Amit Mandoli

Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity

Introduction Monocytes circulate in the bloodstream for up to 3 to 5 days. Concomitantly, immunological imprinting of either tolerance (immunosuppression) or trained immunity (innate immune memory) determines the functional fate of monocytes and monocyte-derived macrophages, as observed after infection or vaccination. The epigenome, DNase I accessibility, and transcriptome were characterized in purified human circulating monocytes, in vitro differentiated naïve, tolerized (immunosuppression), and trained macrophages (innate immune memory). This allowed the identification of pathways functionally implicated in innate immune memory. This epigenetic signature of human monocyte-to-macrophage differentiation and monocyte training generates hypotheses to understand and manipulate medically relevant immune conditions. Methods Purified circulating monocytes from healthy volunteers were differentiated under the homeostatic macrophage colony-stimulating factor concentrations present in human serum. During the first 24 hours, trained immunity was induced by β-glucan (BG) priming, and postsepsis immunoparalysis was mimicked by exposure to lipopolysaccharide (LPS), generating endotoxin-induced tolerance. Epigenomic profiling of the histone marks H3K4me1, H3K4me3, and H3K27ac, DNase I accessibility, and RNA sequencing were performed at both the start of the experiment (ex vivo monocytes) and at the end of the 6 days of in vitro culture (macrophages). Results Compared with monocytes (Mo), naïve macrophages (Mf ) display a remodeled metabolic enzyme repertoire and attenuated innate inflammatory pathways, most likely necessary to generate functional tissue macrophages. Epigenetic profiling uncovered about 8000 dynamic regions associated with about 11,000 DNase I hypersensitive sites. Changes in histone acetylation identified most dynamic events. Furthermore, these regions of differential histone marks displayed some degree of DNase I accessibility that was already present in monocytes. H3K4me1 mark increased in parallel with de novo H3K27ac deposition at distal regulatory regions; H3K4me1 mark remained even after the loss of H3K27ac, marking decommissioned regulatory elements. β-glucan priming specifically induced about 3000 distal regulatory elements, whereas LPS tolerization induced H3K27ac at about 500 distal regulatory regions. At the transcriptional level, we identified coregulated gene modules during monocyte-to-macrophage differentiation, as well as discordant modules between trained and tolerized cells. These indicate that training likely involves an increased expression of modules expressed in naïve macrophages, including genes that code for metabolic enzymes. On the other hand, endotoxin tolerance involves gene modules that are more active in monocytes than in naïve macrophages. About 12% of known human transcription factors display variation in expression during macrophage differentiation, training, and tolerance. We also observed transcription factor motifs in DNase I hypersensitive sites at condition-specific dynamic epigenomic regions, implying that specific transcription factors are required for trained and tolerized macrophage epigenetic and transcriptional programs. Finally, our analyses and functional validation indicate that the inhibition of cyclic adenosine monophosphate generation blocked trained immunity in vitro and during an in vivo model of lethal Candida albicans infection, abolishing the protective effects of trained immunity. Discussion We documented the importance of epigenetic regulation of the immunological pathways underlying monocyte-to-macrophage differentiation and trained immunity. These dynamic epigenetic elements may inform on potential pharmacological targets that modulate innate immunity. Altogether, we uncovered the epigenetic and transcriptional programs of monocyte differentiation to macrophages that distinguish tolerant and trained macrophage phenotypes, providing a resource to further understand and manipulate immune-mediated responses. A BLUEPRINT of immune cell development To determine the epigenetic mechanisms that direct blood cells to develop into the many components of our immune system, the BLUEPRINT consortium examined the regulation of DNA and RNA transcription to dissect the molecular traits that govern blood cell differentiation. By inducing immune responses, Saeed et al. document the epigenetic changes in the genome that underlie immune cell differentiation. Cheng et al. demonstrate that trained monocytes are highly dependent on the breakdown of sugars in the presence of oxygen, which allows cells to produce the energy needed to mount an immune response. Chen et al. examine RNA transcripts and find that specific cell lineages use RNA transcripts of different length and composition (isoforms) to form proteins. Together, the studies reveal how epigenetic effects can drive the development of blood cells involved in the immune system. Science, this issue 10.1126/science.1251086, 10.1126/science.1250684, 10.1126/science.1251033 Genome-wide approaches analyze human monocyte differentiation in vitro into functional macrophages. Monocyte differentiation into macrophages represents a cornerstone process for host defense. Concomitantly, immunological imprinting of either tolerance or trained immunity determines the functional fate of macrophages and susceptibility to secondary infections. We characterized the transcriptomes and epigenomes in four primary cell types: monocytes and in vitro–differentiated naïve, tolerized, and trained macrophages. Inflammatory and metabolic pathways were modulated in macrophages, including decreased inflammasome activation, and we identified pathways functionally implicated in trained immunity. β-glucan training elicits an exclusive epigenetic signature, revealing a complex network of enhancers and promoters. Analysis of transcription factor motifs in deoxyribonuclease I hypersensitive sites at cell-type–specific epigenetic loci unveiled differentiation and treatment-specific repertoires. Altogether, we provide a resource to understand the epigenetic changes that underlie innate immunity in humans.

