PU.1 Regulates Ig Light Chain Transcription and Rearrangement in Pre-B Cells during B Cell Development.

B cell development and Ig rearrangement are governed by cell type- and developmental stage-specific transcription factors. PU.1 and Spi-B are E26-transformation-specific transcription factors that are critical for B cell differentiation. To determine whether PU.1 and Spi-B are required for B cell development in the bone marrow, Spi1 (encoding PU.1) was conditionally deleted in B cells by Cre recombinase under control of the Mb1 gene in Spib (encoding Spi-B)-deficient mice. Combined deletion of Spi1 and Spib resulted in a lack of mature B cells in the spleen and a block in B cell development in the bone marrow at the small pre-B cell stage. To determine target genes of PU.1 that could explain this block, we applied a gain-of-function approach using a PU.1/Spi-B-deficient pro-B cell line in which PU.1 can be induced by doxycycline. PU.1-induced genes were identified by integration of chromatin immunoprecipitation-sequencing and RNA-sequencing data. We found that PU.1 interacted with multiple sites in the Igκ locus, including Vκ promoters and regions located downstream of Vκ second exons. Induction of PU.1 induced Igκ transcription and rearrangement. Upregulation of Igκ transcription was impaired in small pre-B cells from PU.1/Spi-B-deficient bone marrow. These studies reveal an important role for PU.1 in the regulation of Igκ transcription and rearrangement and a requirement for PU.1 and Spi-B in B cell development.

Identification of a negative regulatory role for spi-C in the murine B cell lineage.

Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib(-/-)Spic(+/-)). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib(-/-) mouse spleens. Spib(-/-)Spic(+/-) B cells had restored proliferation compared with Spib(-/-) B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib(-/-)Spic(+/-) phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib(-/-)Spic(+/-) B cells compared with Spib(-/-) B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function.

PU.1 opposes IL-7-dependent proliferation of developing B cells with involvement of the direct target gene bruton tyrosine kinase.

Deletion of genes encoding the E26 transformation-specific transcription factors PU.1 and Spi-B in B cells (CD19-CreΔPB mice) leads to impaired B cell development, followed by B cell acute lymphoblastic leukemia at 100% incidence and with a median survival of 21 wk. However, little is known about the target genes that explain leukemogenesis in these mice. In this study we found that immature B cells were altered in frequency in the bone marrow of preleukemic CD19-CreΔPB mice. Enriched pro-B cells from CD19-CreΔPB mice induced disease upon transplantation, suggesting that these were leukemia-initiating cells. Bone marrow cells from preleukemic CD19-CreΔPB mice had increased responsiveness to IL-7 and could proliferate indefinitely in response to this cytokine. Bruton tyrosine kinase (BTK), a negative regulator of IL-7 signaling, was reduced in preleukemic and leukemic CD19-CreΔPB cells compared with controls. Induction of PU.1 expression in cultured CD19-CreΔPB pro-B cell lines induced Btk expression, followed by reduced STAT5 phosphorylation and early apoptosis. PU.1 and Spi-B regulated Btk directly as shown by chromatin immunoprecipitation analysis. Ectopic expression of BTK was sufficient to induce apoptosis in cultured pro-B cells. In summary, these results suggest that PU.1 and Spi-B activate Btk to oppose IL-7 responsiveness in developing B cells.

Genome-wide comparison of PU.1 and Spi-B binding sites in a mouse B lymphoma cell line.

BACKGROUND: Spi-B and PU.1 are highly related members of the E26-transformation-specific (ETS) family of transcription factors that have similar, but not identical, roles in B cell development. PU.1 and Spi-B are both expressed in B cells, and have been demonstrated to redundantly activate transcription of genes required for B cell differentiation and function. It was hypothesized that Spi-B and PU.1 occupy a similar set of regions within the genome of a B lymphoma cell line. RESULTS: To compare binding regions of Spi-B and PU.1, murine WEHI-279 lymphoma cells were infected with retroviral vectors encoding 3XFLAG-tagged PU.1 or Spi-B. Anti-FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed. Analysis for high-stringency enriched genomic regions demonstrated that PU.1 occupied 4528 regions and Spi-B occupied 3360 regions. The majority of regions occupied by Spi-B were also occupied by PU.1. Regions bound by Spi-B and PU.1 were frequently located immediately upstream of genes associated with immune response and activation of B cells. Motif-finding revealed that both transcription factors were predominantly located at the ETS core domain (GGAA), however, other unique motifs were identified when examining regions associated with only one of the two factors. Motifs associated with unique PU.1 binding included POU2F2, while unique motifs in the Spi-B regions contained a combined ETS-IRF motif. CONCLUSIONS: Our results suggest that complementary biological functions of PU.1 and Spi-B may be explained by their interaction with a similar set of regions in the genome of B cells. However, sites uniquely occupied by PU.1 or Spi-B provide insight into their unique functions.

