YY1 regulates the germinal center reaction by inhibiting apoptosis

Abstract

The germinal center (GC) reaction produces high-affinity antibodies for a robust adaptive immune response. If dysregulated, the same processes cause GC B cells to become susceptible to lymphomagenesis. It is important to understand how the GC reaction is regulated. In this study, we show that transcription factor YY1 is required to maintain a robust GC reaction in mice. Selective ablation of YY1 significantly decreased in the frequency and number of GC B cells during the GC reaction. This decrease of GC B cells was accompanied by increased apoptosis in these cells. Further, we found that loss of YY1 disrupted the balance between dark zones and light zones, leading to a preferential decrease in dark zone cells. Collectively, these results indicate that YY1 plays an important role in regulating the balance between dark zone and light zone cells in GCs and between survival and death of GC B cells.

Introduction

Germinal centers (GCs) are sites in secondary lymphoid organs where antibody affinity maturation occurs (1). Upon antigen stimulation, naïve B cells interact with T follicular helper cells and become activated to form distinct GCs within the lymphoid follicles (1–3). GC B cells can be detected with cell surface markers CD95, GL7 or peanut agglutinin (PNA) as early as 4 days after antigen encounter. GCs begin to polarize into dark zones (DZ) and light zones (LZ) by day 7 after antigen stimulation. In DZ, GC B cells undergo rapid proliferation and somatic hypermutation (SHM) of the immunoglobulin (Ig) genes. GC B cells in LZ undergo selection for high-affinity antibodies and class switch recombination (CSR) for fine-tuning of Ig effector functions. GC B cells that express Ig with high-affinity for antigen are positively selected and differentiate into memory B cells or plasma cells to produce high-affinity antibodies (1–3).

Although the GC reaction is essential for adaptive immunity, it can also lead to B-cell lymphomagenesis. Both SHM and CSR involve error-prone DNA repair that can target genes other than Ig in GC B cells (4–10), leading to genetic alterations that promote tumorigenesis. Furthermore, GC B cells in DZ are among the fastest dividing mammalian cells with an estimated cell cycle time of 6–12 hours (11–13). Accelerated proliferation of GC B cells is accompanied by attenuation of DNA damage sensing and replication checkpoints (14–17), thus increasing the risk of accumulation of oncogenic mutations. Because of these mutagenic processes, GC B cells are at risk of tumorigenesis. It is not surprising that most non-Hodgkin’s lymphomas are derived from GC B cells or B cells that have passed through GCs (18–22). Therefore, regulation of the GC reaction is critical to our understanding of not only antibody affinity maturation but also pathogenesis of B-cell lymphoma.

A distinct gene expression signature distinguishes GC B cells from other B cell subsets at different developmental stages (23–25), suggesting that specific transcriptional programs play important roles in the GC development. A number of transcription factors and chromatin modifiers that regulate transcription have been found to be required for the GC reaction, including Bcl6 (26–28), c-Myc (29, 30), Ezh2 (31, 32), IRF4 (33, 34) and MEF2C (35). Recently, binding motifs for transcription factor YY1 were found to be significantly enriched in the promoter regions of genes preferentially expressed in GC B cells, suggesting that YY1 regulates the GC reaction (23). However, experimental evidence supporting a role of YY1 in the GC reaction is lacking. YY1 is a GLI-Kruppel class of zinc finger protein that can activate or repress its target genes (36–38). In addition, YY1 has been implicated as the DNA-binding member of the polycomb repressive complex (PRC) to help target PRC to specific regions of chromatin in certain contexts (39). Loss of YY1 has been shown to cause embryonic lethality (40) in a dose-dependent manner (41). Ablation of YY1 in B cells during early B-cell development leads to a blocked transition from progenitor B cells to precursor B cells, partially through impairing chromatin contraction at the Ig heavy chain locus and V(D)J recombination (42). In this report, we deleted YY1 selectively in GC B cells and found that loss of YY1 leads to an impaired GC reaction, indicating that YY1 is indeed an important regulator of the GC reaction.