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease

Summary Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.

Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells

Summary Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

ERG and FLI1 binding sites demarcate targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia

ERG and FLI1 are closely related members of the ETS family of transcription factors and have been identified as essential factors for the function and maintenance of normal hematopoietic stem cells. Here genome-wide analysis revealed that both ERG and FLI1 occupy similar genomic regions as AML1-ETO in t(8;21) AMLs and identified ERG/FLI1 as proteins that facilitate binding of oncofusion protein complexes. In addition, we demonstrate that ERG and FLI1 bind the RUNX1 promoter and that shRNA-mediated silencing of ERG leads to reduced expression of RUNX1 and AML1-ETO, consistent with a role of ERG in transcriptional activation of these proteins. Finally, we identify H3 acetylation as the epigenetic mark preferentially associated with ETS factor binding. This intimate connection between ERG/FLI1 binding and H3 acetylation implies that one of the molecular strategies of oncofusion proteins, such as AML1-ETO and PML-RAR-α, involves the targeting of histone deacetylase activities to ERG/FLI1 bound hematopoietic regulatory sites. Together, these results highlight the dual importance of ETS factors in t(8;21) leukemogenesis, both as transcriptional regulators of the oncofusion protein itself as well as proteins that facilitate AML1-ETO binding.

Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

BackgroundThe impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by activated transcription factors to recruitment of activated transcription factors to pre-established long-range interactions.ResultsUsing circular chromosome conformation capture coupled with next generation sequencing and high-resolution chromatin interaction analysis by paired-end tag sequencing of P300, we observed agonist-induced changes in long-range chromatin interactions, and uncovered interconnected enhancer–enhancer hubs spanning up to one megabase. The vast majority of activated glucocorticoid receptor and nuclear factor kappa-b appeared to join pre-existing P300 enhancer hubs without affecting the chromatin conformation. In contrast, binding of the activated transcription factors to loci with their consensus response elements led to the increased formation of an active epigenetic state of enhancers and a significant increase in long-range interactions within pre-existing enhancer networks. De novo enhancers or ligand-responsive enhancer hubs preferentially interacted with ligand-induced genes.ConclusionsWe demonstrate that, at a subset of genomic loci, ligand-mediated induction leads to active enhancer formation and an increase in long-range interactions, facilitating efficient regulation of target genes. Therefore, our data suggest an active role of signal-dependent transcription factors in chromatin and long-range interaction remodeling.

Context-Selective Death of Acute Myeloid Leukemia Cells Triggered by the Novel Hybrid Retinoid-HDAC Inhibitor MC2392

HDAC inhibitors (HDACi) are widely used in the clinic to sensitize tumorigenic cells for treatment with other anticancer compounds. The major drawback of HDACi is the broad inhibition of the plethora of HDAC-containing complexes. In acute promyelocytic leukemia (APL), repression by the PML-RARα oncofusion protein is mediated by an HDAC-containing complex that can be dissociated by pharmacologic doses of all trans retinoic acid (ATRA) inducing differentiation and cell death at the expense of side effects and recurrence. We hypothesized that the context-specific close physical proximity of a retinoid and HDACi-binding protein in the repressive PML-RARα-HDAC complex may permit selective targeting by a hybrid molecule of ATRA with a 2-aminoanilide tail of the HDAC inhibitor MS-275, yielding MC2392. We show that MC2392 elicits weak ATRA and essentially no HDACi activity in vitro or in vivo. Genome-wide epigenetic analyses revealed that in NB4 cells expressing PML-RARα, MC2392 induces changes in H3 acetylation at a small subset of PML-RARα-binding sites. RNA-seq reveals that MC2392 alters expression of a number of stress-responsive and apoptotic genes. Concordantly, MC2392 induced rapid and massive, caspase-8-dependent cell death accompanied by RIP1 induction and ROS production. Solid and leukemic tumors are not affected by MC2392, but expression of PML-RARα conveys efficient MC2392-induced cell death. Our data suggest a model in which MC2392 binds to the RARα moiety and selectively inhibits the HDACs resident in the repressive complex responsible for the transcriptional impairment in APLs. Our findings provide proof-of-principle of the concept of a context-dependent targeted therapy.

