Evidence of Adaptive Evolution in Wolbachia-Regulated Gene DNMT2 and Its Role in the Dipteran Immune Response and Pathogen Blocking.

Eukaryotic nucleic acid methyltransferase (MTase) proteins are essential mediators of epigenetic and epitranscriptomic regulation. DNMT2 belongs to a large, conserved family of DNA MTases found in many organisms, including holometabolous insects such as fruit flies and mosquitoes, where it is the lone MTase. Interestingly, despite its nomenclature, DNMT2 is not a DNA MTase, but instead targets and methylates RNA species. A growing body of literature suggests that DNMT2 mediates the host immune response against a wide range of pathogens, including RNA viruses. Curiously, although DNMT2 is antiviral in Drosophila, its expression promotes virus replication in mosquito species. We, therefore, sought to understand the divergent regulation, function, and evolution of these orthologs. We describe the role of the Drosophila-specific host protein IPOD in regulating the expression and function of fruit fly DNMT2. Heterologous expression of these orthologs suggests that DNMT2's role as an antiviral is host-dependent, indicating a requirement for additional host-specific factors. Finally, we identify and describe potential evidence of positive selection at different times throughout DNMT2 evolution within dipteran insects. We identify specific codons within each ortholog that are under positive selection and find that they are restricted to four distinct protein domains, which likely influence substrate binding, target recognition, and adaptation of unique intermolecular interactions. Collectively, our findings highlight the evolution of DNMT2 in Dipteran insects and point to structural, regulatory, and functional differences between mosquito and fruit fly homologs.

DNMT3A haploinsufficiency causes dichotomous DNA methylation defects at enhancers in mature human immune cells.

DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations.

Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection.

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin.

Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation.

Foxp3+ regulatory T (Treg) cells restrict immune pathology in inflamed tissues; however, an inflammatory environment presents a threat to Treg cell identity and function. Here, we establish a transcriptional signature of central nervous system (CNS) Treg cells that accumulate during experimental autoimmune encephalitis (EAE) and identify a pathway that maintains Treg cell function and identity during severe inflammation. This pathway is dependent on the transcriptional regulator Blimp1, which prevents downregulation of Foxp3 expression and "toxic" gain-of-function of Treg cells in the inflamed CNS. Blimp1 negatively regulates IL-6- and STAT3-dependent Dnmt3a expression and function restraining methylation of Treg cell-specific conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Consequently, CNS2 is heavily methylated when Blimp1 is ablated, leading to a loss of Foxp3 expression and severe disease. These findings identify a Blimp1-dependent pathway that preserves Treg cell stability in inflamed non-lymphoid tissues.

B cell activation and plasma cell differentiation are inhibited by de novo DNA methylation.

B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cellular division and is linked to DNA hypomethylation. Conversely, little is known about how de novo deposition of DNA methylation affects B cell fate and function. Here we show that genetic deletion of the de novo DNA methyltransferases Dnmt3a and Dnmt3b (Dnmt3-deficient) in mouse B cells results in normal B cell development and maturation, but increased cell activation and expansion of the germinal center B cell and plasma cell populations upon immunization. Gene expression is mostly unaltered in naive and germinal center B cells, but dysregulated in Dnmt3-deficient plasma cells. Differences in gene expression are proximal to Dnmt3-dependent DNA methylation and chromatin changes, both of which coincide with E2A and PU.1-IRF composite-binding motifs. Thus, de novo DNA methylation limits B cell activation, represses the plasma cell chromatin state, and regulates plasma cell differentiation.

Hyperleukocytosis is associated with distinct genetic alterations and is an independent poor-risk factor in de novo acute myeloid leukemia patients.

OBJECTIVES: Acute myeloid leukemia (AML) with hyperleukocytosis (HL) is intuitively thought as a unique group with dismal prognosis. However, comprehensive studies regarding the genetic landscape and clinical outcome in this group of patients are limited. METHODS: A total of 693 newly diagnosed de novo non-M3 AML patients were consecutively enrolled. We compared relevant mutations in 20 genes between AML patients with or without HL and exposed their prognostic implications. RESULTS: Hyperleukocytosis, defined as initial white blood cell counts above 50 000/µL, occurred in 28.9% of AML patients. HL patients had higher incidences of FLT3-ITD, NPM1, DNMT3A, CEBPA, and TET2 mutations. Multivariate analysis demonstrated that HL was an independent poor prognostic factor for overall survival and disease-free survival in total patients, those with intermediate-risk cytogenetics and normal karyotype irrespective of genetic alterations. Intriguingly, HL predicted poor survival in CEBPA double mutated, NPM1 + /FLT3-ITD- and NPM1-/FLT3-ITD- patients. Further, HL patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR) had a significantly longer overall survival and disease-free survival than those without allo-HSCT. CONCLUSIONS: Hyperleukocytosis is an independent poor prognostic factor irrespective of cytogenetics and mutation status. Allo-HSCT in first CR seems to ameliorate the poor prognostic impact of HL.

