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1.
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
2.
Genes Dev ; 33(17-18): 1159-1174, 2019 09 01.
Article in English | MEDLINE | ID: mdl-31371436

ABSTRACT

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.


Subject(s)
Nucleosomes/metabolism , Regulatory Elements, Transcriptional/physiology , Transcription Factors/metabolism , Animals , Cells, Cultured , Chromatin Immunoprecipitation , High-Throughput Nucleotide Sequencing , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Micrococcal Nuclease/metabolism
3.
Genes Dev ; 31(4): 399-412, 2017 02 15.
Article in English | MEDLINE | ID: mdl-28275002

ABSTRACT

Enhancers and promoters that control the transcriptional output of terminally differentiated cells include cell type-specific and broadly active housekeeping elements. Whether the high constitutive activity of these two groups of cis-regulatory elements relies on entirely distinct or instead also on shared regulators is unknown. By dissecting the cis-regulatory repertoire of macrophages, we found that the ELF subfamily of ETS proteins selectively bound within 60 base pairs (bp) from the transcription start sites of highly active housekeeping genes. ELFs also bound constitutively active, but not poised, macrophage-specific enhancers and promoters. The role of ELFs in promoting high-level constitutive transcription was suggested by multiple evidence: ELF sites enabled robust transcriptional activation by endogenous and minimal synthetic promoters, ELF recruitment was stabilized by the transcriptional machinery, and ELF proteins mediated recruitment of transcriptional and chromatin regulators to core promoters. These data suggest that the co-optation of a limited number of highly active transcription factors represents a broadly adopted strategy to equip both cell type-specific and housekeeping cis-regulatory elements with the ability to efficiently promote transcription.


Subject(s)
Gene Expression Regulation/genetics , Genes, Essential/genetics , Macrophages/physiology , Transcription Factors/metabolism , Animals , Chromatin/metabolism , Enhancer Elements, Genetic/genetics , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , Protein Binding , Protein Transport , Transcription Factors/genetics
4.
Nat Immunol ; 18(5): 530-540, 2017 05.
Article in English | MEDLINE | ID: mdl-28288101

ABSTRACT

Stimulation of macrophages with interferon-γ (IFN-γ) and interleukin 4 (IL-4) triggers distinct and opposing activation programs. During mixed infections or cancer, macrophages are often exposed to both cytokines, but how these two programs influence each other remains unclear. We found that IFN-γ and IL-4 mutually inhibited the epigenomic and transcriptional changes induced by each cytokine alone. Computational and functional analyses revealed the genomic bases for gene-specific cross-repression. For instance, while binding motifs for the transcription factors STAT1 and IRF1 were associated with robust and IL-4-resistant responses to IFN-γ, their coexistence with binding sites for auxiliary transcription factors such as AP-1 generated vulnerability to IL-4-mediated inhibition. These data provide a core mechanistic framework for the integration of signals that control macrophage activation in complex environmental conditions.


Subject(s)
Cell Differentiation , Epigenesis, Genetic , Macrophages/physiology , Proto-Oncogene Proteins c-myc/metabolism , Transcriptional Activation , Animals , Cell Line , Gene Expression Regulation , Humans , Interferon Regulatory Factor-1/genetics , Interferon Regulatory Factor-1/metabolism , Interferon-gamma/metabolism , Interleukin-4/metabolism , Mice , Mice, Inbred Strains , Proto-Oncogene Proteins c-myc/genetics , RNA, Small Interfering/genetics , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , Signal Transduction , Transcription Factor AP-1/metabolism
5.
Mol Cell ; 60(3): 460-74, 2015 Nov 05.
Article in English | MEDLINE | ID: mdl-26593720

ABSTRACT

Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1 H3K4 methyltransferases and the nuclear protein phosphatase 1 (PP1) complexes to the initiating Pol II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1, or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes and active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs.


Subject(s)
Cell Nucleus/metabolism , Enhancer Elements, Genetic/physiology , Promoter Regions, Genetic/physiology , RNA Polymerase II/metabolism , RNA, Untranslated/biosynthesis , Transcription Termination, Genetic/physiology , Active Transport, Cell Nucleus/physiology , Animals , Cell Nucleus/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Mice , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , RNA Polymerase II/genetics , RNA, Untranslated/genetics
6.
Mol Cell ; 54(5): 844-857, 2014 Jun 05.
Article in English | MEDLINE | ID: mdl-24813947

ABSTRACT

Transcription factors (TFs) preferentially bind sites contained in regions of computationally predicted high nucleosomal occupancy, suggesting that nucleosomes are gatekeepers of TF binding sites. However, because of their complexity mammalian genomes contain millions of randomly occurring, unbound TF consensus binding sites. We hypothesized that the information controlling nucleosome assembly may coincide with the information that enables TFs to bind cis-regulatory elements while ignoring randomly occurring sites. Hence, nucleosomes would selectively mask genomic sites that can be contacted by TFs and thus be potentially functional. The hematopoietic pioneer TF Pu.1 maintained nucleosome depletion at macrophage-specific enhancers that displayed a broad range of nucleosome occupancy in other cell types and in reconstituted chromatin. We identified a minimal set of DNA sequence and shape features that accurately predicted both Pu.1 binding and nucleosome occupancy genome-wide. These data reveal a basic organizational principle of mammalian cis-regulatory elements whereby TF recruitment and nucleosome deposition are controlled by overlapping DNA sequence features.


