Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.

Cooperation between bHLH transcription factors and histones for DNA access.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

Divergent evolution toward sex chromosome-specific gene regulation in Drosophila.

The dosage compensation complex (DCC) of Drosophila identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the D. melanogaster model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that in D. virilis, a species separated from melanogaster by 40 million years of evolution, all principles are active but contribute differently to X specificity. In melanogaster, the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. In virilis, PionX motifs are abundant and not X-enriched. Accordingly, MSL2 lacks specific recognition. Here, roX2 RNA plays a more instructive role, counteracting a nonproductive interaction of CLAMP and modulating DCC binding selectivity. Remarkably, roX2 triggers a stable chromatin binding mode characteristic of DCC. Evidently, X-specific regulation is achieved by divergent evolution of protein, DNA, and RNA components.

Genome-wide Rules of Nucleosome Phasing in Drosophila.

Regular successions of positioned nucleosomes, or phased nucleosome arrays (PNAs), are predominantly known from transcriptional start sites (TSSs). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs for Drosophila, we applied spectral analysis to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSSs and are strongly enriched for an uncharacterized sequence motif. Through genome-wide reconstitution of physiological chromatin in Drosophila embryo extracts, we uncovered the molecular basis of PNA formation. We identified Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the motif. It also revealed how the global activity of the chromatin remodelers CHRAC/ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. Our work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scale in vitro.

Genomic context-dependent histone H3K36 methylation by three Drosophila methyltransferases and implications for dedicated chromatin readers.

Methylation of histone H3 at lysine 36 (H3K36me3) marks active chromatin. The mark is interpreted by epigenetic readers that assist transcription and safeguard the integrity of the chromatin fiber. The chromodomain protein MSL3 binds H3K36me3 to target X-chromosomal genes in male Drosophila for dosage compensation. The PWWP-domain protein JASPer recruits the JIL1 kinase to active chromatin on all chromosomes. Unexpectedly, depletion of K36me3 had variable, locus-specific effects on the interactions of those readers. This observation motivated a systematic and comprehensive study of K36 methylation in a defined cellular model. Contrasting prevailing models, we found that K36me1, K36me2 and K36me3 each contribute to distinct chromatin states. A gene-centric view of the changing K36 methylation landscape upon depletion of the three methyltransferases Set2, NSD and Ash1 revealed local, context-specific methylation signatures. Set2 catalyzes K36me3 predominantly at transcriptionally active euchromatin. NSD places K36me2/3 at defined loci within pericentric heterochromatin and on weakly transcribed euchromatic genes. Ash1 deposits K36me1 at regions with enhancer signatures. The genome-wide mapping of MSL3 and JASPer suggested that they bind K36me2 in addition to K36me3, which was confirmed by direct affinity measurement. This dual specificity attracts the readers to a broader range of chromosomal locations and increases the robustness of their actions.

Processivity and specificity of histone acetylation by the male-specific lethal complex.

Acetylation of lysine 16 of histone H4 (H4K16ac) stands out among the histone modifications, because it decompacts the chromatin fiber. The metazoan acetyltransferase MOF (KAT8) regulates transcription through H4K16 acetylation. Antibody-based studies had yielded inconclusive results about the selectivity of MOF to acetylate the H4 N-terminus. We used targeted mass spectrometry to examine the activity of MOF in the male-specific lethal core (4-MSL) complex on nucleosome array substrates. This complex is part of the Dosage Compensation Complex (DCC) that activates X-chromosomal genes in male Drosophila. During short reaction times, MOF acetylated H4K16 efficiently and with excellent selectivity. Upon longer incubation, the enzyme progressively acetylated lysines 12, 8 and 5, leading to a mixture of oligo-acetylated H4. Mathematical modeling suggests that MOF recognizes and acetylates H4K16 with high selectivity, but remains substrate-bound and continues to acetylate more N-terminal H4 lysines in a processive manner. The 4-MSL complex lacks non-coding roX RNA, a critical component of the DCC. Remarkably, addition of RNA to the reaction non-specifically suppressed H4 oligo-acetylation in favor of specific H4K16 acetylation. Because RNA destabilizes the MSL-nucleosome interaction in vitro we speculate that RNA accelerates enzyme-substrate turn-over in vivo, thus limiting the processivity of MOF, thereby increasing specific H4K16 acetylation.

The effect of climate change on avian offspring production: A global meta-analysis.

