Immunity, Vol. 15, 545–555, October, 2001, Copyright 2001 by Cell Press

PU.1/Spi-B Regulation of c-rel Is Essentialfor Mature B Cell Survival

of genes. Ets family members are transcription factorsthat mediate binding to a purine-rich GGAA/T core se-quence and are characterized by a winged helix-loop-

Cheng-Jun Hu,1,2 Sridhar Rao,3

Diana L. Ramirez-Bergeron,1,2 Lee Ann Garrett-Sinha,4

Steve Gerondakis,5 Marcus R. Clark,4

helix DNA binding domain (Gunther et al., 1990; Kodan-and M. Celeste Simon1,2,6

dapani et al., 1996; Macleod et al., 1992; Nye et al.,1 Abramson Family Cancer Research Institute1992). Currently, there are more than 25 members of2 Howard Hughes Medical Institutethis family.University of Pennsylvania School of Medicine

PU.1, Spi-B, and Spi-C constitute an Ets subfamily,Philadelphia, Pennsylvania 19104exhibiting structural similarity in several domains such3 Department of Pathologyas their DNA binding and PEST domains (Bemark et al.,4 Department of Medicine1999). The 90% similarity in the DNA binding domain ofUniversity of ChicagoPU.1 and Spi-B make them indistinguishable in termsChicago, Illinois 60637of DNA binding specificity (Rao et al., 1999b). PU.1 is5 The Walter and Eliza Hall Institute of Medical Researchexpressed in most hematopoietic cells, including B lym-The Royal Melbourne Hospitalphocytes, monocytes, granulocytes, megakaryocytes,Parkville, Victoria 3050mast cells, and immature erythroid cells (Hromas et al.,Australia1993; Klemsz et al., 1990; Ray et al., 1992). We demon-strated the essential role of PU.1 in hematopoiesis bygenerating PU.1�/� mice that die in utero and produceSummaryno lymphoid or myeloid cells (Scott et al., 1994). In con-trast to PU.1, Spi-B expression is limited to lymphoidPU.1�/�Spi-B�/� mice exhibit reduced numbers of im-cells; high levels are detected in B cells, and low levelsmature and mature B lymphocytes, which exhibit se-are found in T cells (Chen et al., 1995; Su et al., 1996).vere defects in response to BCR-mediated stimulationInterestingly, expression of Spi-B in B lymphocytes isand poor survival. We found that expression of c-rel,developmental stage dependent, with high levels in im-a member of the Rel/NF-�B family, is dramatically re-mature and mature B cells and low levels in pro-B andduced in PU.1�/�Spi-B�/� splenic B cells. Analysis ofpre-B cells (Anderson et al., 1999; Su et al., 1996). Thisthe murine c-rel promoter identified three PU.1/Spi-Bexpression pattern suggests that Spi-B plays a role inbinding sites critical for c-rel promoter activity. Further-B cell function rather than B cell development. Indeed,more, reintroduction of Rel protein restored wild-typeSpi-B�/� mice produce normal B cell numbers, butB cell numbers to mice reconstituted with PU.1�/�Spi-Spi-B�/� B cells proliferate poorly and die in response toB�/� bone marrow. These findings are the first to dem-BCR crosslinking in vitro (Su et al., 1997). Furthermore,onstrate that a member of the Rel/NF-�B family isimmunized Spi-B�/� mice form smaller splenic germinaldirectly regulated by Ets proteins and dissect the mo-centers with increased numbers of apoptotic B cellslecular basis for the function of two Ets factors, PU.1(Su et al., 1997). Given that Spi-B�/� mice exhibit suchand Spi-B, in promoting B lymphocyte survival.B cell defects, Spi-B must possess a unique functionnot compensated by wild-type levels of PU.1 in lympho-Introductioncytes. However, PU.1 likely masks certain phenotypesin Spi-B�/� mice, due to their identical DNA binding spec-Normal B cell development and response to antigenicificity. Analysis of the role of PU.1 in mature B cells is

stimulation depend on signaling through the B cell anti-prevented by the complete lack of lymphocytes in

gen receptor (BCR) (Kurosaki, 1999). BCR-mediated sig-PU.1�/� mice. In an effort to study PU.1 function in

naling pathways have been defined in substantial detail. B lymphocytes, we crossed PU.1�/� with Spi-B�/� mice toHowever, critical transcription factors and downstream generate PU.1�/�Spi-B�/� mice. Of note, PU.1�/�Spi-B�/�

genes that regulate later stages of B cell differentiation, mice exhibit novel phenotypes with reduced numbersproliferation in response to stimulation, isotype switch- of B220�/IgM� immature and IgM�/IgD� mature B cellsing, and/or antibody secretion remain unclear. Neverthe- in bone marrow, spleen, and lymph nodes (Garrett-Sinhaless, two groups of transcription factors, Rel/NF-�B and et al., 1999). In addition, these B cell functional defectsEts proteins, have been implicated in this process are dramatically more severe than those of Spi-B�/�

(Glimcher and Singh, 1999). Rel/NF-�B family members B lymphocytes in vivo and in vitro (Garrett-Sinha et al.,(Rel, RelA, RelB, NF-�B1, and NF-�B2 in mammals) 1999). These data indicate that both PU.1 and Spi-B areshare a common N-terminal domain (Rel homology do- important for mature B cell function.main) that is important for DNA binding, dimerization, PU.1 has been shown to regulate many genes thatand nuclear import (Baldwin, 1996). While Rel, RelA, and are important for B cell development and BCR-mediatedRelB contain C-terminal transactivation domains, NF-�B1 signaling. These include Ig � heavy chain (Nelsen et al.,and NF-�B2 lack the transactivation activity. These indi- 1993), Ig J chain (Shin and Koshland, 1993), Ig lightvidual proteins interact with each other to form homo- chains (� and �) (Eisenbeis et al., 1993; Pongubala andand heterodimers that control transcription of a variety Atchison, 1991; Pongubala et al., 1992), mb-1 (Ig�) (Hag-

man and Grosschedl, 1992), B29 (Ig�) (Omori and Wall,1993), Btk (Himmelmann et al., 1996), LSP1 (Omori et6 Correspondence: celeste2@mail.med.upenn.edu

