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The Journal of Immunology, 2004, 172: 5427-5440.
Copyright © 2004 by The American Association of Immunologists

Modular Nature of Blimp-1 in the Regulation of Gene Expression during B Cell Maturation1

Roger Sciammas2,* and Mark M. Davis*,{dagger}

* Department of Microbiology and Immunology and {dagger} Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The transcription factor Blimp-1 induces the maturation of B cells into Ab-secreting plasma cells. DNA microarrays were used to analyze the transcription profiles of both Blimp-1-transduced murine B cell lines and the inducible B cell line BCL1. Hundreds of genes were differentially regulated, showing how Blimp-1 both restricts affinity maturation and promotes Ab secretion, homeostasis, migration, and differentiation. Strikingly, when different modes of plasma cell induction are used, very different genetic programs are used, suggesting that the transition from a B cell to plasma cell can occur in multiple ways, perhaps accounting for the different types of Ab-secreting cells observed in vivo. Furthermore, mutagenesis of Blimp-1 reveals multiple effector domains, which regulate distinct genes. This indicates that Blimp-1 subdivides the maturation program into select and tunable pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
B lymphocytes, as members of the adaptive arm of the immune system, produce Ag-specific Abs, which are critical for immunity to infectious agents (1). In response to Ag or the activation of pattern receptors, B cells proliferate, undergo affinity maturation, and ultimately differentiate into plasma cells, the cellular source of serum Abs (2). Plasma cells are specialized cellular factories secreting high levels of Ab, a form derived by differentiation-specific, alternative 3' end processing of the Ig mRNA (3, 4). Although most plasma cells are short-lived, a few persist and secrete Ig by interacting with stromal cells in the lymph nodes or in the bone marrow (2). Individuals with congenital mutations that lead to low levels of serum Abs are highly susceptible to bacterial, viral, and fungal infections (5).

The B lymphoma cell line (BCL1),3 inducible by the cytokines IL-2 and IL-5, has served as a model system to track the genetic changes associated with Ab secretion and plasma cell differentiation (6). Blimp-1 is a zinc finger transcription factor that is inducible in BCL1 cells, myeloid cells, following virus induction in HeLa cells, and in both murine and Xenopus embryogenesis (7, 8, 9, 10). Strikingly, ectopic expression of Blimp-1 in BCL1 cells triggers many changes associated with plasma cell differentiation, including Ab secretion, underscoring the importance of this factor in B cell immunity and indicating that Blimp-1 may serve as a master regulator of plasma cell differentiation (11, 12). Adding to its capabilities, recent work has shown that c-myc, CIIta, Id3, and SpiB can function as Blimp-1 target genes (13, 14, 15).

Blimp-1 contains five consecutive zinc finger motifs of the Krûppel C2H2 type in the C-terminal half of the protein, of which the first two interact with DNA (16). In addition, the C-terminal tail of Blimp-1 is highly acidic, a property of many transcriptional activators. In the middle of the protein is a region rich in prolines, and this region has been shown to have a role in differentiation and transcriptional repression, namely by recruiting members of the Groucho family of corepressors and members of the histone deacetylase families (17, 18, 19, 20). Finally, the N-terminal half of Blimp-1 contains a 120-aa region (the PRD1-BF1 and R1Z region of similarity (PR) domain) that shares significant homology to the Su(var)39-1, Enhancer of Zeste, Trithorax (SET) family of methyltransferases (21).

The physiological changes that accompany plasma cell differentiation range from dramatic reorganization of the endoplasmic reticulum (ER) and high level Ab secretion to specialized cellular homing and adhesion characteristics to cellular senescence, implying a host of genetic changes controlling these events. Therefore, we analyzed the effect of Blimp-1 expression on B cells using mRNA profiling on microarrays (22, 23). This parallels the analysis of Blimp-1-regulated genes by Shaffer et al. (15), but our use of different cellular systems, mutational analysis, and a different microarray platform allows us to make a number of novel observations about Blimp-1 and its role in B cell maturation.

In particular, we have identified a large number (409) of genes differentially expressed upon Blimp-1 transduction in the M12 cell line, consistent with its role as a master regulator. A subset of these genes (125) is regulated in the context of IL-2 and IL-5 stimulation or LPS stimulation of BCL1 cells. In addition, many of the identified genes are represented in the plasma cell genetic program (24). Strikingly, subsets of these genes are regulated with unique temporal or directional characteristics depending on the cellular context, suggesting that aspects of the Blimp-1 maturation program are differentially used depending on the nature of the immune response. Thus, there appear to be a number of routes to arrive at a plasma cell, and it is useful to compare as many as possible to arrive at a complete picture of this process. The genes identified in this comparison show that Blimp-1 modulates a variety of B cell responses, cellular stress, ER functions, adhesion, migration, and transcription factors during maturation. We also used mutagenesis to modify two different regions of Blimp-1: the acid-rich tail portion and the PR domain, which is homologous to histone methyltransferases implicated in chromatin remodeling. In each case, inactivating these portions of Blimp-1 results in it losing its ability to regulate distinct sets of genes (12 and 33, respectively) but not the vast majority. This indicates that Blimp-1 is at least partially modular in nature, with different parts of the same molecule able to control the expression of subsets of genes in an autonomous manner. This may be a general feature of important transcription factors, as it allows whole sets of genes to be gained or lost.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture conditions

BCL1 cells (CW13.20-3B3, ATCC CRL 1669) were obtained from American Type Culture Collection (Manassas, VA). CH12.LX cells were a kind gift of G. Bishop (University of Iowa, Iowa City, IA). M12 and MOPC315J cells have been described previously (25, 26). Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) FCS, 2 mM L-glutamine, 2 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME, 1 mM sodium pyruvate, and 10 mM HEPES. IL-2 and IL-5 supernatant were prepared from cell lines transfected with the respective cDNA and grown in the above described medium (27). Phoenix-E cells were a kind gift from G. Nolan (Stanford University, Palo Alto, CA) and grown in DMEM supplemented as above. Primary B cells were purified from mouse spleens using a negative selection strategy; an Ab mixture consisting of FITC-coupled anti-CD4, 8,11b, and Thy-1.1 (Caltag Laboratories, Burlingame, CA) was bound to cells before separation using magnetic beads that bind FITC (Polysciences, Warrington, PA). Enriched B cells were stimulated with LPS (10 µg/ml) or anti-IgM F(ab')2 (Southern Biotechnology, Birmingham, AL) (5 µg/ml) for 24 h before retroviral transduction. Cells were sorted 2 days following transduction and processed for RNA isolation.

