Regulation of transposable elements via histone modifications

Das Mausgenom besteht ungefähr zu 40% aus repetitiven Elementen (vor allem Transposons), die stillgelegt werden müssen, um die Integrität des Genoms aufrechterhalten zu können. Das wird durch verschiedene Mechanismen gewährleistet, wie zum Beispiel RNA Interferenz, Methylierung der DNA und Modulation der Chromatinstruktur. Die Fragestellung dieser Arbeit war, aufzuklären, inwieweit die Struktur des Chromatins an diesem Prozess beteiligt ist; im Detail sollte untersucht werden, welche Rolle die Azetylierung von Histonen bei der transkriptionellen Kontrolle von Transposons spielt. Histonazetylierung ist normalerweise mit transkriptionell aktivem Chromatin assoziiert und wird durch zwei antagonistisch wirkender Enzymklassen reguliert: Histonazetyltransferasen, die Azetylreste an die Histone anfügen, und Histondeazetylasen (HDAC), die sie wieder wegnehmen. Im Rahmen dieser Arbeit verwendeten wir HDAC-Hemmer bzw. Zellen ohne funktionelles Gen für das Enzym Histondeazetylase 1, um Histonazetylierungsmuster in der Zelle zu verändern. Anschließend wurden die transkriptionelle Aktivität von Transposons, Histonmodifikationsmuster auf Gesamthistonextrakten und Transposon-assoziiertem Chromatin überprüft. Dabei stellten wir eine erhöhte Histonazetylierung von transkriptionell aktivierten Transposons fest. Interessanterweise konnten wir mit dem LTR Retrotransposon VL30 (Virus-like 30S element) ein Transposon identifizieren, das nach Hemmung von Histondeazetylasen ein starke Expression aufwies. Eine Analyse von VL30 Transkripten ergab, dass sich für die erhöhte Transkription eine spezifische Untergruppe der VL30 Elemente verantwortlich zeigte, die durch spezielle Merkmale in der regulatorischen Region gekennzeichnet war. Weiters wurde die transkriptionelle Aktivierung mithilfe von Stimuli (wie Serum oder Anisomycin), die zu einer Aktivierung von MAK Kinasen und zur Phosphorylierung von Histonen führen können, noch verstärkt. Tatsächlich konnten wir nach kombinatorischer Behandlung doppelt modifizierte (azetylierte und phosphorylierte) Histone Proteine am LTR von VL30 Elementen detektieren. Es wird ein Modell vorgeschlagen, wonach bestimmte Histonmodifikationen kooperieren, um ein volle transkriptionelle Aktivierung von VL30 Retrotransposons zu erreichen.A large portion of the mouse genome (~40%) consists of repetitive elements which have to be silenced in order to maintain the integrity of the genome. This task is accomplished by different mechanisms, including RNA interference, DNA methylation and the modulation of chromatin structure. The aim of this work was to gain insight in how far the modulation of chromatin structure contributes to this process; in detail we wanted to clarify the role of histone acetylation in transcriptional control of transposable elements. Acetylation of histones is a post translational modification usually associated with open chromatin and transcriptionally active genes. It is regulated via the action of two antagonising enzyme families: histone acetyltransferases, which add acetyl-moieties to histone proteins and histone deacetylases (HDACs) which remove them. In the course of this work we took advantage of chemical inhibitors of HDACs and a cell system where a major histone deacetylase, HDAC1, was inactivated, to change histone acetylation patterns in the cell. Subsequently we analysed transcriptional activity of transposable elements, global histone modification patterns as well as histone acetylation of transposon-associated chromatin. We could detect increased histone acetylation levels of transcriptionally active transposable elements. Most interestingly we identified the LTR retrotransposon VL30 (Virus-like 30S element) to be highly inducible upon HDAC inhibition. Further analysis of VL30 transcripts revealed, that a distinct subgroup of VL30 elements, associated with a characteristic regulatory region, is mainly responsible for increased transcription. Moreover transcriptional activation was further increased by stimuli (such as serum and anisomycin) which lead to the activation of MAK kinase pathways and can cause phosphorylation of histones. Indeed we could detect double-modified (acetylated and phosphorylated) histone proteins at the LTR of VL30 elements upon combinatorial treatment with HDAC inhibitors and MAK kinase activators. A model is proposed, where two histone modifications cooperate to gain full transcriptional activity of VL30 retrotransposons

Activation of a HERV-H LTR induces expression of an aberrant calbindin protein in human prostate carcinoma cells

