The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1.

Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci.

Pioneer transcription factors (TFs) function as genomic first responders, binding to inaccessible regions of chromatin to promote enhancer formation. The mechanism by which pioneer TFs gain access to chromatin remains an important unanswered question. Here we show that PARP-1, a nucleosome-binding protein, cooperates with intrinsic properties of the pioneer TF Sox2 to facilitate its binding to intractable genomic loci in embryonic stem cells. These actions of PARP-1 occur independently of its poly(ADP-ribosyl) transferase activity. PARP-1-dependent Sox2-binding sites reside in euchromatic regions of the genome with relatively high nucleosome occupancy and low co-occupancy by other transcription factors. PARP-1 stabilizes Sox2 binding to nucleosomes at suboptimal sites through cooperative interactions on DNA. Our results define intrinsic and extrinsic features that determine Sox2 pioneer activity. The conditional pioneer activity observed with Sox2 at a subset of binding sites may be a key feature of other pioneer TFs operating at intractable genomic loci.

The histone deubiquitinase OTLD1 targets euchromatin to regulate plant growth.

Histone monoubiquitination is associated with active chromatin and plays an important role in epigenetic regulation of gene expression in plants. Deubiquitinating enzymes remove the ubiquitin group from histones and thereby contribute to gene repression. The Arabidopsis thaliana genome encodes 50 deubiquitinases, yet only 2 of them-UBP26 and OTLD1, members of the USP/UBP (ubiquitin-specific protease and ubiquitin-binding protein) and OTU (ovarian tumor protease) deubiquitinase families-are known to target histones. Furthermore, UBP26 is the only plant histone deubiquitinase for which the functional role has been characterized in detail. We used gain- and loss-of-function alleles of OTLD1 to examine its role in the plant life cycle and showed that OTLD1 stimulates plant growth, increases cell size, and induces transcriptional repression of five major regulators of plant organ growth and development: GA20OX, WUS, OSR2, ARL, and ABI5 OTLD1 associated with chromatin at each of these target genes and promoted the removal of euchromatic histone acetylation, ubiquitination, and methylation marks. Thus, these data indicate that OTLD1 promotes the concerted epigenetic regulation of a set of genes that collectively limit plant growth.

Regulation of cell differentiation and function by the euchromatin histone methyltranserfases G9a and GLP.

The euchromatin histone methyltransferases (EHMTs) are an evolutionarily conserved protein family that are known for their ability to dimethylate histone 3 at lysine 9 in euchromatic regions of the genome. In mammals there are two EHMT proteins, G9a, encoded by EHMT2, and GLP, encoded by EHMT1. EHMTs have diverse roles in the differentiation of different tissues and cell types and are involved in adult-specific processes like memory, drug addiction, and immune response. This review discusses recent findings from rodent and Drosophila models that are beginning to reveal the broad biological role and complex mechanistic functioning of EHMT proteins.

Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin.

Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼ 90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Histone acetylation: from code to web and router via intrinsically disordered regions.

Structural changes of chromatin, which consists of nucleosomes and nucleosome-associated factors, lead to functional changes that are important determinants of eukaryotic gene regulation. These structural changes are regulated by modifications of histones and DNA, both of which are components of nucleosomes, as well as by replacement of histone variants and the actions of noncoding RNAs. In studies of chromatin modifications, a great deal of attention has been paid to histone acetylation. Progress in understanding this subject has been extensive, including i) elucidation of the relationship of histone acetylation and gene activity; ii) the first isolation of a histonemodifying enzyme; iii) the first identification of a factor that recognizes a modified site; iv) elucidation of the mechanism by which histone modification leads to structural changes in nucleosomes; and v) elucidation of the mechanism of border formation between euchromatin and heterochromatin. Histone acetylation is considered to be fundamental in several fields, including studies of a) the role of chromatin and epigenetics in higher-order biochemical systems such as transcription, DNA replication, and repair; b) biological phenomena such as cell proliferation and differentiation; and c) cancer and aging, potentially leading to clinical applications. In this review, I will discuss the histone code hypothesis, at one time believed to represent a unified theory regarding the functions of histone modification. In addition, I will describe the "modification web theory, " by which the problems in the histone code hypothesis can be overcome, as well as the "signal router theory, " which explains the mechanisms of formation, development, and evolution of the modification web from a structural viewpoint. Lastly, I will illustrate how these novel theories partially explain the robustness of biological systems against various perturbations, and elucidate the strategy that a cell employs to avoid fatal fragility.

Distant positioning of proteasomal proteolysis relative to actively transcribed genes.

While it is widely acknowledged that the ubiquitin-proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.