Materials and Methods

Mouse strains

Mouse strains YY1^Fl/Fl (B6;129S4-Yy1^tm2Yshi/J), AID-Cre (B6.129P2-Aicda^tm2(cre)Mnz/J), mT/mG (B6.129(Cg)-Gt(ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo}/J) and C57BL/6 wild-type were obtained from The Jackson Laboratory. Mouse studies were approved by the Institutional Animal Care and Use Committee of University of Massachusetts Medical School.

Immunization

Sheep red blood cells (1.5x10⁹) (Cocalico Biologicals) were injected intraperitoneally into 8 to 10-week old mice of both sexes. At various days after immunization, spleens were collected for fluorescence-activated cell sorting (FACS) staining, fixed in formalin or frozen in OCT for sectioning.

Flow Cytometry

Spleens were prepared into single cell suspension and red blood cells were lysed in cold distilled water. After filtered through 70-µm nylon mesh and counting using a MACSQuant analyzer (Miltenyi Biotec), cells were incubated with anti-CD16/32 antibody (Bio X Cell) to block Fc receptors and stained with fixable viability dye eFluor 780 (eBioscience). Cells were then stained for 20 min in staining media (Hank’s balanced salt solution, 3% FBS, 0.02% sodium azide, 1 mM EDTA) with primary antibodies including B220-FITC, B220-eVolve 655 (clone RA3–6B2), GL7-eFluor 660, GL7-eFluor 450 (clone GL7), CD95-PE, CD95-APC (clone 15A7) (eBioscience), peanut agglutinin (PNA)-biotin (Vector Laboratories), CD86-Pe-Cy7 (clone GL-1; BioLegend) or CD184-biotin (2b11/CXCR4; BD Biosciences). Cells stained with biotin-labeled antibodies were incubated with streptavidin-eFluor 450 (eBioscience). For intracellular staining, cells were stained with fixable viability dye eFluor 780, permeabilized and fixed using a cytofix/cytoperm plus kit (BD Biosciences) according to manufacturer suggested protocol. Cells were then stained with YY1 antibody (H-414; Santa Cruz Biotechnology) and DyLight 594-conjugated secondary antibody (Jackson ImmunoResearch). Flow cytometry analysis was performed on an LSRII FACS or a FACSAria cell sorter (BD Biosciences), and analyzed using FlowJo software (FlowJo).

Single-cell PCR of floxed and deleted YY1 alleles

GC B cells (B220⁺CD95⁺GL7⁺) were sorted into sterile water (5–10 µl) as one cell per well in 96-well plates. Cells were lysed by 3 freeze-thaw cycles followed by heating to 98°C for 10 minutes. PCR to amply the YY1 locus was performed using Phusion hot start flex DNA polymerase (New England Biolabs) and primers: P1 (5’-ACCTGGTCTATCGAAAGGAAGCAC-3’), P2 (5’-GCTTCGCCTATTCCTCGCTCATAA-3’), and P4 (5’-CCAAAGTTCGAAACCTGCTTTCCT-3’) as described (42).

BrdU incorporation and cell cycle analysis

Mice were injected intraperitoneally with 1 mg BrdU. 6–16 hours later, spleen cells were stained for GC B cells as described above. After being fixed and permeabilized using a BrdU flow kit (BD Biosciences), cells were stained with anti-BrdU-FITC (BD Biosciences) or anti-Ki-67-PerCp-Cy5.5 (Sola15; eBioscience) antibody. Cells stained for Ki-67 were further incubated with DAPI. Stained cells were analyzed by flow cytometry as described above.

Activated Caspase-3 staining

Spleen cells were collected and stained for GC B cells as described above. An active Caspase-3 apoptosis kit and anti-activated caspase-3-PE antibody (BD Biosciences) were used following manufacturer suggested protocol. Stained cells were analyzed by flow cytometry as described above.

