Epigenetics in the formation of pathological aggregates in amyotrophic lateral sclerosis.

The progressive degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is accompanied by the formation of a broad array of cytoplasmic and nuclear neuronal inclusions (protein aggregates) largely containing RNA-binding proteins such as TAR DNA-binding protein 43 (TDP-43) or fused in sarcoma/translocated in liposarcoma (FUS/TLS). This process is driven by a liquid-to-solid phase separation generally from proteins in membrane-less organelles giving rise to pathological biomolecular condensates. The formation of these protein aggregates suggests a fundamental alteration in the mRNA expression or the levels of the proteins involved. Considering the role of the epigenome in gene expression, alterations in DNA methylation, histone modifications, chromatin remodeling, non-coding RNAs, and RNA modifications become highly relevant to understanding how this pathological process takes effect. In this review, we explore the evidence that links epigenetic mechanisms with the formation of protein aggregates in ALS. We propose that a greater understanding of the role of the epigenome and how this inter-relates with the formation of pathological LLPS in ALS will provide an attractive therapeutic target.

Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

Muscle-Specific Pyruvate Kinase Isoforms, Pkm1 and Pkm2, Regulate Mammalian SWI/SNF Proteins and Histone 3 Phosphorylation During Myoblast Differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, Pkm1 and Pkm2, function in glycolysis, but Pkm2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of Pkm1 and Pkm2 during myoblast differentiation. RNA-seq analysis revealed that Pkm2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. Dpf2 and Baf250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for activation of myogenic gene expression during differentiation. Pkm2 also mediated the incorporation of Dpf2 and Baf250a into the regulatory sequences controlling myogenic gene expression. Pkm1 did not affect expression but was required for nuclear localization of Dpf2. Additionally, Pkm2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters, but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for Pkm2 and a novel function for Pkm1 in gene expression and chromatin regulation during myoblast differentiation.

Effects of Chemical inhibition of histone deacetylase proteins in the growth and virulence of Macrophomina phaseolina (Tassi) Goid / Efectos de la inhibición química de las proteínas desacetilasas de histonas en el crecimiento y la virulencia de Macrophomina phaseolina (Tassi) Goid

Abstract Chromatin remodeling enzymes are important "writers'', "readers'' and "erasers'' of the epigenetic code. These proteins are responsible for the placement, recognition, and removal of molecular marks in histone tails that trigger structural and functional changes in chromatin. This is also the case for histone deacetylases (HDACs), i.e., enzymes that remove acetyl groups from histone tails, signaling heterochromatin formation. Chromatin remodeling is necessary for cell differentiation processes in eukaryotes, and fungal pathogenesis in plants includes many adaptations to cause disease. Macrophomina phaseolina (Tassi) Goid. is a nonspe-cific, necrotrophic ascomycete phytopathogen that causes charcoal root disease. M. phaseolina is a frequent and highly destructive pathogen in crops such as common beans (Phaseolus vulgaris L.), particularly under both water and high temperature stresses. Here, we evaluated the effects of the classical HDAC inhibitor trichostatin A (TSA) on M. phaseolina in vitro growth and virulence. During inhibition assays, the growth of M. phaseolina in solid media, as well as the size of the microsclerotia, were reduced (p <0.05), and the colony morphology was remark-ably affected. Under greenhouse experiments, treatment with TSA reduced (p <0.05) fungal virulence in common bean cv. BAT 477. Tests of LIPK, MAC1 and PMK1 gene expression during the interaction of fungi with BAT 477 revealed noticeable deregulation. Our results provide additional evidence about the role of HATs and HDACs in important biological processes of M. phaseolina.

Resumen Las enzimas remodeladoras de la cromatina son «escritores¼, «lectores¼ y «borradores¼ importantes del código epigenético. Estas proteínas son responsables de la localización, el reconocimiento y la remoción de las marcas moleculares sobre las terminaciones de las histonas que desencadenan cambios funcionales y estructurales en la cromatina. Es el caso de las desacetilasas de histonas (HDAC), enzimas que remueven grupos acetilo de las «colas¼ de las histonas, señalizando la formación de heterocromatina. La anterior es una actividad necesaria en los procesos de diferenciación celular de los eucariotas, y se conoce que la patogénesis fúngica en las plantas requiere de adaptaciones diversas para ocasionar enfermedad. Macrophomina phaseolina (Tassi) Goid. es un ascomiceto fitopatógeno, necrótrofo e inespecífico, causante de la pudrición carbonosa. Este es un hongo frecuente y altamente destructivo en cultivos como fríjol común (Phaseolus vulgaris L.), particularmente bajo estrés hídrico y térmico. En este trabajo evaluamos los efectos del inhibidor de HDAC clásicas tricostatina A (TSA) sobre el crecimiento in vitro y la virulencia de M. phaseolina. El TSA redujo el crecimiento de M. phaseolina en medio sólido y el tamano de los microesclerocios (p < 0,05), lo que afectó la morfología colonial. En invernadero, el tratamiento con TSA disminuyó (p<0,05) la gravedad de la infección en la variedad de frijol BAT 477. La expresión de los genes de patogenicidad LIPK, MAC1 y PMK1 durante la interacción del hongo con la planta reveló una desregulación importante. Estos resultados proporcionan evidencia adicional del papel que cumplen las HDAC en la regulación de procesos biológicos fundamentales de M. phaseolina. © 2023 Asociación Argentina de Microbiología. Publicado por Elsevier Espana, S.L.U.

