Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1 - accessibility) in absolute terms as the percentage of cut or uncut molecules, respectively, out of all molecules. The protocol presented here takes this classical approach to the genome-wide level. After exhaustive restriction enzyme digestion of chromatin, DNA is purified, sheared, and converted into libraries for high-throughput sequencing. Bioinformatic analysis counts uncut DNA fragments as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straightforward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments. Our protocol includes two orthogonal approaches to correct for this bias, the "corrected cut-uncut" and the "cut-all cut" method, so that accurate measurements of absolute accessibility or occupancy at restriction sites throughout a genome are possible. The protocol is presented for the example of S. cerevisiae chromatin but may be adapted for any other species.

SUMMARY The core promoter, the region immediately surrounding the transcription start site, plays a central role in setting metazoan gene expression levels, but how exactly it ‘computes’ expression remains poorly understood. To dissect core promoter function, we carried out a comprehensive structure-function analysis to measure synthetic promoters’ activities, with and without an external stimulus (hormonal activation). By using robotics and a dual-luciferase reporter assay, we tested ∼3000 mutational variants representing 19 different Drosophila melanogaster promoter architectures. We explored the impact of different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, positioning, and flanking sequences. We observe strong effects of the mutations on activity, and a linear combination of the individual motif features can largely account for the combinatorial effects on core promoter activity. Our findings shed new light on the quantitative assessment of gene expression, a fundamental process in all metazoans.

There are still many open questions about the sequence preference of nucleosomes and our approach will need to be extended to answer them. Only integrated models that combine the thermodynamics of nucleosome binding with experimental errors can deconvolve the two and learn the true preferences of nucleosomes.

Mapping of nucleosomes, the basic DNA packaging unit in eukaryotes, is fundamental for understanding genome regulation because nucleosomes modulate DNA access by their positioning along the genome. A cell-population nucleosome map requires two observables: nucleosome positions along the DNA ("Where?") and nucleosome occupancies across the population ("In how many cells?"). All available genome-wide nucleosome mapping techniques are yield methods because they score either nucleosomal (e.g., MNase-seq, chemical cleavage-seq) or nonnucleosomal (e.g., ATAC-seq) DNA but lose track of the total DNA population for each genomic region. Therefore, they only provide nucleosome positions and maybe compare relative occupancies between positions, but cannot measure absolute nucleosome occupancy, which is the fraction of all DNA molecules occupied at a given position and time by a nucleosome. Here, we established two orthogonal and thereby cross-validating approaches to measure absolute nucleosome occupancy across the Saccharomyces cerevisiae genome via restriction enzymes and DNA methyltransferases. The resulting high-resolution (9-bp) map shows uniform absolute occupancies. Most nucleosome positions are occupied in most cells: 97% of all nucleosomes called by chemical cleavage-seq have a mean absolute occupancy of 90 ± 6% (±SD). Depending on nucleosome position calling procedures, there are 57,000 to 60,000 nucleosomes per yeast cell. The few low absolute occupancy nucleosomes do not correlate with highly transcribed gene bodies, but correlate with increased presence of the nucleosome-evicting chromatin structure remodeling (RSC) complex, and are enriched upstream of highly transcribed or regulated genes. Our work provides a quantitative method and reference frame in absolute terms for future chromatin studies.

RECQL5 is the sole member of the RECQ family of helicases associated with RNA polymerase II (RNAPII). We now show that RECQL5 is a general elongation factor that is important for preserving genome stability during transcription. Depletion or overexpression of RECQL5 results in corresponding shifts in the genome-wide RNAPII density profile. Elongation is particularly affected, with RECQL5 depletion causing a striking increase in the average rate, concurrent with increased stalling, pausing, arrest, and/or backtracking (transcription stress). RECQL5 therefore controls the movement of RNAPII across genes. Loss of RECQL5 also results in the loss or gain of genomic regions, with the breakpoints of lost regions located in genes and common fragile sites. The chromosomal breakpoints overlap with areas of elevated transcription stress, suggesting that RECQL5 suppresses such stress and its detrimental effects, and thereby prevents genome instability in the transcribed region of genes.

Characterization of the human interactome of chromatin-associated messenger ribonucleoprotein particles identifies DBC1 and a new protein (ZIRD) as subunits of a protein complex (DBIRD) that binds directly to RNAPII, regulates alternative splicing of exons embedded in (A + T)-rich DNA, and whose depletion results in region-specific decreases in transcript elongation. Studies in recent years have shown how the processes involved in making, processing and splicing RNA are linked. Jesper Svejstrup and colleagues now describe a previously unknown protein complex called DBIRD, which links RNA transcript elongation to splicing. This complex binds the RNA polymerase and affects alternative splicing of a subgroup of messenger RNAs by promoting transcription across (A + T)-rich DNA encoding exons. Alternative messenger RNA splicing is the main reason that vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation1,2,3. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle; this is the functional form of the nascent pre-mRNA and determines the fate of the mature transcript4. However, factors that connect the transcribing polymerase with the mRNP particle and help to integrate transcript elongation with mRNA splicing remain unclear. Here we characterize the human interactome of chromatin-associated mRNP particles. This led us to identify deleted in breast cancer 1 (DBC1) and ZNF326 (which we call ZNF-protein interacting with nuclear mRNPs and DBC1 (ZIRD)) as subunits of a novel protein complex—named DBIRD—that binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in (A + T)-rich DNA, and is present at the affected exons. RNA-interference-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. Together, these data indicate that the DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with the regulation of alternative splicing.

I HOPE we may assume, from the paragraph which appears among the “Notes” in your issue of last Thursday (p. 129), that the Fishery Board for Scotland is about to take some active course towards the removal of obstructions to the ascent of salmon up Scottish rivers. When you say the Board “is specially desirous to introduce as soon as possible a fishway at the falls, and this, when done, would open up some 500 miles of excellent fishing and spawning ground,” I hardly think you can be alluding to any one particular river. Am I correct in supposing you refer to the aggregate mileage of rivers in Scotland now closed by natural obstructions, i.e. waterfalls? The Report of the Special Commission to inquire into the condition of the salmon fisheries of Scotland, published in 1871, informed us that the River Tay alone had some 115 miles of river blocked against the salmon by the two natural obstructions of the Tummel Falls and the Falls of Garry on the two important Tay tributaries from which the respective waterfalls are named. If your “Note” meant to include the entire mileage of Scottish rivers seriously affected by artificial dams of a more or less obstructive character (and their name is legion in Scotland), as well as by the natural barriers that occur, I think 500 miles of obstructed fishing and spawning ground is far too low an estimate; it might in fact, I should say, be multiplied at the very least by three. Now that theoretical playthings are being laid aside, and in their place appears a prospect of a more sound, natural, and scientific basis being made the foundation of our future salmon cultivation, the absolute necessity of opening up the natural breeding-beds of the fish will, it is hoped, become patent to every one, and the dream of my old friend the late William J. Ffennel, the father, so to speak, of our modern salmon fishery legislation and salmon river cultivation may at last be realised. “If I live,” he said to me one day (I hardly care to remember how long ago it was, or how soon after he was taken from us), “I shall never rest until every weir and mill-dam in the three countries—England, Ireland, and Scotland—has a thoroughly good and permanent salmon ladder built upon it, or into it, or around it. We have shown we can restore the fisheries; we must now restore the rivers. That, sir, is the true position to take up, and that must be our next aim.” Had Mr. Ffennel lived, river restoration would probably have progressed more than it has during the last decade.