Immunofluorescence and immunohistochemistry staining

Frozen spleen sections (5 µm) were fixed in 4% paraformaldehyde and extracted in 0.5% Triton-X 100. Sections were stained with PNA-biotin and streptavidin-DyLight 594 (Jackson ImmunoResearch). TUNEL staining was performed after PNA surface staining using an in situ cell death detection kit (Roche) following manufacturer’s recommendations. Fluorescence images were obtained using an Axiovert 200 microscope (Carl Zeiss) equipped with a 40x objective and multi-bandpass dichroic and emission filter sets (model 83000; Chroma Technology) set up in a wheel to prevent optical shift. Images were captured with the AxioVision software (Carl Zeiss) and an Orca-ER camera (Hamamatsu Photonics). Formalin fixed spleens were embedded in paraffin and sections stained with PNA-biotin. Immunohistochemistry images were obtained using an Axiovert 40 CFL microscope (Carl Zeiss) equipped with 2.5X and 10X objectives. Images were captured with QCapture Pro 7 software and a QImaging QIClick camera (QImaging).

Cell Culture and viral infection

Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos and cultured as described (43). MEFs were infected with Adeno-Cre-GFP virus (University of Iowa) with a multiplicity of infection of 100. Four days later, cells were harvested for flow cytometry and Western blotting. SU-DHL-6 cells infected with lentiviral shRNA targeting YY1 (V2LHS_219592, V2LHS_389741) and a non-silencing shRNA control (RHS4346) (GE Dharmacon) were described previously (44).

Western blots

Whole cell lysates were isolated using RIPA buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid and 0.02% sodium azide) plus fresh protease inhibitor complete (Roche). Lysates were run on SDS–PAGE Criterion X-gel (Bio-Rad) and transferred to nitrocellulose membranes (GE Osmonics). Membranes were probed with antibodies against YY1 (H414) and β-actin (Santa Cruz Biotechnology). Membranes were visualized using Western lightening chemiluminescence detection (PerkinElmer) and ChemiDoc MP System with Image lab software (Bio-Rad).

Statistical Analysis

Data were presented as mean ± standard deviation. Two-tailed and unpaired Student’s t-test was used for pairwise comparisons with P<0.05 considered statistically significant.

Results

YY1 protein level is increased in GC B cells

Despite the observation that YY1 binding motifs are significantly enriched in the promoter regions of genes preferentially expressed in GC B cells, the YY1 transcript is not changed in GC B cells compared to other B cell subsets (23). As the transcript level of a transcription factor is not necessarily an accurate indicator of its transcriptional activity, we examined the YY1 protein level in GC B cells. We immunized C57BL/6 wild-type mice with sheep red blood cells (sRBCs) to stimulate the GC reaction. At day 10 post immunization when the GC reaction is at the peak, we purified GC B cells (B220⁺CD95⁺GL7⁺) and non-GC B cells (B220⁺CD95⁻GL7⁻) in spleen by FACS for Western blot analysis. We found that YY1 protein was significantly increased (7.2-fold) in GC B cells compared to non-GC B cells (Fig. 1), prompting us to investigate whether YY1 is required for the GC reaction.

FIGURE 1.

YY1 protein is increased in GC B cells. YY1 protein levels are determined by Western blotting in FACS-sorted GC B cells (B220⁺CD95⁺GL7⁺) and non-GC B cells (B220⁺CD95⁻GL7⁻) from spleens of wild-type mice (N=3) at 10 days post sRBC immunization. The relative levels of YY1 protein are quantified using β-actin as a loading control.

