Transient transfection

WEHI-279 MIGR1, MIG-3XFLAG-Spi-B, and MIG-3XFLAG-Spi-C cells were generated as previously described (DeKoter et al, 2010; Xu et al, 2012). Cells in mid to early log phase growth were washed three times with serum-free DMEM (4.5g/L, Wisent Inc, St-Bruno, Quebec) and incubated for 10 minutes at room temperature with 10 μg of each luciferase reporter plasmid and 1 μg of pRL-TK (Promega). Cell-DNA mixtures were electroporated at 220 V and 950 mF using 4-mm gap cuvettes with a GenePulser II with Capacitance Extender (Bio-Rad), incubated at room temperature for 10 min, then transferred to 6-well culture plates in complete DMEM and incubated at 37°C for 24 hours. A Dual-Luciferase Reporter Assay System (Promega) was performed on cell lysates. Light production was measured using a Lumat LB 9507 tube luminometer (Berthold Technologies, Oak Ridge, TN).

3.4.9

ChIP experiments

Chromatin was prepared from enriched B cells as previously described (Sokalski et al, 2011; Xu et al, 2012). In brief, cells were cross-linked and lysed followed by sonication. Sonicated chromatin was incubated overnight at 4°C with anti-Spi-C (generated by Fulkerson lab) and anti-FLAG (M2, Sigma-Aldrich) conjugated to protein G DynaBeads (Invitrogen). Magnetic bead complexes were enriched using a MagneSphere® Technology Magnetic Separation Stand (Promega) and washed. Immunocomplexes were eluted and cross-linked chromatin was reversed overnight at 65°C. DNA was purified using a QIAquick PCR purification kit (Qiagen, Limburg, Netherlands). Enrichment was measured using qPCR of immunoprecipitated DNA using primers indicated in Table EI.

3.4.10 ChIP-Seq Analysis

ChIP-seq experiments were performed using mouse WEHI-279 cells. Cells were spin- infected with a MIGR1 vector for expression of 3XFLAG-PU.1 or 3XFLAG-Spi-B. Immunoprecipitation was performed using anti-FLAG microbeads (M2, Sigma-Aldrich). Purified chromatin was sequenced on an Ilimuna HiSeq at the BC cancer research

agency. Peak finding and data analysis was performed using Galaxy Suite (Blankenberg et al, 2010; Giardine et al, 2005; Goecks et al, 2010). Reads were aligned to the reference genome (NCBI37/mm9), using BOWTIE (Langmead et al, 2009). Peaks were called using MACS version 1.0.1 (Zhang et al, 2008) with default parameters, a P-value cutoff for peak detection of 1e-05 and an effective genome size of 2.7e+9 bp. The ChIP-seq data from this publication have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) and assigned the identifier GSE48891 and GSE58128.

3.4.11 Statistical analysis

Statistical significance was determined using one-way or two-way ANOVA analysis with a Bonferroni post-test unless otherwise indicated. P values ≤0.05 were considered significant. For all figures, *P ≤0.05, **P ≤0.01, and ***≤P 0.001. Statistical analysis was performed using Prism 5.0 (Graph-Pad Software).

Chapter 4

4

Overall discussion and future directions

This thesis describes the ability of the transcription factors PU.1, Spi-B, and Spi- C to control B cell development and proliferative function. The hypothesis that PU.1 and Spi-B were required for positively regulating components of TLR responses was tested. In chapter 2, B cells from PUB (Spi1+/-Spib-/-) micewere shown to be impaired for TLR- mediated proliferation responses. The impairment in PUB B cells was attributed to reduced levels of p50 protein, which is encoded by the Nfkb1 gene. It was demonstrated that both PU.1 and Spi-B are capable of directly binding to the Nfkb1 promoter, and the ETS binding sites within the promoter were required for efficient transcription of the gene. Impaired TLR-mediated proliferation was restored upon retroviral reinsertion of p50 into PUB B cells. These results suggest that Nfkb1 activation by PU.1 and Spi-B is important for TLR-initiated B cell responses.

Chapter 3 characterized the role of Spi-C in B cells. The hypothesis that Spi-C inhibited target genes of PU.1 and Spi-B was tested. By generating the novel mouse model Spib-/-Spic+/-and comparing it to Spib-/- mice, it was determined that Spi-C has a functionally distinct role compared to Spi-B. Spi-C heterozygosity restored many aspects of the Spib-/- phenotype, including a restoration in FO and T2 B cell numbers in the spleen, and restored LPS and anti-IgM-mediated B cell proliferation. Nfkb1 was hypothesized to participate in the mechanism for the Spib-/-Spic+/- phenotype. Transcript levels of Nfkb1 were elevated in Spib-/-Spic+/- B cells compared to Spib-/- B cells. In B cell lines overexpressing Spi-B, Nfkb1 promoter activity was substantially elevated; however, Nfkb1 promoter activity was significantly reduced in B cell lines overexpressing Spi-C. It was demonstrated that Spi-C directly bound to the same sites as PU.1 and Spi-B at the Nfkb1 promoter. Therefore, Spi-C directly opposes Spi-B transcriptional regulation in B cells.

These studies have identified a mechanism by which PU.1, Spi-B, and Spi-C regulate development and proliferative function in B cells. PU.1 and Spi-B are capable of each individually binding directly to the Nfkb1 promoter to activate its expression (Figure

4.1A). In contrast, Spi-C directly binds to the same Nfkb1 promoter region and represses Nfkb1 activation (Figure 4.1B). Since all three transcription factors can bind to the same regions, we hypothesize that Spi-C can function to compete with PU.1 and Spi-B for binding at the same sites within the Nfkb1 promoter. However, the mechanism of activation/repression through direct competition for binding can be complex.

There are many aspects of transcriptional regulation which can be addressed in the future. Repression of Nfkb1 by Spi-C could be mediated through recruitment of other transcription factors or histone modifying proteins which affect the accessibility of the Nfkb1 promoter. Likewise, the activation of Nfkb1 by PU.1 and Spi-B can involve interactions with distal enhancer sites to recruit additional transcription factors to activate gene promoters or modulate chromatin accessibility. A high-throughput method of ChIP- mass spectrometry could identify the plethora of interacting proteins recruited by PU.1, Spi-B, or Spi-C (Wang et al, 2013). Furthermore, using chromosome conformation capture (3C) assays would elucidate the interactions between distal regulatory enhancers or insulators (Hagege et al, 2007). Overall, determining what transcription factors or associated proteins are recruited by PU.1, Spi-B, and Spi-C could improve our understanding of transcriptional regulatory networks.

Figure 4.1. A model of transcriptional regulation of Nfkb1 by PU.1, Spi-B, and Spi-C. (A) PU.1 and Spi-B directly bind to the Nkfb1 promoter to activate transcription. (B) Spi- C can directly bind to the same Nkfb1 promoter region to inhibit its transcription.