Feedback loops in B-cell chromatin regulation as a therapeutic target for modulating antibody production and malignancies: insights from BACH2.

Transgenic expression of BACH1 transcription factor results in megakaryocytic impairment

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.

The epithelial-mesenchymal transition (EMT) is a complex biological program that allows cancer cells to aquire motility and invasiveness. Transcriptional repressor Bach1 (BTB and CNC homology 1) belongs to cap'n'collar basic leucin zipper transcription factors family and represses transcription of stress-responsive genes such as heme oxygenase-1 (Hmox-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) by binding to the MARE (Maf recognition element) sequenceses in the regulatory regions of these genes. We have recently reported that Bach1 also recruites histon deacetylase-1 (HDAC1) to a subset of p53 target genes (Perp1, p21, Noxa, Puma, PAI-1) and contributes to the inhibition of p53-dependent cellular senescence (Dohi Y. et al. Nat Struct Mol Biol. 2008;15(12):1246–54). In the present study we addressed whether Bach1 regulated cells motility and invasiveness, contributing to EMT. First, we analyzed the phenotype of Bach1-deficient (Bach1−/−) mice. We found increased spontaneous motility of immortalized embryonic fibroblasts (MEF) derived from Bach1−/− mice in the wound-healing assay. To identify differential gene expression which might explain enhanced motility of Bach−/− MEFs, we compared gene expression profiles of Bach1−/− and wild type (WT) MEFs. We found that NF-кB essential modulator, NEMO (IKBKG) was highly expressed in Bach1−/− MEFs. Mouse genome database search revealed three MARE sites located in 10 kb upstream of the first exon of NEMO gene. We confirmed that Bach1 bound to these sites in ChIP and EMSA assays and also repressed transcription of the reporter gene construct containing MARE sequences from the NEMO promoter. DNA binding activity of NF-кB, measured by EMSA, and expression of NF-кB-dependent reporter construct were enhanced in Bach1−/− MEFs. To identify Bach1 target genes downstream of NF-кB, we compared mRNA expression profiles of the Bach1−/− and WT MEFs, treated with TNF-α. Genes encoding cytokines, chemokines and matrix metalloproteinases (IL-6, CXCL-1, CXCL-2, MMP2, MMP3, MT2-MMP) were up-regulated in Bach1−/− cells. Interestingly, IL-6 mRNA level changed earlier and more intensively than other factors; moreover, we detected higher level of IL-6 in the culture supernatants from Bach1−/− MEFs. Phoshorylation of STAT3, a target of IL-6 and a regulator of many MMPs and chemokines, was enhanced in Bach1−/− MEFs. RNAi targeting of both NEMO and IL-6 significantly redused motility of Bach1−/− MEFs indicating causative role of the both factors. Thus, Bach1 may regulate cells motility by fine-tuning the expression of chemokines and matrix metaloproteinases via cross-talk with the NF-кB signal transduction pathway in IL-6 dependent manner. Next, we addressed whether the lost of Bach1 in epithelia or stroma would activate EMT program in cancer cells. RNAi targeting of Bach1 in breast cancer cell line MCF-7 resulted in lower E-cadherin expression, reduced number of cell-to-cell, cell-to-substrate contacts and increased spontaneous motility. Co-culture with Bach1−/− but not WT MEFs induced transwell migration and basement membrane matrix invasion of MCF-7 cells. In conclusion, our data indicate that Bach1 regulates cell autonomous and non-autonomous induction of chemokines and extracellular matrix degrading enzymes, inhibiting motility, invasiveness and EMT. Citation Information: Cancer Res 2009;69(23 Suppl):B7.

Identification of genomic regions that control tissue-specific gene expression is currently problematic. ChIP and high-throughput sequencing (ChIP-seq) of enhancer-associated proteins such as p300 identifies some but not all enhancers active in a tissue. Here we show that co-occupancy of a chromatin region by multiple transcription factors (TFs) identifies a distinct set of enhancers. GATA-binding protein 4 (GATA4), NK2 transcription factor-related, locus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), here referred to as "cardiac TFs," have been hypothesized to collaborate to direct cardiac gene expression. Using a modified ChIP-seq procedure, we defined chromatin occupancy by these TFs and p300 genome wide and provided unbiased support for this hypothesis. We used this principle to show that co-occupancy of a chromatin region by multiple TFs can be used to identify cardiac enhancers. Of 13 such regions tested in transient transgenic embryos, seven (54%) drove cardiac gene expression. Among these regions were three cardiac-specific enhancers of Gata4, Srf, and swItch/sucrose nonfermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 (Smarcd3), an epigenetic regulator of cardiac gene expression. Multiple cardiac TFs and p300-bound regions were associated with cardiac-enriched genes and with functional annotations related to heart development. Importantly, the large majority (1,375/1,715) of loci bound by multiple cardiac TFs did not overlap loci bound by p300. Our data identify thousands of prospective cardiac regulatory sequences and indicate that multiple TF co-occupancy of a genomic region identifies developmentally relevant enhancers that are largely distinct from p300-associated enhancers.