Subject(s)
Enhancer Elements, Genetic , Nucleosomes/genetics , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Animals , Base Sequence , Binding Sites , Cells, Cultured , Consensus Sequence , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Mice , Models, Genetic , Nucleosomes/metabolism , Proto-Oncogene Proteins/genetics , RNA, Small Interfering/genetics , Sequence Analysis, DNA , Support Vector Machine , Trans-Activators/genetics
7.
Brief Funct Genomics ; 12(6): 483-8, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23814131

ABSTRACT

Inflammation is a tightly regulated process that is achieved through the specific and controlled activation of innate immune system cells, notably neutrophils, macrophages and dendritic cells. Functional genomics studies in the last years have contributed to an integrated picture of the events controlling macrophage specialization and plasticity. Here we will summarize recent advances in the characterization of the molecular determinants of macrophage functional properties, and specifically how the interplay between genomic and epigenomic information, transcription factors and micro-environmental cues results in a fine-tuned transcriptional response.


Subject(s)
Genomics/methods , Inflammation/genetics , Animals , Humans , Inflammation/immunology , Macrophages/immunology , Macrophages/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
8.
Methods ; 63(1): 66-75, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23542771

ABSTRACT

Mammalian genomes are pervasively transcribed, generating mostly RNAs with no coding potential that display different size, structure and interspecies sequence conservation. A prominent contribution to the ncRNA pool comes from the transcription of cis-regulatory elements, namely promoters, enhancers and locus control regions. While this phenomenon has been extensively documented, possible roles of such ncRNAs in gene regulation are still unclear. Addressing this issue will require experimental strategies dealing with the low abundance of enhancer-templated ncRNAs and aimed at specifically dissecting the relative role of transcription per se vs. RNA products. In this review, we first focus on the identification and characterization of cis-regulatory elements, highlighting the differences between emerging classes of ncRNAs associated to specific chromatin signatures. We then discuss current experimental strategies to dissect the function of nc transcription and computational approaches to the analysis and classification of regulatory sequences identified in next-generation sequencing experiments.


Subject(s)
Computational Biology/methods , Enhancer Elements, Genetic , Gene Expression Regulation , Regulatory Sequences, Nucleic Acid/genetics , Animals , Conserved Sequence/genetics , Genome , Mammals , Promoter Regions, Genetic
9.
Cell Mol Life Sci ; 70(11): 2015-29, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23283301

ABSTRACT

Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.


Subject(s)
Adenylate Kinase/physiology , Forkhead Transcription Factors/physiology , Glucose/metabolism , Mitochondria/metabolism , Sirtuin 3/physiology , Adenylate Kinase/genetics , Adenylate Kinase/metabolism , Animals , Cells, Cultured , DNA, Mitochondrial/metabolism , Electron Transport , Energy Metabolism , Food Deprivation , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , Genome, Mitochondrial , Humans , Male , Mice , Mice, Inbred C57BL , Models, Biological , NIH 3T3 Cells , Sirtuin 3/genetics , Sirtuin 3/metabolism
10.
Cancer Lett ; 324(1): 98-108, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-22579651

ABSTRACT

We recently demonstrated that p38α is required to maintain colorectal cancer (CRC) metabolism, as its inhibition leads to FoxO3A activation, autophagy, cell death, and tumor growth reduction both in vitro and in vivo. Here we show that inhibition of p38α is followed by TRAIL-mediated activation of caspase-8 and FoxO3A-dependent HER3 upregulation with consequent overactivation of the MEK-ERK1/2 survival pathway. p38α and MEK combined inhibition specifically induces apoptosis by enabling TRAIL signaling propagation through t-Bid and caspase-3, and fosters cell death in CRC cells and preclinical mouse models. Current MEK1-directed pharmacological strategies could thus be exploited, in combination with p38α inhibition, to develop new approaches for CRC treatment.