Climate change affects timing of reproduction in many bird species, but few studies have investigated its influence on annual reproductive output. Here, we assess changes in the annual production of young by female breeders in 201 populations of 104 bird species (N = 745,962 clutches) covering all continents between 1970 and 2019. Overall, average offspring production has declined in recent decades, but considerable differences were found among species and populations. A total of 56.7% of populations showed a declining trend in offspring production (significant in 17.4%), whereas 43.3% exhibited an increase (significant in 10.4%). The results show that climatic changes affect offspring production through compounded effects on ecological and life history traits of species. Migratory and larger-bodied species experienced reduced offspring production with increasing temperatures during the chick-rearing period, whereas smaller-bodied, sedentary species tended to produce more offspring. Likewise, multi-brooded species showed increased breeding success with increasing temperatures, whereas rising temperatures were unrelated to reproductive success in single-brooded species. Our study suggests that rapid declines in size of bird populations reported by many studies from different parts of the world are driven only to a small degree by changes in the production of young.

Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites.

MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. We explore the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naïve embryonic chromatin. We found that the cooperativity requires physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. We find that the cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in our reconstitution, such as roX2 lncRNA.

Cell-free genomics: transcription factor interactions in reconstituted naïve embryonic chromatin.

Extracts from Drosophila preblastoderm embryos (DREX) form the basis of a powerful in vitro chromatin reconstitution system that assembles entire genomes into complex chromatin with physiological nucleosome spacing and polymer condensation. As the zygotic genome has not yet been activated in preblastoderm embryos, the reconstitution extract lacks endogenous transcription factors (TFs) and the RNA polymerase machinery. At the same time, it contains high levels of ATP-dependent nucleosome sliding enzymes that render the reconstituted chromatin dynamic. The naïve chromatin can be used to determine the intrinsic DNA binding properties of exogenous, usually recombinant TFs (or DNA binding proteins in general) in a complex chromatin context. Recent applications of the system include the description of cooperation and competition of Drosophila pioneer TFs for composite binding sites, and the characterization of nucleosome interactions of mammalian pioneer TFs in the heterologous system.

Global and specific responses of the histone acetylome to systematic perturbation.

Regulation of histone acetylation is fundamental to the utilization of eukaryotic genomes in chromatin. Aberrant acetylation contributes to disease and can be clinically combated by inhibiting the responsible enzymes. Our knowledge of the histone acetylation system is patchy because we so far lacked the methodology to describe acetylation patterns and their genesis by integrated enzyme activities. We devised a generally applicable, mass spectrometry-based strategy to precisely and accurately quantify combinatorial modification motifs. This was applied to generate a comprehensive inventory of acetylation motifs on histones H3 and H4 in Drosophila cells. Systematic depletion of known or suspected acetyltransferases and deacetylases revealed specific alterations of histone acetylation signatures, established enzyme-substrate relationships, and unveiled an extensive crosstalk between neighboring modifications. Unexpectedly, overall histone acetylation levels remained remarkably constant upon depletion of individual acetyltransferases. Conceivably, the acetylation level is adjusted to maintain the global charge neutralization of chromatin and the stability of nuclei.

Structure of the RNA Helicase MLE Reveals the Molecular Mechanisms for Uridine Specificity and RNA-ATP Coupling.

The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. We identified a stable MLE core comprising the DExH helicase module and two auxiliary domains: a dsRBD and an OB-like fold. MLEcore is an unusual DExH helicase that can unwind blunt-ended RNA duplexes and has specificity for uridine nucleotides. We determined the 2.1 Å resolution structure of MLEcore bound to a U10 RNA and ADP-AlF4. The OB-like and dsRBD folds bind the DExH module and contribute to form the entrance of the helicase channel. Four uridine nucleotides engage in base-specific interactions, rationalizing the conservation of uridine-rich sequences in critical roX substrates. roX2 binding is orchestrated by MLE's auxiliary domains, which is prerequisite for MLE localization to the male X chromosome. The structure visualizes a transition-state mimic of the reaction and suggests how eukaryotic DEAH/RHA helicases couple ATP hydrolysis to RNA translocation.

Extinction of high-power laser radiation under adverse weather conditions.

