YY1 Overexpression Negatively Impacts B Cells Figure 6. Anti-apoptotic genes NFkB2 and Bcl-xl are down regulated by YY1 in murine pro-B cells but not in murine myeloid cells. (A) NFkB2 and Bcl-xl are down regulated at the transcript level in murine 38B9 pro-B cells. RT-PCR analyses of NFkB2 and Bcl-xl mRNA levels relative to hprt are plotted for MigR1-YY1 or MigR1 transduced 38B9 cells. The data represent the mean and standard error of the mean for three independent experiments. (B) NFkB2 and Bcl-xl are down regulated by YY1 at the protein level in murine 38B9 pro-B cells. Cell lysates from MigR1- YY1 or MigR1 transduced 38B9 cells were immunoblotted for Bcl-xl, NFkB2 and YY1. b-actin was used as a loading control. (C) NFkB2 and Bcl-xl are up regulated at the transcript level in murine myeloid cell line 32D. GFP positive MigR1-YY1 and MigR1 transduced murine myeloid cell line 32D samples were isolated by FACS. Levels of NF-kB2 and Bcl-xl transcripts were determined by RT-PCR and hprt was used as an internal control and for normalization. The data represent the mean and standard error of the mean from three independent experiments. (D) GFP positive MigR1-YY1 and MigR1 transduced murine myeloid cell line 32D samples were isolated by FACS. Cell lysates were made from sorted cells and immunoblotted for NFkB2, Bcl-xl and YY1. b-actin was used as a loading control. Mean and standard error of the mean are shown. Asterisks indicate significant differences between samples at P,0.05. doi:10.1371/journal.pone.0030656.g006 YY1 is a PcG protein and these factors function in the stable silencing of specific gene sets through chromatin modifications and dynamic reprogramming of epigenetic states. Many PcG proteins including Bmi-1, EZH2, Rae28, and Mel18 are involved in HSC function [22,23], and forced expression of PcG protein EZH2 can prevent HSC exhaustion [22]. YY1 is the only mammalian PcG protein with specific DNA binding ability, and YY1 can recruit PcG proteins to specific DNA sites causing subsequent trimethyla- tion of histone H3 on lysine 27 [18,20]. Thus, forced expression of YY1 might change gene expression profiles by altering global chromatin structure. Dysfunction of PcG proteins has been argued to contribute to the generation of cancer stem cells implying that PcG proteins provide potential targets for anticancer therapy [49,50,51]. For instance, loss or knock-down of PcG protein Ezh2 causes loss of cell growth and the transformed phenotype of prostate cancer cells [52]. Thus, the enrichment of cells expressing high levels of YY1 in the LT-HSC cell population argue that YY1 might be involved in cancer stem cell function and therefore could be a potential therapeutic target for some hematopoietic malignancies. The finding that transcription factor YY1 differentially controls hematopoietic development provides rationale for multiple future studies on the effect of YY1 on stem cell biology, lineage development, and growth control. Materials and Methods All work involving animals complied with all federal, state, and local laws, and was done with approval and oversight of the University of Pennsylvania Institutional Animal Care and Use Committee under protocol 803080. Retroviral constructs The flag-tagged YY1 cDNA sequence encoding the complete 414 amino acid protein was cloned into the HpaI site of GFP- expressing MSCV-IRES-GFP vector (MigR1) by blunt end ligation. High titer retroviral supernatants were prepared following transient transfection of HEK293 cells. Retroviral envelopes with ecotropic host ranges (pHIT123, for murine cells and pHIT 456 for human cells) were used. Bone marrow transduction and transplantation Bone marrow cells were harvested from 6 to 8 week-old C57BL/6 or mb1-CRE yy1f/f mice 4 days after intravenous injection of 250 mg/kg 5-fluorouracil (5-FU). Cells were cultured overnight in DMEM plus 10% fetal bovine serum and L- Glutamine, with IL-3 (6 ng/ml), IL-6 (5 ng/ml), and SCF (100 ng/ml), then washed and resuspended in retroviral superna- tant containing polybrene (4 mg/ml) and the same cytokine PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e30656