Anders Wallenius

B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins

Upon encountering antigens, B-lymphocytes can adapt to produce a highly specific and potent antibody response. Somatic hypermutation, which introduces point mutations in the variable regions of antibody genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both somatic hypermutation and CSR. The mutagenic AID enzyme has to be tightly controlled. Here, we show that engagement of the membrane-bound antibodies of the B-cell receptor (BCR), which signals that good antibody affinity has been reached, inhibits AID gene expression and that calcium (Ca2+) signaling is essential for this inhibition. Moreover, we show that overexpression of the Ca2+ sensor protein calmodulin inhibits AID gene expression, and that the transcription factor E2A is required for regulation of the AID gene by the BCR. E2A mutated in the binding site for calmodulin, and thus showing calmodulin-resistant DNA binding, makes AID expression resistant to the inhibition through BCR activation. Thus, BCR activation inhibits AID gene expression through Ca2+/calmodulin inhibition of E2A.

TRAF6 Stimulates the Tumor-Promoting Effects of TGFβ Type I Receptor Through Polyubiquitination and Activation of Presenilin 1

Preventing the cleavage of the TGFβ type I receptor by presenilin 1 could limit prostate tumor growth. Blocking Oncogenic Receptor Cleavage The surface receptor for transforming growth factor–β (TGFβ) can be cleaved, generating a fragment of the receptor containing the intracellular domain that enters the nucleus and mediates transcription of genes that encode tumor-promoting factors. Gudey et al. found that presenilin 1, a component of the γ-secretase complex, which cleaves various transmembrane proteins, also targeted the TGFβ receptor. TGFβ treatment increased the abundance and activity of presenilin 1 and the production of the receptor intracellular domain, which transcriptionally activated genes involved in invasion and metastasis. Treatment of mice bearing tumors formed from prostate cancer cells with a γ-secretase inhibitor decreased the production of the intracellular domain of the TGFβ receptor and tumor growth. Thus, blocking γ-secretase activity could attenuate the tumor-promoting effects of TGFβ signaling and decrease the growth of prostate tumors. Transforming growth factor–β (TGFβ) can be both a tumor promoter and suppressor, although the mechanisms behind the protumorigenic switch remain to be fully elucidated. The TGFβ type I receptor (TβRI) is proteolytically cleaved in the ectodomain region. Cleavage requires the combined activities of tumor necrosis factor (TNF) receptor–associated factor 6 (TRAF6) and TNF-α–converting enzyme (TACE). The cleavage event occurs selectively in cancer cells and generates an intracellular domain (ICD) of TβRI, which enters the nucleus to mediate gene transcription. Presenilin 1 (PS1), a γ-secretase catalytic core component, mediates intramembrane proteolysis of transmembrane receptors, such as Notch. We showed that TGFβ increased both the abundance and activity of PS1. TRAF6 recruited PS1 to the TβRI complex and promoted lysine-63–linked polyubiquitination of PS1, which activated PS1. Furthermore, PS1 cleaved TβRI in the transmembrane domain between valine-129 and isoleucine-130, and ICD generation was inhibited when these residues were mutated to alanine. We also showed that, after entering the nucleus, TβRI-ICD bound to the promoter and increased the transcription of the gene encoding TβRI. The TRAF6- and PS1-induced intramembrane proteolysis of TβRI promoted TGFβ-induced invasion of various cancer cells in vitro. Furthermore, when a mouse xenograft model of prostate cancer was treated with the γ-secretase inhibitor DBZ {(2S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-propionamide}, generation of TβRI-ICD was prevented, transcription of the gene encoding the proinvasive transcription factor Snail1 was reduced, and tumor growth was inhibited. These results suggest that γ-secretase inhibitors may be useful for treating aggressive prostate cancer.

Uracil-DNA Glycosylase in Base Excision Repair and Adaptive Immunity SPECIES DIFFERENCES BETWEEN MAN AND MOUSE

Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung+/+ and Ung−/− backcrossed mice. Interestingly, human cells displayed ∼15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was ∼8-fold higher in mouse cells, constituting ∼50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung−/− mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity.

Initiation of Antigen Receptor-Dependent Differentiation into Plasma Cells by Calmodulin Inhibition of E2A

Differentiation of B lymphocytes into Ab-secreting plasmablasts and plasma cells is Ag driven. The interaction of Ag with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. However, not much is known about the coupling between BCR activation and the shift in transcription factor network from that of a B cell to that of ASC differentiation. Our genome-wide analysis shows that Ab-secreting cell differentiation of mouse B cells is induced by BCR activation through very fast regulatory events from the BCR. We identify activation of IFN regulatory factor-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression as immediate early events. Furthermore, the transcription factor E2A is required for the rapid key down-regulations after BCR activation, and the Ca2+ sensor protein calmodulin has the corresponding regulatory effect as BCR activation. Moreover, mutants in the calmodulin binding site of E2A show that Ca2+ signaling through calmodulin inhibition of E2A is essential for the rapid down-regulation of immediate early genes after BCR activation in initiation of plasma cell differentiation.

