The next-generation sequencing-chess problem.

The development of next-generation sequencing (NGS) technologies paved the way for studying the spatiotemporal coordination of cellular processes along the genome. However, data sets are commonly limited to a few time points, and missing information needs to be interpolated. Most models assume that the studied dynamics are similar between individual cells, so that a homogeneous cell culture can be represented by a population-wide average.

A genome-wide comprehensive analysis of nucleosome positioning in yeast.

In eukaryotic cells, the one-dimensional DNA molecules need to be tightly packaged into the spatially constraining nucleus. Folding is achieved on its lowest level by wrapping the DNA around nucleosomes. Their arrangement regulates other nuclear processes, such as transcription and DNA repair.

Functional interplay between Mediator and RSC chromatin remodeling complex controls nucleosome-depleted region maintenance at promoters.

Chromatin organization is crucial for transcriptional regulation in eukaryotes. Mediator is an essential and conserved co-activator thought to act in concert with chromatin regulators. However, it remains largely unknown how their functions are coordinated.

A quantitative modelling approach for DNA repair on a population scale.

The great advances of sequencing technologies allow the in vivo measurement of nuclear processes-such as DNA repair after UV exposure-over entire cell populations. However, data sets usually contain only a few samples over several hours, missing possibly important information in between time points. We developed a data-driven approach to analyse CPD repair kinetics over time in Saccharomyces cerevisiae.

Genomic analysis of Rad26 and Rad1-Rad10 reveals differences in their dependence on Mediator and RNA polymerase II.

Mediator is a conserved coregulator playing a key role in RNA polymerase (Pol) II transcription. Mediator also links transcription and nucleotide excision repair (NER) via a direct contact with Rad2/ERCC5(XPG) endonuclease. In this work, we analyzed the genome-wide distribution of Rad26/ERCC6(CSB) and Rad1-Rad10/ERCC4(XPF)-ERCC1, addressing the question of a potential link of these proteins with Mediator and Pol II in yeast Our genomic analyses reveal that Rad1-Rad10 and Rad26 are present on the yeast genome in the absence of genotoxic stress, especially at highly transcribed regions, with Rad26 binding strongly correlating with that of Pol II.

Microfluidic platform for monitoring mutation accumulation.

Mutations in DNA have large-ranging consequences, from evolution to disease. Many mechanisms contribute to mutational processes such as dysfunctions in DNA repair pathways and exogenous or endogenous mutagen exposures. Model organisms and mutation accumulation (MA) experiments are indispensable to study mutagenesis.

Glucocorticoids delay RAF-induced senescence promoted by EGR1.

Expression of hyperactive RAF kinases, such as the oncogenic B-RAF-V600E mutant, in normal human cells triggers a proliferative arrest that blocks tumor formation. We discovered that glucocorticoids delayed the entry into senescence induced by B-RAF-V600E in human fibroblasts, and allowed senescence bypass when the cells were regularly passaged, but that they did not allow proliferation of cells that were already senescent. Transcriptome and siRNA analyses revealed that the EGR1 gene is one target of glucocorticoid action.

Functional interplay between Mediator and RNA polymerase II in Rad2/XPG loading to the chromatin.

Transcription and maintenance of genome integrity are fundamental cellular functions. Deregulation of transcription and defects in DNA repair lead to serious pathologies. The Mediator complex links RNA polymerase (Pol) II transcription and nucleotide excision repair via Rad2/XPG endonuclease.

Six domesticated PiggyBac transposases together carry out programmed DNA elimination in .

The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In , PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide.

A mating-type mutagenesis screen identifies a zinc-finger protein required for specific DNA excision events in Paramecium.

In the ciliate Paramecium tetraurelia, functional genes are reconstituted during development of the somatic macronucleus through the precise excision of ∼45 000 single-copy Internal Eliminated Sequences (IESs), thought to be the degenerate remnants of ancient transposon insertions. Like introns, IESs are marked only by a weak consensus at their ends. How such a diverse set of sequences is faithfully recognized and precisely excised remains unclear: specialized small RNAs have been implicated, but in their absence up to ∼60% of IESs are still correctly excised.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

Background: DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression.

A meiosis-specific Spt5 homolog involved in non-coding transcription.

Spt5 is a conserved and essential transcriptional regulator that binds directly to RNA polymerase and is involved in transcription elongation, polymerase pausing and various co-transcriptional processes. To investigate the role of Spt5 in non-coding transcription, we used the unicellular model Paramecium tetraurelia. In this ciliate, development is controlled by epigenetic mechanisms that use different classes of non-coding RNAs to target DNA elimination.

TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements.

Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs).

Local effect of enhancer of zeste-like reveals cooperation of epigenetic and cis-acting determinants for zygotic genome rearrangements.

In the ciliate Paramecium tetraurelia, differentiation of the somatic nucleus from the zygotic nucleus is characterized by massive and reproducible deletion of transposable elements and of 45,000 short, dispersed, single-copy sequences. A specific class of small RNAs produced by the germline during meiosis, the scnRNAs, are involved in the epigenetic regulation of DNA deletion but the underlying mechanisms are poorly understood. Here, we show that trimethylation of histone H3 (H3K27me3 and H3K9me3) displays a dynamic nuclear localization that is altered when the endonuclease required for DNA elimination is depleted.

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion.

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic.

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events.