Selective deletion of YY1 in GC B cells using AID-Cre

Constitutive deletion of YY1 leads to embryonic lethality (40), and ablation of YY1 in B cells using a floxed YY1 allele (YY1^Fl) and Mb1-Cre blocks the transition from progenitor B cells to precursor B cells (42). As this block prevents the formation of mature B cells in secondary lymphoid organs, the use of Mb1-Cre or CD19-Cre (45) to delete YY1 in B cells is not suitable to study the role of YY1 in the GC reaction. To ablate YY1 selectively in GC B cells, we used AID-Cre, in which the Cre gene is inserted to replace exon 1 of the Aicda gene encoding activation-induced cytidine deaminase (AID) (7). As AID expression is induced to high levels in GC B cells (46), AID-Cre mediates recombination between loxP sites in GC B cells, and the resulting recombined allele is carried over when GC B cells differentiate into memory or plasma B cells (7, 46). We crossed the AID-Cre mice with a Cre reporter mT/mG (mTomato/mGFP) mouse strain (47) to track cells that had undergone Cre-mediated recombination at ROSA26 at various time points after immunization with sRBCs. The frequency of GC B cells (B220⁺CD95⁺GL7⁺) was increased significantly between day 4 and day 5 post immunization (Supplemental Fig. 1A, 1E). Coinciding with this increase in GC B cells, we found a substantial increase in GFP⁺ cells, in particular the mG⁺mT⁺ population (Supplemental Fig. 1B). These double positive cells are newly recombined as mGFP was expressed but mTomato protein was not degraded yet. Within the GC B cell population, we found a dramatic increase in the newly recombined mG⁺mT⁺ cells between day 4 and day 5 (Supplemental Fig. 1C, 1G), suggesting that AID-Cre is active as early as day 4 in GC B cells. In contrast, very few GFP⁺ cells were found in non-GC B cells (B220⁺CD95⁻GL7⁻) or non-B cells (B220⁻) (Supplemental Fig. 1D, 1F), indicating very low levels of AID-Cre activity in other subsets of cells.

Deletion of YY1 leads to an impaired GC reaction

To investigate the role of YY1 in the GC reaction, we generated YY1^Fl/Fl; AID-Cre (referred as YY1^CKO hereafter) mice and compared them with YY1^Fl/Fl alone, AID-Cre alone or C57BL/6 wild-type mice for the GC reaction in response to sRBC immunization. We examined GC B cells (B220⁺CD95⁺GL7⁺ or B220⁺PNA⁺) by flow cytometry at different time points after immunization. Day 4 post immunization represents the earliest time point that newly formed GC B cells can be reliably detected by flow cytometry using cell surface markers. Day 6 is a time during which GCs are still maturing, whereas day 10 is at the peak of the GC reaction (1–3).

After sRBC immunization, B220⁺ populations remained largely unchanged (Fig. 2). Starting at day 4 post immunization, we observed a gradual increase in the frequency and total number of GC B cells (B220⁺CD95⁺GL7⁺) in spleens of wild-type, YY1^Fl/Fl or AID-Cre control mice compared to unimmunized mice (Fig. 2). The frequency of GC B cells in YY1^Fl/Fl mice was lower than wild-type or AID-Cre mice (Fig. 2B-2D), suggesting that the floxed YY1 allele may be hypomorphic in the GC reaction. However, a significant decrease in the frequency of GC B cells was observed in YY1^CKO mice compared to YY1^Fl/Fl, AID-Cre or wild-type mice at day 4 (Fig. 2B). This decrease resulted in 30–70% reduction of total GC B cells in spleen of YY1^CKO mice compared to control mice. Significant decreases in the frequency and number of GC B cells in YY1^CKO mice were observed at day 6 and day 10 post immunization (Fig. 2C, 2D). Further, at day 10 post immunization, we found that the frequency and number of GC B cells in YY1^CKO mice continued to decrease (by 3-fold) compared to day 6, while control mice had largely maintained their GC B cells (Fig. 2D). The decrease in GC B cells in YY1^CKO mice was similar when we used B220⁺PNA⁺ for GC B cells (Supplemental Fig. 2) or stained with PNA in immunohistochemistry for the presence of GCs (Fig. 3). Collectively, these data indicate that loss of YY1 leads to an impaired GC reaction.

FIGURE 2.

Loss of YY1 leads to an impaired GC reaction. (A) Representative flow cytometry analyses of GC B cells based on B220⁺CD95⁺GL7⁺ staining at day 10 post sRBC immunization. Frequency of each gated population as a percent of displayed cells is shown. (B-D) Percent of B cells (B220⁺), GC B cells (B220⁺CD95⁺GL7⁺) in live B cells and total number of GC B cells in spleens of mouse at (B) day 4 (4 independent experiments), (C) day 6 (8 independent experiments) and (D) day 10 (7 independent experiments) post sRBC immunization. Each point represents data from a single mouse. Horizontal bar indicates mean of data. Statistical significance is determined by two-tailed unpaired Student’s t-test (*: P<0.05, **: P<0.01, ***: P<0.001).

