Increased Expression of PcG Protein YY1 Negatively Regulates B Cell Development while Allowing Accumulation of Myeloid Cells and LT-HSC Cells

Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs

YY1 controls immunoglobulin class switch recombination and nuclear aid levels

YY1 is a multifunctional zinc finger transcription factor that regulates activation, repression and transcriptional initiation of many genes. YY1 functions in diverse biological processes, including embryonic development, cell cycle, Polycomb Group function, lineage differentiation, X-chromosome inactivation, imprinting and cancer. Recently, a new role for YY1 was described in early B cell development. Ablation of transcription factor YY1 in the B cell lineage results in arrest at the pro-B cell stage, reduced immunoglobulin locus contraction, and reduced rearrangement of distal variable genes. However, the role of YY1 in late B cell functions is unknown. Activation induced deaminase (AID) normally expressed in activated germinal center B cells is required for late stage B cell functions including somatic hypermutation (SHM) and class switch recombination (CSR). AID deaminates cytosines on DNA to inititate SHM and CSR. AID must be transported to the nucleus to mediate its activities, but the mechanism of AID nuclear localization is unclear. To investigate role of YY1 in late B cell development we employed a conditional knockout system using mature splenic B cells purified from yy1flox/flox mice treated with recombinant TAT-CRE protein ex vivo. In this thesis we show that YY1 ablation interferes with CSR. Loss of YY1 does not affect B cell proliferation, levels of switch region germline transcripts, or expression of the Aicda or IgM genes. However, YY1 physically interacts with AID and controls nuclear accumulation of AID. AID also interacts with PU.1 and PAX5 but these transcription factors do not affect AID nuclear localization. We propose that YY1 regulates CSR by controlling amounts of AID in the nucleus. We show for the first time that YY1 plays a novel role in regulating CSR

PU.1 Can Recruit BCL6 to DNA To Repress Gene Expression in Germinal Center B Cells ▿

BCL6 is a transcriptional repressor crucial for germinal center formation. BCL6 represses transcription by a variety of mechanisms by binding to specific DNA sequences or by recruitment to DNA by protein interactions. We found that BCL6 can inhibit activities of the immunoglobulin kappa (Igκ) intron and 3′ enhancers. At the Igκ 3′ enhancer, BCL6 repressed enhancer activity through the PU.1 binding site. We found that BCL6 physically interacted with PU.1 in vivo and in vitro, and the results of sequential chromatin immunoprecipitation assays and transient-expression assays suggested that BCL6 recruitment to the Igκ and Igλ 3′ enhancers occurred via PU.1 interaction. By computational studies, we identified genes that are repressed in germinal center cells and whose promoters contain conserved PU.1 binding sites in mouse and human. We found that many of these promoters bound to both PU.1 and BCL6 in vivo. In addition, BCL6 knockdown resulted in increased expression of a subset of these genes, demonstrating that BCL6 is involved in their repression. The recruitment of BCL6 to promoter regions by PU.1 represents a new regulatory mechanism that expands the number of genes regulated by this important transcriptional repressor

Transduction with MigR1-FlagYY1 results in selective disadvantage in the B cell lineage in vitro and in vivo, but not in myeloid cells.

<p>(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. <i>E</i>, 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.</p

High levels of YY1 induce apoptosis in B cells but not in myeloid cells.

<p>(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. <i>C</i>, 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. β-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 GFP− 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-AAD−) 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. β-actin was used as a loading control. The upper band indicates the Flag-tagged exogenous YY1 and the lower band is endogenous YY1.</p

Cells expressing high levels of YY1 are enriched in the LT-HSC cell population.

<p>Chimeric mice were made as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030656#pone-0030656-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030656#pone-0030656-g002" target="_blank">2</a>. (A) MigR1-FlagYY1 reconstituted mice show a relative enrichment in the LSK fraction. MigRI and MigR1-FlagYY1 reconstituted bone marrow cells were sorted into GFP− and GFP+ populations and the percentage of LSK cells in each fraction are plotted. (B) YY1 expressing cells are enriched in the LT-HSC fraction but not in the ST-HSC, CMP, or GMP fractions. In (A) and (B), cell fractions marked with two asterisks show significant differences between MigRI and MigR1-FlagYY1 populations at P<0.01.</p

Anti-apoptotic genes NFκB2 and Bcl-xl are down regulated by YY1 in murine pro-B cells but not in murine myeloid cells.

<p>(A) NFκB2 and Bcl-xl are down regulated at the transcript level in murine 38B9 pro-B cells. RT-PCR analyses of NFκB2 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) NFκB2 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, NFκB2 and YY1. β-actin was used as a loading control. (C) NFκB2 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 NFκB2, Bcl-xl and YY1. β-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.</p

High levels of YY1 do not affect myeloid lineage development.

<p>Chimeric mice were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030656#pone-0030656-g001" target="_blank">Fig. 1</a>. Recipient mice were subject to analysis at 10 weeks, 14 weeks, 18 weeks or 26 weeks post transplantation. (A) The top panel shows the cell gating strategy. Bone marrow cells were evaluated for the presence of CMP (lin⁻ c-kit ^hi Sca⁻ CD16/32^lo CD34⁺), GMP (lin⁻ c-kit ^hi Sca⁻ CD16/32^hi CD34⁺), and myeloid (Gr-1⁺CD11b⁺) cells. GFP+ percentages are listed in each cell compartment in MigR1 or MigR1-FlagYY1 reconstituted mice 10 weeks (left panel) or 26 weeks (right panel) post reconstitution. (B) GFP+ percentages of MigR1-FlagYY1 reconstituted mice are the same as MigR1 control mice in myeloid lineage cells. GFP+ percentages are shown for individual mice in the LSK, CMP, GM and myeloid cells in reconstituted mice 14 or 18 weeks post reconstitution. Each data point represents a GFP+ percentage of an individual mouse. (C) GFP MFI remains stable throughout myeloid cell development in MigR1-FlagYY1 reconstituted mice. GFP MFI of bone marrow LSK, CMP, GMP and myeloid 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. (D) Cells expressing high levels of YY1 experience negative selection in the B cell lineage but not the myeloid lineage. GFP+ percentages of CMP, GMP, myeloid, pro-B, pre-B, immature B and re-circulating B cells are normalized to the GFP+ percentage of bone marrow LSK cells. Mean and standard error of the mean are shown. In B, C, and D, 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.</p