Las terapias epigenéticas, más allá de los biológicos en el tratamiento de la artritis reumatoide / Epigenetic therapies, a step beyond biologics for rheumatoid arthritis

La artritis reumatoide ha experimentado en la última década una revolución terapéutica, derivada del conocimiento de los procesos patogénicos y favorecida por el desarrollo de la tecnología necesaria para distribuir tratamientos moleculares. Las nuevas terapias permiten diferenciar subtipos de pacientes según la respuesta clínica y además mejoran nuestra comprensión de la enfermedad. Ello hace vaticinar la llegada de nuevas generaciones de moléculas para un tratamiento individualizado. Uno de los campos hacia donde se dirigen las investigaciones es la epigenética. Los mecanismos de regulación epigenéticos son interruptores que deciden cuándo y cómo expresar determinados genes en cada célula. Actuando como vigilantes de una expresión génica inapropiada, protegen al organismo del desarrollo de tumores. La principal ventaja de los tratamientos epigenéticos podría ser su selectividad por las células que muestran patrones epigenéticos alterados, por lo que el reto es identificar estas alteraciones entre los pacientes con artritis reumatoide. Aunque debe establecerse su perfil de seguridad, parece probable el uso de terapias epigenéticas en las enfermedades autoinmunes (AU)

Over the last decade, the management of rheumatoid arthritis has evolved as a result of both the understanding of disease-related processes and the availability of the necessary high-throughput technology to provide patients with molecule-based therapies. New therapies allow the classification of patients into subsets as regards clinical response, at the same time adding to our knowledge of rheumatoid arthritis pathogenesis. New generations of molecules will likely soon be ready for “a la carte” treatment of patients. A promising field of research is epigenetics. Epigenetic regulatory mechanisms switch on and off the transcription of specific genes in individual cells. Acting as observers on non-adequate gene expression, these mechanisms yield protection against the development of tumours. The major achievement of epigenetic therapies could be their selective action on cells with altered epigenetic programs, and it is our challenge to recognize these alterations among patients with rheumatoid arthritis. Although safety concerns may arise, epigenetic drugs will likely be used to treat autoimmune diseases (AU)

Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions.

How DNA repair machineries detect and access, within the context of chromatin, lesions inducing little or no distortion of the DNA structure is a poorly understood process. Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway. We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin. The underlying mechanism does not require direct interaction of the protein with the oxidized base, however, the release of the protein from the chromatin fraction requires completion of repair. Inducing chromatin compaction by sucrose results in a complete but reversible inhibition of the in vivo repair of 8-oxoguanine. We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions.

Protein acetylation sites mediated by Schistosoma mansoni GCN5.

The transcriptional co-activator GCN5, a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcription activation. As in other eukaryotes, the DNA from the parasite Schistosome mansoni is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Using a series of synthetic peptides we determined that Lys-14 of histone H3 was acetylated by the recombinant SmGCN5-HAT domain. SmGCN5 was also able to acetylate schistosome non-histone proteins, such as the nuclear receptors SmRXR1 and SmNR1, and the co-activator SmNCoA-62. Electron microscopy revealed the presence of SmGCN5 protein in the nuclei of vitelline cells. Within the nucleus, SmGCN5 was found to be located in interchromatin granule clusters (IGCs), which are transcriptionally active structures. The data suggest that SmGCN5 is involved in transcription activation.

A two-way street: LSD1 regulates chromatin boundary formation in S. pombe and Drosophila.

Two recent studies in Molecular Cell (Lan et al., 2007; Rudolph et al., 2007) implicate histone demethylation by LSD1 in the regulation of boundaries between silenced and active chromatin domains in both fission yeast and flies, but by distinct mechanisms.

Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells.

In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a(-/-) embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a(-/-) cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a(-/-) cells. These data indicate that G9a site-selectively contributes to DNA methylation.

The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development.

Mammalian G9a is a histone H3 Lys-9 (H3-K9) methyltransferase localized in euchromatin and acts as a co-regulator for specific transcription factors. G9a is required for proper development in mammals as g9a-/g9a- mice show growth retardation and early lethality. Here we describe the cloning, the biochemical and genetical analyses of the Drosophila homolog dG9a. We show that dG9a shares the structural organization of mammalian G9a, and that it is a multi-catalytic histone methyltransferase with specificity not only for lysines 9 and 27 on H3 but also for H4. Surprisingly, it is not the H4-K20 residue that is the target for this methylation. Spatiotemporal expression analyses reveal that dG9a is abundantly expressed in the gonads of both sexes, with no detectable expression in gonadectomized adults. In addition we find a low but clearly observable level of dG9a transcript in developing embryos, larvae and pupae. Genetic and RNAi experiments reveal that dG9a is involved in ecdysone regulatory pathways.

Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9.

Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing. G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and is essential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals, called GLP/Eu-HMTase1. Here we show that GLP is also important for H3-K9 methylation of mouse euchromatin. GLP-deficiency led to embryonic lethality, a severe reduction of H3-K9 mono- and dimethylation, the induction of Mage-a gene expression, and HP1 relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency. Furthermore, we show that G9a and GLP formed a stoichiometric heteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo, likely through the formation of higher-order heteromeric complexes.