Mechanistic analyses based on improved imaging techniques have begun to explore the biological implications of chromatin movement within the nucleus. Studies in both prokaryotes and eukaryotes have ...shed light on what regulates the mobility of DNA over long distances. Interestingly, in eukaryotes, genomic loci increase their movement in response to double-strand break induction. Break mobility, in turn, correlates with the efficiency of repair by homologous recombination. We review here the source and regulation of DNA mobility and discuss how it can both contribute to and jeopardize genome stability.
The factors that sequester transcriptionally repressed heterochromatin at the nuclear periphery are currently unknown. In a genome-wide RNAi screen, we found that depletion of S-adenosylmethionine ...(SAM) synthetase reduces histone methylation globally and causes derepression and release of heterochromatin from the nuclear periphery in Caenorhabditis elegans embryos. Analysis of histone methyltransferases (HMTs) showed that elimination of two HMTs, MET-2 and SET-25, mimics the loss of SAM synthetase, abrogating the perinuclear attachment of heterochromatic transgenes and of native chromosomal arms rich in histone H3 lysine 9 methylation. The two HMTs target H3K9 in a consecutive fashion: MET-2, a SETDB1 homolog, mediates mono- and dimethylation, and SET-25, a previously uncharacterized HMT, deposits H3K9me3. SET-25 colocalizes with its own product in perinuclear foci, in a manner dependent on H3K9me3, but not on its catalytic domain. This colocalization suggests an autonomous, self-reinforcing mechanism for the establishment and propagation of repeat-rich heterochromatin.
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► Reduced SAM synthetase levels releases perinuclear arrays of heterochromatin ► Peripheral anchoring of gene arrays and chromosome arms requires H3K9 methylation ► The repressive H3K9me3 mark is established step-wise by the HMTs MET-2 and SET-25 ► H3K9me3 leads to sequestration of SET-25 in perinuclear heterochromatin foci
The identification of two histone methyltransferases that act sequentially on lysine 9 on histone 3 to produce trimethylated lysine 9 reveals a requirement for H3K9me3 for the association of chromatin with the nuclear envelope and suggests the existence of a self-reinforcing loop that ensures robust H3K9 methylation at the nuclear periphery.
Chromatin is organized into higher-order structures that form subcompartments in interphase nuclei. Different categories of specialized enzymes act on chromatin and regulate its compaction and ...biophysical characteristics in response to physiological conditions. We present an overview of the function of chromatin structure and its dynamic changes in response to genotoxic stress, focusing on both subnuclear organization and the physical mobility of DNA. We review the requirements and mechanisms that cause chromatin relocation, enhanced mobility, and chromatin unfolding as a consequence of genotoxic lesions. An intriguing link has been established recently between enhanced chromatin dynamics and histone loss.
The combinatorial action of transcription factors drives cell-type-specific gene expression patterns. However, transcription factor binding and gene regulation occur in the context of chromatin, ...which modulates DNA accessibility. High-resolution chromatin interaction maps have defined units of chromatin that are in spatial proximity, called topologically associated domains (TADs). TADs can be further classified based on expression activity, replication timing, or the histone marks or non-histone proteins associated with them. Independently, other chromatin domains have been defined by their likelihood to interact with non-DNA structures, such as the nuclear lamina. Lamina-associated domains (LADs) correlate with low gene expression and late replication timing. TADs and LADs have recently been evaluated with respect to cell-type-specific gene expression. The results shed light on the relevance of these forms of chromatin organization for transcriptional regulation, and address specifically how chromatin sequestration influences cell fate decisions during organismal development.
Nuclear actin has been implicated in a variety of DNA-related processes including chromatin remodeling, transcription, replication, and DNA repair. However, the mechanistic understanding of actin in ...these processes has been limited, largely due to a lack of research tools that address the roles of nuclear actin specifically, that is, distinct from its cytoplasmic functions. Recent findings support a model for homology-directed DNA double-strand break (DSB) repair in which a complex of ARP2 and ARP3 (actin-binding proteins 2 and 3) binds at the break and works with actin to promote DSB clustering and homology-directed repair. Further, it has been reported that relocalization of heterochromatic DSBs to the nuclear periphery in Drosophila is ARP2/3 dependent and actin–myosin driven. Here we provide an overview of the role of nuclear actin and actin-binding proteins in DNA repair, critically evaluating the experimental tools used and potential indirect effects.
Methods used to study nuclear actin have major caveats that can lead to biased data interpretation.Globular actin in nuclear remodeling complexes and histone acetyltransferases is often neglected due to a focus on filamentous nuclear actin.Recent evidence implicates nuclear actin and actin-binding proteins in resection-dependent DNA repair. The mechanism is unclear.The field needs further development of tools, the use of technical controls, and a systematic evaluation of alternative interpretations to reveal the function of nuclear actin.
Chromatin structure has a crucial role in processes of metabolism, including transcription, DNA replication and DNA damage repair. An evolutionarily conserved variant of histone H2A, called H2AX, is ...one of the key components of chromatin. H2AX becomes rapidly phosphorylated on chromatin surrounding DNA double-strand breaks (DSBs). Recent studies have shown that H2AX and other components of damaged chromatin also become modified by acetylation and ubiquitylation. This review discusses how specific combinations of histone modifications affect the accumulation and function of DNA repair factors (MDC1, RNF8, RNF168, 53BP1, BRCA1) and chromatin remodeling complexes (INO80, SWR1, TIP60-p400) at DSBs. These collectively regulate DSB repair and checkpoint arrest, avoiding genomic instability and oncogenic transformation in higher eukaryotes.
Chromatin is organized and compacted in the nucleus through the association of histones and other proteins, which together control genomic activity. Two broad types of chromatin can be distinguished: ...euchromatin, which is generally transcriptionally active, and heterochromatin, which is repressed. Here we examine the current state of our understanding of repressed chromatin in
, focusing on roles of histone modifications associated with repression, such as methylation of histone H3 lysine 9 (H3K9me2/3) or the Polycomb Repressive Complex 2 (MES-2/3/6)-deposited modification H3K27me3, and on proteins that recognize these modifications. Proteins involved in chromatin repression are important for development, and have demonstrated roles in nuclear organization, repetitive element silencing, genome integrity, and the regulation of euchromatin. Additionally, chromatin factors participate in repression with small RNA pathways. Recent findings shed light on heterochromatin function and regulation in
, and should inform our understanding of repressed chromatin in other animals.
The accessibility of eukaryotic genomes to the action of enzymes involved in transcription, replication and repair is maintained despite the organization of DNA into nucleosomes. This access is often ...regulated by the action of ATP-dependent nucleosome remodellers. The INO80 class of nucleosome remodellers has unique structural features and it is implicated in a diverse array of functions, including transcriptional regulation, DNA replication and DNA repair. Underlying these diverse functions is the catalytic activity of the main ATPase subunit, which in the context of a multisubunit complex can shift nucleosomes and carry out histone dimer exchange. In vitro studies showed that INO80 promotes replication fork progression on a chromatin template, while in vivo it was shown to facilitate replication fork restart after stalling and to help evict RNA polymerase II at transcribed genes following the collision of a replication fork with transcription. More recent work in yeast implicates INO80 in the general eviction and degradation of nucleosomes following high doses of oxidative DNA damage. Beyond these replication and repair functions, INO80 was shown to repress inappropriate transcription at promoters in the opposite direction to the coding sequence. Here we discuss the ways in which INO80's diverse functions help maintain genome integrity.
This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.
Budding yeast, like other eukaryotes, carries its genetic information on chromosomes that are sequestered from other cellular constituents by a double membrane, which forms the nucleus. An elaborate ...molecular machinery forms large pores that span the double membrane and regulate the traffic of macromolecules into and out of the nucleus. In multicellular eukaryotes, an intermediate filament meshwork formed of lamin proteins bridges from pore to pore and helps the nucleus reform after mitosis. Yeast, however, lacks lamins, and the nuclear envelope is not disrupted during yeast mitosis. The mitotic spindle nucleates from the nucleoplasmic face of the spindle pole body, which is embedded in the nuclear envelope. Surprisingly, the kinetochores remain attached to short microtubules throughout interphase, influencing the position of centromeres in the interphase nucleus, and telomeres are found clustered in foci at the nuclear periphery. In addition to this chromosomal organization, the yeast nucleus is functionally compartmentalized to allow efficient gene expression, repression, RNA processing, genomic replication, and repair. The formation of functional subcompartments is achieved in the nucleus without intranuclear membranes and depends instead on sequence elements, protein-protein interactions, specific anchorage sites at the nuclear envelope or at pores, and long-range contacts between specific chromosomal loci, such as telomeres. Here we review the spatial organization of the budding yeast nucleus, the proteins involved in forming nuclear subcompartments, and evidence suggesting that the spatial organization of the nucleus is important for nuclear function.
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