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Tissue resident macrophages and recruited monocyte-derived macrophages contribute to host defense but also play pathological roles in a diverse range of human diseases. Multiple macrophage phenotypes are often represented in a diseased tissue, but we lack a deep understanding of the mechanisms that control diversification. Here we use a combination of genetic, genomic, and imaging approaches to investigate the origins and epigenetic trajectories of hepatic myeloid cells during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induces striking changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss is compensated by gain of adjacent monocyte derived macrophages that exhibit convergent epigenomes, transcriptomes and functions. NASH-induced changes in Kupffer cell enhancers are driven by AP-1 and Egr factors that reprogram the Kupffer cell specific functions of Liver X receptors. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct programs of gene expression and corresponding phenotypes.

Unique cell type-specific patterns of activated enhancers can be leveraged to interpret non-coding genetic variation associated with complex traits and diseases such as neurological and psychiatric disorders. Here, we have defined active promoters and enhancers for major cell types of the human brain. Whereas psychiatric disorders were primarily associated with regulatory regions in neurons, idiopathic Alzheimer’s disease (AD) variants were largely confined to microglia enhancers. Interactome maps connecting GWAS variants in cell type-specific enhancers to gene promoters revealed an extended microglia gene network in AD. Deletion of a microglia-specific enhancer harboring AD-risk variants ablated BIN1 expression in microglia but not in neurons or astrocytes. These findings revise and expand the genes likely to be influenced by non-coding variants in AD and suggest the probable brain cell types in which they function.One Sentence SummaryIdentification of cell type-specific regulatory elements in the human brain enables interpretation of non-coding GWAS risk variants.

Oxidized phospholipids (OxPLs), which arise due to oxidative stress, are pro-inflammatory and pro-atherogenic, but their roles in non-alcoholic steatohepatitis (NASH) are unknown. Here, we show that OxPLs accumulate in human and mouse NASH. Using a transgenic mouse that expresses a functional single chain variable fragment of E06, a natural antibody that neutralizes OxPLs, we demonstrate the casual role of OxPLs in NASH. Targeting OxPLs in hyperlipidemic Ldlr(−/−) mice improved multiple aspects of NASH, including steatosis, inflammation, fibrosis, hepatocyte death and progression to hepatocellular carcinoma. Mechanistically, we found that OxPLs promote ROS accumulation to induce mitochondrial dysfunction in hepatocytes. Neutralizing OxPLs in AMLN diet-fed Ldlr(−/−) mice reduced oxidative stress, improved hepatic and adipose tissue mitochondrial function and fatty acid oxidation. These results suggest targeting OxPLs may be an effective therapeutic strategy for NASH.

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-β) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

Tissue-resident and recruited macrophages contribute to both host defense and pathology. Multiple macrophage phenotypes are represented in diseased tissues, but we lack deep understanding of mechanisms controlling diversification. Here, we investigate origins and epigenetic trajectories of hepatic macrophages during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induced significant changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss was compensated by gain of adjacent monocyte-derived macrophages that exhibited convergent epigenomes, transcriptomes, and functions. NASH-induced changes in Kupffer cell enhancers were driven by AP-1 and EGR that reprogrammed LXR functions required for Kupffer cell identity and survival to instead drive a scar-associated macrophage phenotype. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct gene expression programs and corresponding functions.

The cell cytoskeleton is composed of microtubules, intermediate filaments, and actin that provide a rigid support structure important for cell shape. However, it is also a dynamic signaling scaffold that receives and transmits complex mechanosensing stimuli that regulate normal physiological and aberrant pathophysiological processes. Studying cytoskeletal functions in the cytoskeleton's native state is inherently difficult due to its rigid and insoluble nature. This has severely limited detailed proteomic analyses of the complex protein networks that regulate the cytoskeleton. Here, we describe a purification method that enriches for the cytoskeleton and its associated proteins in their native state that is also compatible with current mass spectrometry-based protein detection methods. This method can be used for biochemical, fluorescence, and large-scale proteomic analyses of numerous cell types. Using this approach, 2346 proteins were identified in the cytoskeletal fraction of purified mouse embryonic fibroblasts, of which 635 proteins were either known cytoskeleton proteins or cytoskeleton-interacting proteins. Functional annotation and network analyses using the Ingenuity Knowledge Database of the cytoskeletome revealed important nodes of interconnectivity surrounding well-established regulators of the actin cytoskeleton and focal adhesion complexes. This improved cytoskeleton purification method will aid our understanding of how the cytoskeleton controls normal and diseased cell functions.