FIGURE 3.

Loss of YY1 impairs the GC reaction. Representative immunohistochemical staining of spleen sections with PNA at day 10 post immunization at (A) 2.5x and (B) 10x magnification.

GCs are polarized into DZ and LZ during GC reaction. Given the diminished GC reaction in YY1^CKO mice, we wanted to determine if YY1 deletion preferentially altered DZ vs. LZ. As shown in Fig. 6, DZ cells (B220⁺CD95⁺GL7⁺CXCR4^hiCD86^lo) outnumbered LZ cells (B220⁺CD95⁺GL7⁺CXCR4^loCD86^hi) in control mice as previously reported (48). In contrast, a significant decrease in the frequency of DZ cells and a concomitant increase in the frequency of LZ cells were found in YY1^CKO mice at 10 day after immunization compared to control mice (Fig. 4), which resulted in a 2-fold decrease in the ratio of DZ vs. LZ cells. The number of DZ cells in YY1^CKO mice was significantly lower than those in AID-Cre or YY1^Fl/Fl control mice, while the number of LZ cells YY1^CKO mice was not significantly different from that in YY1^Fl/Fl mice (Fig. 4C), suggesting a preferential decrease in DZ cells in YY1^CKO mice. Collectively, these data suggest that YY1 regulates the balance between DZ and LZ cells in GCs.

FIGURE 4.

Loss of YY1 leads to decreased DZ cells. (A) Representative flow cytometry analysis of DZ cells (B220⁺CD95⁺GL7⁺CXCR4^hiCD86^lo) and LZ cells (B220⁺CD95⁺GL7⁺CXCR4^loCD86^hi) in YY1^CKO mice at 10 day after sRBC immunization compared to control mice. Cells were gated on GC B cells (B220⁺CD95⁺GL7⁺). Frequency of each gated population as a percent of displayed cells is shown. (B) Percent of DZ and LZ cells in GC B cells and (C) total number of DZ and LZ cells in YY1^CKO mice (N=6) at 10 day after sRBC immunization compared to control mice (YY1^Fl/Fl: N=5; AID-Cre: N=5). Each point represents data from a single mouse. Horizontal bar indicates mean of data. This figure represents data from 2 independent experiments. Statistical significance is determined by two-tailed unpaired Student’s t-test (***: P<0.001).

Because there were still small numbers of GC B cells in response to sRBC immunization in YY1^CKO mice even at day 10 (Fig. 2), we sought to determine whether these GC B cells had undergone complete deletion of both YY1 alleles, or whether one or both alleles of YY1 still remained intact. To facilitate the characterization of YY1 in GC B cells, we attempted to examine YY1 protein using intracellular flow cytometry. We found that intracellular staining of YY1 was not specific, even though the same antibody detects YY1 protein specifically in Western blotting (Supplemental Fig. 3). Instead, we sorted GC B cells from YY1^CKO mice and used single-cell PCR to determine the status of the YY1 locus (Supplemental Fig. 3). At day 4 post immunization, 73% of GC B cells had both YY1 alleles deleted, whereas 10.8% and 16.2% of GC B cells had only one YY1 allele deleted or no YY1 deletion in YY1^CKO mice, respectively (Table I). Despite increased AID-Cre activity from day 4 to day 10 (Supplemental Fig. 1), the frequency of GC B cells with both YY1 alleles deleted didn’t increase at day 10 (69.6%) compared to day 4. Furthermore, the fraction of GC B cells without YY1 deletion was similar at day 4 (16.2%) and day 10 (21.6%). Collectively, these data indicate that although YY1-null GC B cells are generated, YY1 ablation greatly reduces the magnitude of the GC response.

Table I.

