Menno J. Oudhoff

TNF-α and TLR agonists increase susceptibility to HIV-1 transmission by human Langerhans cells ex vivo

Genital coinfections increase an individuals risk of becoming infected with HIV-1 by sexual contact. Several mechanisms have been proposed to explain this, such as the presence of ulceration and bleeding caused by the coinfecting pathogen. Here we demonstrate that Langerhans cells (LCs) are involved in the increased susceptibility to HIV-1 in the presence of genital coinfections. Although LCs are a target for HIV-1 infection in genital tissues, we found that immature LCs did not efficiently mediate HIV-1 transmission in an ex vivo human skin explant model. However, the inflammatory stimuli TNF-alpha and Pam3CysSerLys4 (Pam3CSK4), the ligand for the TLR1/TLR2 heterodimer, strongly increased HIV-1 transmission by LCs through distinct mechanisms. TNF-alpha enhanced transmission by increasing HIV-1 replication in LCs, whereas Pam3CSK4 acted by increasing LC capture of HIV-1 and subsequent trans-infection of T cells. Genital infections such as Candida albicans and Neisseria gonorrhea not only triggered TLRs but also induced TNF-alpha production in vaginal and skin explants. Thus, during coinfection, LCs could be directly activated by pathogenic structures and indirectly activated by inflammatory factors, thereby increasing the risk of acquiring HIV-1. Our data demonstrate a decisive role for LCs in HIV-1 transmission during genital coinfections and suggest antiinflammatory therapies as potential strategies to prevent HIV-1 transmission.

(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells

Significance Protein methyltransferases constitute an emerging but undercharacterized class of therapeutic targets with diverse roles in normal human biology and disease. Small-molecule “chemical probes” can be powerful tools for the functional characterization of such enzymes, and here we report the discovery of (R)-PFI-2—a first-in-class, potent, highly selective, and cell-active inhibitor of the methyltransferase activity of SETD7 [SET domain containing (lysine methyltransferase) 7]—and two related compounds for control and chemoproteomics studies. We used these compounds to characterize the role of SETD7 in signaling, in the Hippo pathway, that controls cell growth and organ size. Our work establishes a chemical biology tool kit for the study of the diverse roles of SETD7 in cells and further validates protein methyltransferases as a druggable target class. SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2—a first-in-class, potent (Kiapp = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7—and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.

Control of the Hippo Pathway by Set7-Dependent Methylation of Yap

Methylation of nonhistone proteins is emerging as a regulatory mechanism to control protein function. Set7 (Setd7) is a SET-domain-containing lysine methyltransferase that methylates and alters function of a variety of proteins in vitro, but the in vivo relevance has not been established. We found that Set7 is a modifier of the Hippo pathway. Mice that lack Set7 have a larger progenitor compartment in the intestine, coinciding with increased expression of Yes-associated protein (Yap) target genes. Mechanistically, monomethylation of lysine 494 of Yap is critical for cytoplasmic retention. These results identify a methylation-dependent checkpoint in the Hippo pathway.

Methyltransferase G9A regulates T cell differentiation during murine intestinal inflammation

Inflammatory bowel disease (IBD) pathogenesis is associated with dysregulated CD4⁺ Th cell responses, with intestinal homeostasis depending on the balance between IL-17-producing Th17 and Foxp3⁺ Tregs. Differentiation of naive T cells into Th17 and Treg subsets is associated with specific gene expression profiles; however, the contribution of epigenetic mechanisms to controlling Th17 and Treg differentiation remains unclear. Using a murine T cell transfer model of colitis, we found that T cell-intrinsic expression of the histone lysine methyltransferase G9A was required for development of pathogenic T cells and intestinal inflammation. G9A-mediated dimethylation of histone H3 lysine 9 (H3K9me2) restricted Th17 and Treg differentiation in vitro and in vivo. H3K9me2 was found at high levels in naive Th cells and was lost following Th cell activation. Loss of G9A in naive T cells was associated with increased chromatin accessibility and heightened sensitivity to TGF-β1. Pharmacological inhibition of G9A methyltransferase activity in WT T cells promoted Th17 and Treg differentiation. Our data indicate that G9A-dependent H3K9me2 is a homeostatic epigenetic checkpoint that regulates Th17 and Treg responses by limiting chromatin accessibility and TGF-β1 responsiveness, suggesting G9A as a therapeutic target for treating intestinal inflammation.

Histatins Enhance Wound Closure with Oral and Non-oral Cells:

The role of human saliva in oral wound-healing has never been fully elucidated. We previously demonstrated that parotid-salivary histatins enhance in vitro wound closure. The question remains whether other salivary-gland secretions enhance wound closure, and also the effects of histatins on primary and non-oral cells. Since the presence of histatins is not limited to parotid saliva, we expected to observe wound-closure activity of other salivary-gland secretions. However, here we show that non-parotid saliva does not stimulate wound closure, most probably due to the presence of mucins, since the addition of MUC5B to parotid saliva abolished its effect. Furthermore, we found that histatins stimulated wound closure of (primary) cells of both oral and non-oral origin. This suggests that the cellular receptor of histatins is widely expressed and not confined to cells derived from the oral cavity. These findings encourage the future therapeutic application of histatins in the treatment of all kinds of wounds.

SETD7 Controls Intestinal Regeneration and Tumorigenesis by Regulating Wnt/β-Catenin and Hippo/YAP Signaling

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/β-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and β-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of β-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/β-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis.

G9a regulates group 2 innate lymphoid cell development by repressing the group 3 innate lymphoid cell program

Antignano, Zaph, and collaborators show that the lysine methyltransferase G9a plays a critical role in determining the developmental programs of group 2 and 3 innate lymphoid cells.

Requirement for Core 2 O-Glycans for Optimal Resistance to Helminth Infection

The migration of lymphocytes to the small intestine is controlled by expression of the integrin α4β7 and the chemokine receptor CCR9. However, the molecules that specifically regulate migration to the large intestine remain unclear. Immunity to infection with the large intestinal helminth parasite Trichuris muris is dependent upon CD4+ T cells that migrate to the large intestine. We examine the role of specific chemokine receptors, adhesion molecules and glycosyltransferases in the development of protective immunity to Trichuris. Mice deficient in expression of the chemokine receptors CCR2 or CCR6 were resistant to infection with Trichuris. Similarly, loss of CD34, CD43, CD44 or PSGL-1 had no effect on resistance to infection. In contrast, simultaneous deletion of the Core2 β1,6-N-acetylglucosaminyltransferase (C2GnT) enzymes C2GnT1 and C2Gnt2 resulted in delayed expulsion of worms. These results suggest that C2GnT-dependent modifications may play a role in migration of protective immune cells to the large intestine.

Histatins: Multifunctional Salivary Antimicrobial Peptides

In this chapter, we overview the plethora of properties that have been attributed to histatins including tannin binding, microbicidal activity, immunomodulatory activity, and the recently found stimulation of cell migration. Attention is in particular paid to the molecular mechanisms underlying these properties. We conclude that many properties of histatins can be explained by their physicochemical properties, which allows them to bind a variety of negatively charged molecules and surfaces. For instance, their cationic character is crucial for their membrane-disrupting activity, which forms the basis of their antimicrobial activity. The only function that cannot directly be predicted on the basis of their physicochemical features is the enhancement of wound healing which proceeds via canonical receptor-mediated cell signalling.

Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection

The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7 -/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/β-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection.