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BLIMP-1: trigger for differentiation of myeloid lineage

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  • 1. © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES BLIMP-1: trigger for differentiation of myeloid lineage David H. Chang1, Cristina Angelin-Duclos2 and Kathryn Calame1,2 B lymphocyte–induced maturation protein-1 (BLIMP-1 or PRDI-BF1) is induced when bone marrow–derived progenitors differentiate in response to macrophage–colony stimulating factor (M- CSF) and is present in peripheral blood monocytes and granulocytes. BLIMP-1 is also induced during differentiation of U937 and HL-60 cells into macrophages or granulocytes. Induction of BLIMP-1 mRNA during macrophage differentiation of U937 and HL-60 shows a biphasic pattern. Overexpression of BLIMP-1 is sufficient to initiate macrophage differentiation of U937 cells whereas blocking endogenous BLIMP-1 inhibits differentiation. One target of BLIMP-1–dependent transcriptional repression in U937 cells is c-myc, providing an explanation for cessation of cell © 2000 Nature America Inc. • http://immunol.nature.com division. Thus BLIMP-1 is a key regulator of terminal differentiation in two separate hematopoietic lineages: myeloid cells and B lymphocytes. The transcriptional repressor B lymphocyte–induced maturation pro- be induced to differentiate into macrophages by treatment with phor- tein-1 (BLIMP-1) is a 98-kD protein containing five Krüppel-type bol 12-myristate 13-acetate (PMA)10. HL-60 is a pluripotential zinc fingers, originally identified by subtractive cloning of mRNAs promyelocytic cell line that differentiates into macrophages when induced during differentiation of a B cell lymphoma 1 line (BCL1)1. treated with PMA, and into granulocytes when treated with dimethyl Ectopic expression of BLIMP-1 is sufficient to drive terminal differ- sulfoxide (DMSO)11,12,13. We show that BLIMP-1 is induced during entiation of BCL1 lymphoma cells to a plasma cell phenotype1,2. differentiation of U937 and HL-60 cells and is both required and suf- Thus BLIMP-1 is viewed as a master regulator of B cell terminal ficient to trigger differentiation of U937 cells. differentiation. The human homolog of BLIMP-1, PRDI-BF1, was cloned by its Results ability to bind the PRDI site in the human interferon β (IFN-β) pro- BLIMP-1 mRNA induction during myeloid differentiation moter3. BLIMP-1, or PRDI-BF1, is a transcriptional repressor that To determine whether BLIMP-1 is expressed in differentiated associates with human Groucho4 and histone deacetylases5. (For sim- myeloid cells, we investigated BLIMP-1 mRNA in primary mono- plicity, both murine and human proteins are referred as BLIMP-1 cytes and granulocytes purified from human peripheral blood. from here on.) One important target of BLIMP-1 transcriptional Peripheral blood monocytes are post-mitotic but undergo further dif- repression in B cells is the c-myc gene, where BLIMP-1 binds a pre- ferentiation to macrophages upon entry into inflammatory sites and viously identified repressor site6,7,8. Recently, the CIITA gene, encod- tissues14,15,16. BLIMP-1 mRNA was present in both monocytes and ing a coactivator for class II major histocompatibility complex granulocytes purified from healthy donors (Fig. 1a, two are shown). (MHC) gene transcription, was identified as an additional target of We found that BLIMP-1 mRNA was not further induced when periph- BLIMP-1 in B cells8 (and J.F. Piskurich et al., unpublished data). eral blood monocytes were activated by in vitro culture (Fig. 1a, Although the initial report suggested that BLIMP-1 mRNA expres- Donor 1, M5). sion was limited to the B cell lineage1, BLIMP-1 is expressed in a We examined whether BLIMP-1 mRNA is induced during differ- human osteosarcoma line3. In addition, BLIMP-1–null mice die dur- entiation of myeloid progenitors. A population enriched for progeni- ing embryogenesis (M.M. Davis, personal communication) and the tor cells was isolated16 from mouse bone marrow and cultured for 8 Xenopus homolog of BLIMP-1 is required for anterior endomesoder- days in M-CSF to promote macrophage differentiation17. After 8 days mal cell fate and head induction9. These data suggested that BLIMP- of culture in M-CSF most cells were adherent and showed membrane 1 might be expressed and functionally important during terminal dif- ruffling (Fig. 1d). As the cells were heterogeneous, especially at the ferentiation of cells outside the B lymphoid lineage. beginning of the culture