Analysis of Gene Expression and Ig Transcription in PU.1/Spi-B-Deficient Progenitor B Cell Lines

Abstract

A number of presumptive target genes for the Ets-family transcription factor PU.1 have been identified in the B cell lineage. However, the precise function of PU.1 in B cells has not been studied because targeted null mutation of the PU.1 gene results in a block to lymphomyeloid development at an early developmental stage. In this study, we take advantage of recently developed PU.1−/−Spi-B−/− IL-7 and stromal cell-dependent progenitor B (pro-B) cell lines to analyze the function of PU.1 and Spi-B in B cell development. We show that contrary to previously published expectations, PU.1 and/or Spi-B are not required for Ig H chain (IgH) gene transcription in pro-B cells. In fact, PU.1−/−Spi-B−/− pro-B cells have increased levels of IgH transcription compared with wild-type pro-B cells. In addition, high levels of Igκ transcription are induced after IL-7 withdrawal of wild-type or PU.1−/−Spi-B−/− pro-B cells. In contrast, we found that Igλ transcription is reduced in PU.1−/−Spi-B−/− pro-B cells relative to wild-type pro-B cells after IL-7 withdrawal. These results suggest that Igλ, but not IgH or Igκ, transcription, is dependent on PU.1 and/or Spi-B. The PU.1−/−Spi-B−/− pro-B cells have other phenotypic changes relative to wild-type pro-B cells including increased proliferation, increased CD25 expression, decreased c-Kit expression, and decreased RAG-1 expression. Taken together, our observations suggest that reduction of PU.1 and/or Spi-B activity in pro-B cells promotes their differentiation to a stage intermediate between late pro-B cells and large pre-B cells.

Spi-C Has Opposing Effects to PU.1 on Gene Expression in Progenitor B Cells

“…Similar to the phenotype of PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ pro-B cells (29), we found that steady-state levels of sterile transcripts initiating from the IgH intronic enhancer (I ) were increased relative to WT-R1 pro-B cells (Fig. 4A).…”

“…We have previously shown that PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ pro-B cells have a phenotype similar to pre-B cells with increased CD25 and decreased c-Kit surface expression relative to wild-type (WT) pro-B cells. In addition, PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ pro-B cells express increased IgH sterile transcripts relative to WT pro-B cells (29). The PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ phenotype could be recapitulated by ectopic expression of a mutant form of PU.1 lacking an activation domain (29).…”

“…4B). We also examined Fc␥RIIb transcripts, because we had previously observed a reduction in these transcripts in PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ pro-B cells (29) and a corresponding reduction in Fc␥RII/III surface expression (Fig. 3C).…”

“…The following primers were used: I : 5Ј-ACCTGGGAAT GTATGGTTGTGGCTT-3Ј and 5Ј-ATGCAGATCTCTGTTTTTGC CTCC-3Ј (40), Fc␥RIIb: 5Ј-CCCAAGTCCAGCAGGTCTTTACC-3Ј and 5Ј-CCCAATGCCAAGGGAGACTAAAT-3Ј, ␤-actin: 5Ј-TCCTTCGTT GCCGGTCCACA-3Ј and 5Ј-CGTCTCCGGAGTCCATCACA-3Ј, CD25: 5Ј-AGAACGGCACCATCCTAAACT-3Ј and 5Ј-TGCATGTCTGTTGT GGTTTGT-3Ј, c-Kit: 5Ј-CAACAGCAATGGCCTCACGAGT-3Ј and 5Ј-GTGGTACACCTTTGCTCTGCTC-3Ј. Relative quantitation real-time RT-PCR was performed as previously described (29). The primers used for I were the same as for standard RT-PCR above.…”

“…In addition, PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ pro-B cells express increased IgH sterile transcripts relative to WT pro-B cells (29). The PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ phenotype could be recapitulated by ectopic expression of a mutant form of PU.1 lacking an activation domain (29). Based on these observations, the inhibition of PU.1 appears to promote a pre-B cell-like phenotype, which is advanced over the pro-B cell stage.…”