Plasmids, probes, and oligos

The mouse stem cell virus-based retroviral expression plasmid used for expression contains, flanked by long terminal repeats and packaging signals, a multiple cloning site-internal ribosomal entry site (IRES)-neomycin resistance or green fluorescent protein (GFP) gene cassette. N-terminal FLAG-tagged Blimp-1 was subcloned into these vectors. The {Delta} proline-rich region, {Delta} COOH terminus, {Delta} zinc finger 1/2, and {Delta} NH2 terminus constructs have been previously described (18). The PR* Blimp-1 mutant was made using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).

The probe for IgG2a was cloned from a PCR product specific for the C region (accession J00471) (Table I). The probes for the remaining Northern blot experiments were obtained from the I.M.A.G.E. consortium as cDNA clones and the inserts were excised, purified, and labeled before use as a probe (Table II).


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  		Table I. Oligos used in this study

 

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  		Table II. Probes used in this study

 
Retroviral transductions

Undiluted supernatants, supplemented with Polybrene (Sigma-Aldrich, St. Louis, MO) to 4 µg/ml, were used to spin infect cells at 2500 rpm for 1.5 h at 32°C at 0 and 24 h. Cells were washed, plated with fresh medium, and supplemented with Geneticin G418 to 0.8 mg/ml at 24 h (Invitrogen/Life Technologies, Carlsbad, CA). Phoenix-E packaging cells were transiently transfected using the calcium-phosphate precipitation method, and supernatants were harvested at 48 and 72 h posttransfection.

RNA and microarray hybridization, Northerns, and RT-PCR

Total RNA was isolated using TRIzol (Invitrogen/Life Technologies) and used to produce cRNA according to Affymetrix (Santa Clara, CA) protocols. Briefly, 8 µg of total RNA was used to make first strand cDNA, and the total mixture was used to convert into double-stranded cDNA. Following cleanup, the double-stranded cDNA was used to produce cRNA using T7 Megascript (Ambion, Austin, TX) and biotinylated nucleotides, Bio-11-CTP and Bio-16-UTP (Enzo Diagnostics, Farmingdale, NY). cRNA was fragmented, and 15 µg was submitted to Stanford’s Peptide and Nucleic Acid Facility for hybridization, washing, and scanning of Affymetrix MGU74v2 mouse genome arrays. This was accomplished using standard protocols provided by Affymetrix for the execution of the Affymetrix Fluidics Station and Agilent gene array scanner. Arrays were scanned following washing by detection of biotinylated hybridized nucleic acids with streptavidin-PE.

Total (15–20 µg) RNA was fractionated on formaldehyde-denaturing agarose gels and transferred to Hybond-XL (Amersham Pharmacia Biotech, Piscataway, NJ) nylon membranes, according to established protocols (28). Membranes were hybridized using 32P-labeled probes generated by Prime-It II random primer labeling kit (Stratagene). Blots were stripped before reprobing.

Total RNA from sorted, GFP-positive, stimulated, primary B cells was isolated and used to produce cDNA. PCR were performed using the SYBR green PCR kit (Qiagen, Valencia, CA) and the ICycler (Bio-Rad, Hercules, CA). A fixed titration of M12 cDNA was used for each amplicon (Table I) to calculate an arbitrary standard curve that plotted copy number against threshold cycle (Ct). The calculated copy numbers were then normalized against Gapdh.

Microarray analysis

Scanned microarray images were analyzed using Affymetrix 4.0 and 5.0 Analysis Suite software using default analysis parameters. For the triplicate M12 experimental set, Blimp-1, {Delta}P, {Delta}C, and PR* hybridization intensities were globally scaled to a factor of 2500 and compared with a baseline file, the control sample of that transduction group. The data was then exported into a Microsoft Excel spreadsheet for querying, filtering, and annotation. Blimp-1-induced genes were scored by: 1) being present in the Blimp-1 samples (P), 2) exhibiting a confidence of p >= 0.003 for differential expression, and 3) exhibiting an increase in intensity by a fold change of 1.5x, and 4) the intensity was greater than baseline. Blimp-1-repressed genes were scored by: 1) not being absent in the control hybridization (*), 2) exhibiting a confidence of p >= 0.997 for differential expression, and 3) exhibiting a decrease in intensity by a fold change of 1.5x, and 4) the intensity of the control hybridization was greater than baseline. For any gene to be included in the final set, it had to exceed these threshold criteria in one-half of the four replicates and then in two of the triplicate experiments. Fold changes reported are derived from the average change in all conditions across the three replicates. In the instances in which the paired t test revealed mutant-specific regulation, the fold change reported is derived from the average change in all conditions except for the mutant condition. A paired t test was used to identify genes that segregated with the different mutations of Blimp-1 and used the Affymetrix change p value for analysis.

All of the duplicate BCL1 time course samples, including replicate B of the unstimulated sample, were globally scaled to a factor of 2500 and compared with a baseline file, replicate A of the unstimulated samples. Similar querying parameters were applied to this data set. Clustering and graphical representation of the fold changes of differentially expressed genes were performed with Cluster and TreeView software (29).

ELISA, Western blots, and FACS analysis

A total of 2 x 105 cells was plated in 1 ml overnight before harvesting supernatants for IgG-specific sandwich ELISA. Quantities were computed by comparison with a standard mouse IgG (Sigma-Aldrich).

