Complementary strategies for directing in vivo transcription factor binding through DNA binding domains and intrinsically disordered regions.

DNA binding domains (DBDs) of transcription factors (TFs) recognize DNA sequence motifs that are highly abundant in genomes. Within cells, TFs bind a subset of motif-containing sites as directed by either their DBDs or DBD-external (nonDBD) sequences. To define the relative roles of DBDs and nonDBDs in directing binding preferences, we compared the genome-wide binding of 48 (∼30%) budding yeast TFs with their DBD-only, nonDBD-truncated, and nonDBD-only mutants. With a few exceptions, binding locations differed between DBDs and TFs, resulting from the cumulative action of multiple determinants mapped mostly to disordered nonDBD regions. Furthermore, TFs' preferences for promoters of the fuzzy nucleosome architecture were lost in DBD-only mutants, whose binding spread across promoters, implicating nonDBDs' preferences in this hallmark of budding yeast regulatory design. We conclude that DBDs and nonDBDs employ complementary DNA-targeting strategies, whose balance defines TF binding specificity along genomes.

Beyond RNA binding domains: determinants of protein-RNA binding.

RNA binding proteins (RBPs) are composed of RNA binding domains (RBDs) often linked via intrinsically disordered regions (IDRs). Structural and biochemical analysis have shown that disordered linkers contribute to RNA binding by orienting the adjacent RBDs and also characterized certain disordered repeats that directly contact the RNA. However, the relative contribution of IDRs and predicted RBDs to the in-vivo binding pattern is poorly explored. Here, we upscaled the RNA tagging method to map the transcriptome-wide binding of sixteen RBPs in budding yeast. We then performed extensive sequence mutations to distinguish binding determinants within predicted RBDs and the surrounding IDRs in eight of these. The majority of the predicted RBDs tested were not individually essential for mRNA binding, while multiple IDRs that lacked predicted RNA binding potential appeared essential for binding affinity or specificity. Our results provide new insights into the function of poorly studied RBPs and emphasize the complex and distributed encoding of RBP-RNA interaction in-vivo.

Intrinsically disordered regions of the Msn2 transcription factor encode multiple functions using interwoven sequence grammars.

Intrinsically disordered regions (IDRs) are abundant in eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as shown for the budding yeast TF Msn2. To examine how IDRs encode both these functions, we compared genomic binding specificity, coactivator recruitment, and gene induction amongst a large set of designed Msn2-IDR mutants. We find that both functions depend on multiple regions across the > 600AA IDR. Yet, transcription activity was readily disrupted by mutations that showed no effect on the Msn2 binding specificity. Our data attribute this differential sensitivity to the integration of a relaxed, composition-based code directing binding specificity with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Therefore, Msn2 utilizes interwoven sequence grammars for encoding multiple functions, suggesting a new IDR design paradigm of potentially general use.

Disordered sequences of transcription factors regulate genomic binding by integrating diverse sequence grammars and interaction types.

Intrinsically disordered regions (IDRs) guide transcription factors (TFs) to their genomic binding sites, raising the question of how structure-lacking regions encode for complex binding patterns. We investigated this using the TF Gln3, revealing sets of IDR-embedded determinants that direct Gln3 binding to respective groups of functionally related promoters, and enable tuning binding preferences between environmental conditions, phospho-mimicking mutations, and orthologs. Through targeted mutations, we defined the role of short linear motifs (SLiMs) and co-binding TFs (Hap2) in stabilizing Gln3 at respiration-chain promoters, while providing evidence that Gln3 binding at nitrogen-associated promoters is encoded by the IDR amino-acid composition, independent of SLiMs or co-binding TFs. Therefore, despite their apparent simplicity, TF IDRs can direct and regulate complex genomic binding patterns through a combination of SLiM-mediated and composition-encoded interactions.

Massively parallel binding assay (MPBA) reveals limited transcription factor binding cooperativity, challenging models of specificity.