BACH1 (BTB and CNC homology 1) is a basic region-leucine zipper (bZip) transcription factor that binds to MAREs (Maf recognition elements) and acts as a repressor of genes that regulate the oxidative stress response. It has been reported that BACH1 is a novel let-7 target and regulates breast cancer metastasis. Here we show that hypoxia induces BACH1 expression by regulating BACH1 promoter activity. In a mouse xenograft model, BACH1 depletion reduces the primary tumor growth of HCT-116 colorectal cancer cells. In addition, knockdown of BACH1 significantly decreases the invasive efficiency of colorectal cancer cells. Finally, we confirm that BACH1 expression is high in the patient samples of colorectal cancer and metastasis, unlike normal colon tissues. These results suggest BACH1 as a novel target for cancer therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3030. doi:1538-7445.AM2012-3030

Persistent Antigen and Germinal Center B Cells Sustain T Follicular Helper Cell Responses and Phenotype

Bach2 is a B cell-specific transcription repressor whose deficiency in mice causes a reduced class switch recombination and a reduced somatic hypermutation of immunoglobulin genes. Little is known about the direct target genes of Bach2 in B cells. By analyzing various B cell and plasma cell lines, we showed that the expression patterns of Bach2 and Blimp-1 (B lymphocyte-induced maturation protein 1), a master regulator of plasma cell differentiation, are mutually exclusive. The reporter gene of the Blimp-1 gene (Prdm1) was repressed by the overexpression of Bach2 in B cell lines. The heterodimer of Bach2/MafK bound to the Maf recognition element located upstream of the Prdm1 promoter in an electrophoretic mobility shift assay. The binding of MafK in B cells to the Prdm1 Maf recognition element was confirmed by chromatin immunoprecipitation assays. When MafK was purified from the BAL17 B cell line, a significant portion of it was present as a heterodimer with Bach2, with no apparent formation of MafK homodimer. These results strongly suggest that Bach2 represses the expression of Blimp-1 together with MafK in B cells prior to plasma cell differentiation. Accordingly, the knockdown of Bach2 mRNA using short hairpin RNA in BAL17 cells resulted in higher levels of Prdm1 expression after the stimulation of B cell receptor by surface IgM cross-linking. Induction of Prdm1 was more robust and faster in primary Bach2-deficient B cells than in wild-type control B cells upon lipopolysaccharide stimulation. Therefore, the Prdm1 regulation in B cells involves the repression by Bach2, which may be cancelled upon terminal plasma cell differentiation.

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

The human immunodeficiency virus, HIV-1, infects human CD4 T cells, including Th1 and Th2 helper cell subsets. The virus relies on host transcription factors, such as NFkB and NFAT for its transcription and expression. It has previously been shown that HIV-1 preferentially replicates in Th2 cells, but the mechanism of action for this finding remains unknown. c-maf is a Th2-restricted transcription factor that is critically important for differentiation along a Th2, but not Th1 lineage, and for transcription of the prototypic Th2 cytokine, IL-4. c-maf directly binds the proximal IL-4 promoter and acts synergistically with a neighboring NFAT site. We have demonstrated at the individual cell level that IL-4 positive cells (Th2) preferentially support HIV-1 replication compared to IFNg positive (Th1) cells. In studying the HIV-1 long terminal repeat (LTR)/promoter sequence, we identified a MARE (maf-recognition element) located just proximal (5') to the dual NFkB/NFAT binding sites. We show that the HIV-1 MARE binds recombinant maf protein and abuts NFAT binding as detected by DNase I in vitro footprinting. In addition, this HIV-1 MARE demonstrates identical mobility shifts compared to the IL-4 promoter MARE in gel-shift assays using nuclear extracts from activated primary human CD4 T cells. Using chromatin immunoprecipitation, we further show that c-maf binds to the HIV-1 LTR in vivo in HIV-1 infected primary human CD4 T cells. Although we have previously shown that both NFAT1 and NFAT2 are capable of transactivating the HIV-1 LTR, we now show for the first time preferential binding in vitro and in vivo of NFAT2 over NFAT1 to the HIV-1 LTR. By comparison, the more abundant NFAT1 family member preferentially binds to the IL-2 promoter. NFAT2 has previously been implicated in Th2 cytokine expression and appears to cooperate with c-maf in binding to the HIV-1 LTR. Functionally, over-expression of cmaf in primary human CD4 T cells cooperatively increases HIV-1 transcription when co-expressed with NFAT1 and 2, and silencing endogenous c-maf expression in primed human CD4 T cells decreases viral transcription. Similarly, over-expression of c-maf alone, or with NFAT1 or 2, increases HIV-1 replication, as measured by intracellular p24/gag expression, in CD4 T cells co-expressing GFP but not in GFP negative/c-maf-negative controls within the same transfected population. Lastly, depletion of c-maf expression by siRNA in primed and HIV-1 infected CD4 T cells decreases p24/gag expression. In summary, the Th2specific transcription factor, c-maf, binds the HIV-1 promoter in cooperation with NFAT transcription factors, primarily NFAT2, to augment HIV-1 transcription and replication in primary human CD4 T cells. These are the first data to mechanistically explain preferential HIV-1 transcription in IL-4 producing (Th2) cells. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes.