Nuclear receptor-binding SET domain protein 1 (NSD1) prototype is a family of mammalian histone methyltransferases (NSD1, NSD2/MMSET/WHSC1, NSD3/WHSC1L1) that are essential in development and are mutated in human acute myeloid leukemia (AML), overgrowth syndromes, multiple myeloma and lung cancers. In AML, the recurring t(5;11)(q35;p15.5) translocation fuses NSD1 to nucleoporin-98 (NUP98). Here, we present the first characterization of the transforming properties and molecular mechanisms of NUP98-NSD1. We demonstrate that NUP98-NSD1 induces AML in vivo, sustains self-renewal of myeloid stem cells in vitro, and enforces expression of the HoxA7, HoxA9, HoxA10 and Meis1 proto-oncogenes. Mechanistically, NUP98-NSD1 binds genomic elements adjacent to HoxA7 and HoxA9, maintains histone H3 Lys 36 (H3K36) methylation and histone acetylation, and prevents EZH2-mediated transcriptional repression of the Hox-A locus during differentiation. Deletion of the NUP98 FG-repeat domain, or mutations in NSD1 that inactivate the H3K36 methyltransferase activity or that prevent binding of NUP98-NSD1 to the Hox-A locus precluded both Hox-A gene activation and myeloid progenitor immortalization. We propose that NUP98-NSD1 prevents EZH2-mediated repression of Hox-A locus genes by colocalizing H3K36 methylation and histone acetylation at regulatory DNA elements. This report is the first to link deregulated H3K36 methylation to tumorigenesis and to link NSD1 to transcriptional regulation of the Hox-A locus.

Severe hypoxia is a common cause of major brain, heart, and kidney injury in adults, children, and newborns. However, mild hypoxia can be protective against later, more severe hypoxia exposure via “hypoxic preconditioning,” a phenomenon that is not yet fully understood. Accordingly, we have established and optimized an embryonic zebrafish model to study hypoxic preconditioning. Using a functional genomic approach, we used this zebrafish model to identify and validate five novel hypoxia-protective genes, including irs2, crtc3, and camk2g2, which have been previously implicated in metabolic regulation. These results extend our understanding of the mechanisms of hypoxic preconditioning and affirm the discovery potential of this novel vertebrate hypoxic stress model.

Homeobox transcription factors Meis1 and Hoxa9 promote hematopoietic progenitor self-renewal and cooperate to cause acute myeloid leukemia (AML). While Hoxa9 alone blocks the differentiation of nonleukemogenic myeloid cell-committed progenitors, coexpression with Meis1 is required for the production of AML-initiating progenitors, which also transcribe a group of hematopoietic stem cell genes, including Cd34 and Flt3 (defined as Meis1-related leukemic signature genes). Here, we use dominant trans-activating (Vp16 fusion) or trans-repressing (engrailed fusion) forms of Meis1 to define its biochemical functions that contribute to leukemogenesis. Surprisingly, Vp16-Meis1 (but not engrailed-Meis1) functioned as an autonomous oncoprotein that mimicked combined activities of Meis1 plus Hoxa9, immortalizing early progenitors, inducing low-level expression of Meis1-related signature genes, and causing leukemia without coexpression of exogenous or endogenous Hox genes. Vp16-Meis1-mediated transformation required the Meis1 function of binding to Pbx and DNA but not its C-terminal domain (CTD). The absence of endogenous Hox gene expression in Vp16-Meis1-immortalized progenitors allowed us to investigate how Hox alters gene expression and cell biology in early hematopoietic progenitors. Strikingly, expression of Hoxa9 or Hoxa7 stimulated both leukemic aggressiveness and transcription of Meis1-related signature genes in Vp16-Meis1 progenitors. Interestingly, while the Hoxa9 N-terminal domain (NTD) is essential for cooperative transformation with wild-type Meis1, it was dispensable in Vp16-Meis1 progenitors. The fact that a dominant transactivation domain fused to Meis1 replaces the essential functions of both the Meis1 CTD and Hoxa9 NTD suggests that Meis-Pbx and Hox-Pbx (or Hox-Pbx-Meis) complexes co-occupy cellular promoters that drive leukemogenesis and that Meis1 CTD and Hox NTD cooperate in gene activation. Chromatin immunoprecipitation confirmed co-occupancy of Hoxa9 and Meis1 on the Flt3 promoter.