AID-Cre mediated deletion of YY1 alleles in GC B cells

Days post immunization	Deletion of both YY1 alleles	Deletion of only one YY1 allele	No YY1 deletion
4	73.0% (54/74)	10.8% (8/74)	16.2% (12/74)
10	69.6% (71/102)	8.8% (9/102)	21.6% (22/102)

YY1 does not affect cell proliferation but prevents apoptosis in GC B cells

The presence of small numbers of GC B cells with both YY1 alleles deleted suggests that YY1 is likely not required for the initiation of the GC reaction. The decrease in DZ cells and the decrease of GC B cells in YY1^CKO mice from day 6 to day 10 (average of 1.5 x 10⁶ cells at day 6 vs. 0.48 x10⁶ cells at day 10) post immunization suggest that impairment of the GC reaction in YY1^CKO mice is due to a defect in maintaining or amplifying GC B cell numbers. We suspected this defect may be the result of altered proliferation and/or apoptosis of GC B cells in YY1^CKO mice. To test this hypothesis, we first examined the consequence of loss of YY1 on cell proliferation and cell cycle. As shown in Fig. 5A, BrdU incorporation was slightly but significantly decreased in GC B cells in YY1^CKO mice compared to control mice (YY1^Fl/Fl or AID-Cre) at day 10 post sRBC immunization. In contrast, proliferation of non-GC B cells (B220⁺CD95⁻GL7⁻) or non-B cells (B220⁻) was low and there was no difference in BrdU incorporation in these cells between YY1^CKO and control mice (Fig. 5A). Interestingly, Ki-67 and DAPI staining indicated there was no significant difference in cell cycle distribution of GC B cells between YY1^CKO and control mice (Fig. 5B, 5C).

FIGURE 5.

Loss of YY1 does not directly affect proliferation of GC B cells. (A) Representative flow cytometry analysis of BrdU incorporation in GC B cells (B220⁺CD95⁺GL7⁺) at day 10 post sRBC immunization in control (YY1^Fl/Fl or AID-Cre), YY1^CKO mice. Negative controls are samples injected with BrdU but stained without anti-BrdU antibody or samples without BrdU injection but stained with anti-BrdU antibody. Average percentages of BrdU incorporation in GC B cells, DZ cells (B220⁺CD95⁺GL7⁺CXCR4^hiCD86^lo), LZ cells (B220⁺CD95⁺GL7⁺CXCR4^loCD86^hi), non-B cells (B220⁻) and non-GC B (B220⁺CD95⁻GL7⁻) cells are shown below. Controls (YY1^Fl/Fl or AID-Cre): N=4, YY1^CKO: N=3. (B) Representative flow cytometry analysis of Ki-67 and DAPI staining of GC B cells at day 10 post immunization and averages of 6 controls (YY1^Fl/Fl or AID-Cre) and 4 YY1^CKO mice are shown at right. (C) Representative cell cycle analysis of GC B cells at day 10 post immunization and averages of 6 controls (YY1^Fl/Fl or AID-Cre) and 4 YY1^CKO mice are shown at right. Error bars are standard deviations. Statistical significance is determined by two-tailed unpaired Student’s t-test.

As DZ cells undergo rapid proliferation (11–13, 48, 49), our finding of altered DZ/LZ ratio in YY1^CKO mice (Fig. 4) prompted us to investigate whether decreased BrdU incorporation in GC B cells in YY1^CKO mice was due to decreased proliferation in DZ and LZ cells, or due to a decrease in DZ cells. We found no significant difference in BrdU incorporation in DZ or LZ cells between YY1^CKO mice and control mice (Fig. 5A). Because LZ cells proliferate slower than DZ cells and incorporate less BrdU (Fig. 5A), these data argue that altered distribution of DZ and LZ cells rather than change in proliferation per se is responsible for the decreased BrdU incorporation in GC B cells of YY1^CKO mice.

