Bach2 Promotes B Cell Receptor-Induced Proliferation of B Lymphocytes and Represses Cyclin-Dependent Kinase Inhibitors.

BTB and CNC homology 2 (Bach2) is a transcriptional repressor that is required for the formation of the germinal center (GC) and reactions, including class switch recombination and somatic hypermutation of Ig genes in B cells, within the GC. Although BCR-induced proliferation is essential for GC reactions, the function of Bach2 in regulating B cell proliferation has not been elucidated. In this study, we demonstrate that Bach2 is required to sustain high levels of B cell proliferation in response to BCR signaling. Following BCR engagement in vitro, B cells from Bach2-deficient (Bach2-/-) mice showed lower incorporation of BrdU and reduced cell cycle progression compared with wild-type cells. Bach2-/- B cells also underwent increased apoptosis, as evidenced by an elevated frequency of sub-G1 cells and early apoptotic cells. Transcriptome analysis of BCR-engaged B cells from Bach2-/- mice revealed reduced expression of the antiapoptotic gene Bcl2l1 encoding Bcl-xL and elevated expression of cyclin-dependent kinase inhibitor (CKI) family genes, including Cdkn1a, Cdkn2a, and Cdkn2b Reconstitution of Bcl-xL expression partially rescued the proliferation defect of Bach2-/- B cells. Chromatin immunoprecipitation experiments showed that Bach2 bound to the CKI family genes, indicating that these genes are direct repression targets of Bach2. These findings identify Bach2 as a requisite factor for sustaining high levels of BCR-induced proliferation, survival, and cell cycle progression, and it promotes expression of Bcl-xL and repression of CKI genes. BCR-induced proliferation defects may contribute to the impaired GC formation observed in Bach2-/- mice.

Phosphorylation of BACH1 switches its function from transcription factor to mitotic chromosome regulator and promotes its interaction with HMMR.

The transcription repressor BACH1 performs mutually independent dual roles in transcription regulation and chromosome alignment during mitosis by supporting polar ejection force of mitotic spindle. We now found that the mitotic spindles became oblique relative to the adhesion surface following endogenous BACH1 depletion in HeLa cells. This spindle orientation rearrangement was rescued by re-expression of BACH1 depending on its interactions with HMMR and CRM1, both of which are required for the positioning of mitotic spindle, but independently of its DNA-binding activity. A mass spectrometry analysis of BACH1 complexes in interphase and M phase revealed that BACH1 lost during mitosis interactions with proteins involved in chromatin and gene expression but retained interactions with HMMR and its known partners including CHICA. By analyzing BACH1 modification using stable isotope labeling with amino acids in cell culture, mitosis-specific phosphorylations of BACH1 were observed, and mutations of these residues abolished the activity of BACH1 to restore mitotic spindle orientation in knockdown cells and to interact with HMMR. Detailed histological analysis of Bach1-deficient mice revealed lengthening of the epithelial fold structures of the intestine. These observations suggest that BACH1 performs stabilization of mitotic spindle orientation together with HMMR and CRM1 in mitosis, and that the cell cycle-specific phosphorylation switches the transcriptional and mitotic functions of BACH1.

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.

Genomewide approaches for BACH1 target genes in mouse embryonic fibroblasts showed BACH1-Pparg pathway in adipogenesis.

The transcription repressor BTB and CNC homology 1 (BACH1) represses genes involved in heme metabolism and oxidative stress response. BACH1 also suppresses the p53-dependent cellar senescence in primary mouse embryonic fibroblasts (MEFs). To investigate the role of BACH1 in MEF other than its known functions, we carried out a genomewide mapping of binding site for BACH1 and its heterodimer partner MAFK in immortalized MEFs (iMEFs) using chromatin immunoprecipitation and next-generation sequencing technology (ChIP-sequence). The comparative analysis of the ChIP-sequence data and DNA microarray data from Bach1-deficient and wild-type (WT) iMEF showed 35 novel candidate target genes of BACH1. Among these genes, five genes (Pparg, Nfia, Ptplad2, Adcy1 and Ror1) were related with lipid metabolism. Bach1-deficient iMEFs showed increased expression of mRNA and protein of PPARγ, which is the key factor of adipogenesis. These cells also showed a concomitant increase in ligand-dependent activation of PPARγ target genes compared with wild-type iMEFs. Moreover, Bach1-deficient iMEFs efficiently differentiated to adipocyte compared with wild-type cells in the presence of PPARγ ligands. Our results suggest that BACH1 regulates expression of adipocyte-related genes including Pparg and potentiates adipocyte differentiation capacity.

MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung.

