Masumichi Saito

BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal center B cells

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center formation and has been linked to lymphomagenesis. BCL6 functions by directly binding to specific DNA sequences and suppressing the transcription of target genes. Here we report an alternative mechanism by which BCL6 controls the transcription of genes lacking a BCL6 binding site and show that this mechanism was required for the prevention of tumor suppressor p53–independent cell cycle arrest in germinal center B cells. BCL6 interacted with the transcriptional activator Miz-1 and, via Miz-1, bound to the promoter and suppressed transcription of the cell cycle arrest gene CDKN1A. Through this mechanism, BCL6 may facilitate the proliferative expansion of germinal centers during the normal immune response and, when deregulated, the pathological expansion of B cell lymphomas.

Genome-wide Identification of Post-translational Modulators of Transcription Factor Activity in Human B-Cells

The ability of a transcription factor (TF) to regulate its targets is modulated by a variety of genetic and epigenetic mechanisms, resulting in highly context-dependent regulatory networks. However, high-throughput methods for the identification of proteins that affect TF activity are still largely unavailable. Here we introduce an algorithm, modulator inference by network dynamics (MINDy), for the genome-wide identification of post-translational modulators of TF activity within a specific cellular context. When used to dissect the regulation of MYC activity in human B lymphocytes, the approach inferred novel modulators of MYC function, which act by distinct mechanisms, including protein turnover, transcription complex formation and selective enzyme recruitment. MINDy is generally applicable to study the post-translational modulation of mammalian TFs in any cellular context. As such it can be used to dissect context-specific signaling pathways and combinatorial transcriptional regulation.

The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry.

After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. The molecular circuits that govern positive selection in the GC are not known. We show here that the GC reaction required biphasic regulation of expression of the cell-cycle regulator c-Myc that involved its transient induction during early GC commitment, its repression by Bcl-6 in DZ B cells and its reinduction in B cells selected for reentry into the DZ. Inhibition of c-Myc in vivo led to GC collapse, which indicated an essential role for c-Myc in GCs. Our results have implications for the mechanism of GC selection and the role of c-Myc in lymphomagenesis.Upon antigenic challenge, B cells enter the dark-zone (DZ) of germinal-centers (GC) to proliferate and hypermutate their immunoglobulin genes. Mutants with increased affinity are positively selected in the light-zone (LZ) to either differentiate into plasma and memory cells, or re-enter the DZ. The molecular circuits governing GC positive selection are not known. We show that the GC reaction requires the biphasic regulation of c-MYC expression, involving its transient induction during early GC commitment, its repression by BCL6 in DZ B cells, and its re-induction in B cells selected for DZ re-entry. Inhibition of MYC in vivo leads to GC collapse, indicating an essential role in GCs. These results have implications for the mechanism of GC selection and the role of MYC in lymphomagenesis.

Integrated biochemical and computational approach identifies BCL6 direct target genes controlling multiple pathways in normal germinal center B cells

BCL6 is a transcriptional repressor required for mature B-cell germinal center (GC) formation and implicated in lymphomagenesis. BCL6s physiologic function is only partially known because the complete set of its targets in GC B cells has not been identified. To address this issue, we used an integrated biochemical-computational-functional approach to identify BCL6 direct targets in normal GC B cells. This approach includes (1) identification of BCL6-bound promoters by genome-wide chromatin immunoprecipitation, (2) inference of transcriptional relationships by the use of a regulatory network reverse engineering approach (ARACNe), and (3) validation of physiologic relevance of the candidate targets down-regulated in GC B cells. Our approach demonstrated that a large set of promoters (> 4000) is physically bound by BCL6 but that only a fraction of them is repressed in GC B cells. This set of 1207 targets identifies several cellular functions directly controlled by BCL6 during GC development, including activation, survival, DNA-damage response, cell cycle arrest, cytokine signaling, Toll-like receptor signaling, and differentiation. These results define a broad role of BCL6 in preventing centroblasts from responding to signals leading to exit from the GC before they complete the phase of proliferative expansion and of antibody affinity maturation.

