The PcG protein Bmi-1 is necessary for hematopoietic stem cell (HSC) self-renewal and can control cell proliferation [17,21,22,23]. Similarly, the PcG protein EZH2 can prevent HSC exhaustion [22] whereas Mel18 negatively regulates HSC self-renewal [24]. As YY1 is a PcG protein, it may also play important roles in HSC biology, but such roles have never been explored. B cell development involves progression from Lin2Sca-1+c-Kit+ (LSK) progenitor cells through common lymphoid progenitors, pro-B, pre-B, immature B, mature B, and plasma cell stages. Transcription factors regulate cell fate determination through a complex network required for development of early progenitors, and for B cell lineage commitment, maturation, proliferation and survival [8,25,26,27,28]. YY1 has long been believed to play an important role in immunoglobulin (Ig) gene regulation because it binds to numerous Ig enhancer elements including the Igk 39 enhancer, the heavy chain intron enhancer, and the heavy chain 39 enhancer [29,30]. The Shi laboratory showed that YY1 is also a critical regulator of B cell development [31], as conditional knock out of the yy1 gene in the B cell lineage results in an early B cell defect with an almost complete block at the pro-B cell stage, impaired VH to DHJH recombination, and reduced Ig locus contraction [31,32]. Since conditional yy1 gene knock-out in the B cell lineage profoundly impacts B cell development, various dosages of YY1 impact gene expression and development, and multiple PcG proteins where previously demonstrated to play crucial roles for hematopoietic stem cell self-renewal, we sought to explore the impact of YY1 over-expression on hematopoietic development. We utilized a mouse bone marrow reconstitution system to explore whether elevated levels of YY1 would impact hematopoietic cell development and growth. We found that elevated YY1 expression resulted in a progressive selective disadvantage as cells matured to the pro-B, pre-B, immature B and re-circulating B cell stages, but had no detrimental effect on bone marrow myeloid lineage cells. On the contrary, we observed an enrichment of YY1-expressing cells in the Lineage2, Sca-1hi, c-Kithi cell fraction, containing HSCs. This enrichment was particularly visible within the long- term HSC (LT-HSC) compartment. YY1 over-expression in B cell lines inhibited survival by inducing apoptosis, whereas apoptosis was not observed in a myeloid line. Two anti-apoptotic genes, Bcl- xl and NFkB2, were down regulated by elevated levels of YY1 in B cell lines but not in a myeloid cell line confirming the pro- apoptotic effect of YY1 in the B cell lineage. Results Conditional YY1 inactivation in the B cell lineage results in developmental arrest at the pro-B cell stage [31]. Haplo- insufficiency of YY1 can lead to numerous developmental defects and two-fold changes in YY1 expression can alter the regulation of many genes [4,5]. As the impact of elevated YY1 expression on the B cell lineage is unknown, we set out to assess the effects of elevated YY1 expression on B cell development using a bone marrow transduction and reconstitution approach. For these studies, we transduced C57BL/6 bone marrow progenitors with retroviral vector alone (MigR1) or a retrovirus expressing Flag- tagged YY1 (MigRI-FlagYY1). The MigR1 vector allows us to track levels of YY1 expression by monitoring levels of green fluorescent protein (GFP) expression from a bicistronic RNA containing an internal ribosomal entry site. Thus, levels of GPF expression correlate with levels of YY1 expression when using this vector (Fig. S1A). In addition, we compared the level of YY1 expression in B lineage 38B9 pro-B cells and myeloid lineage 32D YY1 Overexpression Negatively Impacts B Cells cells. Cells were transduced with MigR1-FlagYY1 vector and sorted into identical GFP low and GFP high gated fractions. Equivalent amounts of lysates were analyzed on the same gel by western blot with anti-YY1 antibody and anti-actin to serve as a loading control so we could directly compare YY1 levels in the two lineages. We found that myeloid cells expressed higher levels of endogenous YY1 compared to B lineage cells (Fig. S1B left, lanes 1 and 4). As expected, transduction with MigR1-FlagYY1 resulted in increased YY1 expression with the highest levels in the GPF high fractions (Fig. S1B lanes 2,3,5,6). Quantitation of these data are shown in Fig. 1B, right. Levels of total YY1 expression are overall similar in the two lineages, but are higher in the myeloid cells due to their higher level of endogenous YY1. MigR1-Flag-YY1 or MigR1 transduced bone marrow cells were transplanted into lethally irradiated C57BL/6 mice. Reconstituted mice were subject to analysis from 10 weeks to 26 weeks post injection. Both MigR1 and MigR1-Flag-YY1 transduced bone marrow cells yielded efficient reconstitution efficiency at 10 weeks post injection as evidenced by the high percentage of GFP positive cells in the Lin2Scahi c-Kithi (LSK) fraction (Fig. 1A). We isolated GFP+ lymphoid cells by FACS from the blood of MigR1-FlagYY1 reconstituted mice, and performed western blots to determine the level of exogenous YY1 protein compared to endogenous YY1 protein. In blood lymphocytes, exogenous Flag-tagged YY1 protein (Fig. S1C left, upper band) was expressed at an equivalent level compared with endogenous YY1 (Fig. S1C left, lower band). Exogenous expression levels were similar to levels observed in transduced 32D myeloid cells (Fig. S1C, right). These experiments (Figure S1) show that cells expressing MigR1-FlagYY1 reconstitute well in recipient mice, that expression of YY1 is elevated in MigRI-FlagYY1 transduced cells, that myeloid and B lineage cells express similar levels of transduced YY1, and that GFP levels correlate with YY1 expression levels. YY1 over-expression results in a B lymphoid-specific selective disadvantage Between 10 to 26 weeks post bone marrow reconstitution, recipient mouse bone marrow and spleen cells were stained and analyzed by flow cytometry. For MigR1 reconstituted mice, the percentage of GFP positive cells remained stable when comparing bone marrow pro-B (B220+CD43+CD19+AA4.1+ IgM2), pre-B (B220+CD432 IgM2AA4.1+), immature B (B220+CD432Ig- M + AA4.1+) and re-circulating B cells (B220+CD432IgM+AA4.12) (Fig. 1A, left panel). However, as early as 10 weeks post reconstitution, in MigR1-FlagYY1 reconstituted mice, there was a progressive decrease in percentage of GFP positive cells from 75% in LSK (Lin2Scahic-Kit hi) cells, to 64% in pro-B, 54% in pre-B, 50% in immature B, and 24% in re-circulating B cells, respectively (Figure 1A, left panel, and Fig. 1B). In addition, there was a significant and progressive leftward shift of GFP signal in pro-B, pre-B, immature B, and re-circulating B cell pools indicating a selection against high level YY1 expression in the B cell lineage (Fig. 1A, left panel, and Fig. 1C). Consistent with less pronounced effects of YY1 expression in early B lineage cells, there was no noticeable difference in VDJ rearrangement of proximal and distal VH genes in MigR1-FlagYY1 transduced animals compared to MigR1 vector alone (Fig. S2). At 26 weeks post reconstitution, there continued to be a pronounced selection against cells expressing high levels of YY1 in the B cell lineage as evidenced by an even more obvious decrease of GFP percentages as well as leftward shifts of GFP signals (Fig. 1A, right panel). MigR1-FlagYY1 reconstituted mice had consistently lower percentages of GFP+ cells in bone marrow pro- B, pre-B, immature B and re-circulating B cells compared with PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30656