From day 6 to day 10 post immunization, GC B cells in YY1^CKO mice continued to decrease (1.5 x 10⁶ vs 0.48 x10⁶) suggesting that cell death is responsible for the loss of GC B cells in YY1^CKO mice. We investigated whether loss of YY1 in GC B cells activates apoptosis by staining cells with an apoptotic marker activated caspase-3 (50, 51). As shown in Fig. 6A, activated caspase-3 was increased more than two-fold in GC B cells in YY1^CKO mice compared to the YY1^Fl/Fl and AID-Cre controls. This increase in apoptosis was limited to GC B cells, as non-GC-B cells and non-B cells displayed very low activated caspase-3 staining and there was no difference between control and YY1^CKO mice (Fig. 6A, 6B). In addition, we used TUNEL staining to detect late stages of apoptosis and found that apoptosis was significantly increased in GC B cells (PNA⁺ cells in follicles) in YY1^CKO mice compared to control mice (Fig. 6C, 6D). Together these data indicate that loss of YY1 in GC B cells leads to an increase in apoptosis and therefore is required for the proper maintenance of a robust GC reaction.

FIGURE 6.

Loss of YY1 leads to increased apoptosis. (A) Representative flow cytometry analysis of activated Caspase-3 staining in GC B cells (B220⁺CD95⁺GL7⁺) at day 10 post sRBC immunization. Samples stained without Caspase-3 antibody were used as negative controls and samples from a mouse irradiated (5 Gy) were used as positive controls for Caspase-3 staining. (B) Average percentages of Caspase-3 positive population in non-B cells (B220⁻), non-GC B cells (B220⁺CD95⁻GL7⁻) and GC B cells. Controls (YY1^Fl/Fl or AID-Cre): N=6, YY1^CKO: N=4. (C) Representative immunofluorescent staining of TUNEL (green), PNA (red) and DAPI (blue) of spleen sections. (D) Averages of TUNEL positive cells as a percent of PNA positive cells in controls (YY1^Fl/Fl or AID-Cre: N=5) and YY1^CKO mice (N=3). About 600–800 PNA⁺ cells were counted per genotype. Error bars are standard deviations. Statistical significance is determined by two-tailed unpaired Student’s t-test.

Discussion

In this report, we found that YY1 expression was increased in GC B cells. Selective deletion of YY1 in GC B cells using AID-Cre significantly impaired the GC reaction as indicated by decreased frequency and number of GC B cells in spleen in response to sRBC immunization. The decrease in GC B cells was observed as early as day 4 post sRBC immunization and exacerbated throughout the GC reaction (Figs. 2, 3 and Supplemental Fig. 2). Previously, YY1 has been implicated as a regulator of the GC reaction based on the enrichment of YY1 target genes in the GC B cell-specific transcriptional signature (23). Now our studies provide experimental evidence indicating that YY1 is required for a normal robust GC reaction. Because the AID-Cre allele that we used is activated in GCs around day 4 after antigen encounter (Supplemental Fig. 1), we cannot completely rule out the possibility that deleting YY1 in naïve mature B cells before antigen encounter will affect the commitment of these cells to become GC B cells. However, our finding that ∼70% of the remaining GC B cells in YY1^CKO mice at day 4 or day 10 after immunization had both YY1 alleles deleted (Table I) suggests that YY1 is not necessarily required for the initiation of the GC reaction, but rather is required to maintain a robust GC reaction. A recent study using Cγ1-Cre to selectively delete YY1 in GC B cells also found that YY1 is required for the development of GC B cells (52).

To understand the defects in the GC reaction in the absence of YY1, we investigated whether loss of YY1 affected proliferation of GC B cells, as YY1 has been shown to regulate proliferation in MEFs or HeLa cells (41). We found that GC B cells in YY1^CKO mice showed a significant decrease in BrdU incorporation compared to GC B cells in control mice (Fig. 5A). However, Ki-67 and DAPI staining indicated there was no significant difference in cell cycle distribution in GC B cells between YY1^CKO and control mice (Fig. 5B, 5C), suggesting that YY1 does not directly affect proliferation of GC B cells. This finding is consistent with a previous report that ablation of YY1 in splenic B cells activated by lipopolysaccharide ex vivo does not alter cell division and proliferation (53). We further found that loss of YY1 resulted in a decrease of DZ cells and a concomitant increase in LZ cells (Fig. 4). In DZ, GC B cells undergo rapid cell proliferation (13, 48, 49). We found that there was no significant difference in BrdU incorporation in DZ or LZ cells between YY1^CKO mice and control mice. These results argue that YY1 does not directly affect proliferation of GC B cells, but rather the altered distribution of DZ and LZ cells is responsible for the decreased BrdU incorporation in GC B cells of YY1^CKO mice.