Immunity546

al., 1997), and CD72 (Ying et al., 1998). Given that Spi-Bbinds to PU.1 sites in all target genes examined thusfar, Spi-B presumably also regulates PU.1 target genesin B cells. Therefore, mutation of Spi-B in combinationwith reduced levels of PU.1 might decrease expressionof such B cell targets. However, the PU.1/Spi-B tar-get genes listed above are expressed normally inPU.1�/�Spi-B�/� B cells (Garrett-Sinha et al., 1999), indi-cating that one or more novel PU.1/Spi-B target gene(s)may be downregulated in PU.1�/�Spi-B�/� mice. Analysisof downstream BCR signaling pathway effectors foundreduced phosphorylation of BLNK and PLC�, but normalsyk activity, suggesting that a novel factor couplingsyk to downstream components is misexpressed inPU.1�/�Spi-B�/� B cells (Garrett-Sinha et al., 1999).

To identify novel PU.1/Spi-B target genes in B lympho-cytes, we performed a subtractive hybridization usingRNA prepared from purified splenic PU.1�/�Spi-B�/�

and PU.1�/�Spi-B�/� B cells (Rao et al., 1999a). We reporthere that c-rel, a Rel/NF-�B family member, is dramati-cally downregulated in mutant B cells. Furthermore,analysis of the murine c-rel promoter identified threePU.1/Spi-B sites that are important for c-rel promoteractivity in B cells. Both PU.1 and Spi-B transactivatethe c-rel promoter. These results strongly suggest thatc-rel is a direct target of both PU.1 and Spi-B. Reintro-duction of Rel protein restores wild-type B cell numbersto mice reconstituted with PU.1�/�Spi-B�/� bone mar-row, indicating that misexpression of Rel is largely re-sponsible for the B cell survival defect detected inPU.1�/�Spi-B�/� mice. Collectively, these findings estab-

Figure 1. Northern Blot Analysis of 3H12 in Splenic B Cells andlish that PU.1, Spi-B, and Rel are critical for regulatingHematopoietic Cell Lines

immature and mature B cell survival.(A) Northern blot analysis of 3H12 expression in purified splenic B cells(more than 90% B220� based on flow cytometry) from PU.1�/�

Results Spi-B�/�, PU.1�/�Spi-B�/�, PU.1�/�Spi-B�/�, and PU.1�/�Spi-B�/�

mice. The same blot was then stripped and reprobed with an actinprobe for RNA loading control. The 3H12 expression level is shownClone 3H12 Is Underexpressed in PU.1�/�Spi-B�/�

as a percentage of wild-type cells after normalization for RNA load-Splenic B Cells and Encodes c-rel, a Membering. Similar results were obtained from multiple mice.of the Rel/NF-�B Family(B) Expression of 3H12 in murine hematopoietic cell lines using

To identify novel PU.1/Spi-B target gene(s) in B lym- Northern blot analysis of total RNA prepared from pro-B (NFS 70 c/phocytes, we performed a PCR-based subtractive 10), pre-B (70 z/3 and 38 B9), immature B (WeHi 231), mature B

(A20), mature T (RMA), immature T (EL-4), macrophage (J774.1),hybridization using mRNA prepared from purifiederythroid (MEL), and fibroblast (3T3) cells. 28S rRNA is shown as awild-type and PU.1�/�Spi-B�/� splenic B cells as de-loading control.scribed previously (Rao et al., 1999a). Three PCR frag-

ments corresponding to genes underexpressed inPU.1�/�Spi-B�/� B cells were confirmed via Northern blot

correlating well with the expression of PU.1 and Spi-Banalysis of splenic B cell RNA from multiple mice (n (see Discussion). To determine the identity of 3H12, a4 for each genotype). One gene, P2Y10, a mouse hetero-320-bp cDNA obtained from our subtractive hybridiza-trimeric G protein-coupled heptahelical receptor, hastion studies was used to probe a mouse splenocytebeen previously described (Rao et al., 1999a). AnothercDNA library, resulting in two overlapping clones (1.7clone, “3H12”, was also expressed at low levels inand 2.1 kb). Combination of the data from these 1.7-kbPU.1�/�Spi-B�/� splenic B cells. As shown in Figure 1A,and 2.1-kb clones with cDNAs generated by 3 and 53H12 was downregulated in splenic B cells obtainedRACE determined that 3H12 was identical to mousefrom both PU.1�/�Spi-B�/� and PU.1�/�Spi-B�/� mice atc-rel. Thus, we determined that c-rel, like P2Y10, is down-approximately 50% of the levels of wild-type mice.regulated in PU.1�/�Spi-B�/� B cells.Moreover, the expression of 3H12 was further de-

creased (13% of wild-type) in PU.1�/�Spi-B�/� B cells,indicating that both PU.1 and Spi-B are important for The Murine c-rel Promoter Includes Multiple PU.1

and/or Spi-B Binding Sitesthe level of 3H12 mRNA. Northern blot analysis of totalRNA from multiple cell lines indicated that 3H12 is ex- Both PU.1 and Spi-B transcription factors are expressed

in immature and mature B lymphocytes (Rao et al.,pressed at high levels in B cells and at low levels in T cells(Figure 1B). The expression of 3H12 in B cells appears 1999a). The low levels of Rel mRNA observed in

PU.1�/�Spi-B�/� B cells imply that PU.1 and Spi-B controlto be highest in immature B lymphocytes, a pattern

PU.1 and Spi-B Regulate c-rel547

Figure 2. The Murine c-rel Promoter Con-tains at Least Three PU.1/Spi-B Binding Sites

(A) Sequences of five potential PU.1/Spi-Bbinding sites in the murine c-rel promoter areshown with GGAA core sequences boxed.PU.1 and Spi-B consensus binding sites arealso shown.(B) EMSAs of three PU.1/Spi-B binding sitesin the c-rel promoter using PU.1 or Spi-B IVTproteins are shown.(C) An EMSA of the �474 PU.1/Spi-B bindingsite using nuclear extracts prepared from A20mature B cells is shown. PI stands for preim-mune serum.

