A Role for Chromatin Remodeling in Cohesin Loading onto Chromosomes.

[EN]Cohesin is a conserved, ring-shaped protein complex that topologically embraces DNA. Its central role in genome organization includes functions in sister chromatid cohesion, DNA repair, and transcriptional regulation. Cohesin loading onto chromosomes requires the Scc2-Scc4 cohesin loader, whose presence on chromatin in budding yeast depends on the RSC chromatin remodeling complex. Here we reveal a dual role of RSC in cohesin loading. RSC acts as a chromatin receptor that recruits Scc2-Scc4 by a direct protein interaction independent of chromatin remodeling. In addition, chromatin remodeling is required to generate a nucleosome-free region that is the substrate for cohesin loading. An engineered cohesin loading module can be created by fusing the Scc2 C terminus to RSC or to other chromatin remodelers, but not to unrelated DNA binding proteins. These observations demonstrate the importance of nucleosome-free DNA for cohesin loading and provide insight into how cohesin accesses DNA during its varied chromosomal activities

Replication-induced DNA secondary structures drive fork uncoupling and breakage

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress

Histone variant macroH2A confers resistance to nuclear reprogramming

Gurdon and collaborators report reversible X chromosome inactivation in epiblast stem cells (EpiSCs) that seems to be determined by macroH2A1 deposition. These findings are of rather general interest as they highlight the epigenetic state of repressed loci as determinant for reprogramming efficiency

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

Evolutionary diversity and developmental regulation of X-chromosome inactivation

X-chromosome inactivation (XCI) results in the transcriptional silencing of one X-chromosome in females to attain gene dosage parity between XX female and XY male mammals. Mammals appear to have developed rather diverse strategies to initiate XCI in early development. In placental mammals XCI depends on the regulatory noncoding RNA X-inactive specific transcript (Xist), which is absent in marsupials and monotremes. Surprisingly, even placental mammals show differences in the initiation of XCI in terms of Xist regulation and the timing to acquire dosage compensation. Despite this, all placental mammals achieve chromosome-wide gene silencing at some point in development, and this is maintained by epigenetic marks such as chromatin modifications and DNA methylation. In this review, we will summarise recent findings concerning the events that occur downstream of Xist RNA coating of the inactive X-chromosome (Xi) to ensure its heterochromatinization and the maintenance of the inactive state in the mouse and highlight similarities and differences between mammals

Epigenetic control of DNA replication dynamics in mammals

One of the most critically important processes in any living organism, essential for development and reproduction, is that of the accurate replication of its genome before each cell division. The process of DNA replication can take place millions of times in a single organism and any mistake, if left unrepaired, is potentially transmitted into the next generation. Errors during replication can result in genetic mutations or karyotype aberrations, both of which can lead to disease or death. The duplication of the genome happens in a well-conserved spatio-temporal manner, a phenomenon implicated in development and disease. This fact indicates that DNA replication needs to be tightly regulated. Further, its precise coordination suggests that distinct genomic regions undergo replication at specific times during S-phase. On the other hand, the regulation of replication is a flexible process throughout development and is, therefore, proposed to be controlled epigenetically. However, the complexity of the mammalian nucleus has hampered the elucidation of how chromatin structure can regulate replication timing. In fact, our understanding of the regulation of replication timing in mammals is restricted to only a few studies with, in part, seemingly contradicting results. In the context of the present thesis, I set out to study the epigenetic mechanisms that control DNA replication dynamics in mammalian cells. To this end, I took advantage of the most prominent example of facultative heterochromatin, the epigenetically silenced X chromosome (Xi) of female mammalian cells, as well as of the mouse chromocenters, formed by clusters of constitutive heterochromatin. To study their particular replication dynamics and the epigenetic mechanisms controlling them, I used a set of genetic (conditional) knockouts, chemical inhibitory treatments and differentiation assays. The latter allowed me to control whole-chromosome inactivation and the subsequent establishment of the corresponding replication pattern, as well as to distinguish the contribution of different epigenetic markers in this process. I visualized the epigenetic changes and their effects on the replication program in situ by immunostainings, also in combination with fluorescence in situ hybridization (FISH), confocal and super resolution light microscopy, as well as in vivo by time-lapse microscopy over peri-ods of up to two days. This approach prompted the development of several tools for live-cell analysis. Using established and new tools, I comprehensively assessed the Xi replication dynamics and the effects of modulating different epigenetic modifications of heterochromatin, their cros-stalk and the subsequent effects on DNA replication timing and was able to show that histone hypoacetylation, a common mark of the Xi and chromocenters, is responsible for the delayed initiation in replication of both heterochromatic regions. Consequently, I propose that histone hyperacetylation, probably due to its opening effect on chromatin structure, renders some genomic regions prone to be bound by initiation factors earlier and / or more abundantly. This preferential binding, e.g. by replication initiation factors, would thus lead to earlier and concomitantly more efficient replication origin firing. Moreover, I discuss the causal relation between transcriptional inactivity and synchronous replication dynamics, a common feature of developmentally opposite systems, such as the mammalian Xi and the embryos of flies and frogs

Epigenetic control of DNA replication dynamics in mammals.

Every time a cell divides it must ensure that its genetic information is accurately duplicated and dis-tributed equally to the two daughter cells. This fundamental biological process is conserved through-out all kingdoms of life and relies on the correct and complete duplication of the DNA before a cell can divide and give rise to other cells or to multicellular organisms. Any mistakes in this process can result in genetic mutations or karyotype aberrations, which may lead to disease or even death. Whereas in prokaryotes the entire genome is replicated from a single origin, the increased genome size and complexity in mammals requires the spatio-temporal coordination of thousands of replica-tion origins. Furthermore, this spatio-temporal order of genome replication changes throughout de-velopment and cellular differentiation. Here we present and discuss current knowledge on the con-trol of DNA replication dynamics in mammals and the role of chromatin modifications in this basic biological process

Spatiotemporal visualization of DNA replication dynamics.

The ability of cells to copy their DNA allows them to transmit their genetic information to their progeny. In such, this central biological process preserves the instructions that direct the entire development of a cell. Earlier biochemical analysis in vitro and genetic analysis in yeast laid the basis of our understanding of the highly conserved mechanism of DNA replication. Recent advances on labeling and live-cell microscopy permit now the dissection of this fundamental process in vivo within the context of intact cells. In this chapter, we describe in detail how to perform multiple DNA replication labeling and detection allowing high spatial resolution imaging, as well as how to follow DNA replication in living cells allowing high temporal resolution imaging