period, immunocytochemistry was per- To explore this possibility we studied cells in the myeloid lineage. formed to identify CD11b+ (the α chain of Mac-1, the complement We investigated the expression of BLIMP-1 during differentiation of receptor 3) cells and to follow expression of BLIMP-1 in this popula- bone marrow progenitors in response to macrophage–colony stimu- tion. CD11b is expressed on early bone marrow progenitors18 and on lating factor (M-CSF also called CSF-1) and in differentiated mono- differentiated macrophages19 but expression is lost in common lym- cytes and granulocytes from peripheral blood. We also took advantage phoid progenitors (CLP) 20 and early pro-B cells21, although it has of two well characterized cell lines that undergo defined programs of been found on a subset of T cell progenitors22. myeloid terminal differentiation. U937 is a promonocytic line that can In the original progenitor-enriched population 66% of the cells Integrated Program in Cellular, Molecular and Biophysical Studies and 2Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 1 10032, USA. Correspondence should be addressed to K.C. (klc1@columbia.edu). http://immunol.nature.com • august 2000 • volume 1 no 2 • nature immunology 169
  • 2. © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES a b Figure 1. BLIMP-1 mRNA is expressed in human peripheral blood mono- c cytes and granulocytes, and induced during differentiation of primary murine macrophages. (a) The expression of BLIMP-1 mRNA in human peripher- al blood monocytes and granulocytes. Human peripheral blood leukocytes from two healthy donors were purified using Histopaque density gradient and adherence sep- aration. Monocytes were 70–80% pure whereas granulocytes were 99.9% pure.Total RNA was analyzed by ribonuclease protection assay with probes protecting BLIMP- 1 mRNA and GAPDH mRNA. (M, purified monocytes; G, purified granulocytes; M5, monocytes after 5 days in vitro culture.) (b–d) Immunocytochemical staining for BLIMP-1 (nuclear red staining) and CD11b (cytoplasmic and cell surface blue stain- ing) in bone marrow–derived progenitors and macrophages. Bone marrow cells were d © 2000 Nature America Inc. • http://immunol.nature.com enriched from progenitors and cultured in M-CSF. (b) Day 0, starting population (66% CD11b+, 5% CD11b+ BLIMP-1+) arrows indicate CD11b+BLIMP-1+ cells. (c) Day 3 (86% CD11b+, 44% CD11b+ BLIMP-1+.) (d) Day 8 (100% CD11b+, 100% CD11b+ BLIMP-1+).At days 0 and 3 staining was performed on cells prepared by cytospin. Day 8 staining was done in situ on cells that adhered to coverslips. Two different fields from each day are shown. expressed CD11b. This number increased to 100% after 8 days of cul- virtually all of them expressed BLIMP-1 and CD11b (Fig. 2c,d). ture in M-CSF, consistent with M-CSF–driven macrophage differenti- The kinetics of BLIMP-1 mRNA induction were monitored in ation. Early in the culture period the cells were not adherent and were PMA- or DMSO-treated U937 and HL-60 cells. BLIMP-1 mRNA therefore prepared for immunocytochemistry by cytospin (which does was low or undetectable before treatment of either U937 or HL-60 not allow analysis of their morphology.) However, at the end of the cells. In U937 cells, BLIMP-1 mRNA (determined by ribonuclease culture, most viable cells became adherent with morphology of typi- protection assay) was induced after 4 h of PMA treatment, subse- cal differentiated macrophages (Fig. 1d). These cells were analyzed quently decreased, then peaked again at the end of the treatment by immunocytochemistry in situ. At day 0, 9% of CD11b+ cells (Fig. 2e). Similarly in HL-60 cells BLIMP-1 mRNA, determined by expressed BLIMP-1 in their nuclei (5% of all cells) (Fig. 1b). After 3 northern blotting, was rapidly induced and peaked in the second hour days of M-CFS treatment the number of CD11b+BLIMP-1+ cells of PMA treatment, decreased, then peaked again late in the treatment increased to 51% (44% of all cells) (Fig.1c). By day 8, 100% of period (Fig. 2f). Three isoforms of BLIMP-1 mRNA observed by CD11b+ cells expressed BLIMP-1 (Fig. 1d). The specificity of our northern blotting differ in their 3′ untranslated sequences antiserum for BLIMP-1 was established by showing that addition of (C. Tunyaplin and K. Calame, unpublished data). When HL-60 cells recombinant BLIMP-1 ablated the signal (data not shown). These data were treated with DMSO (Fig. 2g), BLIMP-1 mRNA stayed low dur- show clearly that BLIMP-1 is induced during differentiation of bone