The propagation of laser radiation over long distances can be significantly affected by atmospheric extinction due to precipitation as well as aerosol particles and molecules. The knowledge of the contribution of precipitation is critical to the operation of a variety of laser-based systems. The study of high-power laser transmission around 1 µm is of particular interest because several atmospheric transmission windows are located in this region. To investigate the effect of adverse weather conditions on laser transmission, free-space laser transmission experiments are conducted on the DLR test range in Lampoldshausen, Germany. A high-power laser with a wavelength of 1.03 µm is used for the transmission measurements in combination with calibrated power monitors. Local weather conditions are continuously monitored by meteorological instruments during the experiments. Extinction coefficients are derived from transmission measurements showing that the extinction for snow is 7 times higher than for rain, and the extinction for drizzle/rain is 4 times higher than for rain at a given precipitation rate of 1 mm/h. For a mixture of rain and snow, the extinction is comparable to that of rain, indicating that the water content strongly influences the optical properties and thus the extinction of laser radiation in mixed precipitation. A good relationship is found between the measured extinction coefficient and visibility for drizzle and rain and a slightly larger scatter of the data for snow. Furthermore, measured extinction coefficients are compared to the extinction coefficients based on the experimental size distributions of precipitation particles and geometric optics. A reasonable agreement is obtained for rain, with no improvement taking the forward-scattering into the detector aperture into account, and a much better agreement is obtained for snow when the forward-scattering contribution is included.

Cell-free genomics reveal intrinsic, cooperative and competitive determinants of chromatin interactions.

Metazoan transcription factors distinguish their response elements from a large excess of similar sequences. We explored underlying principles of DNA shape read-out and factor cooperativity in chromatin using a unique experimental system. We reconstituted chromatin on Drosophila genomes in extracts of preblastoderm embryos, mimicking the naïve state of the zygotic genome prior to developmental transcription activation. We then compared the intrinsic binding specificities of three recombinant transcription factors, alone and in combination, with GA-rich recognition sequences genome-wide. For MSL2, all functional elements reside on the X chromosome, allowing to distinguish physiological elements from non-functional 'decoy' sites. The physiological binding profile of MSL2 is approximated through interaction with other factors: cooperativity with CLAMP and competition with GAF, which sculpts the profile by occluding non-functional sites. An extended DNA shape signature is differentially read out in chromatin. Our results reveal novel aspects of target selection in a complex chromatin environment.

Engineering long spin coherence times of spin-orbit qubits in silicon.

Electron-spin qubits have long coherence times suitable for quantum technologies. Spin-orbit coupling promises to greatly improve spin qubit scalability and functionality, allowing qubit coupling via photons, phonons or mutual capacitances, and enabling the realization of engineered hybrid and topological quantum systems. However, despite much recent interest, results to date have yielded short coherence times (from 0.1 to 1 µs). Here we demonstrate ultra-long coherence times of 10 ms for holes where spin-orbit coupling yields quantized total angular momentum. We focus on holes bound to boron acceptors in bulk silicon 28, whose wavefunction symmetry can be controlled through crystal strain, allowing direct control over the longitudinal electric dipole that causes decoherence. The results rival the best electron-spin qubits and are 104 to 105 longer than previous spin-orbit qubits. These results open a pathway to develop new artificial quantum systems and to improve the functionality and scalability of spin-based quantum technologies.

Telomere length is heritable and genetically correlated with lifespan in a wild bird.

Telomeres are protective caps at the end of eukaryotic chromosomes that shorten with age and in response to stressful or resource-demanding conditions. Their length predicts individual health and lifespan across a wide range of animals, but whether the observed positive association between telomere length and lifespan is environmentally induced, or set at conception due to a shared genetic basis, has not been tested in wild animals. We applied quantitative genetic "animal models" to longitudinal telomere measurements collected over a 10-year period from individuals of a wild seabird (common tern; Sterna hirundo) with known pedigree. We found no variation in telomere shortening with age among individuals at the phenotypic and genetic level, and only a small permanent environmental effect on adult telomere length. Instead, we found telomere length to be highly heritable and strongly positively genetically correlated with lifespan. Such heritable differences between individuals that are set at conception may present a hitherto underappreciated component of variation in somatic state.

Variation and correlation in the timing of breeding of North Atlantic seabirds across multiple scales.