c-rel expression directly or indirectly. Murine and human peted by wild-type P2Y10 oligonucleotides as describedin the Experimental Procedures (Figure 2B). PU.1 bind-c-rel promoters are very conserved and contain a puta-

tive PU.1 binding site at �548 (Grumont et al., 1993; ing may be stronger than that of Spi-B at sites �474and �413. To further assess PU.1 and Spi-B bindingViswanathan et al., 1996). To determine if c-rel is likely

to be a direct target of PU.1 and/or Spi-B, a 1432-bp to the �474, �413, and �92 c-rel sites, EMSAs wereperformed using nuclear extracts from A20 mature B cellsmurine c-rel promoter fragment was further investigated

for potential PU.1/Spi-B binding site(s). Out of 15 poten- known to express both PU.1 and Spi-B proteins (Figure2C and data not shown). When the putative binding sitetial Ets GGAA or AGAA binding sites within the proximal

c-rel promoter region, 5 sites are consistent with a PU.1/ at �474 was incubated with A20 nuclear extract, twocomplexes (A and B) were noted (lane 1). Complex A isSpi-B consensus site and adhere to the rule that C at

�5, �4, �3 and T at �2, �1, and �1 is prohibited (Ray- specific for the PU.1/Spi-B binding site, since it wasoutcompeted by wild-type but not mutated cold �474Gallet et al., 1995) (Figure 2A). To assess whether PU.1

and/or Spi-B bind these sites, electrophoretic mobility oligonucleotide (GGGAAG to TCCGGT) (lanes 2 and 3).In direct contrast, the nonspecific nuclear complex Bshift assays (EMSAs) were performed using equal

amounts of in vitro-translated (IVT) PU.1 and Spi-B pro- was diminished by both wild-type and mutated coldcompetitor. To determine if nuclear PU.1 and/or Spi-Bteins (see Experimental Procedures). Three sites at

�474, �413, and �92 relative to the transcription start proteins are responsible for the formation of complexA, a “supershift” experiment was performed using anti-site appeared to be binding sites for both IVT PU.1 and

Spi-B (Figure 2B). However, no DNA-protein complexes Spi-B or anti-PU.1 antibodies. The addition of preim-mune serum (lane 4) and anti-Spi-B antibodies (lane 5)were observed when sites �548 and �404 were used

in EMSAs (data not shown). Binding at sites �474, �413, did not ablate complex A; however, incubation of nuclearextract with anti-PU.1 antibody (lane 6) completely elimi-and �92 is specific for PU.1/Spi-B recognition elements,

since DNA-protein complexes were specifically outcom- nated this complex. These results indicate that PU.1

Immunity548

protein was solely responsible for complex A in A20nuclear extracts. The failure to detect a nuclear Spi-B-DNA complex may result from lower levels of Spi-Bprotein in A20 cells (Rao et al., 1999a) and/or loweraffinity of the �474 site for Spi-B protein (see Figure 2B).Similar results were observed when PU.1/Spi-B sites at�413 and �92 were used for EMSAs (data not shown).

PU.1/Spi-B Binding Sites Are Important for c-relPromoter ActivityTo directly assess the functional importance of thesethree PU.1/Spi-B binding sites for c-rel promoter activ-ity, a 893-bp c-rel promoter fragment ( from �634 to �259)was cloned into a promoterless human growth hormonereporter gene, generating the pRelGH plasmid (Figure3A). This fragment contains previously identified bindingsites for transcription factor Oct-1/2, NF-�B, EGR-1, andSp-1 and exhibits high levels of promoter activity inB cells (Grumont et al., 1993). Subsequently, all threePU.1/Spi-B binding sites in pRelGH were mutated, re-sulting in triple-mutated pRelGH (tmRelGH) (Figure 3A).Mutation of these three binding sites reduced promoteractivity to 50% of the wild-type promoter in A20 B lym-phocytes (Figure 3B). These results are significant giventhat binding sites for Oct-1/2, NF-�B, EGR-1, and Sp-1remain intact. In contrast, no change in promoter activitywas observed between pRelGH and tmRelGH in ery-throid cells MEL and 3T3 fibroblasts that express noPU.1/Spi-B protein (Figure 3B and data not shown).These data showed that PU.1 and/or Spi-B binding sitesare important for c-rel promoter activity in B cells. Tofurther analyze the role of PU.1 and Spi-B in c-rel tran-scription, cotransfection experiments were performed

Figure 3. Three PU.1/Spi-B Binding Sites in the c-rel Promoter Are(Figure 3C). Transfection of 3T3 cells with pRelGH and Important for c-rel Promoter Activitywild-type PU.1 or Spi-B expression vectors increased

(A) A schematic diagram of the c-rel promoter reporter is shownreporter activity 3- to 4-fold relative to empty vector with three PU.1/Spi-B binding sites indicated. In tmRelGH, three(pcDNA3). However, transfection of mutant PU.1 or Spi-B PU.1/Spi-B binding sites were mutated. The arrow indicates the

transcription start site.lacking Ets DNA binding domains (PU.1�Ets or Spi-B�Ets)(B) A functional examination of PU.1/Spi-B binding sites for c-relor transcriptional activation domains (PU.1�TA or Spi-promoter activity using transient transfection of the wild-type c-relB�TA) did not enhance c-rel promoter activity. We con-promoter (pRelGH) or the promoter with PU.1/Spi-B sites mutatedclude that both PU.1 and Spi-B proteins can induce the(tmRelGH) in several cell lines. The data represent results obtained

c-rel promoter. from four independent experiments.(C) PU.1 and Spi-B transactivate the c-rel promoter reporter in 3T3cells. PU.1 or Spi-B expression vectors (or mutants lacking transacti-Expression of Rel Protein Restores B Cell Numbersvation or Ets DNA binding domains) were tranfected into 3T3 cellsin Mice Reconstituted with PU.1�/�Spi-B�/�

with the wild-type c-rel promoter reporter. The data represent meanBone Marrowfold induction in comparison to the empty pcDNA3 vector from four

Functional analysis of murine Rel by gene-targeted independent experiments.mutation has demonstrated a defect in c-rel�/� B cellproliferation and survival (Kontgen et al., 1995; Tumanget al., 1998), similar to the phenotype observed in duced high titers of virus-expressing Rel protein, as

demonstrated by Western blot analysis of MIGR-Rel-PU.1�/�Spi-B�/� mice (Garrett-Sinha et al., 1999). The dra-matically reduced expression of Rel in PU.1�/�Spi-B�/� infected 3T3 cells (data not shown).

