TANK Binding Kinase 1 Promotes BACH1 Degradation through Both Phosphorylation-Dependent and -Independent Mechanisms without Relying on Heme and FBXO22.

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.

Accelerated plasma-cell differentiation in Bach2-deficient mouse B cells is caused by altered IRF4 functions.

Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.

BACH1 Expression Is Promoted by Tank Binding Kinase 1 (TBK1) in Pancreatic Cancer Cells to Increase Iron and Reduce the Expression of E-Cadherin.

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species and promotes metastasis of various cancers including pancreatic ductal adenocarcinoma (PDAC). However, it is not clear how BACH1 is regulated in PDAC cells. Knockdown of Tank binding kinase 1 (TBK1) led to reductions of BACH1 mRNA and protein amounts in AsPC-1 human PDAC cells. Gene expression analysis of PDAC cells with knockdown of TBK1 or BACH1 suggested the involvement of TBK1 and BACH1 in the regulation of iron homeostasis. Ferritin mRNA and proteins were both increased upon BACH1 knockdown in AsPC-1 cells. Flow cytometry analysis showed that AsPC-1 cells with BACH1 knockout or knockdown contained lower labile iron than control cells, suggesting that BACH1 increased labile iron by repressing the expression of ferritin genes. We further found that the expression of E-cadherin was upregulated upon the chelation of intracellular iron content. These results suggest that the TBK1-BACH1 pathway promotes cancer cell metastasis by increasing labile iron within cells.

Chromatin Protein PC4 Orchestrates B Cell Differentiation by Collaborating with IKAROS and IRF4.

The chromatin protein positive coactivator 4 (PC4) has multiple functions, including chromatin compaction. However, its role in immune cells is largely unknown. We show that PC4 orchestrates chromatin structure and gene expression in mature B cells. B-cell-specific PC4-deficient mice show impaired production of antibody upon antigen stimulation. The PC4 complex purified from B cells contains the transcription factors (TFs) IKAROS and IRF4. IKAROS protein is reduced in PC4-deficient mature B cells, resulting in de-repression of their target genes in part by diminished interactions with gene-silencing components. Upon activation, the amount of IRF4 protein is not increased in PC4-deficient B cells, resulting in reduction of plasma cells. Importantly, IRF4 reciprocally induces PC4 expression via a super-enhancer. PC4 knockdown in human B cell lymphoma and myeloma cells reduces IKAROS protein as an anticancer drug, lenalidomide. Our findings establish PC4 as a chromatin regulator of B cells and a possible therapeutic target adjoining IKAROS in B cell malignancies.

Transcription Factor IRF8 Governs Enhancer Landscape Dynamics in Mononuclear Phagocyte Progenitors.

Monocytes and dendritic cells (DCs), mononuclear phagocytes essential for immune responses, develop from hematopoietic stem cells via monocyte-DC progenitors (MDPs). The molecular basis of their development remains unclear. Because promoter-distal enhancers are key to cell fate decisions, we analyzed enhancer landscapes during mononuclear phagocyte development in vivo. Monocyte- and DC-specific enhancers were gradually established at progenitor stages before the expression of associated genes. Of the transcription factors predicted to bind to these enhancers, IRF8, essential for monocyte and DC development, was found to be required for the establishment of these enhancers, particularly those common to both monocyte and DC lineages. Although Irf8-/- mononuclear phagocyte progenitors, including MDPs, displayed grossly normal gene expression patterns, their enhancer landscapes resembled that of an upstream progenitor population. Our results illustrate the dynamic process by which key transcription factors regulate enhancer formation and, therefore, direct future gene expression to achieve mononuclear phagocyte development.

S-Adenosylmethionine Synthesis Is Regulated by Selective N6-Adenosine Methylation and mRNA Degradation Involving METTL16 and YTHDC1.

S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N6-methyladenosine (m6A) in the 3' UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3' UTR. Knockdown of METTL16 and the m6A reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3' UTR revealed that these m6As were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.

A Bach2-Cebp Gene Regulatory Network for the Commitment of Multipotent Hematopoietic Progenitors.

Hematopoietic stem cell and multipotent progenitor (MPP) commitment can be tuned in response to an infection so that their differentiation is biased toward myeloid cells. Here, we find that Bach2, which inhibits myeloid differentiation in common lymphoid progenitors, represses a cohort of myeloid genes and activates those linked to lymphoid function. Bach2 repressed both Cebpb and its target Csf1r, encoding C/EBPß and macrophage colony-stimulating factor receptor (M-CSFr), respectively, whereas C/EBPß repressed Bach2 and activated Csf1r. Bach2 and C/EBPß further bound to overlapping regulatory regions at their myeloid target genes, suggesting the presence of a gene regulatory network (GRN) with mutual repression between these factors and a feedforward loop leading to myeloid gene regulation. Lipopolysaccharide reduced the expression of Bach2, resulting in enhanced myeloid differentiation. The Bach2-C/EBPß GRN pathway thus tunes MPP commitment to myeloid and lymphoid lineages both under normal conditions and after infection.

Epigenetic Regulation of the Blimp-1 Gene (Prdm1) in B Cells Involves Bach2 and Histone Deacetylase 3.

B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation. The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports reactions such as class switch recombination of the antibody genes. We found that histones H3 and H4 around the Prdm1 intron 5 Maf recognition element were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its expression. Conversely, methylation of H3-K9 was lower in X63/0 cells than BAL17 cells. Purification of the Bach2 complex in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin ß-like 1X-linked (Tbl1x), and RAP1-interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding of HDAC3 and Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased Prdm1 mRNA expression. Bach2 is suggested to cooperate with HDAC3-containing co-repressor complexes in B cells to regulate the stage-specific expression of Prdm1 by writing epigenetic modifications at the Prdm1 locus.