DNA-binding domains (DBDs) within transcription factors (TFs) recognize short sequence motifs that are highly abundant in genomes. In vivo, TFs bind only a small subset of motif occurrences, which is often attributed to the cooperative binding of interacting TFs at proximal motifs. However, large-scale testing of this model is still lacking. Here, we describe a novel method allowing parallel measurement of TF binding to thousands of designed sequences within yeast cells and apply it to quantify the binding of dozens of TFs to libraries of regulatory regions containing clusters of binding motifs, systematically mutating all motif combinations. With few exceptions, TF occupancies were well explained by independent binding to individual motifs, with motif cooperation being of only limited effects. Our results challenge the general role of motif combinatorics in directing TF genomic binding and open new avenues for exploring the basis of protein-DNA interactions within cells.

A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress.

Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.

Rtt109 promotes nucleosome replacement ahead of the replication fork.

DNA replication perturbs chromatin by triggering the eviction, replacement, and incorporation of nucleosomes. How this dynamic is orchestrated in time and space is poorly understood. Here, we apply a genetically encoded sensor for histone exchange to follow the time-resolved histone H3 exchange profile in budding yeast cells undergoing slow synchronous replication in nucleotide-limiting conditions. We find that new histones are incorporated not only behind, but also ahead of the replication fork. We provide evidence that Rtt109, the S-phase-induced acetyltransferase, stabilizes nucleosomes behind the fork but promotes H3 replacement ahead of the fork. Increased replacement ahead of the fork is independent of the primary Rtt109 acetylation target H3K56 and rather results from Vps75-dependent Rtt109 activity toward the H3 N terminus. Our results suggest that, at least under nucleotide-limiting conditions, selective incorporation of differentially modified H3s behind and ahead of the replication fork results in opposing effects on histone exchange, likely reflecting the distinct challenges for genome stability at these different regions.

The molecular grammar of protein disorder guiding genome-binding locations.

Intrinsically disordered regions (IDRs) direct transcription factors (TFs) towards selected genomic occurrences of their binding motif, as exemplified by budding yeast's Msn2. However, the sequence basis of IDR-directed TF binding selectivity remains unknown. To reveal this sequence grammar, we analyze the genomic localizations of >100 designed IDR mutants, each carrying up to 122 mutations within this 567-AA region. Our data points at multivalent interactions, carried by hydrophobic-mostly aliphatic-residues dispersed within a disordered environment and independent of linear sequence motifs, as the key determinants of Msn2 genomic localization. The implications of our results for the mechanistic basis of IDR-based TF binding preferences are discussed.

Nucleosome retention by histone chaperones and remodelers occludes pervasive DNA-protein binding.

DNA packaging within chromatin depends on histone chaperones and remodelers that form and position nucleosomes. Cells express multiple such chromatin regulators with overlapping in-vitro activities. Defining specific in-vivo activities requires monitoring histone dynamics during regulator depletion, which has been technically challenging. We have recently generated histone-exchange sensors in Saccharomyces cerevisiae, which we now use to define the contributions of 15 regulators to histone dynamics genome-wide. While replication-independent exchange in unperturbed cells maps to promoters, regulator depletions primarily affected gene bodies. Depletion of Spt6, Spt16 or Chd1 sharply increased nucleosome replacement sequentially at the beginning, middle or end of highly expressed gene bodies. They further triggered re-localization of chaperones to affected gene body regions, which compensated for nucleosome loss during transcription complex passage, but concurred with extensive TF binding in gene bodies. We provide a unified quantitative screen highlighting regulator roles in retaining nucleosome binding during transcription and preserving genomic packaging.

Rtt109 slows replication speed by histone N-terminal acetylation.

The wrapping of DNA around histone octamers challenges processes that use DNA as their template. In vitro, DNA replication through chromatin depends on histone modifiers, raising the possibility that cells modify histones to optimize fork progression. Rtt109 is an acetyl transferase that acetylates histone H3 before its DNA incorporation on the K56 and N-terminal residues. We previously reported that, in budding yeast, a wave of histone H3 K9 acetylation progresses ∼3-5 kb ahead of the replication fork. Whether this wave contributes to replication dynamics remained unknown. Here, we show that the replication fork velocity increases following deletion of RTT109, the gene encoding the enzyme required for the prereplication H3 acetylation wave. By using histone H3 mutants, we find that Rtt109-dependent N-terminal acetylation regulates fork velocity, whereas K56 acetylation contributes to replication dynamics only when N-terminal acetylation is compromised. We propose that acetylation of newly synthesized histones slows replication by promoting replacement of nucleosomes evicted by the incoming fork, thereby protecting genome integrity.