Meis1 is a homeodomain transcription factor coexpressed with Hoxa9 in most human acute myeloid leukemias (AMLs). In mouse models of leukemia produced by Hoxa9, Meis1 accelerates leukemogenesis. Because Hoxa9 immortalizes myeloid progenitors in the absence of Meis1 expression, the contribution of Meis1 toward leukemia remains unclear. Here, we describe a cultured progenitor model in which Meis1 programs leukemogenicity. Progenitors immortalized by Hoxa9 in culture are myeloid-lineage restricted and only infrequently caused leukemia after more than 250 days. Coexpressed Meis1 programmed rapid AML-initiating character, maintained multipotent progenitor potential, and induced expression of genes associated with short-term hematopoietic stem cells (HSCs), such as FLT3 and CD34, whose expression also characterizes the leukemia-initiating stem cells of human AML. Meis1 leukemogenesis functions required binding to Pbx, binding to DNA, and a conserved function of its C-terminal tail. We hypothesize that Meis1 is required for the homing and survival of leukemic progenitors within their hematopoietic niches, functions mediated by HSC-specific genes such as CD34 and Fms-like tyrosine kinase 3 (FLT3), respectively. This is the first example of a transcription factor oncoprotein (Meis1) that establishes expression of a tyrosine kinase oncoprotein (FLT3), and explains their coexpression in human leukemia. This cultured progenitor model will be useful to define the genetic basis of leukemogenesis involving Hoxa9 and Meis1.

Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer.

The genes encoding Hoxa9 and Meis1 are transcriptionally coactivated in a subset of acute myeloid leukemia (AML) in mice. In marrow reconstitution experiments, coexpression of both genes produces rapid AML, while neither gene alone generates overt leukemia. Although Hoxa9 and Meis1 can bind DNA as heterodimers, both can also heterodimerize with Pbx proteins. Thus, while their coactivation may result from the necessity to bind promoters as heterodimers, it may also result from the necessity of altering independent biochemical pathways that cooperate to generate AML, either as monomers or as heterodimers with Pbx proteins. Here we demonstrate that constitutive expression of Hoxa9 in primary murine marrow immortalizes a late myelomonocytic progenitor, preventing it from executing terminal differentiation to granulocytes or monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3. This immortalized phenotype is achieved in the absence of endogenous or exogenous Meis gene expression. The Hoxa9-immortalized progenitor exhibited a promyelocytic transcriptional profile, expressing PU.1, AML1, c-Myb, C/EBP alpha, and C/EBP epsilon as well as their target genes, the receptors for GM-CSF, G-CSF, and M-CSF and the primary granule proteins myeloperoxidase and neutrophil elastase. G-CSF obviated the differentiation block of Hoxa9, inducing neutrophilic differentiation with accompanying expression of neutrophil gelatinase B and upregulation of gp91phox. M-CSF also obviated the differentiation block, inducing monocytic differentiation with accompanying expression of the macrophage acetyl-low-density lipoprotein scavenger receptor and F4/80 antigen. Versions of Hoxa9 lacking the ANWL Pbx interaction motif (PIM) also immortalized a promyelocytic progenitor with intrinsic biphenotypic differentiation potential. Therefore, Hoxa9 evokes a cytokine-selective block in differentiation by a mechanism that does not require Meis gene expression or interaction with Pbx through the PIM.

Differentiation mechanisms and inflammatory functions of neutrophils and macrophages are usually studied by genetic and biochemical approaches that require costly breeding and time-consuming purification to obtain phagocytes for functional analysis. Because Hox oncoproteins enforce self-renewal of factor-dependent myeloid progenitors, we queried whether estrogen-regulated Hoxb8 (ER-Hoxb8) could immortalize macrophage or neutrophil progenitors that would execute normal differentiation and normal innate immune function upon ER-Hoxb8 inactivation. Here we describe methods to derive unlimited quantities of mouse macrophages or neutrophils by immortalizing their respective progenitors with ER-Hoxb8 using different cytokines to target expansion of different committed progenitors. ER-Hoxb8 neutrophils and macrophages are functionally superior to those produced by many other ex vivo differentiation models, have strong inflammatory responses and can be derived easily from embryonic day 13 (e13) fetal liver of mice exhibiting embryonic-lethal phenotypes. Using knockout or small interfering RNA (siRNA) technologies, this ER-Hoxb8 phagocyte maturation system represents a rapid analytical tool for studying macrophage and neutrophil biology.