RNA Sequencing Data-Driven Dissection of Human Plasma Cell Differentiation Reveals New Potential Transcription Regulators

The induction mechanism of heme oxygenase-1 (HO-1) by heat shock (HS) is still unknown. Here, we discovered that HS activates the HO-1 expression in a mouse hepatoma cell line (Hepa 1-6). Knockdown experiments showed that the HS-induced HO-1 expression was dependent on HS factor 1 (HSF1). A chromatin immunoprecipitation (ChIP) assay demonstrated that the HS-activated HSF1 bound to the HS elements (HSEs) in the upstream enhancer 1 region (E1). Unexpectedly, HS also facilitates the BTB and CNC homology 1 (BACH1) binding to the Maf recognition elements (MAREs) in E1. We examined the effects of a catalytically inactive CRISPR-associated 9 nucleases (dCas9) with short guide RNAs (sgRNAs), and demonstrated that the HSF1 binding to HSEs in E1 was indispensable for the HS-induced HO-1 expression. Heme treatment (HA) dissociates BACH1 from MAREs and facilitated the binding of nuclear factor-erythroid-2-related factor 2 (NRF2) to MAREs. Following treatment with both HS and HA, the HO-1 induction and the HSF1 binding to HSEs in E1 were most notably observed. These results indicate that the HS-induced HO-1 expression is dependent on the HSF1 binding to HSEs in E1, although modulated by the BACH1 and NRF2 binding to MAREs within the same E1.

Using genome-wide approaches, we studied the microRNA (miRNA) expression profile during human plasma cell (PC) differentiation induced by stimulation of human blood B cells with T follicular helper cell–dependent signals. Combining the profiles of differentially expressed genes in PC differentiation with gene ontology (GO) analysis revealed that a significant group of genes involved in the transcription factor (TF) activity was preferentially changed. We thus focused on studying the effects of differentially expressed miRNAs on several key TFs in PC differentiation. Cohorts of differentially expressed miRNAs cooperating as miRNA hubs were predicted and validated to modulate key TFs, including a down-regulated miRNA hub containing miR-101-3p, -125b-5p, and -223-3p contributing to induction of PRDM1 as well as an up-regulated miRNA hub containing miR-34a-5p, -148a-3p, and -183-5p suppressing BCL6, BACH2, and FOXP1. Induced expression of NF-κB and PRDM1 during PC differentiation controlled the expression of up- and down-regulated miRNA hubs, respectively. Co-expression of miR-101-3p, -125b-5p, and -223-3p in stimulated B cells showed synergistic effects on inhibition of PC formation, which can be rescued by re-introduction of PRDM1. Together, we catalogue the complex roadmap of miRNAs and their functional interplay in collaboratively directing PC differentiation.

Zinc finger–IRF composite elements bound by Ikaros/IRF4 complexes function as gene repression in plasma cell

Small Maf transcription factors possess a basic region-leucine zipper motif through which they form homodimers or heterodimers with CNC and Bach proteins. Different combinations of small Maf and CNC/Bach protein dimers bind to cis-acting DNA elements, collectively referred to as Maf-recognition elements (MAREs), to either activate or repress transcription. As MAREs defined by function are often divergent from the consensus sequence, we speculated that sequence variations in the MAREs form the basis for selective Maf:Maf or Maf:CNC dimer binding. To test this hypothesis, we analyzed the binding of Maf-containing dimers to variant sequences of the MARE using bacterially expressed MafG and Nrf2 proteins and a surface plasmon resonance-microarray imaging technique. We found that base substitutions in the MAREs actually determined their binding preference for different dimers. In fact, we were able to categorize MAREs into five groups: MafG homodimer-orientd MAREs (Groups I and II), ambivalent MAREs (Group III), MafG:Nrf2 heterodimer-orientd MAREs (Group IV), and silent MAREs (Group V). This study thus manifests that a clear set of rules pertaining to the cis-acting element determine whether a given MARE preferentially associates with MafG homodimer or with MafG:Nrf2 heterodimer.