To examine the in vivo consequences of Rel expres-B cells may be responsible for B cell survival defectsnoted in PU.1�/�Spi-B�/� mice. To determine if the rein- sion in PU.1�/�Spi-B�/� B cells, we performed bone mar-

row engraftment of irradiated Rag-2�/� mice. Bone marrowtroduction of Rel protein would overcome this deficiencyin PU.1�/�Spi-B�/� B cells, we employed a retroviral- cells isolated from PU.1�/�Spi-B�/� or PU.1�/�Spi-B�/�

mice were infected with control MIGR-GFP or MIGR-mediated approach to introduce c-rel into hematopoi-etic cells derived from PU.1�/�Spi-B�/� mice. A Rel cDNA Rel retroviruses and then injected into lethally irradiated

Rag-2�/� mice. PU.1�/�Spi-B�/� mice served as a control,was cloned into the MIGR-1 vector (Pui et al., 1999),encoding a bicistronic mRNA including both Rel and since these mice exhibit normal B cell numbers (Su et

al., 1997) and have similar genetic background togreen fluorescent protein (GFP) (Figure 4A). Transfectionof BOSC 23-packaging cells with MIGR-Rel vector pro- PU.1�/�Spi-B�/� mice. The source of the bone marrow

PU.1 and Spi-B Regulate c-rel549

Figure 4. Reintroduction of Rel Protein Re-stores Bone Marrow B Cell Numbers in MiceReconstituted with PU.1�/�Spi-B�/� BoneMarrow Cells

(A) A diagram of the Rel retrovirus construct.5 LTR-mediated transcription generates abicistronic mRNA, with the GFP readingframe accessed by an internal ribosomal en-try site (IRES).(B) Representative data from a single mouseare shown. The source of bone marrow andthe type of retrovirus with which they wereinfected specified four categories of reconsti-tuted mice; PU.1�/�Spi-B�/�/GFP-reconstitu-ted mice are boxed. The percentages of GFP�

(column I) and B220�/IgM� (column II) cellsfrom total bone marrow were determined byimmunostaining and flow cytometry. GFP�

(column III) or GFP� (column IV) populationswere then analyzed for numbers of B220�/IgM� cells.

and the virus type classified the reconstituted mice into Rel protein has no apparent effect on immature B cellsthat already express nearly normal levels of Rel proteinfour groups (n 3 for each group): PU.1�/�Spi-B�/�/

GFP, PU.1�/�Spi-B�/�/Rel, PU.1�/�Spi-B�/�/GFP, and (see Discussion). To examine Rel function more closely,B cell numbers in GFP� and GFP� bone marrow cellsPU.1�/�Spi-B�/�/Rel. After 8 weeks, reconstituted mice

were sacrificed, and bone marrow, spleen, lymph nodes, were analyzed. All GFP� cells in PU.1�/�Spi-B�/�/Rel andPU.1�/�Spi-B�/�/Rel mice also express Rel. In theand thymus were harvested for FACS analysis of B and

T cells. Representative data are shown in Figures 4B PU.1�/�Spi-B�/�/Rel mice, 12.1% of Rel-expressing cellsare B cells (Figure 4B, column III), compared with onlyand 5. These results were generated from a single mouse

of each experimental group. A total of 10%–35% of cells 4.4% in the Rel-negative cells (Figure 4B, column IV). Inter-estingly, Rel expression in the control PU.1�/�Spi-B�/�/Relfrom each tissue obtained expressed GFP, indicating

that these cells were derived from retrovirus-infected mice did not increase immature B cell numbers (3.2%in GFP� cells versus 5.2% in GFP� cells). Thus, Relbone marrow precursors expressing retrovirus-encoded

genes (Figures 4B and 5, column I; data not shown). expression elevated the B cell numbers exclusively inRag-2�/� mice reconstituted with bone marrow fromAnalysis of total bone marrow by B220/IgM immuno-

staining showed that control PU.1�/�Spi-B�/�/GFP mice PU.1�/�Spi-B�/� mice.As observed in bone marrow, spleens fromcontain 4.7% B220�/IgM� immature B cells (Figure 4B,

column II). However, only 1.5% immature B cells were PU.1�/�Spi-B�/�/GFP mice exhibit reduced numbersof IgM�/B220� B cells (25%) compared to controldetected in PU.1�/�Spi-B�/�/GFP mice, representing a

60% reduction (Figure 4B, column II, boxed). This is con- PU.1�/�Spi-B�/�/GFP mice (51%) (Figure 5, column II).Importantly, this defect was once again corrected insistent with our previous data showing that B220�/IgM�

B cell numbers are reduced in PU.1�/� Spi-B�/� bone mar- PU.1�/-Spi-B�/�/Rel mice (52%). We conclude that Relprotein expression is responsible for this phenotypicrow (Garrett-Sinha et al., 1999). We also observed a reduc-

tion of B220�/IgM� pro- and pre-B cells in reconstituted rescue, since more IgM�/B220� B cells are present inGFP� cells (70%) (Figure 5, column III) than in GFP�PU.1�/�Spi-B�/�/GFP mice (2.2% in PU.1�/�Spi-B�/�/GFP

and 11.4% in PU.1�/�Spi-B�/�/GFP), as seen in the bone cells (51%) (Figure 5, column IV) in the splenocytes ofPU.1�/�Spi-B�/�/Rel mice. A similar pattern was ob-marrow of many PU.1�/�Spi-B�/� mice (Garrett-Sinha et

al., 1999). Importantly, reintroduction of Rel protein cor- served in IgM�/IgD� mature splenic B cells (Figure 5,columns V–VII). As in the bone marrow and spleen, ex-rected B cell number defects in PU.1�/�Spi-B�/�/Rel

mice (5.2% immature B cells and 11.9% pro- and pre- pression of Rel protein in reconstituted PU.1�/�Spi-B�/�

Rel mice also increased the B cell number in lymphB cells) (Figure 4B, column II). Of note, B cell numbersin the control PU.1�/�Spi-B�/�/Rel mice (4.3%) are not nodes (data not shown). However, reintroduction of Rel

protein had no effect on thymic T cell numbers in any ofsignificantly different from those of PU.1�/�Spi-B�/�/GFP mice (4.7%), indicating that forced-expression of the four classes of reconstituted mice (data not shown).