HoxB8 was the first homeobox gene identified as a cause of leukemia. In murine WEHI3B acute myeloid leukemia (AML) cells, proviral integration leads to the expression of both HoxB8 and Interleukin (IL-3). Enforced expression of HoxB8 blocks differentiation of factor-dependent myeloid progenitors, while IL-3 co-expression induces autocrine proliferation and overt leukemogenicity. Previously, we demonstrated that HoxB8 binds DNA cooperatively with members of the Pbx family of transcription factors, and that HoxB8 makes contact with the Pbx homeodomain through a hexameric sequence designated the Pbx-interaction motif (PIM). E2a-Pbx1, an oncogenic derivative of Pbx1, both retains its ability to heterodimerize with Hox proteins and arrest myeloid differentiation. This observation prompts the question of whether E2a-Pbx1 and Hox oncoproteins use endogenous Hox and Pbx proteins, respectively, to target a common set of cellular genes. Here, we use four different models of neutrophil and macrophage differentiation to determine whether HoxB8 needs to bind DNA or Pbx cofactors in order to arrest myeloid differentiation. The ability of HoxB8 to bind DNA or to bind Pbx was essential (1) to block differentiation of factor-dependent myeloid progenitors from primary marrow; (2) to block IL-6-induced monocytic differentiation of M1-AML cells; and (3) to block granulocytic differentiation of GM-CSF-dependent ECoM-G cells. However, while DNA-binding was required, the HoxB8 Pbx-interaction motif was unnecessary for preventing macrophage differentiation of ECoM-M cells. We conclude that HoxB8 prevents differentiation by directly influencing cellular gene expression, and that the genetic context within a cell dictates whether the effect of HoxB8 is dependent on a physical interaction with Pbx proteins.

Variants of the S49 mouse lymphoma cell line exhibit multiple lesions along the pathway of cyclic AMP generation in response to β 2 -adrenergic stimulation. Two such variants, β p and β d , are characterized by decreased receptor binding and mRNA expression, 50% and 25% of wild-type receptor expression, respectively. The rate of β 2 -adrenergic receptor synthesis was measured and found to be decreased in the β d cells vis-a-vis the rate in wild type cells. The molecular mass of the β 2 -adrenergic receptor in the S49 wild-type, β p and β d variant cells was estimated by labeling the receptor with the photoaffinity probe [ 125 I]iodocyanopindololdiazirine. Receptor size was found to be 67 000 and 47 000 Da in the wild-type and 60 000 and 42 000 in the two variant cells. This 6 kDa discrepancy in mass was abolished upon treatment of labeled cell extracts with N -glycosidase F, suggesting the possibility of either N-terminal truncation or altered glycosylation of the receptor in the variant cells. To distinguish between these possibilities, we sequenced the β 2 -adrenergic receptor gene and two kilobases of the 5′-non-coding region. No differences were found in the coding region of the gene from wild-type, β p and β d S49 cells suggesting that both the diminished expression and the decreased size of β 2 -adrenergic receptor in the β p and β d S49 variants are related to impaired glycosylation of the receptor. This hypothesis was substantiated by the reduced retention of the variant cells' β 2 -adrenergic receptor on immobilized WGA. Furthermore, growth of the S49 cells in the presence of the α -mannosidase II inhibitor, swainsonine, preferentially impaired the ability of the receptors derived from the variant cells to bind to WGA. These results imply that altered expression and glycosylation of G-protein-linked receptors occur as a consequence of one or more mutations outside the receptor's open reading frame. ©1997 Elsevier Science B.V. All rights reserved.

MEIS1 and Hoxa9 are homeobox transcription factors that promote self-renewal in hematopoietic progenitors. MEIS1 does not induce leukemia, but cooperates strongly with Hoxa9 to produce acute myeloid leukemia (AML). Previously, we demonstrated that Hoxa9 blocks differentiation of myeloid progenitors that do not express MEIS1 and do not induce leukemic. Coexpression of MEIS1 causes transcription of genes that segregate with the leukemia-initiating subset of human AML blasts, such as CD34 and FLT3. We designate these genes as leukemic stem cell genes, or LSC genes. MEIS1 promoted LCS gene transcription by a mechanism that requires interaction with PBX and DNA, and that also requires a short MEIS1 C-terminal transactivation domain (CTD). Here we use a dominant transactivating or transrepressing form of MEIS1 to determine whether the activation or repression function of Pbx:MEIS1 complexes is sufficient to cause myeloid leukemia in combination with coexpressed Hoxa9. Surprisingly, fusion of MEIS1 to the Vp16 transactivation domain (but not the engrailed transrepression domain) produced an autonomous oncoprotein that immortalized progenitors and caused myeloid leukemias without the need for coexpressed exogenous or endogenous Hox genes. Like MEIS1, Vp16MEIS1 required binding to Pbx and DNA for immortalization; however, the CTD was not necessary in the context of Vp16MEIS1. This suggests that the CTD participates in target gene activation in AML blasts, a function replaced by Vp16 in its absence. Retroviral expression of Hoxa9 or Hoxa7 induced a further, strong, transcriptional upregulation of LSC genes in Vp16MEIS1 progenitors and elevated their leukemic potential to the level of bona fide AML blasts. These data suggest that transactivation is the essential function of Pbx:MEIS1 complexes in AML, and that HOX proteins cooperate with Pbx:MEIS1 complexes to activate transcription of early progenitor genes whose expression is required for human AML.