Genome-wide analysis of histone modifications by ChIP-on-chip

Post-translational modifications to histone proteins regulate the packaging of genomic DNA into chromatin, gene activity and other functions of the genome. They are understood to play key roles in embryonic development and disease pathogenesis. Recent advances in technology have made it possible to analyze chromatin structure genome-wide in mammalian cells. Global patterns of histone modifications can be observed using a technique called ChIP-on-chip, which combines the specificity of chromatin immunoprecipitation with the unbiased, high-throughput capabilities of microarrays. The resulting maps provide insight into the functions of, and relationships between, different modifications. Here, we provide validated ChIP-on-chip methods for analyzing histone modification patterns at genome-scale in mammalian cells.     

DNA甲基化对子宫内膜异位症(EMs)的作用及其调控机制

表观遗传通过DNA甲基化、组蛋白修饰、染色质重塑、以及microRNA等调控方式来实现对基因表达、DNA复制和基因组稳定性的控制。DNA甲基化是目前研究的最为广泛的表观遗传修饰方式之一,可调控真核生物的基因表达。DNA甲基化在哺乳动物发育、肿瘤发生发展及人类其他疾病中均发挥着至关重要的作用。DNA甲基化状态的改变已被视为人类肿瘤细胞的生物标志之一。EMs虽是一种良性妇科疾病,但伴有细胞增殖、侵袭性及远处种植转移等肿瘤的特点。最新研究发现,DNA甲基化可能与子宫内膜异位症(EMs)的发生存在密切的关系并认为EMs从根本上是一种表观遗传学疾病。由于表观遗传修饰都是可逆的过程,这就为EMs的治疗提供了一种新的途径。本文就DNA甲基化在EMs中的发生发展中的作用及其调控的分子机制,以及在诊断治疗中作用的最新研究进展做一综述。     

DNA甲基化对子宫内膜异位症（EMs）的作用及其调控机制

表观遗传通过DNA甲基化、组蛋白修饰、染色质重塑、以及microRNA等调控方式来实现对基因表达、DNA复制和基因组稳定性的控制。DNA甲基化是目前研究的最为广泛的表观遗传修饰方式之一,可调控真核生物的基因表达。DNA甲基化在哺乳动物发育、肿瘤发生发展及人类其他疾病中均发挥着至关重要的作用。DNA甲基化状态的改变已被视为人类肿瘤细胞的生物标志之一。EMs虽是一种良性妇科疾病,但伴有细胞增殖、侵袭性及远处种植转移等肿瘤的特点。最新研究发现,DNA甲基化可能与子宫内膜异位症（EMs）的发生存在密切的关系并认为EMs从根本上是一种表观遗传学疾病。由于表观遗传修饰都是可逆的过程,这就为EMs的治疗提供了一种新的途径。本文就DNA甲基化在EMs中的发生发展中的作用及其调控的分子机制,以及在诊断治疗中作用的最新研究进展做一综述。     

Nuclear architecture in the light of gene expression and cell differentiation studies

It is evident that primary DNA sequences, that define genomes, are responsible for genome functions. However, the functional properties of chromatin are additionally regulated by heritable modifications known as epigenetic factors and, therefore, genomes should be also considered with respect to their 'epigenomes'. Nucleosome remodelling, DNA methylation and histone modifications are the most prominent epigenetic changes that play fundamental roles in the chromatin-mediated control of gene expression. Another important nuclear feature with functional relevance is the organization of mammalian chromatin into distinct chromosome territories which are surrounded by the interchromatin compartment that is necessary for transport of regulatory molecules to the targeted DNA. The inner structure of the chromosome territories, as well as the arrangement of the chromosomes within the interphase nuclei, has been found to be non-randomly organized. Therefore, a specific nuclear arrangement can be observed in many cellular processes, such as differentiation and tumour cell transformation.     

Preserving genome integrity and function: the DNA damage response and histone modifications

Modulation of chromatin templates in response to cellular cues, including DNA damage, relies heavily on the post-translation modification of histones. Numerous types of histone modifications including phosphorylation, methylation, acetylation, and ubiquitylation occur on specific histone residues in response to DNA damage. These histone marks regulate both the structure and function of chromatin, allowing for the transition between chromatin states that function in undamaged condition to those that occur in the presence of DNA damage. Histone modifications play well-recognized roles in sensing, processing, and repairing damaged DNA to ensure the integrity of genetic information and cellular homeostasis. This review highlights our current understanding of histone modifications as they relate to DNA damage responses (DDRs) and their involvement in genome maintenance, including the potential targeting of histone modification regulators in cancer, a disease that exhibits both epigenetic dysregulation and intrinsic DNA damage.     

