Demethylase Kdm6a epigenetically promotes IL-6 and IFN-β production in macrophages

Highlights

• Kdm6a promotes IL-6 and IFN-β production during inflammation.

• Kdm6a specifically promotes Il6 transcription via H3K27me3 demethylation.

• Kdm6a promotes IFN-β-specific enhancer-derived RNA (eRNA) transcription.

• Kdm6a promotes MLL4 recruitment for full activating IFN-β-specific enhancer.

Abstract

Molecular regulation of innate signal-initiated proinflammatory cytokine production has been extensively investigated. However, the roles of epigenetic modifiers and their underlying mechanisms in regulating innate inflammatory response and development of autoimmune diseases need to be further understood. Demethylase Kdm6a promotes gene transcription in cell-lineage specification through demethylating histone H3 lysine di/tri-methylation (H3K27me2/3), and loss of Kdm6a results in developmental defects. However, the function of Kdm6a in innate immunity and inflammation remains largely unknown. Here we found that Kdm6a, significantly downregulated via JNK pathway upon innate stimuli, promotes cytokine IL-6 and IFN-β transcription in primary macrophages during innate response. Kdm6a promoted IL-6 expression through demethylating H3K27me3 at promoter in a demethylase enzymatic activity-dependent manner. Interestingly, Kdm6a promoted IFN-β expression independent of its demethylase enzymatic activity, but through increasing transcription of IFN-β-specific enhancer-derived RNA (eRNA) S-IRE1. For the underlying mechanism, Kdm6a interacted with MLL4 and promoted MLL4 recruitment and H3K4me2 level in S-IRE1 region of Ifnb1 gene for full activation of enhancer. Our results reveal a previously unknown role of kdm6a in promoting innate IFN-β gene transcription at enhancer, in addition to demethylation at promoter. The function of Kdm6a in promoting innate inflammatory response also adds insights to better understanding of epigenetic modifiers in inflammatory and autoimmune disease.

Introduction

Epigenetic regulation of immunity and inflammation attracts much attention now. Epigenetic aberrations play important roles in the development of autoimmunity such as Systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and multiple sclerosis (MS) [1], [2], [3]. In particular, aberrant histone modifications are tightly associated with the pathogenesis of multiple human diseases [4], and some of them have been proven to be potential diagnostic biomarkers or therapeutic targets for autoimmunity diseases [1]. Recent breakthroughs in epigenetic regulation in innate immunity and inflammation have extended our understanding through discovery of epigenetic patterns subjected to autoimmune diseases (AID) [5]. Thus, more potential epigenetic modifiers which are involved in regulating innate immunity and inflammation will be identified as critical regulators in the pathogenesis of autoimmune diseases.

Modification of DNA and histone, together with their associated proteins and epigenetic regulators such as non-coding RNAs add an important layer of transcription regulation, in addition to cell-specific transcription factors during cell lineage commitment [6]. Histone methylation is linked to both repression and activation of transcription, determined by the specific lysine (K) residue and its methylation state (mono-, di-, or trimethylation) in histone tails [7]. For example, di- and trimethylation at K4 of histone H3 (H3K4) are associated with gene activation, whereas di- and trimethylation at K27 of histone H3 (H3K27) are associated with gene repression [8], [9]. Histone methylation is a dynamic process, which is mediated by lysine-specific methyl-transferases (KMT) and demethylases (KDM) [10]. How the histone modifiers regulate dynamic histone codes during lineage-specification have been gradually revealed in different biological processes, and development and activation of innate immune system is also in the list [11].

The physiological importance of TLR signaling is underscored by the involvement of TLRs and their major signaling components in human diseases, including cancer and immunological disorders [12], [13]. Regarding the latter, aberrant TLR signals and overexpression of inflammatory cytokines have been associated with various inflammatory diseases, such as endotoxin shock, rheumatoid arthritis (RA), and inflammatory bowel disease (IBD) [14]. Furthermore, excessive type I IFN production can be harmful to the host and gives rise to inflammatory or autoimmune diseases, such as systemic lupus erythematosus (SLE), dermatomyositis (DM), and type 1 diabetes [15], [16], [17]. Both positive and negative regulators of TLR and antiviral signaling pathways have been largely revealed, furthermore, epigenetic regulators, especially the gene-specific chromatin modifiers at inflammatory gene locus have been gradually identified [18], [19]. We previously identified several histone modifiers as important regulators of TLRs signaling in innate immunity, such as H3K4me3 methyl-transferase Ash1l [20], H3K27me3 methyl-transferase Ezh1 [21] and DNA cytosine methyl-transferase Dnmt3a [22]. However, in addition to HDACs identified for repressing cytokine transcription [23], the role of histone methylation modifiers as cytokine gene-specific chromatin regulators, especially for H3K27me2/3, remains to be further understood.

Trimethylation of H3K27 (H3K27me3) is considered a repressive mark important for maintaining embryonic stem (ES) cell pluripotency and plasticity during embryonic development, Polycomb-mediated gene silencing, and X chromosome inactivation [9], [24], [25]. Given the importance of H3K27 tri-methylation, Kdm6a and Jmjd3, members of a subfamily of JmjC-domain-containing proteins have been reported to demethylate H3K27me2/3 [7], [26], [27], [28]. In addition to the JmjC domain, Kdm6a and UTY, but not jmjd3, also contain tetratricopeptide repeats at their N-terminal regions, which are predicted to be protein interaction motifs [26]. The product of Kdm6a gene residing on the X chromosome, also called UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome), mediates escaping of X chromosome inactivation and is ubiquitously expressed in mice and humans [29]. Jmjd3 is firstly identified in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity for macrophage specification and polarization [30], [31]. Kdm6a is shown to be important for maintaining the steady-state levels of H3K27me3 in proliferating cells [27]. Indeed, accumulating evidence shows that gene expression of Kdm6a is down-regulated in myeloid cells in response to LPS stimulation (GSE2002, GSE7768, GSE53986) [32], [33], [34], and Kdm6a expression also displays significantly increase in some autoimmune diseases such as Systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and multiple sclerosis (MS) (GSE10325, GSE10500, GSE26484) [35], [36], [37], [38]. However, whether Kdm6a functions in innate inflammatory responnse and involves development of autoimmune disease remain need to be fully investigated.

In this study, we investigated the role of Kdm6a in the regulation of the innate immune response in macrophages and found that Kdm6a-dependent H3K27me2/3 de-methylation promotes IL-6 transcription, meanwhile Kdm6a promotes IFN-β transcription through promoting full activation of IFN-β-specific enhancer and increasing the enhancer-derived RNA (eRNA) S-IRE1 in a histone demethylase-independent manner. Our results indicate that one epigenetic modifier can promote inflammatory innate responses through classic or non-classic epigenetic manners to regulate different proinflammatory cytokine production.