Nuclear lysates were prepared for Western blotting by lysing cells and isolating nuclei using a lysis buffer containing 12.5 mM Tris, pH 7.5, 12.5 mM KCl, 3.75 mM MgCl2, 0.5% Nonidet P-40 (v/v), 30% sucrose (v/v), and Complete Mini protease inhibitor mixture tablets (Roche, Basel, Switzerland). Nuclei were pelleted for 5 min at 4000 x g and then lysed in whole cell lysis buffer containing high salt: 1% Nonidet P-40 (v/v), 50 mM Tris, pH 7.4, 400 mM NaCl, and 5 mM EDTA. Protein content was quantitated using the protein assay kit (Bio-Rad), and 25 µg was run on SDS-PAGE before transfer to nitrocellulose membranes. Blots were probed with anti-Blimp-1 (12) and developed using HRP-coupled anti-rabbit (Amersham Pharmacia Biotech).

FACS analysis was performed by washing cells in staining buffer consisting of PBS supplemented with 1% BSA and 0.05% sodium azide. Cells were stained with fluorochrome-coupled anti-integrin {alpha}4 Abs (clone P/S2 or R1-2) for 30 min at 4°C and then washed before analysis by FACS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Blimp-1 regulates Ig secretion in a transient assay and at the level of 3' end processing

Cells and conditions were identified for the efficient induction of secreted Ig by ectopic Blimp-1 expression in a high throughput transient assay. A retrovirus-based expression system was used to express Blimp-1 and includes, 3' to Blimp-1, an IRES-aminoglycoside phosphotransferase gene for selection (neomycin selection). With this approach, increased Ig secretion is observed in BCL1, WEHI 231, M12, and 2PK-3 cell lines (data not shown). Of these, the most robust induction of secreted Ig was seen in M12 cells (>=10-fold), and thus these were chosen for further analysis (Fig. 1A). Northern analysis demonstrates that Blimp-1-positive cells show a dramatic increase in the ratio of secreted to membrane Ig mRNA isoforms, indicating that the regulation of Ig transcripts by Blimp-1 occurs at the transcriptional level (Fig. 1B). Interestingly, there is also an increase in total Ig mRNA, suggesting that in addition to the processing events that lead to the secreted transcripts, Blimp-1 also increases the abundance of Ig mRNA.



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  		FIGURE 1. Ig secretion is induced by Blimp-1 and occurs at the posttranscriptional level. A, ELISA-based quantitation of Ig is shown from duplicate experiments of Blimp-1 or control-transduced M12 cell supernatants. B, Northern analysis of M12 cells mock treated or transduced with control or Blimp-1 retrovirus and probed for IgG. Comparison of RNA isolated from cytoplasmic or nuclear fractions does not show differential processing.

 
Microarray analysis of Blimp-1-expressing cells

To define the nature of Blimp-1-driven B cell maturation, as well as the effects of Blimp-1 on secretion, we used Affymetrix microarray technology to define the expression changes induced by Blimp-1. We examined three types of Blimp-1-expressing B cells with the microarrays. First, Blimp-1 was expressed in the mature B lymphoma line, M12, as described above. Second, cytokine- and LPS-stimulated BCL1 cells were surveyed by microarrays to compare which genes Blimp-1 regulates in the context of different physiological stimuli that each lead to plasma cell differentiation in vivo. Analysis of ectopic Blimp-1 expression in BCL1 cells was not pursued because the effect of Blimp-1 on Ig secretion, although consistent, was never greater than 2-fold compared with controls. Affymetrix mouse genome U74 v2 microarrays (chips A, B, and C) that contain oligonucleotide probes for >36,000 target RNAs, as well as specificity and hybridization controls, were used.

The M12 lymphoma line. It was determined that at 75 h following transduction, ~40% of the M12 cells survive neomycin selection, and the Blimp-1 protein levels of the population approach those of cytokine-stimulated BCL1 cells (data not shown). This served as the earliest possible time point to assay the transcriptional changes induced by Blimp-1. Analysis of selected M12 cells indicates that Blimp-1-expressing cells grow moderately slower than controls and that a minority are annexin V positive (13%), indicating that M12 cells are tolerant to the toxic effects observed in other B cell lines (15, 18). Triplicate hybridizations were performed from both independent transductions and separate passages. Overall, ~30% of the genes on the arrays were scored as expressed by the Affymetrix analysis algorithm. Differentially expressed genes were identified by hybridization quality, consistency in at least two of three replicates, significance of differential expression of p <= 0.003, and expression level changes of >=1.5-fold between Blimp-1 and control-transduced cells. Although the fold change criterion is conservative, it is likely that differential expression is a consequence of the dose of ectopically expressed Blimp-1, and thus consistency between experiments was emphasized above a high-fold change threshold. Blimp-1 expression is absent in wild-type M12 cells, and this was confirmed by RT-PCR (data not shown). The microarray analysis also scored the expression of CIIta as absent; however, as predicted from the known functions of Blimp-1, appropriate induction of the Ig genes and repression of c-myc occurred.

Blimp-1 expression results in the differential expression of 409 genes and includes several noteworthy batteries of genes (Table III and Supplementary Web Fig. 1). These include those that have documented functions or are related in sequence to characterized genes, whereas unknown genes constitute uncharacterized expressed sequence tag clusters. Analysis of the differentially expressed genes yielded 158 known and 152 unknown induced genes, and 49 known and 50 unknown repressed genes. The large number of unannotated expressed sequence tag clusters suggests that aspects of the differentiation program remain to be characterized.