MLL-AF9 and MLL-AF4 oncofusion proteins bind a distinct enhancer repertoire and target the RUNX1 program in 11q23 acute myeloid leukemia

In 11q23 leukemias, the N-terminal part of the mixed lineage leukemia (MLL) gene is fused to >60 different partner genes. In order to define a core set of MLL rearranged targets, we investigated the genome-wide binding of the MLL-AF9 and MLL-AF4 fusion proteins and associated epigenetic signatures in acute myeloid leukemia (AML) cell lines THP-1 and MV4-11. We uncovered both common as well as specific MLL-AF9 and MLL-AF4 target genes, which were all marked by H3K79me2, H3K27ac and H3K4me3. Apart from promoter binding, we also identified MLL-AF9 and MLL-AF4 binding at specific subsets of non-overlapping active distal regulatory elements. Despite this differential enhancer binding, MLL-AF9 and MLL-AF4 still direct a common gene program, which represents part of the RUNX1 gene program and constitutes of CD34+ and monocyte-specific genes. Comparing these data sets identified several zinc finger transcription factors (TFs) as potential MLL-AF9 co-regulators. Together, these results suggest that MLL fusions collaborate with specific subsets of TFs to deregulate the RUNX1 gene program in 11q23 AMLs.

The oncofusion protein FUS-ERG targets key hematopoietic regulators and modulates the all-trans retinoic acid signaling pathway in t(16;21) acute myeloid leukemia.

The ETS transcription factor ERG has been implicated as a major regulator of both normal and aberrant hematopoiesis. In acute myeloid leukemias harboring t(16;21), ERG function is deregulated due to a fusion with FUS/TLS resulting in the expression of a FUS–ERG oncofusion protein. How this oncofusion protein deregulates the normal ERG transcription program is unclear. Here, we show that FUS–ERG acts in the context of a heptad of proteins (ERG, FLI1, GATA2, LYL1, LMO2, RUNX1 and TAL1) central to proper expression of genes involved in maintaining a stem cell hematopoietic phenotype. Moreover, in t(16;21) FUS–ERG co-occupies genomic regions bound by the nuclear receptor heterodimer RXR:RARA inhibiting target gene expression and interfering with hematopoietic differentiation. All-trans retinoic acid treatment of t(16;21) cells as well as FUS–ERG knockdown alleviate the myeloid-differentiation block. Together, the results suggest that FUS–ERG acts as a transcriptional repressor of the retinoic acid signaling pathway.

The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs

The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic transcription factors LYL1 and LMO2. In contrast, ERG, FLI1, TAL1, and RUNX1 bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. We find that expression of wild-type transcription factors RUNX1 and ERG in AML is required to prevent this oncogene overexpression. Together our results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells.

AML associated oncofusion proteins PML-RARA, AML1-ETO and CBFB-MYH11 target RUNX/ETS-factor binding sites to modulate H3ac levels and drive leukemogenesis

Chromosomal translocations are one of the hallmarks of acute myeloid leukemia (AML), often leading to gene fusions and expression of an oncofusion protein. Over recent years it has become clear that most of the AML associated oncofusion proteins molecularly adopt distinct mechanisms for inducing leukemogenesis. Still these unique molecular properties of the chimeric proteins converge and give rise to a common pathogenic molecular mechanism. In the present study we compared genome-wide DNA binding and transcriptome data associated with AML1-ETO, CBFB-MYH11 and PML-RARA oncofusion protein expression to identify unique and common features. Our analyses revealed targeting of oncofusion binding sites to RUNX1 and ETS-factor occupied genomic regions. In addition, it revealed a highly comparable global histone acetylation pattern, similar expression of common target genes and related enrichment of several biological pathways critical for maintenance of AML, suggesting oncofusion proteins deregulate common gene programs despite their distinct binding signatures and mechanisms of action.