Immunity550

Figure 5. Reintroduction of Rel Protein Restores Splenic B Cell Numbers to Mice Reconstituted with PU.1�/�Spi-B�/� Bone Marrow Cells

The arrangement of data is identical to that of Figure 4; besides the B220�/IgM� immature B cells, IgM�/IgD� mature B cells are also shown.

Moreover, no difference was observed in T cell numbers PU.1/Spi-B target genes underlying these functions re-mained unclear. We now report that c-rel is directly regu-between GFP� and GFP� thymic cells. Therefore, Rel

protein appears to specifically promote B cell survival. lated by PU.1 and Spi-B in B cells. Importantly, expressionof Rel in mice reconstituted with PU.1�/� Spi-B�/� bonemarrow cells overcame the B cell survival defect, dem-Enforced Bcl-2 Expression Promotes Survival

of PU.1�/�Spi-B�/� B Cells onstrating that misexpression of c-rel in PU.1�/�Spi- B�/�

B cells accounts for a major portion of this phenotype.To determine whether the reduced survival ofPU.1�/�Spi-B�/� B cells was in part due to low levels ofBcl-family members, we tested whether this defect c-rel Appears to Be a Direct Target Gene

of Both PU.1 and Spi-Bcould be overcome by enforced expression of a Bcl-2transgene (Strasser et al., 1991). Wild-type and Rel/NF-�B family members play important roles in B cell

development, differentiation, and survival (GerondakisPU.1�/�Spi-B�/� mice were crossed to mice that carry aBcl-2 transgene under the control of the Ig heavy chain et al., 1999). Induction of Rel/NF-�B proteins during pri-

mary B cell differentiation is developmental-stage de-enhancer (E�) and SV40 promoter (see ExperimentalProcedures). Results from representative mice gener- pendent, as RelA/p50 dimers are expressed predomi-

nantly in pro-B and pre-B cells, Rel/p50 in immature andated in these experiments are presented in Figure 6.Enforced Bcl-2 expression increased the number of mature B cells, and RelB/p52 in plasmacytes (Grumont

and Gerondakis, 1994; Kistler et al., 1998; Liou et al.,B220�/IgM� and IgM�/IgD� B cells in PU.1�/�Spi-B�/�

bone marrow to numbers comparable to wild-type mice 1994; Miyamoto et al., 1994; Weih et al., 1994). However,it is interesting that PU.1, Spi-B, and Rel levels increase(Figure 6). The Bcl-2 transgene also elevated B220�/

IgM� and IgM�/IgD� B cell numbers in PU.1�/�Spi-B�/� concomitantly when primary pre-B cells undergo matu-ration to immature IgM� B cells (Anderson et al., 1999;spleens (Figure 6) and lymph nodes (data not shown).

However, unlike transduction of c-rel, the Bcl-2 trans- Grumont and Gerondakis, 1994; Kistler et al., 1998). Themurine c-rel promoter has previously been partiallygene increased B cell numbers in both control and mu-

tant animals (compare Figure 5 with Figure 6). These characterized (Grumont et al., 1993). A GC-rich 200-ntregion upstream of the major transcription start site wasresults suggest a nonspecific general effect of Bcl-2 on

survival (Strasser et al., 1991) and contrast with those shown to contain c-rel promoter activity in T cells. Sur-prisingly, this promoter fragment is not sufficient forof c-rel. Bcl-2 can exert its effect independently of any

cofactors. In direct contrast, Rel must heterodimerize transcription in B cells (Grumont et al., 1993). In contrast,elements located between nucleotides �640 and �202with other NF-kB family members, and its activity

is therefore dependent on other proteins (i.e., NF-�B1, are critical for c-rel promoter activity in B cells (Grumontet al., 1993). Interestingly, two newly identified PU.1/NF-�B2, I�B, etc.)Spi-B binding sites (�474 and �413) are within thisregion. This region also contains two Rel/NF-�B sitesDiscussion(from �411 to �402 and from �260 to �251) and an Octsite (from �438 to �431). Although these Rel/NF-�B andOur previous analysis of PU.1�/�Spi-B�/� mice showed

that expression of both PU.1 and Spi-B is critical for late- Oct sites are functionally important in promoter-drivenreporter assays (Grumont et al., 1993), the relative im-stage B cell survival and proliferative responses to BCR

stimulation (Garrett-Sinha et al., 1999). However, the portance of each of these transcription factors in con-

PU.1 and Spi-B Regulate c-rel551

Figure 6. Overexpression of Bcl-2 Gene Increased the B Cell Numbers in PU.1�/�Spi-B�/� Bone Marrow and Spleen

PU.1�/�Spi-B�/�/Bcl-2 mice were generated by crossing PU.1�/�Spi-B�/� mice with Bcl-2 transgenic mice. Two- to three-month-old animalswere sacrificed, and single-cell suspensions were prepared from the bone marrow and spleen for flow cytometry analysis of IgM�/B220� orIgM�/IgD� B cells.

trolling Rel expression in B cells in vivo needs to be vivo B cell survival defect (Su et al., 1997). However, afurther decrease to 10%–13% clearly leads to the sur-determined. In contrast, PU.1 and Spi-B are critical for

Rel mRNA expression in IgM� B cells, since Rel expres- vival phenotype noted in PU.1�/�Spi-B�/� cells. Reintro-duction of Rel protein into PU.1�/�Spi-B�/� lymphocytession is dramatically reduced in PU.1�/�Spi-B�/� B cells

in vivo (Figure 1A). In vitro transient transfection assays via the retroviral reconstitution system overcame thisB cell number defect. Moreover, there is a significant(Figures 3B and 3C) further emphasized the importance

of PU.1 and Spi-B in regulating c-rel. First, although the increase in immature and mature B cells in Rel-express-ing cells in comparison to Rel-nonexpressing cells.mutated promoter still contained functional NF-�B and