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  		Table III. Categories of genes regulated by Blimp-1 in M12 Cells; assessed by Affymetrix microarray analysisa

 
To substantiate the microarray data and the criteria imposed on the data sets, Northern blot analysis was performed for selected genes, and for comparison included RNA from two inducible B cell lines, BCL1 and CH12.LX, and from the plasmacytoma, MOPC315J (Fig. 2). In each case, Northern analysis qualitatively confirmed the microarray data. Even Bcl6 repression, which was calculated as 1.5-fold repressed by the Affymetrix algorithm, was readily detected by Northern analysis (Fig. 2B; compare hybridizations between empty vector- and Blimp-1-transduced samples in the Bcl6 blot from both experiments 1 and 2).



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  		FIGURE 2. Northern blot analysis of select Blimp-1-regulated genes. A–F, Comparison of RNA from separate M12 transductions (Expt. 1 or 2), MOPC plasmacytoma cells, and stimulated/unstimulated BCL1 and CH12.LX cells. The probes are indicated on the right of the blot.

 
To authenticate the effect of Blimp-1 under different conditions, we used real-time RT-PCR to measure the transcript levels for some of these up-regulated genes in primary B cells stimulated with LPS or LPS + anti-IgM F(ab')2, transducing the latter with control or Blimp-1-IRES-GFP retroviruses. This strategy is used to induce a population of cycling cells that fail to up-regulate endogenous Blimp-1 expression, providing a modality to examine the effects of ectopic Blimp-1 expression. The efficiency of transduction ranged from 10 to 15%, and following sorting of the GFP-positive cells, we found a 5- to 10-fold increase in IgM secretion (data not shown). Of 11 transcripts measured, 3 did not change and 8 increased in the presence of ectopic Blimp-1 (Fig. 3B). Interestingly, this pattern correlated with a change in transcript levels between LPS and LPS + anti-IgM F(ab')2, such that the eight transcripts that were increased also increased in LPS vs LPS + anti-IgM F(ab')2 and the three static genes also did not change in LPS vs LPS + anti-IgM F(ab')2 either. This is interesting because LPS induces endogenous Blimp-1 expression and activity and thus suggests that the regulation of the three static genes is subject to additional layers of regulation that are not influenced by Blimp-1 (endogenous or ectopic) during this stage of activation. Interestingly, these three genes are up-regulated in differentiated plasma cells (24), indicating that these changes are plasma cell specific, and, considering that two of these genes are up-regulated in both the M12 and BCL1 cells, perhaps are influenced by Blimp-1 at later points in the differentiation pathway. Interestingly, Ckip1, although repressed in Blimp-1-transduced M12 cells, is up-regulated in both LPS vs LPS + anti-IgM F(ab')2-treated primary cells and in Blimp-1-transduced LPS + anti-IgM F(ab')2 cells. Because this effect is Blimp-1 specific in each of these systems, it suggests that some genes are differentially required in different cellular contexts and that Blimp-1 transcriptional activity can be modulated depending on the nuclear milieu. Therefore, Blimp-1 up-regulates several plasma cell-specific genes in Ig-secreting and differentiating plasmablastic cells.1



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  		FIGURE 3. Genes induced by Blimp-1 in primary mouse B cells. A, Control or Blimp-1-expressing M12 and LPS + anti-IgM F(ab')2-stimulated primary cells were stained with integrin {alpha}4 Abs and analyzed by flow cytometry. B, Real-time PCR-computed Ct values of different transcript levels between LPS, LPS + anti-IgM F(ab')2, and LPS + anti-IgM F(ab')2 transduced with control, or Blimp-1 retroviruses. Increased fold change is shown as computed from normalized Ct values. Ct values were normalized to GAPDH using a standard curve, and the average from duplicate reactions is shown; the SD is shown in a smaller font. Data from two independent experiments are shown in which percentage of GFP-positive cells (15 and 6%) and fold induction of IgM secretion (5x and 9x) were measured in each. Asterisks denote measurements in which the SD is too great to compute a fold change.

 
Microarray analysis of inducible BCL1 differentiation. The M12 data set was compared with microarray data generated by analyzing BCL1 cells stimulated with the cytokines IL-2 and IL-5 or by the mitogen LPS, to induce Blimp-1 expression, differentiate, and secrete Ig. Blimp-1 expression is synergistically induced by IL-2 and IL-5 or by LPS, and both lead to sustained protein expression that peaks at 12 h following stimulation (data not shown). Therefore, a time course analysis of gene expression was performed following stimulation and resulted in the differential expression (1.5-fold) of ~1450 probe sets compared with the unstimulated samples. In addition to a sizeable mitogenic program of gene expression, many genes with roles associated with the physiological changes that accompany BCL1 differentiation are regulated.

As expected, Blimp-1, J chain, and Ig were appropriately induced, and CIIta was repressed. Expression of c-myc was stabilized at the first time point at 12 h postcytokine stimulation, as previously described (13). Approximately 28% of the Blimp-1-regulated genes in the M12 expression experiments are differentially regulated by cytokine- or LPS-driven BCL1 differentiation (Fig. 4). The majority of these genes fit the expectation of being regulated at peak Blimp-1 protein expression (12 h), coregulated by LPS, and maintained during the late phase of differentiation (48 h); however, a few do not (Fig. 4). These outliers suggest that those genes are subject to additional levels of differentiation-specific regulation. Interestingly, c-myc is induced by LPS at 12 h, suggesting that Blimp-1 is not a dominant repressor of this locus. As these genes are regulated in both systems, the nature of coregulators and/or the nature of promoter occupancy in these cells may be influencing the transcriptional output.



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  		FIGURE 4. Control of Blimp-1-regulated genes by IL-2/5 or LPS during 48 h of BCL1 differentiation. TreeView analysis displays genes (y-axis) with up-regulated, down-regulated, or unchanged genes color coded as red, green, or black, respectively. The expression change of each gene is represented by the ratio of gene intensity in the stimulated samples over unstimulated sample (replicate A). The x-axis denotes replicate and time following stimulation. LPS responses were interrogated only at the 12-h time point; this represents the peak of Blimp-1 expression for LPS and IL-2/5. The degree of color represents a scale between –8.5- and 34.6-fold changes.

