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1.
Mol Syst Biol ; 20(6): 626-650, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38724853

ABSTRACT

More than 500 kinases are implicated in the control of most cellular process in mammals, and deregulation of their activity is linked to cancer and inflammatory disorders. 80 clinical kinase inhibitors (CKIs) have been approved for clinical use and hundreds are in various stages of development. However, CKIs inhibit other kinases in addition to the intended target(s), causing both enhanced clinical effects and undesired side effects that are only partially predictable based on in vitro selectivity profiling. Here, we report an integrative approach grounded on the use of chromatin modifications as unbiased, information-rich readouts of the functional effects of CKIs on macrophage activation. This approach exceeded the performance of transcriptome-based approaches and allowed us to identify similarities and differences among CKIs with identical intended targets, to recognize novel CKI specificities and to pinpoint CKIs that may be repurposed to control inflammation, thus supporting the utility of this strategy to improve selection and use of CKIs in clinical settings.


Subject(s)
Epigenome , Protein Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Humans , Animals , Mice , Macrophage Activation/drug effects , Macrophage Activation/genetics , Macrophages/drug effects , Macrophages/metabolism
2.
Cancer Cell ; 42(4): 662-681.e10, 2024 Apr 08.
Article in English | MEDLINE | ID: mdl-38518775

ABSTRACT

Intratumor morphological heterogeneity of pancreatic ductal adenocarcinoma (PDAC) predicts clinical outcomes but is only partially understood at the molecular level. To elucidate the gene expression programs underpinning intratumor morphological variation in PDAC, we investigated and deconvoluted at single cell level the molecular profiles of histologically distinct clusters of PDAC cells. We identified three major morphological and functional variants that co-exist in varying proportions in all PDACs, display limited genetic diversity, and are associated with a distinct organization of the extracellular matrix: a glandular variant with classical ductal features; a transitional variant displaying abortive ductal structures and mixed endodermal and myofibroblast-like gene expression; and a poorly differentiated variant lacking ductal features and basement membrane, and showing neuronal lineage priming. Ex vivo and in vitro evidence supports the occurrence of dynamic transitions among these variants in part influenced by extracellular matrix composition and stiffness and associated with local, specifically neural, invasion.


Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/pathology , Carcinoma, Pancreatic Ductal/pathology , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Basement Membrane/metabolism , Nervous System
3.
Sci Adv ; 10(13): eadk5386, 2024 Mar 29.
Article in English | MEDLINE | ID: mdl-38536927

ABSTRACT

While pancreatic ductal adenocarcinomas (PDACs) are addicted to KRAS-activating mutations, inhibitors of downstream KRAS effectors, such as the MEK1/2 kinase inhibitor trametinib, are devoid of therapeutic effects. However, the extensive rewiring of regulatory circuits driven by the attenuation of the KRAS pathway may induce vulnerabilities of therapeutic relevance. An in-depth molecular analysis of the transcriptional and epigenomic alterations occurring in PDAC cells in the initial hours after MEK1/2 inhibition by trametinib unveiled the induction of endogenous retroviruses (ERVs) escaping epigenetic silencing, leading to the production of double-stranded RNAs and the increased expression of interferon (IFN) genes. We tracked ERV activation to the early induction of the transcription factor ELF3, which extensively bound and activated nonsilenced retroelements and synergized with IRF1 (interferon regulatory factor 1) in the activation of IFNs and IFN-stimulated genes. Trametinib-induced viral mimicry in PDAC may be exploited in the rational design of combination therapies in immuno-oncology.