Timing of breeding, an important driver of fitness in many populations, is widely studied in the context of global change, yet despite considerable efforts to identify environmental drivers of seabird nesting phenology, for most populations we lack evidence of strong drivers. Here we adopt an alternative approach, examining the degree to which different populations positively covary in their annual phenology to infer whether phenological responses to environmental drivers are likely to be (a) shared across species at a range of spatial scales, (b) shared across populations of a species or (c) idiosyncratic to populations. We combined 51 long-term datasets on breeding phenology spanning 50 years from nine seabird species across 29 North Atlantic sites and examined the extent to which different populations share early versus late breeding seasons depending on a hierarchy of spatial scales comprising breeding site, small-scale region, large-scale region and the whole North Atlantic. In about a third of cases, we found laying dates of populations of different species sharing the same breeding site or small-scale breeding region were positively correlated, which is consistent with the hypothesis that they share phenological responses to the same environmental conditions. In comparison, we found no evidence for positive phenological covariation among populations across species aggregated at larger spatial scales. In general, we found little evidence for positive phenological covariation between populations of a single species, and in many instances the inter-year variation specific to a population was substantial, consistent with each population responding idiosyncratically to local environmental conditions. Black-legged kittiwake Rissa tridactyla was the exception, with populations exhibiting positive covariation in laying dates that decayed with the distance between breeding sites, suggesting that populations may be responding to a similar driver. Our approach sheds light on the potential factors that may drive phenology in our study species, thus furthering our understanding of the scales at which different seabirds interact with interannual variation in their environment. We also identify additional systems and phenological questions to which our inferential approach could be applied.

PionX sites mark the X chromosome for dosage compensation.

The rules defining which small fraction of related DNA sequences can be selectively bound by a transcription factor are poorly understood. One of the most challenging tasks in DNA recognition is posed by dosage compensation systems that require the distinction between sex chromosomes and autosomes. In Drosophila melanogaster, the male-specific lethal dosage compensation complex (MSL-DCC) doubles the level of transcription from the single male X chromosome, but the nature of this selectivity is not known. Previous efforts to identify X-chromosome-specific target sequences were unsuccessful as the identified MSL recognition elements lacked discriminative power. Therefore, additional determinants such as co-factors, chromatin features, RNA and chromosome conformation have been proposed to refine targeting further. Here, using an in vitro genome-wide DNA binding assay, we show that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. MSL2, the male-specific organizer of the complex, uses two distinct DNA interaction surfaces-the CXC and proline/basic-residue-rich domains-to identify complex DNA elements on the X chromosome. Specificity is provided by the CXC domain, which binds a novel motif defined by DNA sequence and shape. This motif characterizes a subclass of MSL2-binding sites, which we name PionX (pioneering sites on the X) as they appeared early during the recent evolution of an X chromosome in D. miranda and are the first chromosomal sites to be bound during de novo MSL-DCC assembly. Our data provide the first, to our knowledge, documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome. They highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology.

Two-step mechanism for selective incorporation of lncRNA into a chromatin modifier.

The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, we developed vitRIP, an unbiased, transcriptome-wide in vitro assay that reveals RNA binding specificity. We found that MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.

Structural basis of X chromosome DNA recognition by the MSL2 CXC domain during Drosophila dosage compensation.

The male-specific lethal dosage compensation complex (MSL-DCC) selectively assembles on the X chromosome in Drosophila males and activates gene transcription by twofold through histone acetylation. An MSL recognition element (MRE) sequence motif nucleates the initial MSL association, but how it is recognized remains unknown. Here, we identified the CXC domain of MSL2 specifically recognizing the MRE motif and determined its crystal structure bound to specific and nonspecific DNAs. The CXC domain primarily contacts one strand of DNA duplex and employs a single arginine to directly read out dinucleotide sequences from the minor groove. The arginine is flexible when bound to nonspecific sequences. The core region of the MRE motif harbors two binding sites on opposite strands that can cooperatively recruit a CXC dimer. Specific DNA-binding mutants of MSL2 are impaired in MRE binding and X chromosome localization in vivo. Our results reveal multiple dynamic DNA-binding modes of the CXC domain that target the MSL-DCC to X chromosomes.

Progressive dosage compensation during Drosophila embryogenesis is reflected by gene arrangement.

In Drosophila melanogaster males, X-chromosome monosomy is compensated by chromosome-wide transcription activation. We found that complete dosage compensation during embryogenesis takes surprisingly long and is incomplete even after 10 h of development. Although the activating dosage compensation complex (DCC) associates with the X-chromosome and MOF acetylates histone H4 early, many genes are not compensated. Acetylation levels on gene bodies continue to increase for several hours after gastrulation in parallel with progressive compensation. Constitutive genes are compensated earlier than developmental genes. Remarkably, later compensation correlates with longer distances to DCC binding sites. This time-space relationship suggests that DCC action on target genes requires maturation of the active chromosome compartment.