Regulation of the Wnt signaling pathway during myogenesis by the mammalian SWI/SNF ATPase BRG1.

Skeletal muscle differentiation is a tightly regulated process, and the importance of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling family for regulation of genes involved in skeletal myogenesis is well-established. Our prior work showed that bromodomains of mSWI/SNF ATPases BRG1 and BRM contribute to myogenesis by facilitating the binding of mSWI/SNF enzymes to regulatory regions of myogenic and other target genes. Here, we report that pathway analyses of differentially expressed genes from that study identified an additional role for mSWI/SNF enzymes via the regulation of the Wnt signaling pathway. The Wnt pathway has been previously shown to be important for skeletal muscle development. To investigate the importance of mSWI/SNF enzymes for the regulation of the Wnt pathway, individual and dual knockdowns were performed for BRG1 and BRM followed by RNA-sequencing. The results show that BRG1, but not BRM, is a regulator of Wnt pathway components and downstream genes. Reactivation of Wnt pathway by stabilization of ß-catenin could rescue the defect in myogenic gene expression and differentiation due to BRG1 knockdown or bromodomain inhibition using a specific small molecule inhibitor, PFI-3. These results demonstrate that BRG1 is required upstream of ß-catenin function. Chromatin immunoprecipitation of BRG1, BRM and ß-catenin at promoters of Wnt pathway component genes showed binding of BRG1 and ß-catenin, which provides further mechanistic insight to the transcriptional regulation of these genes.

Effects of chemical inhibition of histone deacetylase proteins in the growth and virulence of Macrophomina phaseolina (Tassi) Goid.

Chromatin remodeling enzymes are important "writers", "readers" and "erasers" of the epigenetic code. These proteins are responsible for the placement, recognition, and removal of molecular marks in histone tails that trigger structural and functional changes in chromatin. This is also the case for histone deacetylases (HDACs), i.e., enzymes that remove acetyl groups from histone tails, signaling heterochromatin formation. Chromatin remodeling is necessary for cell differentiation processes in eukaryotes, and fungal pathogenesis in plants includes many adaptations to cause disease. Macrophomina phaseolina (Tassi) Goid. is a nonspecific, necrotrophic ascomycete phytopathogen that causes charcoal root disease. M. phaseolina is a frequent and highly destructive pathogen in crops such as common beans (Phaseolus vulgaris L.), particularly under both water and high temperature stresses. Here, we evaluated the effects of the classical HDAC inhibitor trichostatin A (TSA) on M. phaseolinain vitro growth and virulence. During inhibition assays, the growth of M. phaseolina in solid media, as well as the size of the microsclerotia, were reduced (p<0.05), and the colony morphology was remarkably affected. Under greenhouse experiments, treatment with TSA reduced (p<0.05) fungal virulence in common bean cv. BAT 477. Tests of LIPK, MAC1 and PMK1 gene expression during the interaction of fungi with BAT 477 revealed noticeable deregulation. Our results provide additional evidence about the role of HATs and HDACs in important biological processes of M. phaseolina.

Epigenetics and Early Development.

The epigenome controls all aspect of eukaryotic development as the packaging of DNA greatly affects gene expression. Epigenetic changes are reversible and do not affect the DNA sequence itself but rather control levels of gene expression. As a result, the science of epigenetics focuses on the physical configuration of chromatin in the proximity of gene promoters rather than on the mechanistic effects of gene sequences on transcription and translation. In the present review we discuss three prominent epigenetic modifications, DNA methylation, histone methylation/acetylation, and the effects of chromatin remodeling complexes. Specifically, we introduce changes to the methylated state of DNA through DNA methyltransferases and DNA demethylases, discuss the effects of histone tail modifications such as histone acetylation and methylation on gene expression and present the functions of major ATPase subunit containing chromatin remodeling complexes. We also introduce examples of how changes in these epigenetic factors affect early development in humans and mice. In summary, this review provides an overview over the most important epigenetic mechanisms and provides examples of the dramatic effects of epigenetic changes in early mammalian development.