Subject(s)
Carcinoma, Pancreatic Ductal , Endogenous Retroviruses , Pancreatic Neoplasms , Humans , Endogenous Retroviruses/genetics , Signal Transduction , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism
4.
Mol Cell ; 84(5): 967-980.e10, 2024 Mar 07.
Article in English | MEDLINE | ID: mdl-38242130

ABSTRACT

Histone-modifying enzymes depend on the availability of cofactors, with acetyl-coenzyme A (CoA) being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA-producing enzymes translocate to the nucleus suggests that high concentrations of locally synthesized metabolites may impact acylation of histones and other nuclear substrates, thereby controlling gene expression. Here, we show that 2-ketoacid dehydrogenases are stably associated with the Mediator complex, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in lipopolysaccharide-stimulated macrophages, inhibited the activity of Mediator-associated 2-ketoacid dehydrogenases. Elevation of NO levels and the disruption of Mediator complex integrity both affected de novo histone acetylation within a shared set of genomic regions. Our findings indicate that the local supply of acetyl-CoA generated by 2-ketoacid dehydrogenases bound to Mediator is required to maximize acetylation of histone tails at sites of elevated HAT activity.


Subject(s)
Histones , Nitric Oxide , Histones/genetics , Histones/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Nitric Oxide/metabolism , Mediator Complex/metabolism , Oxidoreductases/metabolism
5.
Genes Dev ; 36(7-8): 414-432, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35361678

ABSTRACT

Six methyltransferases divide labor in establishing genomic profiles of histone H3 lysine 9 methylation (H3K9me), an epigenomic modification controlling constitutive heterochromatin, gene repression, and silencing of retroelements. Among them, SETDB1 is recruited to active chromatin domains to silence the expression of endogenous retroviruses. In the context of experiments aimed at determining the impact of SETDB1 on stimulus-inducible gene expression in macrophages, we found that loss of H3K9me3 caused by SETDB1 depletion was associated with increased recruitment of CTCF to >1600 DNA binding motifs contained within SINE B2 repeats, a previously unidentified target of SETDB1-mediated repression. CTCF is an essential regulator of chromatin folding that restrains DNA looping by cohesin, thus creating boundaries among adjacent topological domains. Increased CTCF binding to SINE B2 repeats enhanced insulation at hundreds of sites and increased loop formation within topological domains containing lipopolysaccharide-inducible genes, which correlated with their impaired regulation in response to stimulation. These data indicate a role of H3K9me3 in restraining genomic distribution and activity of CTCF, with an impact on chromatin organization and gene regulation.


Subject(s)
Chromatin , Gene Silencing , Heterochromatin , Methylation , Retroelements
6.
PLoS Biol ; 20(2): e3001538, 2022 02.
Article in English | MEDLINE | ID: mdl-35143476

ABSTRACT

Within the immune system, microRNAs (miRNAs) exert key regulatory functions. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9-mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Cell Proliferation/genetics , Gene Expression Regulation , Intercellular Signaling Peptides and Proteins/genetics , MicroRNAs/genetics , Regulatory Factor X Transcription Factors/genetics , 3' Untranslated Regions/genetics , Blotting, Western , CD4-Positive T-Lymphocytes/cytology , Cells, Cultured , Chromatin Immunoprecipitation Sequencing/methods , HEK293 Cells , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Jurkat Cells , Lymphocyte Activation/genetics , Regulatory Factor X Transcription Factors/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics
7.
EMBO Rep ; 22(9): e53251, 2021 09 06.
Article in English | MEDLINE | ID: mdl-34328708

ABSTRACT

Macrophages react to microbial and endogenous danger signals by activating a broad panel of effector and homeostatic responses. Such responses entail rapid and stimulus-specific changes in gene expression programs accompanied by extensive rewiring of metabolism, with alterations in chromatin modifications providing one layer of integration of transcriptional and metabolic regulation. A systematic and mechanistic understanding of the mutual influences between signal-induced metabolic changes and gene expression is still lacking. Here, we discuss current evidence, controversies, knowledge gaps, and future areas of investigation on how metabolic and transcriptional changes are dynamically integrated during macrophage activation. The cross-talk between metabolism and inflammatory gene expression is in part accounted for by alterations in the production, usage, and availability of metabolic intermediates that impact the macrophage epigenome. In addition, stimulus-inducible gene expression changes alter the production of inflammatory mediators, such as nitric oxide, that in turn modulate the activity of metabolic enzymes thus determining complex regulatory loops. Critical issues remain to be understood, notably whether and how metabolic rewiring can bring about gene-specific (as opposed to global) expression changes.