Epigenetic events in malignant melanoma

Irreversible changes in the DNA sequence, including chromosomal deletions or amplification, activating or inactivating mutations in genes, have been implicated in the development and progression of melanoma. However, increasing attention is being turned towards the participation of 'epigenetic' events in melanoma progression that do not affect DNA sequence, but which nevertheless may lead to stable inherited changes in gene expression. Epigenetic events including histone modifications and DNA methylation play a key role in normal development and are crucial to establishing the correct program of gene expression. In contrast, mistargeting of such epigenetic modifications can lead to aberrant patterns of gene expression and loss of anti-cancer checkpoints. Thus, to date at least 50 genes have been reported to be dysregulated in melanoma by aberrant DNA methylation and accumulating evidence also suggests that mistargetting of histone modifications and altered chromatin remodeling activities will play a key role in melanoma. This review gives an overview of the many different types of epigenetic modifications and their involvement in cancer and especially in melanoma development and progression.     

Roles for nutrients in epigenetic events

The field of epigenetics is the study of modifications of DNA and DNA-binding proteins that alter the structure of chromatin without altering the nucleotide sequence of DNA; some of these modifications may be associated with heritable changes in gene function. Nutrients play essential roles in the following epigenetic events. First, folate participates in the generation of S-adenosylmethionine, which acts as a methyl donor in the methylation of cytosines in DNA; methylation of cytosines is associated with gene silencing. Second, covalent attachment of biotin to histones (DNA-binding proteins) plays a role in gene silencing and in the cellular response to DNA damage. Third, tryptophan and niacin are converted to nicotinamide adenine dinucleotide, which is a substrate for poly(ADP-ribosylation) of histones and other DNA-binding proteins; poly(ADP-ribosylation) of these proteins participates in DNA repair and apoptosis. Here we present a novel procedure to map nutrient-dependent epigenetic marks in the entire genomes of any given species: the combined use of chromatin immunoprecipitation assays and DNA microarrays. This procedure is also an excellent tool to map the enzymes that mediate modifications of DNA and DNA-binding proteins in chromatin. Given the tremendous opportunities offered by the combined use of chromatin immunoprecipitation assays and DNA microarrays, the nutrition community can expect seeing a surge of information related to roles for nutrients in epigenetic events.     

Histone modifications in DNA damage response

DNA damage is a relatively common event in eukaryotic cell and may lead to genetic mutation and even cancer. DNA damage induces cellular responses that enable the cell either to repair the damaged DNA or cope with the damage in an appropriate way. Histone proteins are also the fundamental building blocks of eukaryotic chromatin besides DNA, and many types of post-translational modifications often occur on tails of histones. Although the function of these modifications has remained elusive, there is ever-growing studies suggest that histone modifications play vital roles in several chromatin-based processes, such as DNA damage response. In this review, we will discuss the main histone modifications, and their functions in DNA damage response.     

Epigenetic control of plant immunity

In eukaryotic genomes, gene expression and DNA recombination are affected by structural chromatin traits. Chromatin structure is shaped by the activity of enzymes that either introduce covalent modifications in DNA and histone proteins or use energy from ATP to disrupt histone–DNA interactions. The genomic ‘marks’ that are generated by covalent modifications of histones and DNA, or by the deposition of histone variants, are susceptible to being altered in response to stress. Recent evidence has suggested that proteins generating these epigenetic marks play crucial roles in the defence against pathogens. Histone deacetylases are involved in the activation of jasmonic acid‐ and ethylene‐sensitive defence mechanisms. ATP‐dependent chromatin remodellers mediate the constitutive repression of the salicylic acid‐dependent pathway, whereas histone methylation at the WRKY70 gene promoter affects the activation of this pathway. Interestingly, bacterial‐infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genomic lesions.     

YTHDF1 facilitates PRC1-mediated H2AK119ub in human ES cells

Polycomb repressive complexes (PRCs) play critical roles in cell fate decisions during normal development as well as disease progression through mediating histone modifications such as H3K27me3 and H2AK119ub. How exactly PRCs recruited to chromatin remains to be fully illuminated. Here, we report that YTHDF1, the N6-methyladenine (m⁶A) RNA reader that was previously known to be mainly cytoplasmic, associates with RNF2, a PRC1 protein that mediates H2AK119ub in human embryonic stem cells (hESCs). A portion of YTHDF1 localizes in the nuclei and associates with RNF2/H2AK119ub on a subset of gene loci related to neural development functions. Knock-down YTHDF1 attenuates H2AK119ub modification on these genes and promotes neural differentiation in hESCs. Our findings provide a noncanonical mechanism that YTHDF1 participates in PRC1 functions in hESCs.     