Prostaglandin E2 Leads to the Acquisition of DNMT3A-Dependent Tolerogenic Functions in Human Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) and dendritic cells (DCs) arise from common progenitors. Tumor-derived factors redirect differentiation from immune-promoting DCs to tolerogenic MDSCs, an immunological hallmark of cancer. Indeed, in vitro differentiation of DCs from human primary monocytes results in the generation of MDSCs under tumor-associated conditions (PGE2 or tumor cell-conditioned media). Comparison of MDSC and DC DNA methylomes now reveals extensive demethylation with specific gains of DNA methylation and repression of immunogenic-associated genes occurring in MDSCs specifically, concomitant with increased DNA methyltransferase 3A (DNMT3A) levels. DNMT3A downregulation erases MDSC-specific hypermethylation, and it abolishes their immunosuppressive capacity. Primary MDSCs isolated from ovarian cancer patients display a similar hypermethylation signature in connection with PGE2-dependent DNMT3A overexpression. Our study links PGE2- and DNMT3A-dependent hypermethylation with immunosuppressive MDSC functions, providing a promising target for therapeutic intervention.

T Cell Exhaustion: An Epigenetically Imprinted Phenotypic and Functional Makeover.

A recent article in Cell demonstrates that the absence of a single DNA methyltransferase, Dnmt3a, prevents cytotoxic T cells from acquiring the hypofunctional or exhausted phenotype typically seen in chronic viral infections and tumors. Upon establishing a causal relationship between exhaustion-associated epigenetic changes and reduced CD8+ T cell function, the authors provided mechanistic evidence that exhaustion constitutes a specific differentiation program.

ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing.

Hypomorphic mutations in DNA-methyltransferase DNMT3B cause majority of the rare disorder Immunodeficiency, Centromere instability and Facial anomalies syndrome cases (ICF1). By unspecified mechanisms, mutant-DNMT3B interferes with lymphoid-specific pathways resulting in immune response defects. Interestingly, recent findings report that DNMT3B shapes intragenic CpG-methylation of highly-transcribed genes. However, how the DNMT3B-dependent epigenetic network modulates transcription and whether ICF1-specific mutations impair this process remains unknown. We performed a transcriptomic and epigenomic study in patient-derived B-cell lines to investigate the genome-scale effects of DNMT3B dysfunction. We highlighted that altered intragenic CpG-methylation impairs multiple aspects of transcriptional regulation, like alternative TSS usage, antisense transcription and exon splicing. These defects preferentially associate with changes of intragenic H3K4me3 and at lesser extent of H3K27me3 and H3K36me3. In addition, we highlighted a novel DNMT3B activity in modulating the self-regulatory circuit of sense-antisense pairs and the exon skipping during alternative splicing, through interacting with RNA molecules. Strikingly, altered transcription affects disease relevant genes, as for instance the memory-B cell marker CD27 and PTPRC genes, providing us with biological insights into the ICF1-syndrome pathogenesis. Our genome-scale approach sheds light on the mechanisms still poorly understood of the intragenic function of DNMT3B and DNA methylation in gene expression regulation.

Non-small-cell lung cancer-induced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation.

