YY1 Overexpression Negatively Impacts B Cells Figure 4. Transduction with MigR1-FlagYY1 results in selective disadvantage in the B cell lineage in vitro and in vivo, but not in myeloid cells. (A) Murine pro-B cell line 38B9, pre-B cell line 3-1, plasmacytoma cell line S194 and Il-7 cultured murine bone marrow cells were retrovirally infected with MigR1-FlagYY1 or MigR1 empty vector. Percentage of GFP positive cells was checked every two days by flow cytometry. Percentages of GFP positive cells were normalized to the level at 48 hours post-transduction. (B) Murine myeloid cell line 32D was transduced by MigR1-FlagYY1 or MigR1 retrovirus vectors. Percentage of GFP positive cells was checked every other day by flow cytometry for 10 days. Percentages of GFP positive cells were normalized to the level on day 4 post-transduction. (C) Myc3 B-lymphoma cells were transduced with MigR1-YY1 or MigR1 control vector. Percentage of GFP positive cells was checked every two days by flow cytometry. Percentages of GFP positive cells were normalized to the level on day 2 post-transduction. (D) Two days post infection, GFP+ percentages of Myc-3 cells were monitored by flow cytometry (pre-injection) and transduced Myc3 cells were then injected into syngeneic mice subcutaneously. The number of GFP positive Myc3 cells in each sample was then analyzed after tumor development two weeks post-injection. Percentages of GFP positive cells were normalized to the level on day 2 post- transduction. E, Human pre-B ALL cell lines 697 and JM-1 were transduced by MigR1-YY1 and MigR1 retrovirus vectors. Percentages of GFP positive cells were normalized to levels on day 4 post-transduction. The data represent the mean and standard error of the mean from three independent experiments. In each panel, 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.g004 rearrangement. On the contrary, cells overexpressing YY1 in the B cell lineage progress past the pro-B cell stage and show normal VH gene rearrangement. However, YY1 overexpressing cells show reduced numbers of B cells at later stages of B cell development and show a progressive selection against cells expressing high levels of YY1. The reduction is most significant past the pro-B cell stage. Our microarray data indicate that YY1 overexpression impacts apoptosis related genes as well as growth control genes. Perhaps most relevant is the ability of YY1 to inhibit expression of cyclin D3. This cyclin is very important for early B cell development and particularly for the proliferative expansion of cells between the pro-B and pre-B cell stages [41]. Knock-out of cyclin D3 causes a significant drop in cells progressing to the pre-B cell stage resulting in reduced cell numbers past the pro-B cell stage [41]. This is similar to the drop we observe in our YY1 overexpression studies. Thus, YY1 may inhibit cyclin D3 expression leading to reduction in the proliferative burst and reduced differentiation to the pre-B cell stage. PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30656