Our finding that YY1 regulates the relative distribution of DZ and LZ in GCs is intriguing as not much is known about how GC polarity is regulated. A large body of work supports a model in which GC B cells transit between DZ and LZ to undergo SHM in DZ and affinity selection as well as CSR in LZ (1, 18). It has been shown that DZ cells express high levels of CXCR4 and CXCR4 deficiency leads to an absence of DZ without altering the size and number of GCs (54, 55). More recently, two groups independently found that FOXO1 and phosphoinositide-3 kinase (PI3K) play a critical role in GC polarity (56, 57). FOXO1 is highly expressed in DZ, and its activity is down-regulated in LZ. Deletion of FOXO1 or activation of PI3K results in a loss of DZ with LZ-only GCs, partly due to down-regulation of CXCR4 (56, 57). In addition, a small number of LZ cells were found to express FOXO1 and c-Myc (56). C-Myc is required to initiate the GC reaction and the re-entry of LZ cells into DZ for additional rounds of SHM (29, 30). FOXO1 is likely involved in both regulation of targets necessary for the formation of DZ and the cyclic re-entry of LZ cells into DZ. The latter function is possibly through up-regulation of c-Myc. YY1 has been found to transactivate c-Myc (58, 59) in splenic B cells (44). It is plausible that YY1-mediated transactivation of c-Myc plays a similar role in the re-entry of LZ cells into the DZ. Loss of YY1 would impair the re-entry of LZ cells, leading to decreased DZ cells in YY1^CKO mice.

At day 10 post immunization, the frequency and number of GC B cells in YY1^CKO mice continued to decrease by 3-fold compared to day 6, while control mice had largely maintained their GC B cells (Fig. 2C). We found that apoptosis was increased in GC B cells in the absence of YY1 (Fig. 6), providing a plausible mechanism for the reduction of GC B cells in YY1^CKO mice. The GC-specific transcriptional profile is enriched for genes involved in cell death (23). In particular, the Bcl-2 family anti-apoptotic protein Mcl1, which is upregulated in GC B cells, is a potential transactivation target of YY1 (23). Mcl1 has been shown to be the pro-survival factor in GCs. It is required for survival of GC B cells and essential for GC formation (60). It will be interesting to investigate whether Mcl1 is the critical downstream target through which YY1 regulates the survival of GC B cells.

The GC reaction is not only critical to produce high affinity antibodies for a robust adaptive immune response, but also can lead to pathogenesis of B-cell lymphoma. Because of their high proliferation rate and highly mutagenic processes, GC B cells are the susceptible targets of B-cell malignancies. Most non-Hodgkin’s lymphomas are derived from GC B cells or B cells that have passed through GCs (18–22). Our finding that dysregulation of YY1 leads to an impaired GC reaction suggests a potential oncogenic role for YY1 in lymphomagenesis. Consistent with this notion, the expression of YY1 is increased significantly in human diffuse large B cell lymphoma, Burkitt’s lymphoma and follicular lymphoma compared to reactive lymph nodes or normal B cells (61–63). Further, high levels of YY1 expression correlates with a worse survival prognosis in human diffuse large B cell lymphoma and follicular lymphoma patients (61, 63).

Abbreviations used in this article

AID

activation-induced cytidine deaminase

CSR

class switch recombination

DZ

dark zone

FACS

fluorescence-activated cell sorting

GC

germinal center

Ig

immunoglobulin

LZ

light zone

MEF

mouse embryonic fibroblasts

mT/mG

mTomato/mGFP

PNA

peanut agglutinin

PRC

polycomb repressive complex

SHM

somatic hyper mutation

sRBCs

sheep red blood cells