Surprising connections between DNA binding and function for the near-complete set of yeast transcription factors.

DNA sequence-specific transcription factors (TFs) modulate transcription and chromatin architecture, acting from regulatory sites in enhancers and promoters of eukaryotic genes. How TFs locate their DNA targets and how multiple TFs cooperate to regulate individual genes is still unclear. Most yeast TFs are thought to regulate transcription via binding to upstream activating sequences, situated within a few hundred base pairs upstream of the regulated gene. While this model has been validated for individual TFs and specific genes, it has not been tested in a systematic way with the large set of yeast TFs. Here, we have integrated information on the binding and expression targets for the near-complete set of yeast TFs. While we found many instances of functional TF binding sites in upstream regulatory regions, we found many more instances that do not fit this model. In many cases, rapid TF depletion affects gene expression where there is no detectable binding of that TF to the upstream region of the affected gene. In addition, for most TFs, only a small fraction of bound TFs regulates the nearby gene, showing that TF binding does not automatically correspond to regulation of the linked gene. Finally, we found that only a small percentage of TFs are exclusively strong activators or repressors with most TFs having dual function. Overall, our comprehensive mapping of TF binding and regulatory targets have both confirmed known TF relationships and revealed surprising properties of TF function.

Broad compatibility between yeast UAS elements and core promoters and identification of promoter elements that determine cofactor specificity.

Three classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA, and Mediator (MED) Tail, but whether this dependence is determined by the core promoter, upstream activating sequences (UASs), or other gene features is unclear. Also unclear is whether UASs can broadly activate transcription from the different promoter classes. Here, we measure transcription and cofactor specificity for thousands of UAS-core promoter combinations and find that most UASs broadly activate promoters regardless of regulatory class, while few display strong promoter specificity. However, matching UASs and promoters from the same gene class is generally important for optimal expression. We find that sensitivity to rapid depletion of MED Tail or SAGA is dependent on the identity of both UAS and core promoter, while dependence on TFIID localizes to only the promoter. Finally, our results suggest the role of TATA and TATA-like promoter sequences in MED Tail function.

Efficient data reduction for time-of-flight neutron scattering experiments on single crystals.

Event-mode data collection presents remarkable new opportunities for time-of-flight neutron scattering studies of collective excitations, diffuse scattering from short-range atomic and magnetic structures, and neutron crystallography. In these experiments, large volumes of the reciprocal space are surveyed, often using different wavelengths and counting times. These data then have to be added together, with accurate propagation of the counting errors. This paper presents a statistically correct way of adding and histogramming the data for single-crystal time-of-flight neutron scattering measurements. In order to gain a broader community acceptance, particular attention is given to improving the efficiency of calculations.

Yeast Mediator facilitates transcription initiation at most promoters via a Tail-independent mechanism.

Mediator (MED) is a conserved factor with important roles in basal and activated transcription. Here, we investigate the genome-wide roles of yeast MED by rapid depletion of its activator-binding domain (Tail) and monitoring changes in nascent transcription. Rapid Tail depletion surprisingly reduces transcription from only a small subset of genes. At most of these Tail-dependent genes, in unperturbed conditions, MED is detected at both the UASs and promoters. In contrast, at most Tail-independent genes, we find MED primarily at promoters but not at the UASs. These results suggest that MED Tail and activator-mediated MED recruitment regulates only a small subset of genes. Furthermore, we define three classes of genes that differ in PIC assembly pathways and the requirements for MED Tail, SAGA, TFIID, and BET factors Bdf1/2. Our combined results have broad implications for the roles of MED, other coactivators, and mechanisms of transcriptional regulation at different gene classes.

BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae.

Human bromodomain and extra-terminal domain (BET) family members are promising targets for therapy of cancer and immunoinflammatory diseases, but their mechanisms of action and functional redundancies are poorly understood. Bdf1/2, yeast homologues of the human BET factors, were previously proposed to target transcription factor TFIID to acetylated histone H4, analogous to bromodomains that are present within the largest subunit of metazoan TFIID. We investigated the genome-wide roles of Bdf1/2 and found that their important contributions to transcription extend beyond TFIID function as transcription of many genes is more sensitive to Bdf1/2 than to TFIID depletion. Bdf1/2 co-occupy the majority of yeast promoters and affect preinitiation complex formation through recruitment of TFIID, Mediator, and basal transcription factors to chromatin. Surprisingly, we discovered that hypersensitivity of genes to Bdf1/2 depletion results from combined defects in transcription initiation and early elongation, a striking functional similarity to human BET proteins, most notably Brd4. Our results establish Bdf1/2 as critical for yeast transcription and provide important mechanistic insights into the function of BET proteins in all eukaryotes.