The human genome contains around 500 000 LTR retrotransposons including endogenous retroviruses (HERVs). Most of these LTR elements are silenced by epigenetic conditions, but may be reactivated by environmental factors such as chemicals, radiation or exogenous viruses. We have analyzed a HERV-H element located upstream of the human calbindin gene (CALB-1) that leads to expression of a truncated calbindin protein by alternative splicing in a human prostate carcinoma cell line (PC3). PC3 cells are polyploid with one or two complete alleles of chromosome 8, and four to five pieces of the 8q-arm, all containing the CALB-1 locus. Analysis of these loci did not reveal substantial alterations on DNA sequence level, such as modification of splice sites, suggesting an epigenetic activation of the HERV-H LTR. Therefore, we compared the DNA methylation status of this LTR in PC3 cells and in three prostate carcinoma cell lines not expressing the truncated calbindin protein. We found that the HERV-H LTR is hypomethylated in two cell lines including PC3. Chromatin immunoprecipitation (ChIP) analysis, however, revealed that the chromatin is associated with active marks (acetylated histone H4 and tri-methylated lysine4 at histone H3) at this locus only in PC3 cells. This data suggests that reactivation of the HERV-H LTR requires at least two steps: demethylation of DNA and modification of histones. There is evidence that expression of the truncated calbindin prevents apoptosis and may thus contribute to the malignant phenotype of PC3 cells

Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans

Although histone deacetylases are generally known as pro-tumourigenic factors, loss of HDAC1 is here shown to promote proliferation and inhibit differentiation in a mouse teratoma model, at least partly via regulation of the transcription factor SNAIL1

Epigenetic dysregulation of eukaryotic initiation factor 3 subunit E (eIF3E) by lysine methyltransferase REIIBP confers a pro-inflammatory phenotype in t(4;14) myeloma

REIIBP is a lysine methyltransferase aberrantly expressed through alternative promoter usage of NSD2 locus in t(4;14)-translocated multiple myeloma (MM). Clinically, t(4;14) translocation is an adverse prognostic factor found in approximately 15% of MM patients. The contribution of REIIBP relative to other NSD2 isoforms as a dependency gene in t(4;14)-translocated MM remains to be evaluated. Here, we demonstrated that despite homology with NSD2, REIIBP displayed distinct substrate specificity by preferentially catalyzing H3K4me3 and H3K27me3, with little activity on H3K36me2. Furthermore, REIIBP was regulated through microRNA by EZH2 in a Dicer-dependent manner, exemplifying a role of REIIBP in SET-mediated H3K27me3. Chromatin immunoprecipitation sequencing revealed chromatin remodeling characterized by changes in genome-wide and loci-specific occupancy of these opposing histone marks, allowing a bidirectional regulation of its target genes. Transcriptomics indicated that REIIBP induced a pro-inflammatory gene signature through upregulation of TLR7, which in turn led to B-cell receptor-independent activation of BTK and driving NFkB-mediated production of cytokines such as IL-6. Activation of this pathway is targetable using Ibrutinib and partially mitigated bortezomib resistance in a REIIBP xenograft model. Mechanistically, REIIBP upregulated TLR7 through eIF3E, and this relied on eIF3E RNA-binding function instead of its canonical protein synthesis activity, as demonstrated by direct binding to the 3’UTR of TLR7 mRNA. Altogether, we provided a rationale that co-existence of different NSD2 isoforms induced diversified oncogenic programs that should be considered in the strategies for t(4;14)-targeted therapy

Epigenetic Regulation of a Murine Retrotransposon by a Dual Histone Modification Mark

Large fractions of eukaryotic genomes contain repetitive sequences of which the vast majority is derived from transposable elements (TEs). In order to inactivate those potentially harmful elements, host organisms silence TEs via methylation of transposon DNA and packaging into chromatin associated with repressive histone marks. The contribution of individual histone modifications in this process is not completely resolved. Therefore, we aimed to define the role of reversible histone acetylation, a modification commonly associated with transcriptional activity, in transcriptional regulation of murine TEs. We surveyed histone acetylation patterns and expression levels of ten different murine TEs in mouse fibroblasts with altered histone acetylation levels, which was achieved via chemical HDAC inhibition with trichostatin A (TSA), or genetic inactivation of the major deacetylase HDAC1. We found that one LTR retrotransposon family encompassing virus-like 30S elements (VL30) showed significant histone H3 hyperacetylation and strong transcriptional activation in response to TSA treatment. Analysis of VL30 transcripts revealed that increased VL30 transcription is due to enhanced expression of a limited number of genomic elements, with one locus being particularly responsive to HDAC inhibition. Importantly, transcriptional induction of VL30 was entirely dependent on the activation of MAP kinase pathways, resulting in serine 10 phosphorylation at histone H3. Stimulation of MAP kinase cascades together with HDAC inhibition led to simultaneous phosphorylation and acetylation (phosphoacetylation) of histone H3 at the VL30 regulatory region. The presence of the phosphoacetylation mark at VL30 LTRs was linked with full transcriptional activation of the mobile element. Our data indicate that the activity of different TEs is controlled by distinct chromatin modifications. We show that activation of a specific mobile element is linked to a dual epigenetic mark and propose a model whereby phosphoacetylation of histone H3 is crucial for full transcriptional activation of VL30 elements