 
Notwithstanding the differences in experimental manipulation, lineage, and activation status of the Blimp-1-expressing M12 and BCL1 cell lines, which most likely contribute significantly to gene expression patterns, we expected to observe a greater overlap in the gene expression changes upon Blimp-1-induced maturation. Interestingly, Northern analysis suggests that detection sensitivity or processing of the microarray data may have contributed to the underrepresentation of Blimp-1-regulated genes in the BCL1 differentiation experiments. For instance, Spi-B and Aicda are not detected on the M12 and BCL1 microarrays, respectively. However, when assayed by Northern analysis, both are detected in both cell lines and are repressed by Blimp-1 (Fig. 2, E and B, respectively). Consistent with the lineage differences between the two cell lines, BCL1 cells expressed the key germinal center genes Bcl6 or Aicda at absent to low levels, respectively. Therefore, the calculation of a 28% overlap between the two data sets is an underestimate.

Functional characterization of Blimp-1-dependent gene expression Genes were categorized into groups of similar cellular activities (Table III); however, cohorts of genes could be further categorized into groups representing operative pathways in maturation (denoted in Table III with numerals 1–4). These include B cell response and affinity maturation, migration and adhesion, stress and secretion, and transcription and differentiation factors.

B cell response and affinity maturation. As denoted in Table III, key germinal center and modifier genes of B cell activation are regulated (Aicda, Spi-B, Swap-70, Bcl6, Taci, Irf4). Blimp-1 expression in B cells is induced following B cell activation and persists in the terminally differentiated state. As it is anticipated that Blimp-1 would act to initiate a new gene expression program for B cell maturation, it is striking to see in this study that it also acts to repress major germinal center genes, suggesting that cessation of the previous phase of differentiation is coordinated by a single factor during maturation. Alternatively, Blimp-1 repression of these genes may explain the lack of affinity maturation in T-independent B cell responses, which also induce Blimp-1 expression. In addition, Blimp-1 represses Taci expression, an important negative regulator of B cell maturation, suggesting that Blimp-1 regulates B cell clonal expansion in extrafollicular foci and high-level secretion as plasmablasts (30, 31).

Migration and adhesion. Whereas most plasmablasts are short-lived, a few differentiate into long-lived plasma cells and migrate to specialized niches in the bone marrow using the chemokine receptor Cxcr4 and the integrin receptor Vla4 (32, 33, 34, 35). Whereas Cxcr4 is up-regulated by Blimp-1 (Table III and Fig. 3B), the probe set representing integrin {alpha}4 increased in intensity in some of the experiments. Therefore, we analyzed integrin {alpha}4 protein expression by flow cytometry following expression of Blimp-1 in M12 and primary B cells and confirmed that expression is increased vs control cells (Fig. 3A). In addition, it has been shown that plamablast survival in extrafollicular foci is enhanced by interactions with dendritic cells, suggesting that the observed regulation of adhesion (Cd23, Cd83, Cd166) and cytoskeletal factors (Cbp) may be involved in this process (36). Also interesting is the up-regulation of Reelin (Figs. 2b and 3b). This extracellular matrix protein plays a pivotal role in neuronal cell migration, as shown by Reeler mice, a spontaneous mutant mouse line that suffers from ataxia. Recently, it was found that Reelin is induced in murine plasma cells, suggesting it has a specialized function in Ab-secreting cells (24).

Stress and secretion. Previously, it was observed that ectopic expression of Blimp-1 caused cell cycle arrest and apoptosis, a phenotype most likely downstream of c-myc repression. Indeed, a large set of genes measured as differentially expressed by Blimp-1 are genes apparently involved in stress responses (Table III). However, the observation that Chop10, heat shock protein 70, and other ER-resident processing genes (Table III) are also up-regulated, coupled with the induction of Ig secretion, suggests a role for Blimp-1 and ER stress in facilitating the unfolded protein response (UPR), which is necessary for Ig secretion (37). In fact, Xbp1, a critical UPR transcription factor, is necessary for Ig secretion (38). However, UPR-dependent regulation of Xbp1 splicing is not regulated by Blimp-1 in M12 cells, suggesting a parallel pathway with Xbp1 and the elaboration of a nonclassical UPR (our unpublished data).

Transcription and differentiation factors. A number of transcription factors and mRNA processing factors are also regulated by Blimp-1, suggesting mechanisms by which execution of secondary and tertiary phases of the differentiation program is propagated (Table III). Interestingly, the homeodomain transcription factor Pax5, a B cell lineage identity factor, is not repressed in Blimp-1-expressing M12 cells (Fig. 2E) despite being repressed in cytokine-induced BCL1 cells and other Blimp-1 expression systems (39).

The PR domain of Blimp-1 is required for Ig secretion

The shift from the membrane to the secreted form of the Ig H chain mRNAs is a hallmark of the plasma cell stage of differentiation and is necessary for Ig secretion. It is likely that Blimp-1 is regulating the expression of a gene involved in this switch because expression of a Blimp-1 mutant lacking the first two zinc fingers involved in DNA binding fails to induce the secretory mRNA isoform (Fig. 5A). Furthermore, we and others have shown that a dominant-negative form of Blimp-1, consisting of the minimal DNA-binding zinc finger domain, is sufficient to block cytokine-induced Ig secretion (15, 18). To characterize the relationship of Blimp-1 and Ig secretion, we tested a series of deletion mutants (Fig. 5A). Although the constructs in which an internal portion of the proline-rich region ({Delta}Pro) was deleted (69 aa) or another with the deletion (112 aa) of the C terminus ({Delta}C)-induced Ig secretion, a construct lacking 181 aa of the N terminus ({Delta}N) did not. As the deleted region includes a significant portion of the novel PR domain (21), two highly conserved adjacent residues were changed to proline (N189P and W190P) to introduce a kink in the structure and to inactivate the domain. Functionally, this mutant (PR*) fails to induce Ig secretion despite producing levels of Blimp-1 equivalent to the wild-type construct (Fig. 5, B and C). Thus, the ability of Blimp-1 to induce Ig secretion is dependent on a region with homology to the SET family of methyltransferases.