Patients with non-small-cell lung cancer (NSCLC) have immune defects that are poorly understood. Forkhead box protein P3 (Foxp3) is crucial for immunosuppression by CD4(+) regulatory T cells (Tregs). It is not well known how NSCLC induces Foxp3 expression and causes immunosuppression in tumor-bearing patients. Our study found a higher percentage of CD4(+) Tregs in the peripheral blood of NSCLC compared with healthy donors. NSCLC patients showed demethylation of eight CpG sites within the Foxp3 promoter with methylation ratios negatively correlated with CD4(+)CD25(+)Foxp3(+) T levels. Foxp3 expression in CD4(+) Tregs was directly regulated by Foxp3 promoter demethylation and was involved in immunosuppression by NSCLC. To verify the effect of tumor cells on the phenotype and function of CD4(+) Tregs, we established a coculture system using NSCLC cell line and healthy CD4(+) T cells and showed that SPC-A1 induced IL-10 and TGF-ß1 secretion by affecting the function of CD4(+) Tregs. The activity of DNA methyltransferases from CD4(+) T was decreased during this process. Furthermore, eight CpG sites within the Foxp3 promoter also appeared to have undergone demethylation. Foxp3 is highly expressed in CD4(+) T cells, and this may be caused by gene promoter demethylation. These induced Tregs are highly immunosuppressive and dramatically inhibit the proliferative activity of naïve CD4(+) T cells. Our study provides one possible mechanism describing Foxp3 promoter demethylation changes by which NSCLC down-regulates immune responses and contributes to tumor progression. Foxp3 represents an important target for NSCLC anti-tumor immunotherapy.

Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages.

During development, progenitor cells with binary potential give rise to daughter cells that have distinct functions. Heritable epigenetic mechanisms then lock in gene-expression programs that define lineage identity. Regulation of the gene encoding the T cell-specific coreceptor CD4 in helper and cytotoxic T cells exemplifies this process, with enhancer- and silencer-regulated establishment of epigenetic memory for stable gene expression and repression, respectively. Using a genetic screen, we identified the DNA-methylation machinery as essential for maintaining silencing of Cd4 in the cytotoxic lineage. Furthermore, we found a requirement for the proximal enhancer in mediating the removal of DNA-methylation marks from Cd4, which allowed stable expression of Cd4 in helper T cells. Our findings suggest that stage-specific methylation and demethylation events in Cd4 regulate its heritable expression in response to the distinct signals that dictate lineage 'choice' during T cell development.

De novo DNA Methyltransferases Dnmt3a and Dnmt3b regulate the onset of Igκ light chain rearrangement during early B-cell development.

Immunoglobulin genes V(D)J rearrangement during early lymphopoiesis is a critical process involving sequential recombination of the heavy and light chain loci. A number of transcription factors act together with temporally activated recombinases and chromatin accessibility changes to regulate this complex process. Here, we deleted the de novo DNA methyltransferases Dnmt3a and Dnmt3b in early B cells of conditionally targeted mice, and monitored the process of V(D)J recombination. Dnmt3a and Dnmt3b deletion resulted in precocious recombination of the immunoglobulin κ light chain without impairing the differentiation of mature B cells or overall B-cell development. Ex vivo culture of IL-7 restricted early B-cell progenitors lacking Dnmt3a and Dnmt3b showed precocious Vκ-Jκ rearrangements that are limited to the proximal Vκ genes. Furthermore, B-cell progenitors deficient in Dnmt3a and Dnmt3b showed elevated levels of germline transcripts at the proximal Vκ genes, alterations in methylation patterns at Igκ enhancer sites and increased expression of the transcription factor E2A. Our data suggest that Dnmt3a and Dnmt3b are critical to regulate the onset of Igκ light chain rearrangement during early B-cell development.

Decitabine suspends human CD34+ cell differentiation and proliferation during lentiviral transduction.

Efficient ex vivo transduction of hematopoietic stem cells (HSCs) is encumbered by differentiation which reduces engraftment. We hypothesized that inhibiting DNA methyltransferase with decitabine would block differentiation of transduced CD34+ cells under cytokine stimulation and thus improve transduction efficiency for engrafting HSCs. Human CD34+ cells in cytokine-containing media were treated with or without decitabine for 24 or 48 hours, and then these cells were transduced with a GFP-expressing lentiviral vector. Utilizing decitabine pre-treatment for 48 hours, we observed an equivalent percentage of successfully transduced cells (GFP-positivity) and a higher percentage of cells that retained CD34 positivity, compared to no decitabine exposure. Cell proliferation was inhibited after decitabine exposure. Similar results were observed among CD34+ cells from six different donors. Repopulating activity was evaluated by transplantation into NOD/SCID/IL2Rγnull mice and demonstrated an equivalent percentage of GFP-positivity in human cells from decitabine-treated samples and a trend for higher human cell engraftment (measured 20-24 weeks after transplantation), compared to no decitabine exposure. In conclusion, ex vivo decitabine exposure inhibits both differentiation and proliferation in transduced human CD34+ cells and modestly increases the engraftment ability in xenograft mice, while the transduction efficiency is equivalent in decitabine exposure, suggesting improvement of lentiviral transduction for HSCs.