Functional Regulation of PPARs through Post-Translational Modifications

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily and they are essential regulators of cell differentiation, tissue development, and energy metabolism. Given their central roles in sensing the cellular metabolic state and controlling metabolic homeostasis, PPARs became important targets of drug development for the management of metabolic disorders. The function of PPARs is mainly regulated through ligand binding, which induces structural changes, further affecting the interactions with co-activators or co-repressors to stimulate or inhibit their functions. In addition, PPAR functions are also regulated by various Post-translational modifications (PTMs). These PTMs include phosphorylation, SUMOylation, ubiquitination, acetylation, and O-GlcNAcylation, which are found at numerous modification sites. The addition of these PTMs has a wide spectrum of consequences on protein stability, transactivation function, and co-factor interaction. Moreover, certain PTMs in PPAR proteins have been associated with the status of metabolic diseases. In this review, we summarize the PTMs found on the three PPAR isoforms PPARα, PPARβ/δ, and PPARγ, and their corresponding modifying enzymes. We also discuss the functional roles of these PTMs in regulating metabolic homeostasis and provide a perspective for future research in this intriguing field

Functional regulation of ppars through post-translational modifications

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily and they are essential regulators of cell differentiation, tissue development, and energy metabolism. Given their central roles in sensing the cellular metabolic state and controlling metabolic homeostasis, PPARs became important targets of drug development for the management of metabolic disorders. The function of PPARs is mainly regulated through ligand binding, which induces structural changes, further affecting the interactions with co-activators or co-repressors to stimulate or inhibit their functions. In addition, PPAR functions are also regulated by various Post-translational modifications (PTMs). These PTMs include phosphorylation, SUMOylation, ubiquitination, acetylation, and O-GlcNAcylation, which are found at numerous modification sites. The addition of these PTMs has a wide spectrum of consequences on protein stability, transactivation function, and co-factor interaction. Moreover, certain PTMs in PPAR proteins have been associated with the status of metabolic diseases. In this review, we summarize the PTMs found on the three PPAR isoforms PPARα, PPARβ/δ, and PPARγ, and their corresponding modifying enzymes. We also discuss the functional roles of these PTMs in regulating metabolic homeostasis and provide a perspective for future research in this intriguing field.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Regulation of Thermogenic Adipocyte Differentiation and Adaptive Thermogenesis Through Histone Acetylation

10.3389/fendo.2020.00095Frontiers in Endocrinology119

SIRT1 Interacts with and Deacetylates ATP6V1B2 in Mature Adipocytes

<div><p>SIRT1 plays a key role in maintaining metabolic homeostasis in mammals by directly modulating the activities of various transcription factors and metabolic enzymes through lysine deacetylation. White adipose tissue plays a key role in lipid storage and metabolism. To identify novel molecular targets of SIRT1 in fat cells, we used a non-biased proteomic approach. We identified a number of proteins whose acetylation status was significantly affected by SIRT1 modulator treatment in 3T3-L1 adipocytes. Among them, ATP6V1B2, a subunit of the vacuolar (H⁺)-ATPase, was further shown to be associated with SIRT1 by co-immunoprecipitation assay. Moreover, SIRT1 deacetylates ATP6V1B2 <i>in vitro</i> and <i>in vivo</i>. Taken together, our study demonstrates that ATP6V1B2 is a molecular target of SIRT1 in fat cells and the role of SIRT1 and ATP6V1B2 acetylation in the vacuolar (H⁺)-ATPase function warrants further investigation.</p></div

Proteomic analysis of SIRT1 modulator-treated 3T3-L1 mature adipocytes identified ATP6V1B2 as a potential SIRT1 target.

<p><b>(A)</b> Representative 2D-PAGE gel transfer blot stained with Coomassie blue and utilized in western blot analysis using a pan-acetyl-lysine antibody. <b>(B)</b> Western blot spots corresponding to ATP6V1B2 showed differential acetylation states upon SIRT1 modulator treatments, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133448#pone.0133448.g002" target="_blank">Fig 2A</a>. The spot intensities were quantified and are shown as a bar graph in the right panel. The value for the blank control was arbitrarily set as one. <b>(C)</b> The protein level of ATP6V1B2 was examined by western blot analyses after SIRT1 modulator treatment of 3T3-L1 mature adipocytes. Calnexin was used as a loading control.</p