Generation and timing of graded responses to morphogen gradients.

Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.

Iron affects Ire1 clustering propensity and the amplitude of endoplasmic reticulum stress signaling.

The unfolded protein response (UPR) allows cells to adjust secretory pathway capacity according to need. Ire1, the endoplasmic reticulum (ER) stress sensor and central activator of the UPR is conserved from the budding yeast Saccharomyces cerevisiae to humans. Under ER stress conditions, Ire1 clusters into foci that enable optimal UPR activation. To discover factors that affect Ire1 clustering, we performed a high-content screen using a whole-genome yeast mutant library expressing Ire1-mCherry. We imaged the strains following UPR induction and found 154 strains that displayed alterations in Ire1 clustering. The hits were enriched for iron and heme effectors and binding proteins. By performing pharmacological depletion and repletion, we confirmed that iron (Fe3+) affects UPR activation in both yeast and human cells. We suggest that Ire1 clustering propensity depends on membrane composition, which is governed by heme-dependent biosynthesis of sterols. Our findings highlight the diverse cellular functions that feed into the UPR and emphasize the cross-talk between organelles required to concertedly maintain homeostasis.

Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing.

RATIONALE: Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. OBJECTIVES: To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. METHODS AND RESULTS: Isolated ventilated and perfused lungs from Cox4i2-/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2-/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2-/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2-/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2-/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. CONCLUSIONS: Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs.

Early Identification of Herbicide Stress in Soybean (Glycine max (L.) Merr.) Using Chlorophyll Fluorescence Imaging Technology.

Herbicides may damage soybean in conventional production systems. Chlorophyll fluorescence imaging technology has been applied to identify herbicide stress in weed species a few days after application. In this study, greenhouse experiments followed by field experiments at five sites were conducted to investigate if the chlorophyll fluorescence imaging is capable of identifying herbicide stress in soybean shortly after application. Measurements were carried out from emergence until the three-to-four-leaf stage of the soybean plants. Results showed that maximal photosystem II (PS II) quantum yield and shoot dry biomass was significantly reduced in soybean by herbicides compared to the untreated control plants. The stress of PS II inhibiting herbicides occurred on the cotyledons of soybean and plants recovered after one week. The stress induced by DOXP synthase-, microtubule assembly-, or cell division-inhibitors was measured from the two-leaf stage until four-leaf stage of soybean. We could demonstrate that the chlorophyll fluorescence imaging technology is capable for detecting herbicide stress in soybean. The system can be applied under both greenhouse and field conditions. This helps farmers to select weed control strategies with less phytotoxicity in soybean and avoid yield losses due to herbicide stress.

Rethinking cell growth models.

The minimal description of a growing cell consists of self-replicating ribosomes translating the cellular proteome. While neglecting all other cellular components, this model provides key insights into the control and limitations of growth rate. It shows, for example, that growth rate is maximized when ribosomes work at full capacity, explains the linear relation between growth rate and the ribosome fraction of the proteome and defines the maximal possible growth rate. This ribosome-centered model also highlights the challenge of coordinating cell growth with related processes such as cell division or nutrient production. Coordination is promoted when ribosomes don't translate at maximal capacity, as it allows escaping strict exponential growth. Recent data support the notion that multiple cellular processes limit growth. In particular, increasing transcriptional demand may be as deleterious as increasing translational demand, depending on growth conditions. Consistent with the idea of trade-off, cells may forgo maximal growth to enable more efficient interprocess coordination and faster adaptation to changing conditions.

ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.

Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.

The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites.

Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood. We found that the yeast membrane protein Yet3, the homolog of human BAP31, is localized to multiple ER contact sites. We show that Yet3 interacts with all the enzymes of the post-squalene ergosterol biosynthesis pathway and recruits them to create sterol-rich domains. Increasing sterol levels at ER contacts causes its depletion from the plasma membrane leading to a compensatory reaction and altered cell metabolism. Our data shows that Yet3 provides on-demand sterols at contacts thus shaping organellar structure and function. A molecular understanding of this protein's functions gives new insights into the role of BAP31 in development and pathology.