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  		FIGURE 5. Blimp-1-induced processing of Ig RNA. Blimp-1 induction of 3' end processing of Ig mRNA requires new gene expression and a novel effector region that is related to the SET methyltransferase-chromatin remodeling domain. A, Depicts Ig ELISA results from supernatants of the indicated transductants. B, Shows a Northern blot comparing the ability of wild-type and various mutant Blimp-1-expressing cells to induce the secretory Ig isoform. C, Western blot probed for Blimp-1 showing the expression efficiency of the various Blimp-1 constructs.

 
Genetic dissection of Blimp-1-induced B cell maturation

The microarray experiments indicate that Blimp-1 affects a variety of transcriptional pathways that both induce and repress gene expression at multiple loci. Thus, this series of Blimp-1 mutants, combined with microarray hybridization, provides an opportunity to discover unique genetic pathways dependent on discrete regions of Blimp-1. Interestingly, when the PR* mutant is compared with wild-type Blimp-1, two genes (Rgs2 and Taci) surveyed by Northern analysis are regulated by the PR* mutant as efficiently as wild-type Blimp-1, despite its deficiency in regulating Ig secretion (Fig. 2, A and B) and suggesting that this module operates independently. Therefore, the PR*, {Delta}C, and {Delta}Pro mutants were expressed in M12 cells, and their mRNA expression profiles were compared using microarrays. Strikingly, the PR* and {Delta}C samples exhibit unique expression signatures compared with wild-type Blimp-1 when analyzed using a paired t test, p <= 0.005 (Fig. 6). Specifically, this analysis demonstrates that the PR and C terminus regions of Blimp-1 are each essential for regulating distinct sets of genes.



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  		FIGURE 6. Gene-specific, modular regulation by Blimp-1. A, Cluster analysis of 409 Blimp-1-regulated genes is shown. TreeView depicts gene intensity ratios to control transductants on the y-axis with induced, repressed, and unchanging genes represented by shades of red, green, and black, respectively. Samples shown on the x-axis include three replicates of Blimp-1 and mutant conditions ({Delta}C, {Delta}Pro, PR*). Genes that display PR region (B)- or C-terminal region (C)-dependent regulation are shown. Value of p from the paired t test is shown for Blimp-1-regulated genes that were uniquely regulated by the mutant constructs.

 
Specifically:

PR*. A paired t test finds 33 genes that fail to be regulated by the PR* mutant compared with wild-type, {Delta}C, and {Delta}Pro constructs. The remaining 376 genes are appropriately regulated, indicating that the overall structure and functionality of Blimp-1 have not been disrupted. Two genes, Ell2 and Reelin, were confirmed by Northern analysis (Fig. 2, A and B) and are shown graphically in Fig. 6. Interestingly, with only two exceptions, the PR region-dependent genes are all normally up-regulated by wild-type Blimp-1.

{Delta}C. Similar to the case of the PR*, the {Delta}C mutant is deficient in regulating a unique set of 12 genes. In contrast to PR*, the {Delta}C mutant shows no bias toward up- or down-regulated genes. Included in this set is Taci, an important gene involved in B cell regulation (Figs. 2c and 6).

{Delta}Pro. This internal deletion mutant was first used by Messika et al. (19), who found that it failed to induce cell cycle arrest and apoptosis in immature B cell lines. Subsequently, Ren et al. (19) determined that this region associates with members of the Groucho family of corepressors. Despite these findings, the microarray signature of the {Delta}Pro mutant is indistinguishable from wild-type Blimp-1. Our {Delta}Pro construct might be a hypomorphic allele, as the Groucho interaction site is only partially eliminated by our construct. Therefore, despite having an effect on immature B cells, the proline-rich region of Blimp-1 appears dispensable in M12 cells.

Use of microarrays to explore the genetic basis of Blimp-1 gene regulation was critical to identifying these novel functional regions. Furthermore, this analysis shows that the PR region and the C terminus operate semiautonomously, in that they are dependent on the Blimp-1 structure and yet they do not cooperate with each other, as demonstrated by the mutually exclusive set of downstream genes. This suggests that Blimp-1 operates differently at diverse loci, perhaps by recruiting distinct coregulators or by facilitating unique combinatorial interactions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The timing of Blimp-1 expression during B cell maturation, as well as its ability to repress c-myc and to induce Ig secretion, has long suggested that it is a master regulator of plasma cell maturation. Global expression profiling confirms this hypothesis, as the data presented in this study and elsewhere (15) show that hundreds of genes are responsive to Blimp-1 expression. In general, these are genes involved in the modulation of B cell functions, migration and adhesion, and Ig secretion (both at the transcriptional and posttranslational levels). Furthermore, we have found several genes regulated by Blimp-1 in M12 cells that represent previously unknown contributions to plasmablast regulation (Taci), homing (Reelin, Nude1, Lis, Lsp1), adhesion (Cd83, Cd166), and differentiation (Ell2, Ariadne2, Arkadia). Second, the observation that discrete portions of Blimp-1 direct specific outcomes of the maturation pathway suggests a complexity of differentiation as well as the novel ability of this factor to integrate distinct transcriptional pathways.

