No abstract

Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in diseases such as leukemia. Here, we describe E/L repli-seq, an extension of our repli-chip protocol. E/L repli-seq is a rapid, robust and relatively inexpensive protocol to analyze RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse labeled with BrdU and early and late S phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S phase fractions. The results are comparable to repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single nucleotide polymorphisms (SNP) to analyze RT allelic asynchrony, and for direct comparison to repli-chip data. This protocol can be performed in up to three days prior to sequencing, and requires basic cellular and molecular biology skills and a basic understanding of Unix and R.

INSERM/Reacting, the French Ebola Task Force, and Institut de Recherche pour le Développement.

doi: bioRxiv preprint modifications. However, whether the replication timing is a cause, a consequence, or an independent correlate of chromatin structure is a matter of debate (Rhind and Gilbert, 2013).In budding yeast, where replication origin locations are well defined, origin firing is stochastic, with each origin firing with a specific probability, independent of neighboring origins (Czajkowsky et al., 2008;Yang et al., 2010; de Moura et al., 2010). Such stochastic firing leads to reproducible replication profiles at the population level, because more efficient origins are more likely to fire early and therefore, on average, have early replication times; by contrast, inefficient origins usually fire late or are passively replicated (Rhind et al., 2010). The heterogeneous and inefficient nature of metazoan replication initiation is also consistent with stochastic initiation. Simulations with a stochastic firing model, in which initiation is regulated only by a local-firing-probability function, faithfully reproduce experimental genome-wide replication timing profiles, suggesting that no deterministic timing program is required (Gindin et al., 2014b). Furthermore, if the local initiation rate is predicted by DNase I hypersensitivity, the simulation closely matches experimental results, consistent with the observed correlation between promoters and enhancers, which are DNase I hypersensitive, and initiation frequency (Gindin et al., 2014b). On the other hand, the reproducible replication timing of individual replication domains measured in single cells has led to the suggestion that replication within those domains initiates at defined times in most cells in the population (Dileep and Gilbert, 2018). Furthermore, neighboring initiation sites have been proposed to show both cooperative firing and lateral inhibition (Cayrou et al., 2011;Guilbaud et al., 2011;Löb et al., 2016), neither of which are consistent with strictly stochastic models. Therefore, whether metazoan initiation timing is stochastic or deterministic, or some combination of the two, is still very much an open question (Bechhoefer and Rhind, 2012).A powerful solution to the problems of heterogeneity and low signal-to-noise ratios is single-molecule analysis, which allows the identification of sites of replication initiation on individual DNA fibers (Técher et al., 2013). Traditional single-molecule approaches-such as fiber autoradiography (

DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site-specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles.

A sex-related difference in the impairment of bone mineral characteristics was identified in children with T1DM. Longitudinal studies are required to investigate whether boys may gain slightly less bone mass during skeletal growth.

Chromatin organization and enhancer-promoter contacts establish unique spatiotemporal gene expression patterns in distinct cell types. Non-coding genetic variants can influence cellular phenotypes by modifying higher-order transcriptional hubs and consequently gene expression. To elucidate genomic regulation in human retina, we mapped chromatin contacts at high resolution and integrated with super-enhancers (SEs), histone marks, binding of CTCF and select transcription factors. We show that topologically associated domains (TADs) with central SEs exhibit stronger insulation and augmented contact with retinal genes relative to TADs with edge SEs. Merging genome-wide expression quantitative trait loci (eQTLs) with topology map reveals physical links between 100 eQTLs and corresponding eGenes associated with retinal neurodegeneration. Additionally, we uncover candidate genes for susceptibility variants linked to age-related macular degeneration and glaucoma. Our study of high-resolution genomic architecture of human retina provides insights into genetic control of tissue-specific functions, suggests paradigms for missing heritability, and enables the dissection of common blinding disease phenotypes.

Reactive oxygen species (ROS) are a byproduct of cell metabolism, and can also arise from environmental sources, such as toxins or radiation. Depending on dose and context, ROS have both beneficial and deleterious roles in mammalian development and disease, therefore it is crucial to understand how these molecules are generated, sensed, and detoxified. The question of how oxidative stress connects to the epigenome, in particular, is important yet incompletely understood. Here we show that an epigenetic regulator, the methyl-CpG-binding protein ZBTB38, limits the basal cellular production of ROS, is induced by ROS, and is required to mount a proper response to oxidative stress. Molecularly, these functions depend on a deubiquitinase, USP9X, which interacts with ZBTB38, deubiquitinates it, and stabilizes it. We find that USP9X is itself stabilized by oxidative stress, and is required together with ZBTB38 to limit the basal generation of ROS, as well as the toxicity of an acute oxidative stress. Our data uncover a new nuclear target of USP9X, show that the USP9X/ZBTB38 axis limits, senses and detoxifies ROS, and provide a molecular link between oxidative stress and the epigenome.