Oct sites, c-rel promoter activity was reduced 50% if These results demonstrated that misexpression of Relis the principle cause of B cell hypocellularity inthree PU.1/Spi-B binding sites were eliminated. Second,

PU.1 or Spi-B protein induced wild-type promoter activ- PU.1�/�Spi-B�/� mice (Figures 4B and 5). This is consis-tent with the notion that Rel is an antiapoptotic moleculeity 3- to 4-fold. Rel expression is sensitive to both PU.1

and Spi-B protein levels in B cells: Rel expression is in B cells. Rel has been demonstrated to regulate theexpression of two Bcl-2 family prosurvival genes, A1reduced (50%) in both PU.1�/�Spi-B�/� and PU.1�/�

Spi-B�/� B cells, indicating that wild-type levels of Spi-B and Bcl-xL (Chen et al., 2000; Grumont et al., 1999; Leeet al., 1999; Zong et al., 1999). In particular, inductioncannot compensate for the loss of PU.1 and vise versa.

Consequently, a dramatic reduction was observed in of A1 is critical for B cell survival upon BCR stimulation(Grumont et al., 1999). Misexpression of A1 and Bcl-xLPU.1�/�Spi-B�/� B cells in vivo. Although PU.1 and Spi-B

exhibit highly conserved DNA binding regions, their in c-rel�/� B cells may explain the increased apoptosisupon BCR stimulation. Furthermore, expression oftransactivation domains are unique. Differential binding

affinities or cofactor interactions may explain why both Bcl-2 overcomes apoptosis in resting and activatedc-rel�/� B cells (Grossmann et al., 2000). Of note, overex-proteins are required for Rel expression. We conclude

that both PU.1 and Spi-B regulate Rel expression during pression of Bcl-2 via a B cell-specific transgene inPU.1�/�Spi-B�/� mice also reversed the poor survival ofB lymphopoiesis.mutant lymphocytes and elevated B cell numbers tothose detected in wild-type animals (Figure 6).Rel Expression Overcomes the B Cell Number

Defect in Mice Reconstituted with PU.1�/�Spi-B�/� PU.1�/�Spi-B�/� mice exhibit increased apoptosis insplenic B cells (Garrett-Sinha et al., 1999). DecreasedBone Marrow

PU.1�/�Spi-B�/� mice exhibit reduced numbers of imma- apoptosis upon Rel transduction may explain the rescueof B cell numbers in PU.1�/�Spi-B�/�/Rel-reconstitutedture (B220�/IgM�) and mature (IgM�/IgD�) B cells in

bone marrow and other lymphoid tissues such as the Rag-2�/� mice. Importantly, apoptosis rates in Rel-expressing B cells in PU.1�/�Spi-B�/�/Rel-reconstitutedspleen, lymph nodes, and peripheral blood (Garrett-

Sinha et al., 1999). Interestingly, a 42% reduction in Rel mice were consistently reduced 2%–5% in comparison tonon-Rel-expressing B cells (data not shown). This reducedprotein in Spi-B�/� mice (Figure 1A) fails to yield an in

Immunity552

apoptosis is likely to become significant over a long fectors BLNK and PLC� and poor proliferation (Garrett-Sinha et al., 1999). We have recently shown that a differ-period of time (8 weeks after bone marrow engraftment).

It is interesting to note that GFP intensity in MIGR- ent PU.1/Spi-B target gene, the adaptor protein GrpL,rescues both membrane proximal tyrosine phosphoryla-Rel-infected cells was much lower than that in control

MIGR-GFP cells (Figures 4B and 5, column I). This may tion and BLNK phosphorylation upon BCR stimulationof mutant lymphocytes (L.A.G.-S. et al., unpublished data).reflect selection against cells that express high levels of

Rel protein. Another possibility is that Rel mRNA hinders However, overexpression of GrpL in PU.1�/�Spi-B�/�

B cells fails to overcome the survival defect. Therefore,ribosome access to the internal ribosome entry site(IRES) to translate GFP. We note a reduction in pro- and distinct PU.1/Spi-B target genes likely account for differ-

ent B cell phenotypes noted in mutant mice. Analysispre-B cells in bone marrow of control PU.1�/�Spi-B�/� /Rel mice (8.9% among GFP� cells and 3.8% in GFP� of PU.1�/�Spi-B�/� mice continues to be a useful ap-

proach to identify novel components of BCR-mediatedcells) (Figure 4B, column III and IV). Therefore, high levelsof Rel protein could be toxic for pro- and pre-B cells responses in lymphocytes.that normally express very low levels of Rel (Liou et al.,

Experimental Procedures1994; Miyamoto et al., 1994; Weih et al., 1994). Thisis consistent with high levels of Rel expression being

Splenic B Cell Purification and Northern Blot Analysisassociated with increased apoptosis in the developing B cells were purified from spleens of mice with the appropriateavian embryo and bone marrow cultures (Abbadie et al., genotypes as described (Su et al., 1997). We purified B cells to more1993). Thus, appropriate Rel protein levels are important than 90% homogeneity, as determined by flow cytometry using

B220 immunostaining. Total RNA isolation from primary B cells orfor B cell function; very high, as well as very low, levelsvarious cell lines and Northern blot analysis were performed usingof Rel may promote B cell death. Rel also plays an impor-standard protocol.tant role in controlling B cell cycle progression, but the

molecular mechanisms by which c-rel�/� B cells arecDNA Amplification

blocked in the G1 phase remain obscure (Grumont et al., 3 and 5 RACE of 3H12 clones were performed using the Marathon1998). Interestingly, in comparison with PU.1�/� Spi-B�/� / cDNA Amplification Kit (Clontech). A 1.0-kb 3 RACE product wasGFP mice, B cell numbers in the GFP� population were PCR amplified using a forward gene-specific primer (5-AACCCAC