ing the first 3 days of treatment, then increased and stayed high after marrow CD11b+ progenitors to macrophages in response to M-CSF. 4 days of DMSO treatment. Thus BLIMP-1 mRNA is induced upon differentiation of U937 and HL-60 cell lines into macrophages in BLIMP-1 induction during U937 and HL-60 differentiation response to PMA, and upon differentiation of HL-60 cells into gran- U937 and HL60 cells were used for further analysis of BLIMP-1 ulocytes in response to DMSO. The presence of BLIMP-1 protein induction and function during myeloid differentiation. When U937 (Fig. 2c,d) correlates with the expression of BLIMP-1 mRNA in and HL-60 were grown in the presence of PMA for 3 days almost PMA-treated U937 cells (Fig. 2e). 100% of cells underwent growth arrest, changed from suspension to Based on its activity in B cells7 we speculated that BLIMP-1 might adherent growth (Fig. 2a,b) and expressed macrophage-specific sur- repress transcription of the c-myc gene during monocyte and granulo- face proteins CD11b and CD11c (the integrin α chain of gp150, 95) cyte differentiation. Consistent with this idea, c-Myc steady-state (see Fig. 4a,b)23,24. When HL-60 cells were treated with DMSO they mRNA was initially high but decreased following the first induction ceased to proliferate and, after 6 days of treatment, differentiated into of BLIMP-1 mRNA during differentiation of U937 and HL-60 cells granulocytes as illustrated by smaller cell size, altered morphology to macrophages after PMA treatment (Fig. 2e,f). During differentia- and positive staining by the Nitro-blue tetrazolium–reduction assay tion of HL-60 cells into granulocytes, c-Myc mRNA levels fell before (data not shown)25. induction of BLIMP-1 mRNA, then increased slightly and decreased To investigate whether BLIMP-1 was expressed in differentiated again after the later induction of BLIMP-1 mRNA (Fig. 2g). U937 cells, we performed immunocytochemical staining for nuclear BLIMP-1 and surface CD11b on U937 cells that became adherent Requirement for BLIMP-1 in U937 differentiation after 3 days of PMA treatment. Although the adherent cells displayed To determine the functional importance of BLIMP-1 induction during different degrees of cytoplasmic extension and membrane ruffling, macrophage differentiation we used a truncated form of BLIMP-1, 170 nature immunology • volume 1 no 2 • august 2000 • http://immunol.nature.com
  • 3. © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES a b e 0 0.5 1 2 3 4 6 8 10 12 36 48 72 96 120 hours Blimp-1 Myc GAPDH f 0 0.5 1 2 3 4 5 6 12 24 48 72 hours days Blimp-1 c d Myc GAPDH g 0 0.5 1 2 3 4 5 6 7 Blimp-1 Myc © 2000 Nature America Inc. • http://immunol.nature.com GAPDH Figure 2. BLIMP-1 expression is induced in U937 and HL-60 cells treated with PMA, and in HL-60 cells treated with DMSO. Wright-Giemsa staining of U937 cells (a) before and (b) after 3-day PMA treatment. Untreated cellls were prepared by cytospin. PMA-treated cells were allowed to adhere to cover slips before staining. Immunocytochemical staining for expression of BLIMP-1 (red) and CD11b (blue) of (c) untreated and (d) PMA-treated U937 cells. (e) BLIMP-1, c-Myc and GAPDH mRNAs in U937 cells, at times indicated following PMA treatment, were determined by ribonuclease protection assay. (f) BLIMP-1, c-Myc and GAPDH mRNAs in HL-60 cells, at times indicated following PMA treatment, were determined by northern blotting. (g) BLIMP-1, c-Myc and GAPDH mRNAs in HL-60 cells, at times indicated following DMSO treat- ment, were determined by northern blotting. T-BLIMP, to interfere with endogenous BLIMP-1. T-BLIMP (Fig. 3a) at low concentrations throughout treatment. T-BLIMP also impaired retains the zinc-finger domain that confers DNA binding26 but lacks the the ability of U937 to phagocytose opsonized bacteria. TB-1, TB-2 proline-rich domain required for transcriptional repression4,5. A similar- and TB-4 (low T-BLIMP) had partial phagocytotic activity whereas ly truncated form of BLIMP-1 was used in the inhibition of endogenous TB-3 and TB-5 (high T-BLIMP) had little phagocytotic activity BLIMP-1 function in B cell lines2 and to inhibit BLIMP-1 function in (Fig. 4e—only Mock-1, TB-1, 3 and 5 are shown). Xenopus embryos9. U937 cells were stably transfected with a T-BLIMP Therefore, blocking endogenous BLIMP-1 with T-BLIMP delays expression vector or a vector control. Five T-BLIMP–expressing trans- or inhibits macrophage differentiation of U937. Higher T-BLIMP fectants (TB-1–TB-5) and three mock transfectants (Mock 1–3) were