Calmodulin inhibition of E2A stops expression of surrogate light chains of the pre-B-cell receptor and CD19

To create antibody diversity, B lymphocyte development is characterized by the ordered rearrangement of first immunoglobulin (Ig) heavy chain gene segments and then Ig light-chain gene segments. Early in B-cell development, expression of a pre-B-cell receptor (pre-BCR) composed of membrane-bound Ig heavy chain protein associated with surrogate light-chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces clonal expansion, but it also turns off transcription of the genes for the SLC proteins lambda5 and VpreB, which limits this proliferation. Here we show that signaling from the pre-BCR rapidly down-regulates lambda5 and VpreB and also the co-receptor CD19 in primary pre-B-cells. We show that calcium (Ca(2+)) signaling is essential for this silencing of the SLC and CD19 genes. The SLC genes are activated by the E2A transcription factor, and we show that E2A is required for pre-BCR-mediated regulation of the genes. E2A mutated in its binding site for the Ca(2+) sensor protein calmodulin, and thus with calmodulin-resistant DNA binding, makes lambda5, VpreB and CD19 expression resistant to the inhibition following pre-BCR activation. Thus, Ca(2+) down-regulates SLC and CD19 gene expression upon pre-BCR activation through inhibition of E2A by Ca(2+)/calmodulin.

Regulated intramembrane proteolysis of the TGFβ type I receptor conveys oncogenic signals

Cancer cells produce high levels of TGFβ, a multipotent cytokine. Binding of TGFβ to its cell surface receptors, the transmembrane serine/threonine kinases TβRII and TβRI, causes phosphorylation and activation of intracellular latent Smad transcription factors. Nuclear Smads act in concert with specific transcription factors to reprogram epithelial cells to become invasive mesenchymal cells. TGFβ also propagates non-canonical signals, so it is crucial to have a better understanding of the underlying molecular mechanisms which favor this pathway. Here we highlight our recent discovery that TGFβ promotes the proteolytic cleavage of TβRI in cancer cells, resulting in the liberation and nuclear translocation of its intracellular domain, acting as co-regulator to transcribe pro-invasive genes. This newly identified oncogenic TGFβ pathway resembles the Notch signaling pathway. We discuss our findings in relation to Notch and provide a short overview of other growth factors that transduce signals via nuclear translocation of their cell surface receptors.

Expression and recruitment of uracil-DNA glycosylase are regulated by E2A during antibody diversification.

B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID), which is positively regulated by the transcription factor E2A, is the key enzyme that initiates these processes by deaminating cytosine to uracil in Ig genes. Nuclear uracil-DNA glycosylase (UNG2) is subsequently required for uracil processing in the generation of high affinity antibodies of different isotypes. Here we show that the transcription factor E2A binds to the UNG2 promoter and represses UNG2 expression. Inhibition of E2A by binding of Ca(2+)-activated calmodulin alleviates this repression. Furthermore, we demonstrate that UNG2 preferentially accumulates in regions of the Ig heavy chain (IgH) gene containing AID hotspots. Calmodulin inhibition of E2A strongly enhances this UNG2 accumulation, indicating that it is negatively regulated by E2A as well. We show also that over-expression of E2A can suppress class switch recombination. The results suggest that E2A is a key factor in regulating the balance between AID and UNG2, both at expression and Ig targeting levels, to stimulate Ig diversification and suppress normal DNA repair processes.

Regulation of diversification and affinity maturation of antibodies

B-lymphocytes can modify their immunoglobulin (Ig) genes to generate antibodies with a new isotype and enhanced affinity. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates these processes. How somatic hypermutation (SH) and class switch recombination (CSR) are targeted and regulated to understand how we achieve good antibodies. The trans-acting factors mediating specific targeting of AID and thereby SH and CSR have remained elusive. How AID is recruited was still a big mystery. We show that mutant E2A transcription factor with defect inhibition by the Ca2+ sensor protein calmodulin results in reduced B cell receptor (BCR), IL4- plus CD40 ligand-stimulated CSR to IgE. AID is shown to be together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus in activated mouse splenic B cells. Calmodulin shows proximity with them after BCR stimulation. Direct protein-protein interactions are shown to enable formation of the complex. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks this reduction. Thus, E2A, AID, PAX5 and IRF4 are components of a CSR and SH complex that calmodulin binding redistributes on the Igh locus. We present also that initiation of antibody diversification leads to formation of a mutasome, a complex between many proteins that enable repair at high error rate of the uracils made by AID on Ig genes but not on most other genes. We show also that BCR activation, which signals end of successful SH, reduces interactions between some proteins in the complex and increases other interactions in the complex with varying kinetics. Furthermore, we show increased localization of SH and CSR coupled proteins on switch regions of the Igh locus upon SH/CSR and that BCR signaling differentially change the localization. Note: This work was presented in Joint Event on 22nd Edition of International Conference on Immunology and Evolution of Infectious Diseases & 12th Edition of International Conference on Tissue Engineering and Regenerative Medicine during May 10-11, 2018 Frankfurt, Germany