BCL6 suppression of BCL2 via Miz1 and its disruption in diffuse large B cell lymphoma

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center (GC) formation and whose deregulation by genomic lesions is implicated in the pathogenesis of GC-derived diffuse large B cell lymphoma (DLBCL) and, less frequently, follicular lymphoma (FL). The biological function of BCL6 is only partially understood because no more than a few genes have been functionally characterized as direct targets of BCL6 transrepression activity. Here we report that the anti-apoptotic proto-oncogene BCL2 is a direct target of BCL6 in GC B cells. BCL6 binds to the BCL2 promoter region by interacting with the transcriptional activator Miz1 and suppresses Miz1-induced activation of BCL2 expression. BCL6-mediated suppression of BCL2 is lost in FL and DLBCL, where the 2 proteins are pathologically coexpressed, because of BCL2 chromosomal translocations and other mechanisms, including Miz1 deregulation and somatic mutations in the BCL2 promoter region. These results identify an important function for BCL6 in facilitating apoptosis of GC B cells via suppression of BCL2, and suggest that blocking this pathway is critical for lymphomagenesis.

Genotoxic stress regulates expression of the proto-oncogene Bcl6 in germinal center B cells

Antigen-specific B cells are selected in germinal centers, the structure in which these cells proliferate while accomplishing genome-remodeling processes such as class-switch recombination and somatic hypermutation. These events are associated with considerable genotoxic stress, which cells tolerate through suppression of DNA-damage responses by Bcl-6, a transcription factor required for the formation of germinal centers. Here we show that the expression of Bcl-6 is regulated by DNA damage through a signaling pathway that promotes Bcl-6 degradation. After DNA damage accumulated, the kinase ATM promoted Bcl-6 phosphorylation, leading to its interaction with the isomerase Pin1 and its degradation by the ubiquitin-proteasome system. Because Bcl-6 is required for the maintenance of germinal centers, our findings suggest that the extent of genotoxic stress controls the fate of germinal center B cells by means of Bcl-6.

Small Molecule Inhibition of HIV-1–Induced MHC-I Down-Regulation Identifies a Temporally Regulated Switch in Nef Action

Nef assembles a multi-kinase complex triggering MHC-I down-regulation. We identify an inhibitor that blocks MHC-I down-regulation, identifying a temporally regulated switch in Nef action from directing MHC-I endocytosis to blocking cell surface delivery. These findings challenge current dogma and reveal a regulated immune evasion program.

Multilayered defense in HLA-B51-associated HIV viral control

Polymorphism in the HLA region of a chromosome is the major source of host genetic variability in HIV-1 outcome, but there is limited understanding of the mechanisms underlying the beneficial effect of protective class I alleles such as HLA-B57, -B27, and -B51. Taking advantage of a unique cohort infected with clade B’ HIV-1 through contaminated blood, in which many variables such as the length of infection, the infecting viral strain, and host genetic background are controlled, we performed a comprehensive study to understand HLA-B51–associated HIV-1 control. We focused on the T cell responses against three dominant HLA-B51–restricted epitopes: Gag327-345(NI9) NANPDCKTI, Pol743-751(LI9) LPPVVAKEI, and Pol283-289(TI8) TAFTIPSI. Mutations in all three dominant epitopes were significantly associated with HLA-B51 in the cohort. A clear hierarchy in selection of epitope mutations was observed through epitope sequencing. L743I in position 1 of epitope LI9 was seen in most B51+ individuals, followed by V289X in position 8 of the TI8, and then, A328S, in position 2 of the NI9 epitope, was also seen in some B51+ individuals. Good control of viral load and higher CD4+ counts were significantly associated with at least one detectable T cell response to unmutated epitopes, whereas lower CD4+ counts and higher viral loads were observed in patients who had developed escape mutations in all three epitopes or who lacked T cell responses specific to these epitope(s). We propose that patients with HLA-B51 benefit from having multiple layers of effective defense against the development of immune escape mutations.

Unbiased Analysis of TCRα/β Chains at the Single-Cell Level in Human CD8+ T-Cell Subsets

T-cell receptor (TCR) α/β chains are expressed on the surface of CD8+ T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5′-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8+ subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8+ subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8+ T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains.

Minimum length of homology arms required for effective Red/ET recombination.

The original protocol of Red/ET recombination requires 50-bp sequence homology with target vector on both sides of the DNA fragment. To make it more cost effective, we investigated to determine the minimal length of homology required for the system to work. We found that a homology of 9-bp was sufficient to achieve homologous recombination with more than 50% efficiency.