expression, which is likely to compete more fully with endogenous used for subsequent studies. TB-3 and TB-5 have relatively high levels BLIMP-1, inhibited PMA-induced adherent growth, expression of of T-BLIMP mRNA, whereas TB-1, TB-2 and TB-4 have medium or macrophage surface antigens, and phagocytosis. Lower T-BLIMP low levels of T-BLIMP mRNA (Fig. 3b). expression partially inhibited or delayed macrophage differentiation. After 3 days of PMA treatment almost 100% of U937 and the three mock transfectants became adherent and displayed macrophage-like morphology. For the five T-BLIMP transfectants, however, we a Ac PR Pro ZnF Ac observed variable morphological changes. For TB-3 and TB-5 (high Blimp-1 T-BLIMP), after 3 days of PMA treatment cell growth was evident and 1 399 466 85 less than one-third of the cells became adherent. For TB-1, TB-2 and 6 T-Blimp TB-4 cells (low T-BLIMP), about one-third of cells were still in sus- 465 856 pension while two-thirds became adherent (data not shown). About 80% of cells from U937 and the mock transfectants were positive for b Mock-2 Mock-1 Mock-3 U937 TB-2 TB-3 TB-1 TB-4 TB-5 macrophage-specific esterase (α-naphthyl butyrate esterase) whereas only 25–40% of the five T-BLIMP transfectants were positive for esterase expression after 3 days of PMA treatment (data not shown). T-Blimp In U937 parental cells and mock transfectants, CD11b and CD11c were expressed on 80–90% of the cells after PMA treatment (Fig. GAPDH 4a,b—only Mock-1, TB-1, TB-3 and TB-5 are shown). For TB-3 and 6.0 6.2 8.2 1.0 12.4 TB-5 (high T-BLIMP), 20–30% of cells expressed CD11b and <30% of cells expressed CD11c. For TB-1, TB-2 and TB-4 (low T-BLIMP), Figure 3. U937 stable transfectants expressing T -BLIMP. (a) Structure of wild- about 50% of cells expressed CD11b and 60% expressed CD11c. type BLIMP-1 and T-BLIMP (aa 465–856). The repression domain (hatched box), When CD11b and CD11c were monitored at intervals during PMA which includes the proline-rich domain, lies between aa 399–466. (b) T-BLIMP mRNA treatment of TB-1 (low T-BLIMP), expression of both surface proteins expression level in 5 T-BLIMP transfectants (TB1–5) was determined by ribonuclease protection assay. Protected fragments correspond to T-BLIMP and GAPDH mRNA. was delayed. However, CD11c (and CD11b partially) eventually T-BLIMP mRNA is absent in U937 parental and mock transfectants. The numbers achieved normal levels (Fig. 4c,d—only Mock-1, TB-1 and Tb-3 are under each lane indicate the relative expression of T-BLIMP mRNA, normalized to shown). For TB-3 (high T-BLIMP), both CD11b and CD11c remained GAPDH mRNA, with the expression of T-BLIMP mRNA in TB-4 arbitrarily set as 1.0. http://immunol.nature.com • august 2000 • volume 1 no 2 • nature immunology 171
  • 4. © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES a c Mock-1 TB-1 TB-3 Mock-1 TB-1 TB-3 TB-5 91.9% 43.0% 21.3% 30.6% Counts Counts Cell Cell CD11b CD11b b d Mock-1 TB-1 TB-3 TB-5 Mock-1 TB-1 TB-3 87.7% 61.0% 38.9% 37.6 % Counts Counts Cell Cell CD11c CD11c e © 2000 Nature America Inc. • http://immunol.nature.com Figure 4.T -BLIMP delays or inhibits the expression macrophage-specific surface proteins CD11b and CD11c, and impairs the phagocytotic ability of PMA-treat- ed U937 cells. FACS analysis of Mock-1 and T-BLIMP transfectants for (a) CD11b and (b) CD11c expression was performed. Cells were either untreated (broken line) or treated with PMA for three days (solid line) before FACS analysis. (Percentage of cells in the pop- ulation of PMA-treated cells indicated by the horizontal bar is shown.) Kinetics of induc- Cell Counts tion of surface (c) CD11b and (d) CD11c in Mock-1,TB-1 and TB-3 after PMA treatment was also determined. Cells were analyzed before treatment (thin broken line), after 1 day (thick broken line), 3 days (thin solid line) and 5 days (thick solid line) in PMA. (e) Phagocytic activity of PMA-treated mock transfectant, and three T-BLIMP expressing clones. Cells untreated (thin solid line) and treated with PMA for 3 days (thick solid line) were incubat- ed with FITC-labeled opsonized S. aureus before FACS analysis. Phagocytosis of FITC-labeled S. aureus BLIMP-1 overexpression drives U937 differentiation trol virus–infected cells where less than 0.1% were adherent. Knowing that BLIMP-1 is required for U937 differentiation, we Expression of BLIMP-1 and CD11b was detected in all the adherent wanted to determine whether expression of BLIMP-1 might be suffi- cells from BLIMP-1-GFP virus infection (Fig. 5f,g). This