Renato Ostuni

Latent Enhancers Activated by Stimulation in Differentiated Cells

According to current models, once the cell has reached terminal differentiation, the enhancer repertoire is completely established and maintained by cooperatively acting lineage-specific transcription factors (TFs). TFs activated by extracellular stimuli operate within this predetermined repertoire, landing close to where master regulators are constitutively bound. Here, we describe latent enhancers, defined as regions of the genome that in terminally differentiated cells are unbound by TFs and lack the histone marks characteristic of enhancers but acquire these features in response to stimulation. Macrophage stimulation caused sequential binding of stimulus-activated and lineage-determining TFs to these regions, enabling deposition of enhancer marks. Once unveiled, many of these enhancers did not return to a latent state when stimulation ceased; instead, they persisted and mediated a faster and stronger response upon restimulation. We suggest that stimulus-specific expansion of the cis-regulatory repertoire provides an epigenomic memory of the exposure to environmental agents.

Macrophages and cancer: from mechanisms to therapeutic implications

Infiltration by immune cells is a hallmark of most forms of malignancy. In this context, tumor-associated macrophages (TAMs) represent key regulators of the complex interplay between the immune system and cancer. We discuss evidence indicating that in many settings TAMs fuel, rather than limit, tumor progression, and negatively impact on responses to therapy. We discuss how the unique functional properties of TAMs are shaped by tumor-derived signals, placing TAM development in the context of the broader understanding of the cellular and molecular mechanisms controlling macrophage origin, differentiation, and maintenance in tissues. Finally, we provide examples of how a molecular understanding of the relationships between TAMs and the tumor microenvironment may lead to improved cancer therapies.

The Histone Methyltransferase Wbp7 Controls Macrophage Function through GPI Glycolipid Anchor Synthesis

Histone methyltransferases catalyze site-specific deposition of methyl groups, enabling recruitment of transcriptional regulators. In mammals, trimethylation of lysine 4 in histone H3, a modification localized at the transcription start sites of active genes, is catalyzed by six enzymes (SET1a and SET1b, MLL1-MLL4) whose specific functions are largely unknown. By using a genomic approach, we found that in macrophages, MLL4 (also known as Wbp7) was required for the expression of Pigp, an essential component of the GPI-GlcNAc transferase, the enzyme catalyzing the first step of glycosylphosphatidylinositol (GPI) anchor synthesis. Impaired Pigp expression in Wbp7(-/-) macrophages abolished GPI anchor-dependent loading of proteins on the cell membrane. Consistently, loss of GPI-anchored CD14, the coreceptor for lipopolysaccharide (LPS) and other bacterial molecules, markedly attenuated LPS-triggered intracellular signals and gene expression changes. These data link a histone-modifying enzyme to a biosynthetic pathway and indicate a specialized biological role for Wbp7 in macrophage function and antimicrobial response.

A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages.

The transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8-PU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRF-AP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites.

Cutting Edge: An Inactive Chromatin Configuration at the IL-10 Locus in Human Neutrophils

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modification status at their IL-10 genomic locus. We analyzed posttranslational modifications of histones associated with genes that are active, repressed, or poised for transcriptional activation, including H3K4me3, H4Ac, H3K27Ac, and H3K4me1 marks. Differently from autologous IL-10–producing monocytes, none of the marks under evaluation was detected at the IL-10 locus of resting or activated neutrophils from healthy subjects or melanoma patients. By contrast, increased H3K4me3, H4Ac, H3K4me1, and H3K27Ac levels were detected at syntenic regions of the IL-10 locus in mouse neutrophils. Altogether, data demonstrate that human neutrophils, differently from either monocytes or mouse neutrophils, cannot switch on the IL-10 gene because its locus is in an inactive state, likely reflecting a neutrophil-specific developmental outcome. Implicitly, data also definitively settle a currently unsolved issue on the capacity of human neutrophils to produce IL-10.

Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils

Controversy currently exists about the ability of human neutrophils to produce IL-6. Here, we show that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively kept in an inactive configuration. However, we also show that upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, highly purified neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14u2009kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous TNFα, which prolongs the synthesis of the IκBζ co-activator and sustains C/EBPβ recruitment and histone acetylation at IL-6 regulatory regions. Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells.

Lineages, cell types and functional states: a genomic view

Cellular differentiation progresses through an ordered cascade of events involving cell autonomous and micro-environment regulated expression or activation of transcription factors (TFs). Lineage-determining and stimulus-activated TFs collaborate in specifying the transcriptional programs of differentiating cells through the establishment of appropriate genomic repertoires of active or poised cis-regulatory elements, which can eventually be altered by environmental changes to generate transient or persistent functional states. Here, we rationalize available genomic and functional data into a mechanistic model whereby terminal differentiation proceeds first through the establishment of a regulatory landscape that is broadly shared among all cell types within a given lineage; and then through the selective activation of a more restricted set of regulatory elements that specify the unique transcriptional outputs of individual cell types. In this scheme, the interplay between cell-autonomous and microenvironment-regulated TFs is highly complex, with several documented variants.

Transcriptional control of macrophage diversity and specialization.

The key driving force underlying cell identity is represented by the complex and dynamic interplay between cell‐intrinsic, lineage‐restricted developmental pathways on the one hand, and cell‐extrinsic, tissue‐specific microenvironmental signals on the other. In this context, macrophages are a paradigmatic cell population whose functional specialization in vivo reflects the impact of the local microenvironment on the intrinsic differentiation program, leading to a variety of specialized macrophage types in different tissues and conditions; however, how this is translated into a biological outcome is not appreciably understood. The kind of investigations described in this Viewpoint aim to explore the inner determinants of cell identity and functional diversification at a genomic level; mechanisms that permit plastic cell types, like macrophages, to adapt to different environments.

TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection.

Neutralization or deletion of tumor necrosis factor (TNF) causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1) expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO) synthase (NOS2) was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO inxa0situ. A similar phenotype was seen in L.xa0major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating Txa0cell response (Th2, Treg) was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

Epigenetic regulation of neutrophil development and function.

In addition to performing well-defined effector functions, neutrophils are now recognized as versatile and sophisticated cells with critical immunoregulatory roles. These include the release of a variety of proinflammatory or immunosuppressive cytokines, as well as the expression of genes with regulatory functions. Neutrophils share broad transcriptional features with monocytes, in keeping with the close developmental relation between the two cell types. However, neutrophil-specific gene expression patterns conferring cell type-specific responses to bacterial, viral or fungal components have been identified. Accumulating evidence suggest that these differences reflect the peculiar epigenomic and regulatory landscapes of neutrophils and monocytes, in turn controlled by the specific lineage-determining transcription factors shaping their identity. In this review, we will describe current knowledge on how neutrophil identity and function are controlled at the molecular level, focusing on transcriptional and chromatin regulation of neutrophil development and activation in response to inflammatory stimuli.