Coupling of Aryl Chlorides with Lithium Nucleophiles Enabled by Molecularly Defined Alkynyllithium Palladium Catalysts.

Palladium-catalyzed cross-couplings of aryl chlorides usually call for bulky, electron-rich ligands such as phosphines or heterocyclic carbenes. We have now found that similarly powerful cross-coupling catalysts are obtained by the reaction of palladium salts with alkynyllithium reagents. The species initially formed in this process was characterized as a dilithium tetraalkinyl palladate complex. It catalyzes the coupling of aryl chlorides with the lithium salts of various terminal alkynes to give alkynyl arenes. The isolated Li-alkynyl-Pd complex also efficiently promotes the reaction of aryl, and allyl chlorides with (hetero)aryl-, alkyl-, and allyllithium compounds as well as lithium amines. None of these reactions proceeded in the presence of palladium salts alone. The preparative utility of this approach was demonstrated by the synthesis of 49 molecules, including pharmaceutically relevant compounds.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Measurement of histone replacement dynamics with genetically encoded exchange timers in yeast.

Histone exchange between histones carrying position-specific marks and histones bearing general marks is important for gene regulation, but understanding of histone exchange remains incomplete. To overcome the poor time resolution of conventional pulse-chase histone labeling, we present a genetically encoded histone exchange timer sensitive to the duration that two tagged histone subunits co-reside at an individual genomic locus. We apply these sensors to map genome-wide patterns of histone exchange in yeast using single samples. Comparing H3 exchange in cycling and G1-arrested cells suggests that replication-independent H3 exchange occurs at several hundred nucleosomes (<1% of all nucleosomes) per minute, with a maximal rate at histone promoters. We observed substantial differences between the two nucleosome core subcomplexes: H2A-H2B subcomplexes undergo rapid transcription-dependent replacement within coding regions, whereas H3-H4 replacement occurs predominantly within promoter nucleosomes, in association with gene activation or repression. Our timers allow the in vivo study of histone exchange dynamics with minute time scale resolution.

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.

Accumulation of cis- and trans-regulatory variations is associated with phenotypic divergence of a complex trait between yeast species.

Gene regulatory variations accumulate during evolution and alter gene expression. While the importance of expression variation in phenotypic evolution is well established, the molecular basis remains largely unknown. Here, we examine two closely related yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus, which show phenotypical differences in morphology and cell cycle progression when grown in the same environment. By profiling the cell cycle transcriptome and binding of key transcription factors (TFs) in the two species and their hybrid, we show that changes in expression levels and dynamics of oscillating genes are dominated by upstream trans-variations. We find that multiple cell cycle regulators show both cis- and trans-regulatory variations, which alters their expression in favor of the different cell cycle phenotypes. Moreover, we show that variations in the cell cycle TFs, Fkh1, and Fkh2 affect both the expression of target genes, and the binding specificity of an interacting TF, Ace2. Our study reveals how multiple variations accumulate and propagate through the gene regulatory network, alter TFs binding, contributing to phenotypic changes in cell cycle progression.

Adult Tissue Extracellular Matrix Determines Tissue Specification of Human iPSC-Derived Embryonic Stage Mesodermal Precursor Cells.

The selection of pluripotent stem cell (PSC)-derived cells for tissue modeling and cell therapy will be influenced by their response to the tissue environment, including the extracellular matrix (ECM). Whether and how instructive memory is imprinted in adult ECM and able to impact on the tissue specific determination of human PSC-derived developmentally fetal mesodermal precursor (P-meso) cells is investigated. Decellularized ECM (dECM) is generated from human heart, kidney, and lung tissues and recellularized with P-meso cells in a medium not containing any differentiation inducing components. While P-meso cells on kidney dECM differentiate exclusively into nephronal cells, only beating clusters containing mature and immature cardiac cells form on heart dECM. No tissue-specific differentiation of P-meso cells is observed on endoderm-derived lung dECM. P-meso-derived endothelial cells, however, are found on all dECM preparations independent of tissue origin. Clearance of heparan-sulfate proteoglycans (HSPG) from dECM abolishes induction of tissue-specific differentiation. It is concluded that HSPG-bound factors on adult tissue-derived ECM are essential and sufficient to induce tissue-specific specification of uncommitted fetal stage precursor cells.

Papillary Renal Cell Carcinomas Rewire Glutathione Metabolism and Are Deficient in Both Anabolic Glucose Synthesis and Oxidative Phosphorylation.

Papillary renal cell carcinoma (pRCC) is a malignant kidney cancer with a prevalence of 7-20% of all renal tumors. Proteome and metabolome profiles of 19 pRCC and patient-matched healthy kidney controls were used to elucidate the regulation of metabolic pathways and the underlying molecular mechanisms. Glutathione (GSH), a main reactive oxygen species (ROS) scavenger, was highly increased and can be regarded as a new hallmark in this malignancy. Isotope tracing of pRCC derived cell lines revealed an increased de novo synthesis rate of GSH, based on glutamine consumption. Furthermore, profound downregulation of gluconeogenesis and oxidative phosphorylation was observed at the protein level. In contrast, analysis of the The Cancer Genome Atlas (TCGA) papillary RCC cohort revealed no significant change in transcripts encoding oxidative phosphorylation compared to normal kidney tissue, highlighting the importance of proteomic profiling. The molecular characteristics of pRCC are increased GSH synthesis to cope with ROS stress, deficient anabolic glucose synthesis, and compromised oxidative phosphorylation, which could potentially be exploited in innovative anti-cancer strategies.

VEGF - Supplemented extracellular matrix is sufficient to induce endothelial differentiation of human iPSC.

Extracellular matrix (ECM) provides a scaffold for cells and tissues, but also supports organogenesis and tissue remodeling. The required instructive properties of the ECM to interact with cells depend on matrix architecture, structural proteins and functional matrix components such as growth factors, providing spatial, chemical and functional cues. Decellularized ECM (dECM) has been proposed as an instructive material that promotes tissue regeneration. We investigated the instructive ECM elements preserved in dECM and necessary to promote endothelial differentiation of human induced pluripotent stem cells (hiPSC). We show that detergent-decellularized human kidney ECM remains structurally intact and carries a number of heparin-binding growth factors, including FGF2, VEGF, BMP2, HGF, EGF, PDGF-BB and TGFß, albeit at reduced levels compared to native tissues. Clearance of these heparin-binding factors, or heparan-sulfate proteoclycans from ECM resulted in massively reduced differentiation of hiPSC, suggesting that remaining structural dECM proteins such as laminin, collagen or fibronectin alone are not instructive. In contrast, replenishing dECM with VEGF replaced medium-supplemented VEGF and resulted in more efficient differentiation of hiPSC into endothelial cells, and even in the absence of other culture-supplemented differentiation factors dECM alone was superior to geltrex. In conclusion, conditioning of dECM with specific growth factors acting as functional cues may allow to generate functional niches by selective promotion of cell attachment, survival and differentiation.