is consis- cient to drive macrophage differentiation in this system. A retrovirus tent with the possibility raised by data in Fig. 5a–c that some BLIMP- containing BLIMP-1 cDNA, an internal ribosome entry site (IRES) 1-GFP–infected cells differentiated even though they did not express and cDNA encoding green fluorescent protein (GFP) was engineered. enough GFP to be detected by FACS. However, the BLIMP-1-GFP Expression of both BLIMP-1 and GFP from the bicistronic vector virus–infected cells were generally smaller and had less cytoplasm ensured that cells expressing BLIMP-1 could be identified by virtue and membrane ruffling compared to cells differentiated in response to of GFP expression. Three days after infection of U937 cells with the PMA (see Fig. 2a–d). Thus, overexpression of BLIMP-1 in U937 is BLIMP-1-GFP virus, about 6% of the cells were GFP+; 50% of the sufficient to induce macrophage differentiation as shown by expres- GFP+ cells (3.1% of the total) expressed CD11b and 73% (4.1% of the sion of CD11b and CD11c on the surface, cell adherence and altered total) expressed CD11c (Fig. 5a–c). Some GFP– cells from the morphology. BLIMP-1-GFP virus infection also expressed CD11b (5%) and CD11c (15%) after infection. As GFP expression by the IRES-GFP BLIMP-1 targets c-myc for repression virus is lower than that of the control MFG-GFP virus, these may rep- The correlation between BLIMP-1 mRNA induction and decreased resent infected cells that express insufficient GFP to be detected by c-Myc mRNA during U937 and HL-60 differentiation suggested that fluorescence-activated cell sorting (FACS) but still express sufficient BLIMP-1 may repress c-myc during macrophage differentiation. To BLIMP-1 to induce CD11b and CD11c expression. Consistent with test this possibility directly we determined c-Myc, T-BLIMP and this interpretation, cells infected with control virus encoding only BLIMP-1 mRNA levels in U937 cells expressing T-BLIMP after treat- GFP (MFG-GFP) had minimal expression of CD11b (0.7%) or ment with PMA using riboprobe analysis (Fig. 6a–d). Endogenous CD11c (3.7%) demonstrating that virus infection did not induce BLIMP-1 mRNA was induced in a biphasic manner in controls as expression of these markers. By comparison, when U937 cells were well as T-BLIMP clones; the levels of T-BLIMP mRNA were in the treated for three days with PMA, 87% expressed CD11b and 98% same range as those of endogenous BLIMP-1 mRNA, consistent with expressed CD11c (data not shown). inhibition of BLIMP-1 by T-BLIMP. In Mock-1, c-Myc mRNA In addition to induction of surface proteins, overexpression of decreased normally after 1 day of PMA treatment (Fig. 6a–d). BLIMP-1 was sufficient to cause morphological changes and adher- However, after 3 days of PMA treatment c-Myc mRNA remained high ent growth of U937 cells (Fig. 5d,e). Three days after infection 10% in all T-BLIMP transfectants. After 5 days of PMA treatment, c-Myc of BLIMP-1-GFP–infected cells became adherent, compared to con- mRNA in TB-1 (low T-BLIMP) decreased while c-Myc mRNA in 172 nature immunology • volume 1 no 2 • august 2000 • http://immunol.nature.com
  • 5. © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES a d e 0.54 0.01 0.69 0.04 CD11c CD11b 0.40 0.41 b f g 0.52 0.24 2.43 1.36 CD11b CD11c 29.1 27.3 © 2000 Nature America Inc. • http://immunol.nature.com c 4.51 3.06 15.0 4.11 Figure 5. BLIMP-1 expression is sufficient to drive macrophage differenti- ation of U937 cells. U937 cells were either (a) untreated, (b) infected with con- CD11c CD11b trol virus MFG-GFP or (c) with BLIMP-1-GFP virus for 3 days. Surface expression of CD11b and CD11c was determined by FACS analysis (7-AAD was used to exclude dead cells).The numbers in each box correspond to the percent of total cells in each population.Wright-Giemsa staining of U937 cells 3 days after infection with (d) con- 3.06 1.54 trol virus (prepared by cytospin) or (e) BLIMP-1-GFP virus (cells which adhered to a cover slip). (f,g) Immunocytochemical staining of cells prepared as described in d and e, respectively, for CD11b (blue) and BLIMP-1 (red). GFP GFP TB-3 and TB-5 (high T-BLIMP) remained high. Concomitant with cell development, particularly plasmacytomas1. We have shown here decreased c-Myc mRNA, U937 and mock transfectants underwent that BLIMP-1 mRNA is expressed in peripheral blood monocytes and growth arrest following 3 days of PMA treatment but T-BLIMP trans- granulocytes and is induced during M-CSF–dependent differentiation fectants continued to divide (Fig. 6e). After 5 days of PMA treatment, of bone marrow–derived macrophages, U937 promonocytes treated TB-1 (low T-BLIMP) became growth-arrested while 10% of TB-3 with PMA, and HL-60 promyelocytes treated with either PMA or (high T-BLIMP) cells were still in S phase (data not shown). Thus, DMSO. Thus, BLIMP-1 is clearly induced during differentiation of these data show that BLIMP-1 is required for growth arrest during promyelocytic cells into macrophages or granulocytes. U937 differentiation, and one target of BLIMP-1 repression is the We also performed northern blot analyses of RNA from adult murine c-myc gene. tissues and detected low levels of BLIMP-1 mRNA in many tissues including brain, lung, heart, kidney and testis (D. H. Chang and K. Discussion Calame, unpublished data). Thus it is clear that expression of BLIMP- BLIMP-1 expression not limited to B cell lineage 1 is not limited to the B cell lineage. Our data are consistent with the The original report described BLIMP-1 as a B cell–specific protein expression of BLIMP-1 mRNA in a human osteosarcoma cell line3, with expression restricted to cell lines representing late stages of B with the embryonic lethal phenotype of BLIMP-1–/– mice a e 1.25 1.00 Cells in S phase 0.75 b c d 0.50 0.25 0.00 U937 Mock1 Mock2 TB-1 TB-2 TB-3 TB-4 TB-5 Figure 6. c-myc is a target of BLIMP-1 repression during U937 differentiation. Ribonuclease protection assay for c-Myc, endogenous BLIMP-1 (human homolog) and ectopic T-BLIMP (mouse homolog) mRNAs in (a) Mock-1 (b) TB-1 (c) TB-3 and (d) TB-5 clones treated with PMA at the days indicated. (e) Cell cycle status of U937, mock and T-BLIMP clones. Cells in S phase were quantified using FACS, gating on cells with high BrdU incorporation and propidium iodide content greater than 2n. http://immunol.nature.com • august 2000 • volume 1 no 2 • nature immunology 173
  • 6. © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES (M.M. Davis, personal communication) and with the recent demonstra- esterases were reduced and the cells’ ability to phagocytose was tion that XBLIMP-1 is necessary for Xenopus embryo development9. impaired. These observations establish the importance of BLIMP-1 in full macrophage differentiation of U937 cells. Coupled with demon- BLIMP-1 represses c-myc transcription in U937 cells strated induction of BLIMP-1 mRNA during differentiation of bone Given that BLIMP-1 is induced during myeloid differentiation, it was marrow–derived macrophages, the data strongly suggest a role for important to identify functionally relevant target genes of BLIMP- BLIMP-1 in normal monocyte and macrophage differentiation. 1–dependent repression. The delayed or absent down-regulation of Furthermore, induction of BLIMP-1 mRNA upon differentiation of c-Myc mRNA observed in U937 transfectants expressing T-BLIMP HL-60 cells into granulocytes, and the presence of BLIMP-1 mRNA provides direct evidence that c-myc is a target of BLIMP-1 repression in normal peripheral blood granulocytes, suggest that BLIMP-1 may during U937 differentiation. This is consistent with the previous iden- also be required for granulocyte development. tification of a region on the c-myc promoter, encompassing the Overexpression of BLIMP-1 in U937 cells was sufficient to trigger BLIMP-1 binding site (positioned 290 bp before the P1 site), which differentiation as shown by induction of CD11b and CD11c on the is required for repression of c-myc transcription in U937 cells27. surface, cell adherence, and acquisition of partial macrophage mor- The importance of c-myc repression in myeloid differentiation has phology. However, neither the percentage of cells expressing CD11b been established by many previous studies. Decreased c-Myc corre- or CD11c nor the acquisition of a macrophage morphology was as lates with U937 differentiation27,28, and ectopic expression of v-Myc complete a response to overexpressed BLIMP-1 as that observed in blocks U937 differentiation29,30. As c-Myc promotes cell proliferation response to PMA. This may simply reflect the suboptimal levels of it makes sense that it must be repressed when cells cease division and BLIMP-1 achieved by retroviral transduction. Alternatively BLIMP-1 © 2000 Nature America Inc. • http://immunol.nature.com undergo terminal differentiation. Thus, c-myc is a critically important may drive some, but not all, aspects of U937 differentiation. target of BLIMP-1–dependent transcriptional repression in U937 Transcription factors AP-1, PU.1, NF-κB and IRF-1 are required cells. Furthermore, repression of c-myc by BLIMP-1 in both for U937 differentiation32,33,34 and gene-targeting studies show that macrophages and B cells7 provides a mechanistic link between termi- PU.135, C/EBP36 ICSBP37,38 are necessary for normal macrophage dif- nal differentiation in these two cell lineages. ferentiation. To our knowledge, however, BLIMP-1 is the only tran- However, BLIMP-1–dependent repression of other potential target scription factor capable of triggering macrophage differentiation in genes may be required for full macrophage differentiation. This is U937 cells. The ability of BLIMP-1 to direct macrophage terminal suggested by the biphasic expression pattern of BLIMP-1 mRNA differentiation is strikingly similar to its ability to drive B cell termi- observed during U937 and HL-60 differentiation in response to PMA. nal differentiation1. Taken together, the data suggest BLIMP-1 may For plasma cell differentiation, we have recently shown that repres- play a critical role in terminal differentiation of many cell lineages. sion of c-myc is necessary, but not sufficient, to drive differentiation (K.-I. Lin and K. Calame, unpublished data) and have identified the Methods CIITA gene as an additional target of BLIMP-1 repression (J.F. Cell culture and isolation. U937 (CRL-1593.2) and HL-60 (CCL-240) were purchased Piskurich et al., unpublished data). It will be important to try to iden- from American Type Culture Collection (ATCC, Manassas, VA) and maintained in growth medium (RPMI 1640, Sigma; 10% heat-inactivated fetal bovine serum (FBS), Gemini, tify other BLIMP-1 target genes in myeloid cells. Calabasas, CA; and 20 µg/ml of Gentamicin, Gemini). PMA (Sigma) was dissolved in In contrast to macrophage differentiation, during granulocytic dif- DMSO (Sigma) in a 1 mg/ml stock solution and used at 10 ng/ml for all experiments. To ferentiation of HL-60 cells, induction of BLIMP-1 mRNA did not induce granulocytic differentiation, DMSO (at a final concentration of 1.25%) was added directly to growth media. Ecotropic Phoenix cells were a gift from G.P. Nolan at Stanford correlate with the initial decrease in c-Myc mRNA levels. University and were cultured following Nolan’s Phoenix retroviral producer line protocol Nevertheless this is consistent with a previous study showing that two (http://www.stanford.edu/group/nolan/NL-phnxr.html). mechanisms of transcriptional control operate on c-myc during Peripheral blood monocytes and granulocytes were isolated from healthy donors (New York Blood Center). Mononuclear cells were isolated by density gradient using DMSO-induced differentiation of HL-60 cells: the early phase Histopaque-1077 (Sigma), according to manufacturer’s procedure. Polynuclear cells involves blockage of c-myc transcriptional elongation and the late (granulocytes) were isolated using Histopaque-1119. Purity was determined by FACS phase involves repression of transcription initiation31. Transcription analysis of CD45 (leukocyte common antigen), CD11b (Mac-1) and CD14 (monocyte- specific antigen), and Wright-Giemsa staining. Monocytes were further purified from repression, which correlates with the induction of BLIMP-1 mRNA mononuclear cell pools by adherence separation. The mononuclear cells were resuspend- we observed, is the irreversible terminal step in granulocytic differ- ed in growth medium (RPMI 1640 + 30% heat-inactivated human ultraserum from entiation of HL60 cells. Therefore it is likely that c-myc is also a tar- Gemini, 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate purchased from get of BLIMP-1 during granulocyte differentiation. Sigma). Cells in growth medium were placed in 75-cm2 tissue culture flasks and incubat- ed for 2 h at 37 ˚C with 5% CO2. Nonadherent cells were removed by aspiration and the remaining adherent cells (monocytes) were rinsed twice in RPMI, scraped off by cell BLIMP-1 required for U937 differentiation to macrophages lifter (Costar Scientific, Cambridge, MA) and centrifuged at 1,800 rpm for 5 min. The cell Multiple clones of U937 expressing T-BLIMP failed to respond nor- pellets were washed twice in RPMI then collected and tested for purity by FACS. Murine bone marrow progenitors were purified based on an established protocol16. mally to PMA treatment. The correlation between relative T-BLIMP Bone marrow cells taken from murine femurs and tibia were flushed out and washed by expression and the degree of inhibition of U937 differentiation pro- EMEM (MEM with Earle salt from Gibco-BRL, Gaithersburg, MD, 100 