Subject(s)
Inflammation , Macrophage Activation , Gene Expression , Gene Expression Regulation , Humans , Inflammation/genetics , Macrophage Activation/genetics , Macrophages
8.
J Cell Biol ; 220(3)2021 03 01.
Article in English | MEDLINE | ID: mdl-33566069

ABSTRACT

The commitment of mesenchymal stem cells to preadipocytes is stimulated by hormonal induction. Preadipocytes induced to differentiate repress protein synthesis, remodel their cytoskeleton, and increase mitochondrial function to support anabolic pathways. These changes enable differentiation into mature adipocytes. Our understanding of the factors that coordinately regulate the early events of adipocyte differentiation remains incomplete. Here, by using multipronged approaches, we have identified zinc finger CCCH-type containing 10 (Zc3h10) as a critical regulator of the early stages of adipogenesis. Zc3h10 depletion in preadipocytes resulted in increased protein translation and impaired filamentous (F)-actin remodeling, with the latter detrimental effect leading to mitochondrial and metabolic dysfunction. These defects negatively affected differentiation to mature adipocytes. In contrast, Zc3h10 overexpression yielded mature adipocytes with remarkably increased lipid droplet size. Overall, our study establishes Zc3h10 as a fundamental proadipogenic transcription factor that represses protein synthesis and promotes F-actin/mitochondria dynamics to ensure proper energy metabolism and favor lipid accumulation.


Subject(s)
Actins/metabolism , Adipogenesis , Mitochondria/metabolism , Protein Biosynthesis , RNA-Binding Proteins/metabolism , Actin Cytoskeleton/metabolism , Adipocytes/metabolism , Adipogenesis/genetics , Adipose Tissue, White/metabolism , Animals , Cell Line , Citric Acid Cycle , Cyclic AMP-Dependent Protein Kinases/metabolism , Energy Metabolism/genetics , Gene Expression Regulation , Lipid Metabolism/genetics , Male , Metabolome , Mice, Inbred C57BL , Mitochondrial Dynamics , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Transcriptome/genetics , rho GTP-Binding Proteins/metabolism
9.
EMBO J ; 38(19): e103271, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31529710

ABSTRACT

The association between specific transcription factors (TFs) and defined tissue-specific macrophage phenotypes is far from being univocal. Many TFs that have been associated with tissue-specific macrophages have relatively broad expression profiles suggesting the critical involvement of combinatorial regulation by multiple TFs in bringing about specific phenotypes. In the current issue of The EMBO Journal, Rauschmeier et al (2019) report the identification of the transcriptional repressors BHLHE40 and BHLHE41 as novel regulators of tissue-specific macrophage properties that work both to promote alveolar macrophage (AM) identity and to suppress alternative tissue-specific phenotypes.


Subject(s)
Macrophages, Alveolar , Transcription Factors/genetics , Macrophages , Phenotype
10.
Blood Adv ; 2(20): 2691-2703, 2018 10 23.
Article in English | MEDLINE | ID: mdl-30337297

ABSTRACT

Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds DNA at CArG (CC[A/T]6GG) domains in association with myocardin-family proteins (eg, myocardin-related transcription factor A [MRTFA]) or the ternary complex factor family of E26 transformation-specific (ETS) proteins. In primary hematopoietic cells, knockout of either SRF or MRTFA decreases megakaryocyte (Mk) maturation causing thrombocytopenia. The human erythroleukemia (HEL) cell line mimics the effects of MRTFA on Mk maturation, and MRTFA overexpression (MRTFAOE) in HEL cells enhances megakaryopoiesis. To identify the mechanisms underlying these effects, we performed integrated analyses of anti-SRF chromatin immunoprecipitation (ChIP) and RNA-sequencing data from noninduced and phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])-induced HEL cells, with and without MRTFAOE We found that 11% of genes were upregulated with TPA induction, which was enhanced by MRTFAOE, resulting in an upregulation of 25% of genes. MRTFAOE increased binding of SRF to genomic sites and enhanced TPA-induced expression of SRF target genes. The TPA-induced genes are predicted to be regulated by SRF and ETS factors, whereas those upregulated by TPA plus MRTFAOE lack ETS binding motifs, and MRTFAOE skews SRF binding to genomic regions with CArG sites in regions relatively lacking in ETS binding motifs. Finally, ChIP-polymerase chain reaction using HEL cells and primary human CD34+ cell-derived subpopulations confirms that both SRF and MRTFA have increased binding during megakaryopoiesis at upregulated target genes (eg, CORO1A). We show for the first time that MRTFA increases both the genomic association and activity of SRF and upregulates genes that enhance primary human megakaryopoiesis.