Deletion of genes encoding PU.1 and Spi-B in B cells impairs differentiation and induces pre-B cell acute lymphoblastic leukemia.

The E26 transformation-specific (Ets) transcription factor PU.1 is required to generate lymphoid progenitor cells from hematopoietic stem cells, but it is not required to generate B cells from committed B-cell lineage progenitors. We hypothesized that PU.1 function in B-cell differentiation is complemented by the related Ets transcription factor Spi-B. To test this hypothesis, mice were generated lacking both PU.1 and Spi-B in the B-cell lineage. Unlike mice lacking PU.1 or Spi-B, mice deficient in both PU.1 and Spi-B in the B-cell lineage had reduced frequencies of B cells as well as impaired B-cell differentiation. Strikingly, all PU.1 and Spi-B-deficient mice developed pre-B cell acute lymphoblastic leukemia before 30 weeks of age. Pre-B cells accumulated in the thymus resulting in massive thymic enlargement and dyspnea. These findings demonstrate that PU.1 and Spi-B are essential transcriptional regulators of B-cell differentiation as well as novel tumor suppressors in the B-cell lineage.

Customizing Maxpar Direct Immune Profiling Assay with Additional Surface Marker and Intracellular Cytokine Staining Workflows for Expanded Mass Cytometry Panels.

Mass cytometry, or cytometry by time-of-flight (the basis for Fluidigm® CyTOF® technology), is a system for single-cell detection using antibodies tagged with metal probes. Without the need for compensation, the highly parametric Helios™ mass cytometer has a detection range of 135 distinct mass channels (75-209 Da). Optimized for mass cytometry, the Maxpar® Direct™ Immune Profiling Assay™ is a dry, metal-tagged antibody cocktail for immunophenotyping 37 immune cell populations found in human peripheral blood in a single tube. The Maxpar Direct Assay utilizes 31 mass channels for marker detection and live/dead viability staining, with at least 14 additional marker channels available from the Fluidigm catalog for flexible custom panel design. Here, we describe a workflow combining the assay with additional surface and intracellular cytokine antibodies for peripheral blood mononuclear cell (PBMC) staining using lanthanide-, bismuth-, and cadmium-tagged antibodies.

Mismatch Repair and Colon Cancer: Mechanisms and Therapies Explored.

Colorectal cancer (CRC) remains one of the most prevalent cancers worldwide. In sporadic CRC, mutations frequently occur in the DNA mismatch repair (MMR) pathway. In addition, germline MMR mutations have been linked to Lynch syndrome, the most common form of hereditary CRC. Although genetic mutations, diet, inflammation, and the gut microbiota can influence CRC, it is unclear how MMR deficiency relates to these factors to modulate disease. In this review, the association of MMR to the etiology of CRC is examined, particularly in the context of microRNAs (miRNAs), inflammation, and the microbiome. We also discuss the most current targeted therapies, methods of prevention, and molecular biomarkers against MMR-deficient CRC, all of which are encouraging advancements in the field.

Nfkb1 activation by the E26 transformation-specific transcription factors PU.1 and Spi-B promotes Toll-like receptor-mediated splenic B cell proliferation.

Generation of antibodies against T-independent and T-dependent antigens requires Toll-like receptor (TLR) engagement on B cells for efficient responses. However, the regulation of TLR expression and responses in B cells is not well understood. PU.1 and Spi-B (encoded by Sfpi1 and Spib, respectively) are transcription factors of the E26 transformation-specific (ETS) family and are important for B cell development and function. It was found that B cells from mice knocked out for Spi-B and heterozygous for PU.1 (Sfpi1(+/-) Spib(-/-) [PUB] mice) proliferated poorly in response to TLR ligands compared to wild-type (WT) B cells. The NF-κB family member p50 (encoded by Nfkb1) is required for lipopolysaccharide (LPS) responsiveness in mice. PUB B cells expressed reduced Nfkb1 mRNA transcripts and p50 protein. The Nfkb1 promoter was regulated directly by PU.1 and Spi-B, as shown by reporter assays and chromatin immunoprecipitation analysis. Occupancy of the Nfkb1 promoter by PU.1 was reduced in PUB B cells compared to that in WT B cells. Finally, infection of PUB B cells with a retroviral vector encoding p50 substantially restored proliferation in response to LPS. We conclude that Nfkb1 transcriptional activation by PU.1 and Spi-B promotes TLR-mediated B cell proliferation.