U/ml penicillin vides additional evidence that ectopic T-BLIMP competes with G and 100 µg/ml streptomycin sulfate). Mononuclear bone marrow cells were purified by density gradient (Histopaque-1077), and B and T lymphocytes consecutively depleted endogenous BLIMP-1. As endogenous BLIMP-1 continues to with anti-B220- and anti-Thy1.2-magnetic beads (Dynal, Oslo) following manufacturer’s increase during PMA treatment of the T-BLIMP transfectants, it protocols. The purified mononuclear cells were cultured in EMEM-10 (EMEM with 10% seems likely that the delayed differentiation observed in some clones heat-inactivated FBS) with 20 ng/ml of M-CSF (Sigma). was overridden when the level of endogenous BLIMP-1 became suf- Plasmids and primers. BLIMP-1 cDNA, a gift of M.M. Davis at Stanford University, ficiently high to counter T-BLIMP’s inhibitory effect. was cloned into pBluescript (pSK) (Stratagene, La Jolla, CA). PRDI-BF1 (human All aspects of macrophage differentiation we measured were affect- BLIMP-1 homolog) cDNA, a gift of T. Maniatis at Harvard University, and human Myc ed by T-BLIMP. T-BLIMP–expressing U937 cells failed to down-reg- cDNA (exons 2 and 3) were cloned into pBluescript (pKS). The pKS-PRDI-BF1, pKS-hMYC and pTRI-human glyceraldehyde-3-phosphate dehydrogenase ulate c-Myc mRNA, continued to cycle, and did not become adherent (pTRI-hGAPDH) (Ambion, Austin) were used for making probes for northern blotting following PMA treatment. CD11b, CD11c and macrophage-specific and ribonuclease protection assay. 174 nature immunology • volume 1 no 2 • august 2000 • http://immunol.nature.com
  • 7. © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES The retroviral construct, pMSCV-IRES-GFP and pMFG-GFP were a gift from G.P. bacteria, cells were incubated with red blood cell lysis solution (Sigma) for 5 min, Nolan at Stanford University39. The VSV-G plasmid was a gift of S. Goff at Columbia washed twice with PBS buffer and treated with 20 µl/0.5 ml of Trypan Blue. Cells that University. Ψ−ECO (packaging DNA) was a gift of O. Witte from UCLA. HA-tagged internalized the FITC-labeled bacteria were detected by FACScan. T-BLIMP, a truncated form of (mouse homolog) BLIMP-1 from bp 1612–2788 of the open reading frame, was cloned into pSK and pBABE40,41. pBABE-HA-T-BLIMP and Acknowledgments pBABE were linearized before using for stable transfection. The pSK-HA-T-BLIMP was We thank R. Dalla-Favera, S. Greenberg, C. Schindler, S. Silverstein and K.-I. Lin for used for ribonuclease protection assay to detect T-BLIMP. reading the manuscript and for discussions, D.J. Husemann and J. Rooney for discus- sions and technical advice, G. Cattoretti for technical support. We are grateful to Transfection and infection. Stable transfection was performed by pulsing 0.3 ml of cells members of the Calame laboratory especially J. Liao for technical assistance and J.Yu suspended in growth medium (5–10 × 106 cells/ml), at 240 V, 960 µF (Bio-Rad for providing the T-BLIMP construct. Supported by National Institutes of Health Electroporator, Richmond, CA). After electroporation cells were resuspended in 10 ml of Cancer Biology Training grant 2 T32 CA09503-14 (to D.H.C) and RO1-AI43576 growth medium, transferred to 96-well plates and allowed to recover for 2 days before (to K.C). adding the selection drug, puromycin, at a final concentration of 1 µg/ml. After about 3–4 weeks of drug selection, individual clones were expanded from 96-well plates. 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Identification and characterization of a novel repressor of β- interfer- on gene expression. Genes Dev. 5, 868–879 (1991). described previously43, and used for infecting cells of interest. Cells were analyzed after 4. Ren, B., Chee, K.J., Kim, T.H. & Maniatis, T. PRDI-BF1/Blimp-1 repression is mediated by core- three days of infection. pressors of the Groucho family of proteins. Genes Dev. 13, 125–137 (1999). 5. Yu, J., Angelin-Duclos, C., Greenwood, J., Liao, J. & Calame, K. Transcriptional repression by RNA analysis. Total RNA was prepared by the guanidium thiocyanate procedure as © 2000 Nature America Inc. • http://immunol.nature.com blimp-1 (PRDI-BF1) involves recruitment of histone deacetylase. Mol. Cell Biol. 20, 2592–2603 described43. Ribonuclease protection assay was performed based on the established pro- (2000). tocol44. Antisense cRNA probes were generated using T3 or T7 RNA polymerase 6. Kakkis, E., Riggs, K.J., Gillespie, W. & Calame, K. 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