Immune regulation by low doses of the DNA methyltransferase inhibitor 5-azacitidine in common human epithelial cancers.

Epigenetic therapy is emerging as a potential therapy for solid tumors. To investigate its mechanism of action, we performed integrative expression and methylation analysis of 63 cancer cell lines (breast, colorectal, and ovarian) after treatment with the DNA methyltransferase inhibitor 5-azacitidine (AZA). Gene Set Enrichment Analysis demonstrated significant enrichment for immunomodulatory pathways in all three cancers (14.4-31.3%) including interferon signaling, antigen processing and presentation, and cytokines/chemokines. Strong upregulation of cancer testis antigens was also observed. An AZA IMmune gene set (AIMs) derived from the union of these immunomodulatory pathway genes classified primary tumors from all three types, into "high" and "low" AIM gene expression subsets in tumor expression data from both TCGA and GEO. Samples from selected patient biopsies showed upregulation of AIM genes after treatment with epigenetic therapy. These results point to a broad immune stimulatory role for DNA demethylating drugs in multiple cancers.

Epigenetic dysregulation in salivary glands from patients with primary Sjögren's syndrome may be ascribed to infiltrating B cells.

Sjögren's syndrome (SS) is an autoimmune exocrinopathy characterized by an epithelium injury with dense lymphocytic infiltrates, mainly composed of activated T and B cells. Present at the interface of genetic and environmental risk factors, DNA methylation is suspected to play a key role in SS. To clarify this point, global DNA methylation was tested within salivary gland epithelial cells (SGEC), peripheral T cells and B cells from SS patients. Global DNA methylation was reduced in SGEC from SS patients, while no difference was observed in T and B cells. SGEC demethylation in SS patients was associated with a 7-fold decrease in DNA methyl transferase (DNMT) 1 and a 2-fold increase in Gadd45-alpha expression. The other DNA methylation/demethylation partners, tested by real time PCR (DNMT3a/b, PCNA, UHRF1, MBD2, and MBD4), were not different. Interestingly, SGEC demethylation may be attributed in part to the infiltrating B cells as suspected in patients treated with anti-CD20 antibodies to deplete B cells. Such hypothesis was confirmed using co-culture experiments with human salivary gland cells and B cells. Furthermore, B cell-mediated DNA demethylation could be ascribed to an alteration of the PKC delta/ERK/DNMT1 pathway. As a consequence, part of the SGEC dysfunction in SS may be linked to epigenetic modifications, thus opening new therapeutic perspectives in SS.

A rapid and sensitive assay for DNA-protein covalent complexes in living cells.

A number of proteins form covalent bonds with DNA as obligatory transient intermediates in normal nuclear transactions. Drugs that trap these complexes have proven to be potent therapeutics in both cancer and infectious disease. Nonetheless, current assays for DNA-protein adducts are cumbersome, limiting both mechanistic studies and translational applications. We have developed a rapid and sensitive assay that enables quantitative immunodetection of protein-DNA adducts. This new 'RADAR' (rapid approach to DNA adduct recovery) assay accelerates processing time 4-fold, increases sample throughput 20-fold and requires 50-fold less starting material than the current standard. It can be used to detect topoisomerase 1-DNA adducts in as little as 60 ng of DNA, corresponding to 10 000 human cells. We apply the RADAR assay to demonstrate that expression of SLFN11 does not increase camptothecin sensitivity by promoting accumulation of topoisomerase 1-DNA adducts. The RADAR assay will be useful for analysis of the mechanisms of formation and resolution of DNA-protein adducts in living cells, and identification and characterization of reactions in which covalent DNA adducts are transient intermediates. The assay also has potential application to drug discovery and individualized medicine.

DNA methyl transferase I acts as a negative regulator of allergic skin inflammation.