Subject(s)
Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Aged , Aged, 80 and over , Animals , Apoptosis/drug effects , Apoptosis/genetics , Benzamides/pharmacology , Caspase 8/metabolism , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Diphenylamine/analogs & derivatives , Diphenylamine/pharmacology , Enzyme Inhibitors/pharmacology , Female , HT29 Cells , Humans , Imidazoles/pharmacology , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , Male , Mice , Mice, Nude , Middle Aged , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Mitogen-Activated Protein Kinase 14/metabolism , Phosphorylation , Pyridines/pharmacology , Xenograft Model Antitumor Assays , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
11.
Cell Cycle ; 10(14): 2355-63, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21685725

ABSTRACT

Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G 1- or G 2-specific differentiation checkpoint. In response to genotoxins that induce G 1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G 2 checkpoint relies on the inability of MyoD to bind the chromatin at the G 2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Repair , Gene Expression Regulation , Muscles/metabolism , Myoblasts/drug effects , Animals , Antineoplastic Agents/toxicity , Cell Line , Chromatin/metabolism , DNA Damage , G1 Phase , G2 Phase , Mice , MyoD Protein/antagonists & inhibitors , MyoD Protein/metabolism , Myoblasts/cytology , Myoblasts/metabolism , Oxidants/toxicity , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-abl/metabolism
12.
EMBO Rep ; 12(2): 164-71, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21212806

ABSTRACT

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.


Subject(s)
Muscle Development/drug effects , Mutagens/toxicity , MyoD Protein/metabolism , Myogenic Regulatory Factor 5/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Biological Evolution , Cell Cycle Proteins/metabolism , Cell Differentiation , Cells, Cultured , Coculture Techniques , Cross-Linking Reagents/toxicity , DNA Damage , DNA-Binding Proteins/metabolism , Etoposide/toxicity , Female , Gene Knockdown Techniques , Methyl Methanesulfonate/toxicity , Mice/embryology , Mitomycin/toxicity , MyoD Protein/genetics , Myogenic Regulatory Factor 5/genetics , Phosphorylation , Pregnancy , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-abl/physiology , RNA Interference , Somites/drug effects , Somites/metabolism , Tumor Suppressor Proteins/metabolism
13.
J Cell Physiol ; 213(3): 642-8, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17894406

ABSTRACT

Studies on DNA damage responses in proliferating cells have revealed the relationship between sensing and repair of the DNA lesions and the regulation of the cell cycle, leading to the discovery and molecular characterization of the DNA damage-activated cell cycle checkpoints. Much less is known about the DNA damage response in progenitors of differentiated cells, in which cell cycle arrest is a critical signal to trigger the differentiation program, and in terminally differentiated cells, which are typically post-mitotic. How DNA lesions are detected, processed and repaired in these cells, the functional impact of DNA damage on transcription of differentiation-specific genes, how these events are coordinated at the molecular level, the consequence of defective DNA damage response on tissue-specific functions and its potential relationship with age-related diseases are currently open questions. In particular the biological complexity inherent to the global genome reprogramming of tissue progenitors, such as embryonic or adult stem cells, suggests the importance of an accurate DNA damage response at the transcription level in these cells to ensure the genomic integrity of regenerating tissues.


Subject(s)
Cell Differentiation/genetics , DNA Damage/genetics , Animals , Humans , Models, Biological
14.
Proc Natl Acad Sci U S A ; 103(30): 11160-5, 2006 Jul 25.
Article in English | MEDLINE | ID: mdl-16847267

ABSTRACT

The MAGE gene family is characterized by a conserved domain (MAGE Homology Domain). A subset of highly homologous MAGE genes (group A; MAGE-A) belong to the chromosome X-clustered cancer/testis antigens. MAGE-A genes are normally expressed in the human germ line and overexpressed in various tumor types; however, their biological function is largely unknown. Here we present evidence indicating that MageA2 protein, belonging to the MAGE-A subfamily, confers wild-type-p53-sensitive resistance to etoposide (ET) by inducing a novel p53 inhibitory loop involving recruitment of histone deacetylase 3 (HDAC3) to MageA2/p53 complex, thus strongly down-regulating p53 transactivation function. In fact, enhanced MageA2 protein levels, in addition to ET resistance, correlate with impaired acetylation of both p53 and histones surrounding p53-binding sites. Association between MAGE-A expression levels and resistance to ET treatment is clearly shown in short-term cell lines obtained from melanoma biopsies harboring wild-type-p53, whereas cells naturally, or siRNA-mediated expressing low MAGE-A levels, correlate with enhanced p53-dependent sensitivity to ET. In addition, combined trichostatin A/ET treatment in melanoma cells expressing high MAGE-A levels reestablishes p53 response and reverts the chemoresistance.


Subject(s)
Antigens, Neoplasm/physiology , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Histone Deacetylases/metabolism , Melanoma-Specific Antigens/biosynthesis , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/metabolism , Apoptosis , Cell Line, Tumor , DNA Damage , Histone Deacetylases/chemistry , Humans , Hydroxamic Acids/pharmacology , Melanoma/metabolism , Melanoma-Specific Antigens/metabolism , Protein Structure, Tertiary
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