H2A.Z depletion impairs proliferation and viability but not DNA double-strand breaks repair in human immortalized and tumoral cell lines

In mammalian cells, DNA double-strand breaks (DSB) can be repaired by 2 main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). To give access to DNA damage to the repair machinery the chromatin structure needs to be relaxed, and chromatin modifications play major roles in the control of these processes. Among the chromatin modifications, changes in nucleosome composition can influence DNA damage response as observed with the H2A.Z histone variant in yeast. In mammals, p400, an ATPase of the SWI/SNF family able to incorporate H2A.Z in chromatin, was found to be important for histone ubiquitination and BRCA1 recruitment around DSB or for HR in cooperation with Rad51. Recent data with 293T cells showed that mammalian H2A.Z is recruited to DSBs and is important to control DNA resection, therefore participating both in HR and NHEJ. Here we show that depletion of H2A.Z in the osteosarcoma U2OS cell line and in immortalized human fibroblasts does not change parameters of DNA DSB repair while affecting clonogenic ability and cell cycle distribution. In addition, no recruitment of H2A.Z around DSB can be detected in U2OS cells either after local laser irradiation or by chromatin immunoprecipitation. These data suggest that the role of H2A.Z in DSB repair is not ubiquitous in mammals. In addition, given that important cellular parameters, such as cell viability and cell cycle distribution, are more sensitive to H2A.Z depletion than DNA repair, our results underline the difficulty to investigate the role of versatile factors such as H2A.Z.     

Advances into understanding metabolites as signaling molecules in cancer progression

Recent years have seen a great expansion in our knowledge of the roles that metabolites play in cellular signaling. Structural data have provided crucial insights into mechanisms through which amino acids are sensed. New nutrient-coupled protein and RNA modifications have been identified and characterized. A growing list of functions has been ascribed to metabolic regulation of modifications such as acetylation, methylation, and glycosylation. A current challenge lies in developing an integrated understanding of the roles that metabolic signaling mechanisms play in physiology and disease, which will inform the design of strategies to target such mechanisms. In this brief article, we review recent advances in metabolic signaling through post-translational modification during cancer progression, to provide a framework for understanding signaling roles of metabolites in the context of cancer biology and illuminate areas for future investigation.     

Poly-ADP-Ribose (PAR) as an epigenetic flag

Epigenetics is the study of hereditable chromatin modifications, such as DNA methylation, histone modifications, and nucleosome-remodelling, which occur without alterations to the DNA sequence. The establishment of different epigenetic states in eukaryotes depends on regulatory mechanisms that induce structural changes in chromatin in response to environmental and cellular cues. Two classes of enzymes modulate chromatin accessibility: chromatin-covalent modifiers and ATP-dependent chromatin remodelling complexes. The first class of enzymes catalyzes covalent modifications of DNA as well as the amino- and carboxy-terminal tails of histones, while the second uses the energy of ATP hydrolysis to reposition nucleosomes along the chromatin fibers or to incorporate histone variants. Thus, epigenetic modifications are reversible nuclear reactions. In the last decade, many studies have strongly indicated that alterations in epigenetic modifications may contribute to the onset and progression of a variety of human diseases such as cancer. Therefore, the enzymes responsible for these chromatin changes are becoming attractive therapeutic targets.     

Functional and physical interaction of yeast Mgs1 with PCNA: impact on RAD6-dependent DNA damage tolerance

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.     

Effects of histone acetylation and CpG methylation on the structure of nucleosomes

Nucleosomes are the fundamental packing units of the eukaryotic genome. A nucleosome core particle comprises an octameric histone core wrapped around by ~147bp DNA. Histones and DNA are targets for covalent modifications mediated by various chromatin modification enzymes. These modifications play crucial roles in various gene regulation activities. A group of common hypotheses for the mechanisms of gene regulation involves changes in the structure and structural dynamics of chromatin induced by chromatin modifications. We employed single molecule fluorescence methods to test these hypotheses by monitoring the structure and structural dynamics of nucleosomes before and after histone acetylation and DNA methylation, two of the best-conserved chromatin modifications throughout eukaryotes. Our studies revealed that these modifications induce changes in the structure and structural dynamics of nucleosomes that may contribute directly to the formation of open or repressive chromatin conformation.