The role of DNA methyl transferase I (DNMT1) in allergic inflammation was investigated. Antigen stimulation decreased expression of DNMT1 in rat basophilic leukemia cells (RBL2H3). The down regulation of DNMT1 induced expression of histone deacetylase 3 (HDAC3). HDAC3 was necessary for allergic skin inflammation, such as such as triphasic cutaneous reaction and passive cutaneous anaphylaxis. The down regulation of DNMT1 resulted from activation of PKC and rac1 which were necessary for proteasome-dependent ubiquitination of DNMT1 by antigen stimulation. N-acetyl-L-cysteine, an inhibitor of reactive oxygen species production, exerted negative effects on allergic skin inflammation. Antigen stimulation led to increased expression of Tip60, a histone acetyl transferase. Wild type, but not mutant form, Tip60 decreased expression of DNMT1 while increasing expression of HDAC3, suggesting role for acetylation in ubiquitin-dependent proteasomal degradation of DNMT1. In vivo down regulation of DNMT1 increased ear thickness, typical of allergic skin inflammation, induced vascular leakage and promoted angiogenesis in BALB/c mouse. The down regulation of DNMT1 enhanced angiogenic potential of rat aortic endothelial cells (RAEC) accompanied by activation of VEGR-2 and induced interaction between VEGR-2 and syk in RAEC. The enhanced angiogenic potential of RAEC was associated with the induction of VEGF by down regulation of DNMT1 in RBL2H3 cells. The down regulation of DNMT1 induced leukocytes-endothelial cell interaction and expression of various adhesion molecules. Aspirin exerted a negative effect on allergic skin inflammation by indirect regulation on DNMT1 via Tip60. Taken together, these results suggest novel role for DNMT1 in allergic skin inflammation.

cAMP response element modulator α controls IL2 and IL17A expression during CD4 lineage commitment and subset distribution in lupus.

Appropriate expression of IL-2 plays a central role during the priming and differentiation of T cells. A tight balance between IL-2 and the effector cytokine IL-17A is essential for immune homeostasis. Epigenetic mechanisms have been documented as a key component of cytokine regulation during lineage commitment. The molecular mechanisms that induce chromatin remodeling are less well understood. We investigated epigenetic regulators that mediate the diametric expression of IL-2 and IL-17A in naive, central memory, and effector memory CD4(+) T cells. We demonstrate that cAMP response modulator (CREM)α contributes to epigenetic remodeling of IL2 in effector memory T cells through the recruitment of DNMT3a. CREMα also reduces CpG-DNA methylation of the IL17A promoter. CREMα expression is regulated at the epigenetic level by CpG-DNA methylation, which allows increased CREMα expression in effector memory CD4(+) T cells. T cells from patients with systemic lupus erythematosus (SLE) express increased levels of CREMα and exhibit a phenotype that is similar to effector memory CD4(+) T cells with epigenetically predetermined expression patterns of IL-2 and IL-17A. We conclude that CREMα mediates epigenetic remodeling of the IL2 and IL17A gene during T-cell differentiation in favor of effector memory T cells in health and disease.

DNMT3B (C46359T) polymorphisms and immunoexpression of DNMT3b and DNMT1 proteins in oral lichen planus.

OBJECTIVE: To investigate the DNMT3B (C46359T) polymorphism and immunoexpression of DNMT3b and DNMT1 in oral lichen planus (OLP) compared to a control group. METHODS: We aimed to investigate the DNMT3B (C46359T) polymorphism and immunoexpression of DNMT3b and DNMT1 in OLP (n = 32), comparing it with oral mucosa (control; n = 24). The DNMT3B (C46359T) polymorphism was analyzed using the RFLP-PCR and DNMT1, and DNMT3a proteins were identified using immunohistochemistry. We also compared the DNMT3B expression in OLP and oral inflammatory fibrous hyperplasia (OIFH), another oral inflammatory disease. Differences between the groups were determined by specific statistical analyses. RESULTS: The CT genotype of DNMT3B was associated with OLP development (p = 0.012). Increased expression of DNMT3B and DNMT1 was observed in OLP compared to the control group (p = 0.014 and p = 0.001, respectively). A significant increase in DNMT3B protein levels was observed in the genotype CT in DNMT3B (C46359T) polymorphisms (p = 0.045). No DNMT3B expression differences between OLP and OIFH were observed. CONCLUSIONS: Our data show that the DNMT3B (C46359T) polymorphism is associated with OLP development. Furthermore, increased expression of the enzyme DNMT3B, an epigenetic-associated protein, is present in OLP.

Epigenetic regulation of human ß-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria.

Gingival epithelia utilize multiple signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. In this study we report that DNA methyltransferase (DNMT1) and histone deacetylase expression were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and nonpathogen Fusobacterium nucleatum. Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human ß-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5'-azacytidine increased hBD2 and CCL20 expression in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.