Subject(s)
Megakaryocytes/metabolism , Serum Response Factor/genetics , Trans-Activators/genetics , Humans , Trans-Activators/metabolism
11.
Mol Cell ; 65(6): 1081-1095.e5, 2017 Mar 16.
Article in English | MEDLINE | ID: mdl-28286024

ABSTRACT

We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.


Subject(s)
Chromatin Assembly and Disassembly , Chromatin/enzymology , Extracellular Signal-Regulated MAP Kinases/metabolism , Histones/metabolism , Serum Response Factor/metabolism , TCF Transcription Factors/metabolism , Transcription, Genetic , Animals , Cell Line , Chromatin/drug effects , Chromatin/genetics , Chromatin Assembly and Disassembly/drug effects , Early Growth Response Protein 1/genetics , Early Growth Response Protein 1/metabolism , Enzyme Activation , Mice , Mice, Knockout , Mutation , Phosphorylation , RNA Interference , Serum Response Factor/genetics , Signal Transduction , TCF Transcription Factors/genetics , Tetradecanoylphorbol Acetate/pharmacology , Transcription Initiation Site , Transcription, Genetic/drug effects , Transfection , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-1/metabolism
12.
Genes Dev ; 31(23-24): 2361-2375, 2017 12 01.
Article in English | MEDLINE | ID: mdl-29317486

ABSTRACT

Both the MRTF-SRF and the YAP-TEAD transcriptional regulatory networks respond to extracellular signals and mechanical stimuli. We show that the MRTF-SRF pathway is activated in cancer-associated fibroblasts (CAFs). The MRTFs are required in addition to the YAP pathway for CAF contractile and proinvasive properties. We compared MRTF-SRF and YAP-TEAD target gene sets and identified genes directly regulated by one pathway, the other, or both. Nevertheless, the two pathways exhibit mutual dependence. In CAFs, expression of direct MRTF-SRF genomic targets is also dependent on YAP-TEAD activity, and, conversely, YAP-TEAD target gene expression is also dependent on MRTF-SRF signaling. In normal fibroblasts, expression of activated MRTF derivatives activates YAP, while activated YAP derivatives activate MRTF. Cross-talk between the pathways requires recruitment of MRTF and YAP to DNA via their respective DNA-binding partners (SRF and TEAD) and is therefore indirect, arising as a consequence of activation of their target genes. In both CAFs and normal fibroblasts, we found that YAP-TEAD activity is sensitive to MRTF-SRF-induced contractility, while MRTF-SRF signaling responds to YAP-TEAD-dependent TGFß signaling. Thus, the MRF-SRF and YAP-TEAD pathways interact indirectly through their ability to control cytoskeletal dynamics.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cancer-Associated Fibroblasts/physiology , Cytoskeleton/metabolism , DNA-Binding Proteins/metabolism , Mammary Neoplasms, Animal/physiopathology , Phosphoproteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins , Cell Line, Tumor , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Mice , Phosphoproteins/genetics , Signal Transduction , TEA Domain Transcription Factors , Trans-Activators/genetics , Transcriptional Activation/genetics , Transforming Growth Factor beta1/metabolism , YAP-Signaling Proteins
13.
Mol Cell ; 64(6): 1048-1061, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27867007

