YY1 Overexpression Negatively Impacts B Cells Figure 5. High levels of YY1 induce apoptosis in B cells but not in myeloid cells. (A) MigR1-FlagYY1 and MigR1 transduced 38B9 cells were stained with propidium iodide and the percentage of cells at each phase of the cell cycle were determined by flow cytometry. C, Cleaved caspase 3 is up regulated in MigR1-FlagYY1 transduced pro-B cell line. MigR1-YY1 and MigR1 transduced 38B9 cell lysates were immunoblotted for cleaved caspase 3 and YY1. b-actin was used as a loading control. (B) YY1 overexpression does not change the percentage of cycling cells. 38B9 cells were transduced with MigR1-YY1, sorted by FACS into GFP+ and GFP2 populations and incubated with Brdu to measure DNA synthesis. (C) Murine pro-B cell line 38B9 was infected with MigR1-YY1 or MigR1 vectors such that 90% of cells were transduced. Cells were stained with Annexin V-APC and 7- AAD, and early apoptotic cells (Annexin-V+ and 7-AAD2) were determined by flow cytometry 48 hours post infection. Percentages of apoptotic cells were normalized to percentage of apoptotic cells of MigR1 vector only infected cells. Mean and standard error of the mean are shown. The asterisk indicates significant differences between MigRI and MigR1-FlagYY1 populations at P,0.05. (D) Cleaved caspase 3 is not up regulated in a MigR1- FlagYY1 transduced myeloid cell line. Murine myeloid cell line 32D was transduced with MigR1-FlagYY1 or MigR1 vectors and GFP positive cells were isolated by FACS. Cell lysates made from sorted cells were immunoblotted for cleaved caspase 3 and YY1. b-actin was used as a loading control. The upper band indicates the Flag-tagged exogenous YY1 and the lower band is endogenous YY1. doi:10.1371/journal.pone.0030656.g005 In addition, cyclin D3 is required for germinal center development and expansion of late stage germinal center B cells [42,43]. Interestingly, cyclin D3 knock-out does not impact cell proliferation in vitro, but results in failed proliferative expansion of late stage germinal center B cells [42,43]. Thus, the impact of YY1 overexpression on reduced B cell numbers past the pro-B cell stage may at least partially relate to inhibition of cyclin D3 which is exquisitely important for two proliferative expressions in the B cell lineage. Others have also shown that modulation of YY1 expression leads to altered expression of genes involved in cell cycle regulation, mitosis and cytokinesis, DNA replication, apoptosis, and cell growth [5]. For instance, complete ablation or knock- down of YY1 leads to growth inhibition in embryonic fibroblasts, HeLa cells, DT40 cells, and embryonic carcinoma cells, [5,12,44]. However, YY1 expression can similarly regulate cell growth by interaction with Rb [8,12,44]. YY1 can also impact senescence by regulating p16INK4a [45], and can increase HoxB7 expression directly involved in tumor progression [46]. The negative impact of YY1 on B cell development could also be due to disrupted stoichiometry of YY1 in multiple protein complexes such as the PcG and INO-80 complexes, or due to interactions with regulatory proteins such as p53, c-myc, histone acetyltransferases, and histone deacetylases [2,12,17,47]. Thus, over-expression of YY1 could influence protein complex formation leading to alterations in cell function. The negative impact of elevated YY1 levels on B cell development could also be the consequence of growth inhibition. However, our in vitro data did not reveal changes in cell cycle progression. Instead, a fraction of cells underwent apoptosis. Our in vitro results are most consistent with YY1 expression inducing increased apoptosis in B cells, but not in myeloid cells. Our study is the first to show that YY1 has a pro-apoptotic function in B cells. In vivo, additional YY1 effects on stem cells and lineage differentiation are likely to be important. Unlike the case in B cells, YY1 expression resulted in enrichment of phenotypically defined LT-HSCs and granulocyte lineage cells. By 26 weeks post reconstitution, cells expressing high levels of YY1 constituted 90% of the LSK population and 91% of the bone marrow Gr-1+ population. The percentage of YY1-expressing reconstituted LT- HSCs and myeloid cells was substantially higher compared with MigR1 vector reconstituted LT-HSC indicating a positive selection for high levels of YY1 expression. These are interesting results because they suggest that elevated levels of YY1 expression might be beneficial for HSC function and may enhance differentiation to the myeloid lineage at expense of the B lymphoid lineage. This is reminiscent of a similar effect by transcription factor PU.1 [48]. Epigenetic states are implicated in controlling various HSC populations such as LT-HSC and ST-HSC, and YY1 expression may function to stabilize the LT-HSC chromatin phenotype. Alternatively, YY1 might function as a traditional transcriptional activator or repressor to regulate specific genes involved in cell cycle control and stem cell function. In preliminary studies we found that YY1 expression correlated with elevated c-kit expression in LSK cells (unpublished data). Thus, YY1 might regulate genes involved in maintenance of the HSC phenotype and self-renewal. Clinical strategies to enhance YY1 expression could thus preserve, or even enhance, hematopoietic reconstitution. PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e30656