 cells (Fig. 4D). We also tested two human pre-B acute lymphoblastic leukemia (ALL) lines (697 and JM-1) to determine whether they would also respond to elevated YY1 expression similar to murine B cell lines. The two cell lines were transduced with MigR1 or MigR1-FlagYY1 and the percentage of GFP-positive cells was plotted over 10 days. Again, there was a drop in GFP-positive cells over time in the YY1- elevated samples, similar to the case with the murine B lymphoma cell lines (Fig. 4E). Thus, in all cases, YY1 expression resulted in a disadvantage among B lineage cells, but had no impact on myeloid cells. PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30656