ABSTRACT

The ERK-regulated ternary complex factors (TCFs) act with the transcription factor serum response factor (SRF) to activate mitogen-induced transcription. However, the extent of their involvement in the immediate-early transcriptional response, and their wider functional significance, has remained unclear. We show that, in MEFs, TCF inactivation significantly inhibits over 60% of TPA-inducible gene transcription and impairs cell proliferation. Using integrated SRF ChIP-seq and Hi-C data, we identified over 700 TCF-dependent SRF direct target genes involved in signaling, transcription, and proliferation. These also include a significant number of cytoskeletal gene targets for the Rho-regulated myocardin-related transcription factor (MRTF) SRF cofactor family. The TCFs act as general antagonists of MRTF-dependent SRF target gene expression, competing directly with the MRTFs for access to SRF. As a result, TCF-deficient MEFs exhibit hypercontractile and pro-invasive behavior. Thus, competition between TCFs and MRTFs for SRF determines the balance between antagonistic proliferative and contractile programs of gene expression.


Subject(s)
Fibroblasts/metabolism , Gene Expression Regulation , Serum Response Factor/genetics , Ternary Complex Factors/genetics , Animals , Base Sequence , Cell Line , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Gene Expression Profiling , Genetic Complementation Test , Humans , Mice , Serum Response Factor/metabolism , Signal Transduction , Ternary Complex Factors/antagonists & inhibitors , Ternary Complex Factors/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription, Genetic , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-1/metabolism
14.
Genes Dev ; 28(9): 943-58, 2014 May 01.
Article in English | MEDLINE | ID: mdl-24732378

ABSTRACT

The transcription factor SRF (serum response factor) recruits two families of coactivators, the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors), to couple gene transcription to growth factor signaling. Here we investigated the role of the SRF network in the immediate transcriptional response of fibroblasts to serum stimulation. SRF recruited its cofactors in a gene-specific manner, and virtually all MRTF binding was directed by SRF. Much of SRF DNA binding was serum-inducible, reflecting a requirement for MRTF-SRF complex formation in nucleosome displacement. We identified 960 serum-responsive SRF target genes, which were mostly MRTF-controlled, as assessed by MRTF chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) and/or sensitivity to MRTF-linked signals. MRTF activation facilitates RNA polymerase II (Pol II) recruitment or promoter escape according to gene context. MRTF targets encode regulators of the cytoskeleton, transcription, and cell growth, underpinning the role of SRF in cytoskeletal dynamics and mechanosensing. Finally, we show that specific activation of either MRTFs or TCFs can reset the circadian clock.


Subject(s)
Actins/metabolism , Fibroblasts/physiology , Serum/metabolism , Signal Transduction , Transcription, Genetic/genetics , Animals , CLOCK Proteins/genetics , Circadian Clocks/genetics , Mice , Mitogen-Activated Protein Kinases/metabolism , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Serum Response Factor/metabolism
15.
Oncotarget ; 1(4): 278-88, 2010 Aug.
Article in English | MEDLINE | ID: mdl-21304178

ABSTRACT

MYCN is a member of the MYC family of oncoproteins frequently amplified or overexpressed in aggressive, paediatric tumours of the nervous system. In this study we have identified the gene B-MYB, encoding the transcription factor also known as MYBL2, as a downstream target of MYCN. Using multiple in silico databases we show that expression of B-MYB significantly correlates with that of MYCN in neuroblastoma patients. MYCN binds to and activates the B-MYB gene in vivo and in vitro. Blunting B-MYB expression by RNA interference causes reduced proliferation of MYCN amplified, but not MYCN-non amplified, neuroblastoma cell lines, indicating that tumour cells are addicted to B-MYB in a MYCN dependent manner. Notably, B-MYB binds in vivo to the MYCN amplicon and is required for its expression. We conclude that MYCN and B-MYB are engaged in a reciprocal regulatory loop whose pharmacological targeting could be beneficial to patients with the aggressive forms of cancer in which MYCN is amplified.


Subject(s)
Cell Cycle Proteins/genetics , Gene Expression Regulation, Neoplastic , Neuroblastoma/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Trans-Activators/genetics , Blotting, Western , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Chromatin Immunoprecipitation , Gene Expression , Humans , N-Myc Proto-Oncogene Protein , Neuroblastoma/metabolism , Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA Interference , Trans-Activators/metabolism
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