Recently, a study analyzing the gene expression profile of Blimp-1-induced B cell maturation in human cells describes many of the same genetic pathways seen in this study, such as repression of affinity maturation, and induction of stress and cell cycle control (15). However, a number of important differences should be noted. First, the ratio of induced to repressed genes is dramatically different, with Shaffer et al. (15) finding an overwhelming repressive effect of Blimp-1 (only 15% induced genes), whereas in the data described in this work using murine cells, the majority (75%) of the genes are induced. Partly due to this fact, only ~20% of the differentially expressed genes are shared by the two studies. To be sure that the genes we have identified in this study are expressed in the normal B cells transitioning to plasma cells, we assayed a subset (11) of our induced genes in Blimp-1-transduced stimulated primary B cells and found that they were up-regulated in that cellular context as well (Fig. 3B). Second, numerous Blimp-1-regulated genes identified in M12 cells were also found in differentiating BCL1 cells (Fig. 4). Additionally, recent microarray studies of Underhill et al. (24) reveal that many of the Blimp-1-regulated genes identified in M12 cells comprise the gene expression profile of plasma cells. Thus, approximately half (210) of the Blimp-1 genes that we discuss in this work were analyzed in that study, and of these, 91 show plasma cell-specific expression (Supplementary Web Fig. 2). The remaining genes include 25 that appear to be expressed only in M12 cells, 39 that are expressed by the primary B cells but not differentially expressed, and 54 that are differentially expressed in a different direction. Due to experimental design, the remaining Blimp-1 genes were not surveyed by Underhill et al. (24). Overall, we find differentiation-specific expression of 127 genes, identified in our M12 Blimp-1 expression experiments, using independent B cell maturation systems. Notably, the majority of these genes are induced in untransformed plasma cells, indicating that the Blimp-1 genetic program is not strictly repressive in nature.

As most of these genes were not scored as regulated in the Shaffer et al. (15) study, there are very significant differences between these two studies that need to be taken into account in assessing the role of Blimp-1 in B cell maturation. One likely answer to this question is that all of the cells studied in this work were robust in their secretion of Ig, a hallmark of plasma cell induction, whereas the cells used by Shaffer et al. (15) do not secrete Ig in response to Blimp-1 expression. Consistent with this interpretation is that in addition to the induction of the secreted isoform of Ig mRNA, we see many genes involved in vesicular transport and ER function induced in Blimp-1-expressing M12 cells, not revealed in the Shafer et al. study (15). In contrast, a number of B cell-specific genes are not differentially expressed in this study as compared with Shaffer et al. (15). In fact, Shaffer et al. report that a number of B cell-specific genes are reduced in expression, possibly a consequence of observing repression of the B cell-specific transcription factors Ebf and Pax5 (15, 39). Although Pax5 is repressed during cytokine-mediated BCL1 differentiation, ectopic expression of Blimp-1 in M12 cells does not result in Pax5 repression (Fig. 2E), suggesting that repression at the Pax5 locus is a complicated process that may be combinatorially dependent on Blimp-1 and the nuclear milieu. Finally, this study shows the regulation of genes known to be important for the maturation and homing of plasmablasts (Irf4, Taci, Cxcr4, Vla4) that are not identified by Shaffer et al. (15).

How could these results vary so widely? Although some of these differences are most likely due to the block in Ig secretion in the cells used by Shafer et al., others may be the result of differences in the transformation status of the cell lines used (the human B cell lines used by Shaffer et al. (15) are EBV transformed, whereas the murine BCL1 and M12 lymphomas arose spontaneously), the amount of Blimp-1 produced in the two transduction systems, or differences in the microarray platforms. In fact, Shaffer et al. chose not to report on the nature of unannotated transcripts that they observe to be regulated by Blimp-1 (15). This is significant because 50% of the genes in this report are of that category, precluding a complete comparison. Also possible is that this heterogeneity reflects something fundamental about the plasticity of the different gene sets affected by Blimp-1 expression. This interpretation is supported by the analysis of Blimp-1-regulated genes, identified in the M12 cells, during BCL1 differentiation. In this regard, we observe temporal and stimulus-specific dynamics of gene expression that suggest that different aspects of the maturational program can be modified by lineage- or stimulus-specific contexts. In addition, 54 (of the 210) genes that are differentially expressed in both the M12 and normal plasma cell experiments performed by Underhill et al. (24) exhibit differences in directionality, suggesting that stage-specific regulation can modify the differentiation program. Indeed, the normal plasma cells are Ig secreting, noncycling, and fully differentiated as compared with the Ig-secreting and -cycling plasmablasts cells used in the M12, BCL1, and Shaffer et al. (15) experiments. Finally, the plasma cells analyzed by Underhill et al. (24) also have many of the gene expression characteristics observed by Shaffer et al. (15), supporting a model in which both the genes we describe in this work and those reported by Shaffer et al. represent different aspects of normal plasma cell maturation.

The differentially expressed genes common to multiple settings of Blimp-1 expression and action are candidates for being direct targets of this molecule. In fact, one of these is SpiB, which Shaffer et al. have shown to be directly repressed (15). Also interesting is the fact that a host of transcription factors is regulated by Blimp-1, including Bcl6, Irf4, and SpiB, with roles in B cell maturation, indicating that secondary transcriptional pathways are being triggered (40, 41, 42).

Ig mRNA processing

The mechanism of how a B cell shifts from membrane to secreted Ig mRNA isoforms during maturation remains unknown. However, the data presented in this work demonstrate that Blimp-1 transcriptional activity plays an important role in this shift, as a mutant lacking the DNA binding domain fails to induce Ig secretion (Fig. 5A). Second, we also show that the PR region of Blimp-1 is necessary for the production of secreted Ig mRNA. The polyadenylation factor CstF64 has previously been implicated in regulating Ig mRNA processing (43). However, analysis of Blimp-1-induced M12 cells or stimulated BCL1 cells shows that it is not differentially expressed (data not shown). As CstF64 is not posttranslationally modified in Ig-secreting cells (44), and yet has been shown to be genetically essential for secretory Ig mRNA processing, it seems likely that Blimp-1 is instead regulating the transcription of a gene that functions to localize or augment the activity of CstF64.

Modular nature of Blimp-1 transcriptional regulation

In this study, we delineate two regions of Blimp-1 that have a unique impact on gene regulation, the PR and C-terminal regions. Discrete sets of genes are sensitive to mutations in either of the domains, indicating that separate, semiautonomous regulatory pathways are operating at different loci. This observation is internally controlled in this study, as the microarrays simultaneously measured many hundreds of genes that are not affected by the mutations, indicating that expression level, stability, DNA binding, or effectiveness of the different forms of Blimp-1 are not responsible for selective gene regulation (Fig. 6). Furthermore, as mutations in these domains do not affect all differentially expressed genes, it is likely that other regions of Blimp-1 contribute to gene regulation (17, 19, 20).