GCATGTCCTCATCATC-3) and AP1. The 0.5–3.0-kb 5 RACE prod-significantly increased in PU.1�/�Spi-B�/� /Rel-reconsti-ucts were generated using a reverse gene-specific primer (5-AGtuted mice (Figure 4B, column IV and Figure 5, columnCACCAGTTAGGCCAGAGTGTACG-3) and AP1. The specific PCRIV and VII), indicating that Rel may regulate other B cellproduct was gel purified and cloned into TA vector PCR 2.1-TOPO

genes that promote B cell survival in a paracrine fashion. (Invitrogen). These clones were sequenced and blast searchedAnother possibility is that some GFP� cells actually ex- against GenBank.press transduced Rel protein. Given the low levels ofGFP expression by cells transduced with the MIGR-Rel Electrophoretic Mobility Shift Assays (EMSA)

PU.1 and Spi-B IVT proteins were prepared using TNT Quick Cou-retrovirus, it is probably impossible to completely gatepled Transcription/Translation System (Promega). The amount ofout infected cells.S35-methionine-labeled PU.1 and Spi-B IVT proteins were measuredby SDS-PAGE and quantification using a Molecular Dynanic Phos-

Rel Misexpression May Not Fully Account phoImager, taking into consideration the number of methioninesfor PU.1�/�Spi-B�/� Phenotype in PU.1 and Spi-B. A20 nuclear extracts (NEs) were prepared as

previously described (Rao et al., 1999a). Mixing equal molar PU.1Although reintroduction of Rel protein overcame theor Spi-B IVT proteins or 8 �g A20 NE with 500,000 cpm double-B cell number defect in our bone marrow reconstitutionstrand 32P-labeled oligonucleotide initiated the DNA-protein bindingassay, misexpression of Rel alone may not explain thereaction. For competition experiments, cold competitor (200-fold

phenotype of reduced B cell numbers in PU.1�/�Spi-B�/�molar excess) was added with radioactive oligonucleotide simulta-

mice. It is likely that Rel target genes also play a role in neously. For “supershifts”, preimmune or antiserum was preincu-this phenomenon. Furthermore, mice lacking single Rel/ bated with NEs for 15 min on ice, followed by the addition of a

radioactive probe. DNA-protein complexes were resolved in a 6%NF-�B transcription factors (including c-rel) exhibit nopolyacrylamide gel using 0.25� TBE. For EMSA, the following oligo-impairment of B cell development, but mutation in multi-nucleotides were used: P2Y10 WT: 5-CGAAAGAGGAAGTAGAGC-3;ple Rel/NF-�B family members leads to B cell defects.P2Y10 MT: 5-CGAAAGTCCGGTTAGAGC-3; -474 WT: 5-GCTC

NF-�B1�/�NF-�B2�/� mice exhibit a block in B cell devel- TGGAGGGGAAGTGCGG-3; -474 MT: 5-GCTCTGGAGTCCGGTTGopment at the immature B cell stage (Franzoso et al., CGG-3; -412 WT: 5-GCGAAATCGGGAAGAGGGG-3; -412 MT:1997; Iotsova et al., 1997). NF-�B1�/�RelA�/� mice exhibit 5-GCGAAATCTCCGGTAGGGG-3; �92 WT: 5-CGGAGCCGGGAA

AGAAGAGG-3; �92 MT: 5-CGGAGCCTCCGGTGAAGAGG-3. Thea block in B cell precursors (Horwitz et al., 1997). Ofunderlined sequence corresponding to the PU.1/Spi-B core bindingnote, mice reconstituted with c-rel�/�RelA�/� fetal liverelements represents the difference between WT and mutant.cells exhibit reduced numbers of immature B cells (50%

of wild-type) and mature B cells (20% of wild-type)Transient Transfections

(Grossmann et al., 1999), a pattern also observed in The c-rel promoter reporter construct, pRelGH, was constructed byPU.1�/�Spi-B�/� mice. Our preliminary data indicate inserting the c-rel promoter fragment into the XbaI-BamHI site of

the promoterless reporter pØGH (Nichols Institution), which containsnormal mRNA levels of RelA, NF-�B1, and I�B-� ina human growth hormone cDNA. The c-rel promoter fragment inPU.1�/�Spi-B�/� splenic B cells. While RelA RNA levelspRelGH was excised from HP-cat plasmid (Grumont et al., 1993)appear normal in mutant B cells, it seems possible that(GenBank accession number X70690) using a Vent DNA polymerase-RelA function is abnormal in PU.1�/�Spi-B�/� mice.based PCR protocol (Hu and Gupta, 2000) via the following primer

PU.1�/�Spi-B�/� mice not only exhibit reduced num- pairs: 5-ACCTCTAGA(XbaI)CATTTATACAGAATGGGGTTA-3,5-ACbers of B cells, but the surviving B cells display defective CGGATCC(BamHI)AAGGCAGCCAGTCAGTCAGTC-3. The same

Vent-PCR protocol was used to mutate three PU.1/Spi-B bindingBCR-mediated phosphorylation of the downstream ef-

PU.1 and Spi-B Regulate c-rel553

sites in pRelGH by amplifying the full-length plasmid with mutations V (Caltag). The labeled cells were analyzed by FACS Calibur (BectonDickinson) using four color setting.(indicated by lowercase letters) incorporated into primers as shown:

-474A: 5-ggaCTCCAGAGCACCCGGACAACTC-3; -474B: 5-ggtTGCGGACAGCCAGCGCCC-3; -413A: 5-ggtAGGGGGAACCACCGGT Generation and Analysis of PU.1�/�Spi-B�/�/Bcl-2 Animals

Transgenic mice (strain E�-Bcl-2-22) expressing the human Bcl-2CGAAAAC-3; -413B: 5-ggaGATTTCGCACATGCAAATCTCCGC-3;�92A: 5-ggtGAAGAGGAGGCCCGGGGGTGGTCCGAG-3; �93B: cDNA under the control of the SV40 promoter and the immuno-

globulin heavy chain enhancer were obtained from the Jackson5-ggaGGCTCCTCTGGGTTTGCGGCTCACACC-3.Transfection of 3T3 cells with reporter and PU.1 or Spi-B expres- Laboratory (Strasser et al., 1991). These animals were crossed to

PU.1�/�Spi-B�/� mice to generate PU.1�/�Spi-B�/�/Bcl-2 animals.sion vectors (Rao et al., 1999b) was performed with SuperFect (Qia-gen) according to the manufacture’s protocol. A total of 5 � 104 Two- to three-month-old animals were sacrificed, and single-cell

suspensions were prepared from the bone marrow and spleen. Forcells in 60-mm dishes were transfected with 1 �g pRelGH, 1 �greference plasmid MSV �-galactosidase, and 3 �g PU.1 or Spi-B ex- flow cytometry analysis, each sample was stained with FITC-labeled

anti-IgM monoclonal antibody and a PE-labeled monoclonal anti-pression plasmid. After 48 hr transfection, supernatants were collectedfor human growth hormone assays (Nichols Institute) and cells were body (B220 or IgDa plus IgDb).lysed for �-galactosidase activity (Promega) to monitor transfectionefficiency. Human growth hormone activity was normalized to �-galac- Acknowledgmentstosidase activity.