The Transcription Factor Bach2 Is Phosphorylated at Multiple Sites in Murine B Cells but a Single Site Prevents Its Nuclear Localization.

The transcription factor Bach2 regulates the immune system at multiple points, including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells are still unclear. Here, we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of the B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in the mature B cell line BAL17. Among the sites identified, serine 535 (Ser-535) was critical for the regulation of Bach2 because a single mutation of Ser-535 abolished cytoplasmic accumulation of Bach2, promoting its nuclear accumulation in pre-B cells, whereas Ser-509 played an auxiliary role. Bach2 repressor activity was enhanced by the Ser-535 mutation in B cells. These results suggest that the PI3K-Akt-mTOR pathway inhibits Bach2 by both repressing its expression and inducing its phosphorylation in B cells.

Orchestration of plasma cell differentiation by Bach2 and its gene regulatory network.

Bach2 is a basic region-leucine zipper (bZip) transcription factor that forms heterodimers with small Maf oncoproteins and binds to target genes, thus repressing their expression. Bach2 is required for class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes in activated B cells. Bach2 represses the expression of Prdm1 encoding Blimp-1 repressor and thereby inhibits terminal differentiation of B cells to plasma cells. This causes a delay in the induction of Prdm1, thereby securing a time window for the expression of Aicda encoding activation-induced cytidine deaminase (AID) required for both CSR and SHM. Based on the characteristics of a gene regulatory network (GRN) involving Bach2 and Prdm1 and its dynamics, a 'delay-driven diversity' model was introduced to explain the responses of activated B cells. Bach2 is also required for the proper differentiation and function of peripheral T cells. In the absence of Bach2, CD4(+) T cells show increased differentiation to effector cells producing higher levels of Th2-related cytokines, such as IL-4 and IL-10, and a reduction in the generation of regulatory T cells. Bach2 represses many genes in T cells, including Prdm1, suggesting that the Bach2-Prdm1 pathway is also important in maintaining the homeostasis of T cells. Furthermore, Bach2 is essential for the function of alveolar macrophages. Therefore, Bach2 orchestrates both acquired and innate immunity at multiple points. Its connection with disease is also reviewed in this report.

Orchestrating B cell lymphopoiesis through interplay of IL-7 receptor and pre-B cell receptor signalling.

The development of B cells is dependent on the sequential DNA rearrangement of immunoglobulin loci that encode subunits of the B cell receptor. The pathway navigates a crucial checkpoint that ensures expression of a signalling-competent immunoglobulin heavy chain before commitment to rearrangement and expression of an immunoglobulin light chain. The checkpoint segregates proliferation of pre-B cells from immunoglobulin light chain recombination and their differentiation into B cells. Recent advances have revealed the molecular circuitry that controls two rival signalling systems, namely the interleukin-7 (IL-7) receptor and the pre-B cell receptor, to ensure that proliferation and immunoglobulin recombination are mutually exclusive, thereby maintaining genomic integrity during B cell development.

Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4.

The transcription factor IRF4 regulates immunoglobulin class switch recombination and plasma cell differentiation. Its differing concentrations appear to regulate mutually antagonistic programs of B and plasma cell gene expression. We show IRF4 to be also required for generation of germinal center (GC) B cells. Its transient expression in vivo induced the expression of key GC genes including Bcl6 and Aicda. In contrast, sustained and higher concentrations of IRF4 promoted the generation of plasma cells while antagonizing the GC fate. IRF4 cobound with the transcription factors PU.1 or BATF to Ets or AP-1 composite motifs, associated with genes involved in B cell activation and the GC response. At higher concentrations, IRF4 binding shifted to interferon sequence response motifs; these enriched for genes involved in plasma cell differentiation. Our results support a model of "kinetic control" in which signaling-induced dynamics of IRF4 in activated B cells control their cell-fate outcomes.

A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation.

The molecular crosstalk between the interleukin 7 receptor (IL-7R) and the precursor to the B cell antigen receptor (pre-BCR) in B lymphopoiesis has not been elucidated. Here we demonstrate that in pre-B cells, the IL-7R but not the pre-BCR was coupled to phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt; signaling by this pathway inhibited expression of recombination-activating gene 1 (Rag1) and Rag2. Attenuation of IL-7 signaling resulted in upregulation of the transcription factors Foxo1 and Pax5, which coactivated many pre-B cell genes, including Rag1, Rag2 and Blnk. Induction of Blnk (which encodes the signaling adaptor BLNK) enabled pre-BCR signaling via the signaling molecule Syk and promoted immunoglobulin light-chain rearrangement. BLNK expression also antagonized Akt activation, thereby augmenting the accumulation of Foxo1 and Pax5. This self-reinforcing molecular circuit seemed to sense limiting concentrations of IL-7 and functioned to constrain the proliferation of pre-B cells and trigger their differentiation.

Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6.

B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.

Architecture and dynamics of the transcription factor network that regulates B-to-plasma cell differentiation.

Upon antigen stimulation, B lymphoid cells undergo terminal differentiation into antibody-secreting plasma cells. This process accompanies drastic changes in cell functions such as a loss of B-cell identity, induction of secretory apparatus, and an extremely increased transcription of antibody genes. These changes are the result of re-wiring of a transcription factor network in B and plasma cells. While the transcription repressor Blimp-1 induces plasma cell differentiation, another repressor Bach2 has emerged as a negative regulator of Blimp-1 in B cells. These two transcription factors, together with other several factors, appear to constitute a main transcriptional regulatory network for the terminal differentiation process of plasma cells from B cells.