The PR domain was first identified through sequence comparisons as a highly homologous region in the 5' region of two zinc finger transcription factors, Blimp-1 (PRDI-BF1) and RIZ, and represents a growing family (45, 46). Strikingly, sequence similarities indicate that this collection of PR regions comprises a subfamily of SET domains that regulate gene expression by inducing chromatin remodeling (21, 47). In fact, SET domains appear to function as lysine methyltransferases specific for histone tails (48), suggesting a scenario in which Blimp-1 affects expression, through methylation, of a gene or genes necessary for Ig mRNA processing and plasma cell differentiation.

About half of the PR region-specific genes are in or subject to the secretory pathway, the extensive development of which is a hallmark of plasma cells. However, in contrast to Ig secretion, two of the PR domain-sensitive genes, Ell2 and Reelin, do not display complex banding patterns on Northern blots, ruling out the regulation of all PR domain-dependent genes by alternative 3' end processing (Fig. 2, A and B, respectively). The differential regulation of Ell2 is the only known transcription factor sensitive to the PR* mutant, suggesting that regulation of the PR set of genes is dictated by Blimp-1 and/or Ell2 (or combinations thereof). The capacity of Blimp-1 to induce Ig secretion maps genetically to both the DNA binding and PR regions, indicating that PR genes are candidates for mediating this event. Because the switch in Ig secretion occurs at the transcriptional level (Fig. 1B), we tested the ability of Ell2 to induce Ig secretion by enforced expression in M12 cells and found that it was not sufficient (data not shown), indicating that one (or combination) of the remaining PR region genes is responsible. Finally, it is interesting that the majority (31 of 33) of genes affected by the PR* mutant are normally induced by wild-type Blimp-1. Perhaps, Blimp-1 PR region-dependent methylation of target proteins induces expression or stability of this subset of genes involved in the maturation program.

With respect to the C terminus mutation, we find that two important modulators of B cell activation (Blnk-s and Taci) are affected, suggesting that this transcriptional pathway regulates genes involved in plasmablast homeostasis. Homology-based searches (basic local alignment search tool) of the C terminus of Blimp-1 do not reveal any similarities with domains or proteins of known function. The most prominent feature is an enrichment of acidic residues characteristic of transcriptional activation domains. Interestingly, the ratio of repressed to induced genes that are subject to C terminus regulation is inversely related to that seen in the total set of Blimp-1 genes, suggesting that, in contrast to the PR domain, this region is mainly involved in repression. Recent data have shown that Blimp-1 interacts and cooperates with the Sp1 family in transcriptional regulation and that the C-terminal domain is a vital component of this process (J. Chi and M. Davis, in preparation). Therefore, a transcriptional complex consisting of Blimp-1 and Sp1 family members may regulate C-terminal region-dependent genes.

Modular domains can synergize with each other to coordinate one function, such as Sp1 (49), or they can act semiautonomously in separate pathways, such as the transcription factor Rest/Nrsf, which has been shown to regulate a few distinct genes via the N or C terminus (50). Perhaps, segregating functions through modular organization may be a feature of proteins operating at important regulatory intersections, such as Blimp-1.

In conclusion, analysis of the Blimp-1-induced transcriptome strongly supports the hypothesis that Blimp-1 is a master regulatory gene that influences many of the attributes of plasma cells. In addition, the many genes that have emerged from this analysis, particularly the unexpected ones, significantly add to our view of this important maturational step and its genetic control. Furthermore, the studies presented in this work together with those of Shafer et al. (15) highlight the wide variability in gene expression that can be seen in different cellular or stimulatory contexts and the need for caution in interpreting the results in any one system. The biological meaning of this diversity in gene expression may be especially relevant as distinct types of Ab-secreting cells reside in different tissues and express different surface markers (51, 52). Another important point is the finding of a surprising modularity in the regulatory ability of Blimp-1, with at least two different regions, the PR domain and the C terminus, governing small, but distinct subsets of genes. The scale of this effect has not previously been seen in transcription factors, and it suggests that some of the complexity involved in the regulation of so many genes is at least partially delegated to specific parts of the same molecule.


   Acknowledgments
 
We thank J.-T. Chi, J. Huppa, M. Kuhns, I. Olave, D. Ron, H. Singh, and L. Wu for many useful discussions and/or for comments on the manuscript. We especially thank R. Glynne for advice regarding analysis of the microarray data. We are grateful to Elizabeth Zuo and the Peptide and Nucleic Acid Facility at Stanford for their expert Affymetrix GeneChip probe array sample hybridization and image acquisition. We thank Stephanie Wheaton for expert secretarial assistance.


   Footnotes
 
1 R.S. was a Basso/Garavano Fellow of the Leukemia and Lymphoma Society, and M.M.D. is an investigator of the Howard Hughes Medical Institute. This work was supported by grants to M.M.D. from the National Institutes of Health and the Howard Hughes Medical Institute. Back

2 Address correspondence and reprint requests to Dr. Roger Sciammas at the current address: Department of Molecular Genetics and Cell Biology, MC1028, N112, 5841 S. Maryland Avenue, Chicago, IL 60637. E-mail address: rsciamma{at}uchicago.edu Back

3 Abbreviations used in this paper: BCL1, B cell line; Ct, threshold cycle; ER, endoplasmic reticulum; GFP, green fluorescence protein; IRES, internal ribosomal entry site; PR, PRD1-BF1 and R1Z region of similarity; SET, Su(var)39-1, Enhancer of Zeste, Trithorax; UPR, unfolded protein response. Back

Received for publication December 3, 2003. Accepted for publication February 19, 2004.


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