Expanding the genetic and phenotypic spectrum of CHD2-related disease: From early neurodevelopmental disorders to adult-onset epilepsy.

CHD2 encodes the chromodomain helicase DNA-binding protein 2, an ATP-dependent enzyme that acts as a chromatin remodeler. CHD2 pathogenic variants have been associated with various early onset phenotypes including developmental and epileptic encephalopathy, self-limiting or pharmacoresponsive epilepsies and neurodevelopmental disorders without epilepsy. We reviewed 84 previously reported patients carrying 76 different CHD2 pathogenic or likely pathogenic variants and describe 18 unreported patients carrying 12 novel pathogenic or likely pathogenic variants, two recurrent likely pathogenic variants (in two patients each), three previously reported pathogenic variants, one gross deletion. We also describe a novel phenotype of adult-onset pharmacoresistant epilepsy, associated with a novel CHD2 missense likely pathogenic variant, located in an interdomain region. A combined review of previously published and our own observations indicates that although most patients (72.5%) carry truncating CHD2 pathogenic variants, CHD2-related phenotypes encompass a wide spectrum of conditions with developmental delay/intellectual disability (ID), including prominent language impairment, attention deficit hyperactivity disorder and autistic spectrum disorder. Epilepsy is present in 92% of patients with a median age at seizure onset of 2 years and 6 months. Generalized epilepsy types are prevalent and account for 75.5% of all epilepsies, with photosensitivity being a common feature and adult-onset nonsyndromic epilepsy a rare presentation. No clear genotype-phenotype correlation has emerged.

The Roles of Chromatin Accessibility in Regulating the Candida albicans White-Opaque Phenotypic Switch.

Candida albicans, a diploid polymorphic fungus, has evolved a unique heritable epigenetic program that enables reversible phenotypic switching between two cell types, referred to as "white" and "opaque". These cell types are established and maintained by distinct transcriptional programs that lead to differences in metabolic preferences, mating competencies, cellular morphologies, responses to environmental signals, interactions with the host innate immune system, and expression of approximately 20% of genes in the genome. Transcription factors (defined as sequence specific DNA-binding proteins) that regulate the establishment and heritable maintenance of the white and opaque cell types have been a primary focus of investigation in the field; however, other factors that impact chromatin accessibility, such as histone modifying enzymes, chromatin remodelers, and histone chaperone complexes, also modulate the dynamics of the white-opaque switch and have been much less studied to date. Overall, the white-opaque switch represents an attractive and relatively "simple" model system for understanding the logic and regulatory mechanisms by which heritable cell fate decisions are determined in higher eukaryotes. Here we review recent discoveries on the roles of chromatin accessibility in regulating the C. albicans white-opaque phenotypic switch.

Regulation of the Mammalian SWI/SNF Family of Chromatin Remodeling Enzymes by Phosphorylation during Myogenesis.

Myogenesis is the biological process by which skeletal muscle tissue forms. Regulation of myogenesis involves a variety of conventional, epigenetic, and epigenomic mechanisms that control chromatin remodeling, DNA methylation, histone modification, and activation of transcription factors. Chromatin remodeling enzymes utilize ATP hydrolysis to alter nucleosome structure and/or positioning. The mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) family of chromatin remodeling enzymes is essential for myogenesis. Here we review diverse and novel mechanisms of regulation of mSWI/SNF enzymes by kinases and phosphatases. The integration of classic signaling pathways with chromatin remodeling enzyme function impacts myoblast viability and proliferation as well as differentiation. Regulated processes include the assembly of the mSWI/SNF enzyme complex, choice of subunits to be incorporated into the complex, and sub-nuclear localization of enzyme subunits. Together these processes influence the chromatin remodeling and gene expression events that control myoblast function and the induction of tissue-specific genes during differentiation.

Mechanisms of Schwann cell plasticity involved in peripheral nerve repair after injury.

The great plasticity of Schwann cells (SCs), the myelinating glia of the peripheral nervous system (PNS), is a critical feature in the context of peripheral nerve regeneration following traumatic injuries and peripheral neuropathies. After a nerve damage, SCs are rapidly activated by injury-induced signals and respond by entering the repair program. During the repair program, SCs undergo dynamic cell reprogramming and morphogenic changes aimed at promoting nerve regeneration and functional recovery. SCs convert into a repair phenotype, activate negative regulators of myelination and demyelinate the damaged nerve. Moreover, they express many genes typical of their immature state as well as numerous de-novo genes. These genes modulate and drive the regeneration process by promoting neuronal survival, damaged axon disintegration, myelin clearance, axonal regrowth and guidance to their former target, and by finally remyelinating the regenerated axon. Many signaling pathways, transcriptional regulators and epigenetic mechanisms regulate these events. In this review, we discuss the main steps of the repair program with a particular focus on the molecular mechanisms that regulate SC plasticity following peripheral nerve injury.