For A20 and MEL cell transfection, 20 �g pRelGH or tmRelGH along We thank Fredrick G. Karnell and Cynthia Clendenin for technicalassistance, Brian Keith, Gwenn Danet, and Andy Arsham for helpfulwith 2 �g reference plasmid MSV �-galactosidase was mixed with 107

cells in 0.4 ml PBS in a 0.4-cm cuvette (Bio-RAD). Electroporation discussions, and Francine Tucker for preparation of the manuscript.This work was supported by grant #52094 (M.C.S.) from the Nationalwas executed at 275V, 960 �F using a Bio-Rad Gene-Pulser. After

48 hr transfection, supernatants and cells were collected for human Institutes of Health and the Abramson Family Cancer Research Insti-tute. M.C.S. is an investigator of the Howard Hughes Medical In-growth hormone and �-galactosidase assays, respectively.stitute.

Generation of Recombinant Retroviruses Received May 22, 2001; revised August 2, 2001.The murine stem cell virus (MSCV) vector, MIGR-1, containing aninternal ribosome entry site (IRES) and “humanized” GFP cDNA was Referencesa gift of Dr. Warren Pear (University of Pennsylvania, Philadelphia,PA). The 1.8-kb murine Rel cDNA was PCR amplified from SP65- Abbadie, C., Kabrun, N., Bouali, F., Smardova, J., Stehelin, D., Van-Rel (Grumont and Gerondakis, 1990) using primers 5-ACCAGATCT denbunder, B., and Enrietto, P.J. (1993). High levels of c-rel expres-(BglII)CGAGCCATGGCCTCGAGTGGATAT-3 and 5-ACCGAATTC sion are associated with programmed cell death in the developing(EcoRI)AACTTATATTTTAAAAAAACCATATGTGAAGGGATCAC-3. avian embryo and in bone marrow cells in vitro. Cell 75, 899–912.The Rel cDNA fragment was cloned into BglII/EcoRI-digested

Anderson, M.K., Hernandez-Hoyos, G., Diamond, R.A., and Roth-MIGR-1 vector to generate MIGR-Rel.enberg, E.V. (1999). Precise developmental regulation of Ets familyRetrovirus was produced by transfecting the ecotropic BOSC-23-transcription factors during specification and commitment to thepackaging cell line with MIGR-1 or MIGR-Rel plasmid (Pear et al.,T cell lineage. Development 126, 3131–3148.1993). Virus titer was determined by infecting 105 NIH 3T3 cells andBaldwin, A.S., Jr. (1996). The NF-kappa B and I kappa B proteins:by FACS analysis of GFP� cells after 2 days of infection. Westernnew discoveries and insights. Annu. Rev. Immunol. 14, 649–683.blot analysis was used to determine Rel protein expression of the

MIGR-Rel virus with rabbit anti-mouse Rel antibody (SC-71, Santa Bemark, M., Martensson, A., Liberg, D., and Leanderson, T. (1999).Cruz). Spi-C, a novel Ets protein that is temporally regulated during

B lymphocyte development. J. Biol. Chem. 274, 10259–10267.

Chen, C., Edelstein, L.C., and Gelinas, C. (2000). The Rel/NF-kappaBBone Marrow Reconstitution Experimentsfamily directly activates expression of the apoptosis inhibitorThe generation of Spi-B�/� and PU.1�/�Spi-B�/� mice has been de-Bcl-x(L). Mol. Cell. Biol. 20, 2687–2695.scribed previously (Garrett-Sinha et al., 1999; Su et al., 1997). Bone

marrow cells were harvested from the tibia and femur of 7-week-old, Chen, H.M., Zhang, P., Voso, M.T., Hohaus, S., Gonzalez, D.A.,Glass, C.K., Zhang, D.E., and Tenen, D.G. (1995). Neutrophils andfemale Spi-B�/� or PU.1�/�Spi-B�/� mice 5 days after they received 5

mg/per mouse 5-fluorouracil (Pharmacia) via tail injection. Bone monocytes express high levels of PU.1 (Spi-1) but not Spi-B. Blood85, 2918–2928.marrow cells were cultured at a concentration of 4 � 106 cell/ml in

DMEM with 15% FBS, 5% WEHI-conditioned medium, 6 ng/ml Eisenbeis, C.F., Singh, H., and Storb, U. (1993). PU.1 is a componentmouse recombinant IL-3, 10 ng/ml mouse recombinant IL-6, 100 of a multiprotein complex which binds an essential site in the murineng/ml mouse recombinant stem cell factor, 100 U/ml streptomycin immunoglobulin lambda 2–4 enhancer. Mol. Cell. Biol. 13, 6452–and penicillin each, and 2 mMol/L L-glutamine (GIBCO). After over- 6461.night stimulation, nonadherent bone marrow cells were spin infected Franzoso, G., Carlson, L., Xing, L., Poljak, L., Shores, E.W., Brown,with control MIGR-GFP virus or MIGR-Rel virus in the medium as K.D., Leonardi, A., Tran, T., Boyce, B.F., and Siebenlist, U. (1997).described above in addition to 4 �g/ml polybrene (Sigma). A second Requirement for NF-kappaB in osteoclast and B-cell development.spin infection was performed after 16 hr. After 6 hr incubation, bone Genes Dev. 11, 3482–3496.marrow cells were washed to remove free virus and were suspended

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