Laparoendoscopic single-site (LESS) varicocelectomy with reusable components: comparison with the conventional laparoscopic technique.

BACKGROUND: This study aimed to compare laparoendoscopic single-site varicocelectomy (LESSV) with multiport laparoscopic varicocelectomy (MLV) in terms of intraoperative parameters and postoperative outcomes. METHODS: A retrospective case-control study investigated 10 male adolescents and 89 adults who underwent either LESSV or MLV at the authors' center. The reusable X-Cone single port was inserted transumbilically. A 5-mm 30° telescope was used together with a straight and a prebent laparoscopic instrument. The MLV procedure was performed using two 5-mm ports and one 10-mm port. RESULTS: Between January 2009 and November 2012, 20 patients underwent LESSV and 79 patients underwent MLV. The demographic data were comparable between the two groups. The mean operating time was 59.1 ± 15.5 min for LESSV and 51.2 ± 14.4 min for MLV (P = 0.04). In the LESSV group, no conversion to MLV was necessary. The hospital stay was 1.6 ± 0.7 days in the LESSV group versus 1.8 ± 0.5 days in the MLV group (P = 0.17). The postoperative pain scores did differ between the two groups. By day 2, significantly more patients in the LESSV group than in the MLV group fully recovered their normal physical activity (P = 0.02). Comparison of pre- and postoperative values showed relief of testicular pain and improvement of semen parameters for the majority of the patients. The overall incidence of complications was distributed equally between the two groups as follows: paresthesia of the upper thigh (8 %), wound infection (5 %), epididymitis (3 %) and hydrocele (4 %). All the patients in the LESSV group were fully satisfied with their cosmetic results compared with only 76 % of the patients in the MLV group (P = 0.01). CONCLUSIONS: The LESSV procedure performed with the reusable X-Cone is as safe and efficient as MLV. After LESSV, the parameters measuring postoperative patient satisfaction are significantly improved. Given its reusable components, including prebent laparoscopic instruments, the X-Cone platform is a cost-effective alternative to disposable or homemade single ports.

Histone exchange sensors reveal variant specific dynamics in mouse embryonic stem cells.

Eviction of histones from nucleosomes and their exchange with newly synthesized or alternative variants is a central epigenetic determinant. Here, we define the genome-wide occupancy and exchange pattern of canonical and non-canonical histone variants in mouse embryonic stem cells by genetically encoded exchange sensors. While exchange of all measured variants scales with transcription, we describe variant-specific associations with transcription elongation and Polycomb binding. We found considerable exchange of H3.1 and H2B variants in heterochromatin and repeat elements, contrasting the occupancy and little exchange of H3.3 in these regions. This unexpected association between H3.3 occupancy and exchange of canonical variants is also evident in active promoters and enhancers, and further validated by reduced H3.1 dynamics following depletion of H3.3-specific chaperone, HIRA. Finally, analyzing transgenic mice harboring H3.1 or H3.3 sensors demonstrates the vast potential of this system for studying histone exchange and its impact on gene expression regulation in vivo.

Evolution of binding preferences among whole-genome duplicated transcription factors.

Throughout evolution, new transcription factors (TFs) emerge by gene duplication, promoting growth and rewiring of transcriptional networks. How TF duplicates diverge was studied in a few cases only. To provide a genome-scale view, we considered the set of budding yeast TFs classified as whole-genome duplication (WGD)-retained paralogs (~35% of all specific TFs). Using high-resolution profiling, we find that ~60% of paralogs evolved differential binding preferences. We show that this divergence results primarily from variations outside the DNA-binding domains (DBDs), while DBD preferences remain largely conserved. Analysis of non-WGD orthologs revealed uneven splitting of ancestral preferences between duplicates, and the preferential acquiring of new targets by the least conserved paralog (biased neo/sub-functionalization). Interactions between paralogs were rare, and, when present, occurred through weak competition for DNA-binding or dependency between dimer-forming paralogs. We discuss the implications of our findings for the evolutionary design of transcriptional networks.