Epigenetic Regulation by ATP-Dependent Chromatin-Remodeling Enzymes: SNF-ing Out Crosstalk.

Cells utilize precise mechanisms to access genomic DNA with spatiotemporal accuracy. ATP-dependent chromatin-remodeling enzymes (also known simply as "remodelers") comprise a specialized class of enzymes that is intimately involved in genomic organization and accessibility. Remodelers selectively position nucleosomes to either alleviate chromatin compaction or achieve genomic condensation locally, based on a multitude of cellular signals. By dictating nucleosome position, remodelers control local euchromatic and heterochromatic states. These activities govern the accessibility of regulatory regions like promoters and enhancers to transcription factors, RNA polymerases, and coactivators or -repressors. As studies unravel the complexities of epigenetic topography, evidence points to a chromatin-based interactome where regulators interact competitively, cooperatively, and/or codependently through physical and functional means. These types of interactions, or crosstalk, between remodelers raise important questions for tissue development. Here, we briefly review the evidence for remodeler interactions and argue for additional studies examining crosstalk.

MITF interacts with the SWI/SNF subunit, BRG1, to promote GATA4 expression in cardiac hypertrophy.

The transcriptional regulation of pathological cardiac hypertrophy involves the interplay of transcription factors and chromatin remodeling enzymes. The Microphthalmia-Associated Transcription Factor (MITF) is highly expressed in cardiomyocytes and is required for cardiac hypertrophy. However, the transcriptional mechanisms by which MITF promotes cardiac hypertrophy have not been elucidated. In this study, we tested the hypothesis that MITF promotes cardiac hypertrophy by activating transcription of pro-hypertrophy genes through interactions with the SWI/SNF chromatin remodeling complex. In an in vivo model of cardiac hypertrophy, expression of MITF and the BRG1 subunit of the SWI/SNF complex increased coordinately in response to pressure overload. Expression of MITF and BRG1 also increased in vitro when cardiomyocytes were stimulated with angiotensin II or a ß-adrenergic agonist. Both MITF and BRG1 were required to increase cardiomyocyte size and activate expression of hypertrophy markers in response to ß-adrenergic stimulation. We detected physical interactions between MITF and BRG1 in cardiomyocytes and found that they cooperate to regulate expression of a pro-hypertrophic transcription factor, GATA4. Our data show that MITF binds to the E box element in the GATA4 promoter and facilitates recruitment of BRG1. This is associated with enhanced expression of the GATA4 gene as evidenced by increased Histone3 lysine4 tri-methylation (H3K4me3) on the GATA4 promoter. Thus, in hypertrophic cardiomyoctes, MITF is a key transcriptional activator of a pro-hypertrophic gene, GATA4, and this regulation is dependent upon the BRG1 component of the SWI/SNF complex.

Modulation of Brahma expression by the mitogen-activated protein kinase/extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation.

Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.

Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages.

Resolvin D1 (RvD1; 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) is an endogenous immunoresolvent that regulates acute inflammation and orchestrates resolution. Here, we investigated anti-inflammatory and proresolving actions of RvD1 after oral administration. RvD1 rapidly accumulated in the mouse plasma after oral delivery and dose-dependently (1-100 ng/mouse) reduced leukocyte infiltration in zymosan A-induced acute peritonitis. Using mathematical resolution indices, RvD1 reduced Ψmax by ∼50%, shortened the resolution interval by 3 h, and significantly reduced total leukocyte (by ∼30-45%) and polymorphonuclear neutrophil (by ∼40-55%) accumulation when administered at the peak of peritonitis. RvD1 also improved course and outcome of severe peritonitis, shifting it toward resolution. In peritoneal macrophages (MΦs) from the resolution phase of peritonitis, RvD1 down-regulated (by 2- to 3-fold) select genes that control gene transcription, namely coactivator-associated arginine methyltransferase 1 (CARM1), and downstream genes, such as colony-stimulating factor 3, intercellular adhesion molecule 1, and monocyte inflammatory protein 2, which promote neutrophil infiltration and reduce MΦ phagocytosis. Congruently, CARM1 knockdown in human and murine MΦs induced a proresolving phenotype, recapitulating in vivo actions of RvD1. These results establish novel properties of RvD1 and demonstrate that RvD1 modifies the transcription control machinery in MΦs, as part of its mechanisms of action during the resolution of acute inflammation.-Recchiuti, A., Codagnone, M., Pierdomenico, A. M., Rossi, C., Mari, V. C., Cianci, E., Simiele, F., Gatta, V., Romano, M. Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages.