Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

Genomics in the long-read sequencing era.

Long-read sequencing (LRS) technologies have provided extremely powerful tools to explore genomes. While in the early years these methods suffered technical limitations, they have recently made significant progress in terms of read length, throughput, and accuracy and bioinformatics tools have strongly improved. Here, we aim to review the current status of LRS technologies, the development of novel methods, and the impact on genomics research. We will explore the most impactful recent findings made possible by these technologies focusing on high-resolution sequencing of genomes and transcriptomes and the direct detection of DNA and RNA modifications. We will also discuss how LRS methods promise a more comprehensive understanding of human genetic variation, transcriptomics, and epigenetics for the coming years.

Mistimed origin licensing and activation stabilize common fragile sites under tight DNA-replication checkpoint activation.

Genome integrity requires replication to be completed before chromosome segregation. The DNA-replication checkpoint (DRC) contributes to this coordination by inhibiting CDK1, which delays mitotic onset. Under-replication of common fragile sites (CFSs), however, escapes surveillance, resulting in mitotic chromosome breaks. Here we asked whether loose DRC activation induced by modest stresses commonly used to destabilize CFSs could explain this leakage. We found that tightening DRC activation or CDK1 inhibition stabilizes CFSs in human cells. Repli-Seq and molecular combing analyses showed a burst of replication initiations implemented in mid S-phase across a subset of late-replicating sequences, including CFSs, while the bulk genome was unaffected. CFS rescue and extra-initiations required CDC6 and CDT1 availability in S-phase, implying that CDK1 inhibition permits mistimed origin licensing and firing. In addition to delaying mitotic onset, tight DRC activation therefore supports replication completion of late origin-poor domains at risk of under-replication, two complementary roles preserving genome stability.

OKseqHMM: a genome-wide replication fork directionality analysis toolkit.

During each cell division, tens of thousands of DNA replication origins are co-ordinately activated to ensure the complete duplication of the human genome. However, replication fork progression can be challenged by many factors, including co-directional and head-on transcription-replication conflicts (TRC). Head-on TRCs are more dangerous for genome integrity. To study the direction of replication fork movement and TRCs, we developed a bioinformatics toolkit called OKseqHMM (https://github.com/CL-CHEN-Lab/OK-Seq, https://doi.org/10.5281/zenodo.7428883). Then, we used OKseqHMM to analyse a large number of datasets obtained by Okazaki fragment sequencing to directly measure the genome-wide replication fork directionality (RFD) and to accurately predict replication initiation and termination at a fine resolution in organisms including yeast, mouse and human. We also successfully applied our analysis to other genome-wide sequencing techniques that also contain RFD information (e.g. eSPAN, TrAEL-seq). Our toolkit can be used to predict replication initiation and fork progression direction genome-wide in a wide range of cell models and growth conditions. Comparing the replication and transcription directions allows identifying loci at risk of TRCs, particularly head-on TRCs, and investigating their role in genome instability by checking DNA damage data, which is of prime importance for human health.

Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing.

Studying the dynamics of genome replication in mammalian cells has been historically challenging. To reveal the location of replication initiation and termination in the human genome, we developed Okazaki fragment sequencing (OK-seq), a quantitative approach based on the isolation and strand-specific sequencing of Okazaki fragments, the lagging strand replication intermediates. OK-seq quantitates the proportion of leftward- and rightward-oriented forks at every genomic locus and reveals the location and efficiency of replication initiation and termination events. Here we provide the detailed experimental procedures for performing OK-seq in unperturbed cultured human cells and budding yeast and the bioinformatics pipelines for data processing and computation of replication fork directionality. Furthermore, we present the analytical approach based on a hidden Markov model, which allows automated detection of ascending, descending and flat replication fork directionality segments revealing the zones of replication initiation, termination and unidirectional fork movement across the entire genome. These tools are essential for the accurate interpretation of human and yeast replication programs. The experiments and the data processing can be accomplished within six days. Besides revealing the genome replication program in fine detail, OK-seq has been instrumental in numerous studies unravelling mechanisms of genome stability, epigenome maintenance and genome evolution.

DNA methylation and hydroxymethylation characterize the identity of D1 and D2 striatal projection neurons.

Neuronal DNA modifications differ from those in other cells, including methylation outside CpG context and abundant 5-hydroxymethylation whose relevance for neuronal identities are unclear. Striatal projection neurons expressing D1 or D2 dopamine receptors allow addressing this question, as they share many characteristics but differ in their gene expression profiles, connections, and functional roles. We compare translating mRNAs and DNA modifications in these two populations. DNA methylation differences occur predominantly in large genomic clusters including differentially expressed genes, potentially important for D1 and D2 neurons. Decreased gene body methylation is associated with higher gene expression. Hydroxymethylation differences are more scattered and affect transcription factor binding sites, which can influence gene expression. We also find a strong genome-wide hydroxymethylation asymmetry between the two DNA strands, particularly pronounced at expressed genes and retrotransposons. These results identify novel properties of neuronal DNA modifications and unveil epigenetic characteristics of striatal projection neurons heterogeneity.

GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.

A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2'-O-Methyl RNAs.

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNAs such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another level of difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the "TruSeq (TS)" protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol "TS5," which showed the best overall performance. We also provide guidelines for bioinformatics analysis of the sequencing data.

Transcriptome architecture and regulation at environmental transitions in flavobacteria: the case of an important fish pathogen.

The family Flavobacteriaceae (phylum Bacteroidetes) is a major component of soil, marine and freshwater ecosystems. In this understudied family, Flavobacterium psychrophilum is a freshwater pathogen that infects salmonid fish worldwide, with critical environmental and economic impact. Here, we report an extensive transcriptome analysis that established the genome map of transcription start sites and transcribed regions, predicted alternative sigma factor regulons and regulatory RNAs, and documented gene expression profiles across 32 biological conditions mimicking the pathogen life cycle. The results link genes to environmental conditions and phenotypic traits and provide insights into gene regulation, highlighting similarities with better known bacteria and original characteristics linked to the phylogenetic position and the ecological niche of the bacterium. In particular, osmolarity appears as a signal for transition between free-living and within-host programs and expression patterns of secreted proteins shed light on probable virulence factors. Further investigations showed that a newly discovered sRNA widely conserved in the genus, Rfp18, is required for precise expression of proteases. By pointing proteins and regulatory elements probably involved in host-pathogen interactions, metabolic pathways, and molecular machineries, the results suggest many directions for future research; a website is made available to facilitate their use to fill knowledge gaps on flavobacteria.

Contrasting Gene Decay in Subterranean Vertebrates: Insights from Cavefishes and Fossorial Mammals.

Evolution sometimes proceeds by loss, especially when structures and genes become dispensable after an environmental shift relaxes functional constraints. Subterranean vertebrates are outstanding models to analyze this process, and gene decay can serve as a readout. We sought to understand some general principles on the extent and tempo of the decay of genes involved in vision, circadian clock, and pigmentation in cavefishes. The analysis of the genomes of two Cuban species belonging to the genus Lucifuga provided evidence for the largest loss of eye-specific genes and nonvisual opsin genes reported so far in cavefishes. Comparisons with a recently evolved cave population of Astyanax mexicanus and three species belonging to the Chinese tetraploid genus Sinocyclocheilus revealed the combined effects of the level of eye regression, time, and genome ploidy on eye-specific gene pseudogenization. The limited extent of gene decay in all these cavefishes and the very small number of loss-of-function mutations per pseudogene suggest that their eye degeneration may not be very ancient, ranging from early to late Pleistocene. This is in sharp contrast with the identification of several vision genes carrying many loss-of-function mutations in ancient fossorial mammals, further suggesting that blind fishes cannot thrive more than a few million years in cave ecosystems.

Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide.

Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription-replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription-replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs.

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the 'TruSeq (TS)' protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol 'TS5', which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

The Third Revolution in Sequencing Technology.

Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives.

The evolution of the temporal program of genome replication.

Genome replication is highly regulated in time and space, but the rules governing the remodeling of these programs during evolution remain largely unknown. We generated genome-wide replication timing profiles for ten Lachancea yeasts, covering a continuous evolutionary range from closely related to more divergent species. We show that replication programs primarily evolve through a highly dynamic evolutionary renewal of the cohort of active replication origins. We found that gained origins appear with low activity yet become more efficient and fire earlier as they evolutionarily age. By contrast, origins that are lost comprise the complete range of firing strength. Additionally, they preferentially occur in close vicinity to strong origins. Interestingly, despite high evolutionary turnover, active replication origins remain regularly spaced along chromosomes in all species, suggesting that origin distribution is optimized to limit large inter-origin intervals. We propose a model on the evolutionary birth, death, and conservation of active replication origins.

Evidence for late Pleistocene origin of Astyanax mexicanus cavefish.

BACKGROUND: Cavefish populations belonging to the Mexican tetra species Astyanax mexicanus are outstanding models to study the tempo and mode of adaptation to a radical environmental change. They are currently assigned to two main groups, the so-called "old" and "new" lineages, which would have populated several caves independently and at different times. However, we do not have yet accurate estimations of the time frames of evolution of these populations. RESULTS: We reanalyzed the geographic distribution of mitochondrial and nuclear DNA polymorphisms and we found that these data do not support the existence of two cavefish lineages. Using IMa2, a program that allows dating population divergence in addition to demographic parameters, we found that microsatellite polymorphism strongly supports a very recent origin of cave populations (< 20,000 years). We identified a large number of single-nucleotide polymorphisms (SNPs) in transcript sequences of pools of embryos (Pool-seq) belonging to Pachón cave population and a surface population from Texas. Based on summary statistics that can be computed with this SNP data set together with simulations of evolution of SNP polymorphisms in two recently isolated populations, we looked for sets of demographic parameters that allow the computation of summary statistics with simulated populations that are similar to the ones with the sampled populations. In most simulations for which we could find a good fit between the summary statistics of observed and simulated data, the best fit occurred when the divergence between simulated populations was less than 30,000 years. CONCLUSIONS: Although it is often assumed that some cave populations have a very ancient origin, a recent origin of these populations is strongly supported by our analyses of independent sets of nuclear DNA polymorphism. Moreover, the observation of two divergent haplogroups of mitochondrial and nuclear genes with different geographic distributions support a recent admixture of two divergent surface populations, before the isolation of cave populations. If cave populations are indeed only several thousand years old, many phenotypic changes observed in cavefish would thus have mainly involved the fixation of genetic variants present in surface fish populations and within a very short period of time.

Complete Sequence of the Intronless Mitochondrial Genome of the Saccharomyces cerevisiae Strain CW252.

The mitochondrial genomes of Saccharomyces cerevisiae strains contain up to 13 introns. An intronless recombinant genome introduced into the nuclear background of S. cerevisiae strain W303 gave the S. cerevisiae CW252 strain, which is used to model mitochondrial respiratory pathologies. The complete sequence of this mitochondrial genome was obtained using a hybrid assembling methodology.

Systematic comparison of small RNA library preparation protocols for next-generation sequencing.

BACKGROUND: Next-generation sequencing technologies have revolutionized the study of small RNAs (sRNAs) on a genome-wide scale. However, classical sRNA library preparation methods introduce serious bias, mainly during adapter ligation steps. Several types of sRNA including plant microRNAs (miRNA), piwi-interacting RNAs (piRNA) in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants contain a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This inhibits 3' adapter ligation and makes library preparation particularly challenging. To reduce bias, the NEBNext kit (New England Biolabs) uses polyethylene glycol (PEG), the NEXTflex V2 kit (BIOO Scientific) uses both randomised adapters and PEG, and the novel SMARTer (Clontech) and CATS (Diagenode) kits avoid ligation altogether. Here we compared these methods with Illumina's classical TruSeq protocol regarding the detection of normal and 2' OMe RNAs. In addition, we modified the TruSeq and NEXTflex protocols to identify conditions that improve performance. RESULTS: Among the five kits tested with their respective standard protocols, the SMARTer and CATS kits had the lowest levels of bias but also had a strong formation of side products, and as a result performed relatively poorly with biological samples; NEXTflex detected the largest numbers of different miRNAs. The use of a novel type of randomised adapters called MidRand-Like (MRL) adapters and PEG improved the detection of 2' OMe RNAs both in the TruSeq as well as in the NEXTflex protocol. CONCLUSIONS: While it is commonly accepted that biases in sRNA library preparation protocols are mainly due to adapter ligation steps, the ligation-free protocols were not the best performing methods. Our modified versions of the TruSeq and NEXTflex protocols provide an improved tool for the study of 2' OMe RNAs.

Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile.

In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.

Replication landscape of the human genome.

Despite intense investigation, human replication origins and termini remain elusive. Existing data have shown strong discrepancies. Here we sequenced highly purified Okazaki fragments from two cell types and, for the first time, quantitated replication fork directionality and delineated initiation and termination zones genome-wide. Replication initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that often abut transcribed genes, and terminates dispersively between them. Replication fork progression is significantly co-oriented with the transcription. Initiation and termination zones are frequently contiguous, sometimes separated by regions of unidirectional replication. Initiation zones are enriched in open chromatin and enhancer marks, even when not flanked by genes, and often border 'topologically associating domains' (TADs). Initiation zones are enriched in origin recognition complex (ORC)-binding sites and better align to origins previously mapped using bubble-trap than λ-exonuclease. This novel panorama of replication reveals how chromatin and transcription modulate the initiation process to create cell-type-specific replication programs.

Nucleoid organization in the radioresistant bacterium Deinococcus radiodurans.

Processes favoring the exceptional resistance to genotoxic stress of Deinococcus radiodurans are not yet completely characterized. It was postulated that its nucleoid and chromosome(s) organization could participate in the DNA double strand break repair process. Here, we investigated the organization of chromosome 1 by localization of three chromosomal loci including oriC, Ter and a locus located in its left arm. For this purpose, we used a ParB-parS system to visualize the position of the loci before and after exposure to γ-rays. By comparing the number of fluorescent foci with the number of copies of the studied loci present in the cells measured by quantitative polymerase chain reaction (qPCR), we demonstrated that the 4-10 copies of chromosome 1 per cell are dispersed within the nucleoid before irradiation, indicating that the chromosome copies are not prealigned. Chromosome segregation is progressive but not co-ordinated, allowing each locus to be paired with its sister during part of the cell cycle. After irradiation, the nucleoid organization is modified, involving a transient alignment of the loci in the late stage of DNA repair and a delay of segregation of the Ter locus. We discuss how these events can influence DNA double strand break repair.