In the lung, heme oxygenase-1 (HO-1) is developmentally regulated, with its highest expression in the first days of life. In addition, neonatal mice have limited HO-1 induction in hyperoxia compared with adults. However, few reports have addressed the functional effect of microRNAs (miRNAs) in the regulation of HO-1 in vivo. The aims of the present study were to characterize changes in lung miRNA expression during postnatal development and in response to hyperoxic exposure, and to identify miRNAs that target lung HO-1 gene expression. Neonatal (<12 h old) and adult (2 mo old) mice were exposed to room air or hyperoxia (95% oxygen) for 72 h. TaqMan low-density array rodent miRNA assays were used to calculate miRNA expression changes between control and hyperoxia groups in neonatal and adult lungs. In neonates, we identified miR-196a, which binds to the 3'-untranslated region of the transcriptional repressor BTB and CNC homology 1 (Bach1) and regulates its expression, and subsequently leads to higher levels of lung HO-1 mRNA compared with levels in adults. Despite the increase at baseline, miR-196a was degraded in hyperoxia resulting in limited HO-1 induction in neonatal mice lungs. Furthermore, the developmental differences in lung HO-1 gene expression can be explained in part by the variation in miRNA-196a and its effect on Bach1. This report is the first to show developmental differences in lung miR-196a and its effect on Bach1 and HO-1 expression at baseline and in hyperoxia.

BACH1 Promotes Pancreatic Cancer Metastasis by Repressing Epithelial Genes and Enhancing Epithelial-Mesenchymal Transition.

Pancreatic ductal adenocarcinoma (PDAC) is among the cancers with the poorest prognoses due to its highly malignant features. BTB and CNC homology 1 (BACH1) has been implicated in RAS-driven tumor formation. We focused on the role of BACH1 in PDAC, more than 90% of which have KRAS mutation. Knockdown of BACH1 in PDAC cell lines reduced cell migration and invasion, in part, by increasing E-cadherin expression, whereas its overexpression showed opposite effects. BACH1 directly repressed the expression of FOXA1 that is known to activate the expression of CDH1 encoding E-cadherin and to inhibit epithelial-to-mesenchymal transition. BACH1 also directly repressed the expression of genes important for epithelial cell adhesion including CLDN3 and CLDN4. In a mouse orthotopic implantation model, BACH1 was required for the high metastatic ability of AsPC-1 cells. IHC analysis of clinical specimens with a newly developed anti-BACH1 mAb revealed that high expression of BACH1 is a poor prognostic factor. These results suggest that the gene regulatory network of BACH1 and downstream genes including CDH1 contribute to the malignant features of PDAC by regulating epithelial-to-mesenchymal transition. SIGNIFICANCE: Greater understanding of the gene regulatory network involved in epithelial-to-mesenchymal transition of pancreatic cancer cells will provide novel therapeutic targets and diagnostic markers.

Bach1 deficiency and accompanying overexpression of heme oxygenase-1 do not influence aging or tumorigenesis in mice.

Oxidative stress contributes to both aging and tumorigenesis. The transcription factor Bach1, a regulator of oxidative stress response, augments oxidative stress by repressing the expression of heme oxygenase-1 (HO-1) gene (Hmox1) and suppresses oxidative stress-induced cellular senescence by restricting the p53 transcriptional activity. Here we investigated the lifelong effects of Bach1 deficiency on mice. Bach1-deficient mice showed longevity similar to wild-type mice. Although HO-1 was upregulated in the cells of Bach1-deficient animals, the levels of ROS in Bach1-deficient HSCs were comparable to those in wild-type cells. Bach1(-/-); p53(-/-) mice succumbed to spontaneous cancers as frequently as p53-deficient mice. Bach1 deficiency significantly altered transcriptome in the liver of the young mice, which surprisingly became similar to that of wild-type mice during the course of aging. The transcriptome adaptation to Bach1 deficiency may reflect how oxidative stress response is tuned upon genetic and environmental perturbations. We concluded that Bach1 deficiency and accompanying overexpression of HO-1 did not influence aging or p53 deficiency-driven tumorigenesis. Our results suggest that it is useful to target Bach1 for acute injury responses without inducing any apparent deteriorative effect.

Identification of senescence-associated genes and their networks under oxidative stress by the analysis of Bach1.

Cellular senescence is induced in response to DNA damage, caused by genotoxic stresses, including oxidative stress, and serves as a barrier against malignant transformation. Tumor-suppressor protein p53 induces genes critical for implementing cellular senescence. However, the identities of p53 target genes and other regulators that achieve senescence under oxidative stress remain to be elucidated. Effector genes for oxidative stress-induced cellular senescence were sought, based on the fact that transcription factor Bach1 inhibits this response by impeding the transcriptional activity of p53. pRb became hypophosphorylated more rapidly in Bach1-deficient MEFs than in wild-type cells, suggesting that pRb activation was involved in their senescence. Bach1-deficient MEFs bypassed the senescence state when the expression of a subset of p53 target genes, including p21, Pai1, Noxa, and Perp, was simultaneously reduced by using RNAi. Combined knockdown of p21 and pRb resulted in vigorous re-proliferation. These results suggest that oxidative stress-induced cellular senescence is registered by multiple p53 target genes, which arrest proliferation redundantly, in part by activating pRb. Our elucidations contrast with previous reports describing monopolistic regulations of senescence by single p53 target genes.