When a healthy cell creates new proteins, it activates a standard two-step biological manufacturing process. Firstly, DNA is transcribed from a specific gene to generate a strand of messenger RNA, or mRNA. Next, this mRNA molecule is translated to create the final protein product. This process of converting DNA into mRNA is supported by a series of helper proteins, including proteins from the bromodomain and extra-terminal domain (BET) family. Cancer cells can become 'addicted' to the process of converting DNA into RNA, leading to the overproduction of mRNA molecules, uncontrolled cell growth and tumor formation. Knocking out BET helper proteins could potentially bring cancer cells under control by halting transcription and preventing tumor growth. However, the precise ways in which BET helper proteins regulate transcription are currently poorly understood, and therefore developing rational ways to target them is a challenge. Building on their previous work, Donczew and Hahn have investigated how two BET helper proteins, Bdf1 and Bdf2, help to regulate transcription in budding yeast. Using a range of genomic techniques, Donczew and Hahn found that Bdf1 and Bdf2 had important roles for initiating transcription and elongating mRNA molecules. Both BET proteins were also involved in recruiting other protein factors to help with the transcription process, including TFIID and Mediator. Based on these findings, it is likely that cooperation between BET proteins, TFIID and Mediator represents a common pathway through which gene expression is regulated across all eukaryotic organisms. Both Bdf1 and Bdf2 were also found to provide the same functions in yeast as similar BET proteins in humans. Using this robust yeast model system to perform further detailed studies of BET factors could therefore provide highly relevant information to expand our understanding of human biology and disease. Ultimately, this research provides important insights into how two members of the BET family of helper proteins contribute to the control of transcription in yeast. This information could be used to guide the design of new drugs for cancer therapy that target not only BET proteins themselves but also other proteins they recruit, including TFIID and Mediator. Such targeted drugs would be expected to be more harmful for cancer cells than for healthy cells, which could reduce unwanted side effects.

Mediator subunit Med15 dictates the conserved "fuzzy" binding mechanism of yeast transcription activators Gal4 and Gcn4.

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein-protein interaction.

The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning.

The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates Pol II to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in vitro in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning.

Characteristics and Outcomes of In-Hospital Cardiac Arrest Events During the COVID-19 Pandemic: A Single-Center Experience From a New York City Public Hospital.

BACKGROUND: Patients hospitalized for severe coronavirus disease 2019 (COVID-19) infection are at risk for in-hospital cardiac arrest (IHCA). It is unknown whether certain characteristics of cardiac arrest care and outcomes of IHCAs during the COVID-19 pandemic differed compared with a pre-COVID-19 period. METHODS: All patients who experienced an IHCA at our hospital from March 1, 2020 through May 15, 2020, during the peak of the COVID-19 pandemic, and those who had an IHCA from January 1, 2019 to December 31, 2019 were identified. All patient data were extracted from our hospital's Get With The Guidelines-Resuscitation registry, a prospective hospital-based archive of IHCA data. Baseline characteristics of patients, interventions, and overall outcomes of IHCAs during the COVID-19 pandemic were compared with IHCAs in 2019, before the COVID-19 pandemic. RESULTS: There were 125 IHCAs during a 2.5-month period at our hospital during the peak of the COVID-19 pandemic compared with 117 IHCAs in all of 2019. IHCAs during the COVID-19 pandemic occurred more often on general medicine wards than in intensive care units (46% versus 33%; 19% versus 60% in 2019; P<0.001), were overall shorter in duration (median time of 11 minutes [8.5-26.5] versus 15 minutes [7.0-20.0], P=0.001), led to fewer endotracheal intubations (52% versus 85%, P<0.001), and had overall worse survival rates (3% versus 13%; P=0.007) compared with IHCAs before the COVID-19 pandemic. CONCLUSIONS: Patients who experienced an IHCA during the COVID-19 pandemic had overall worse survival compared with those who had an IHCA before the COVID-19 pandemic. Our findings highlight important differences between these 2 time periods. Further study is needed on cardiac arrest care in patients with COVID-19.

A High-Throughput Screen for Transcription Activation Domains Reveals Their Sequence Features and Permits Prediction by Deep Learning.

Acidic transcription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that promote their dynamic behavior, "fuzzy" interactions, and target specificity. We screened a large set of random 30-mer peptides for AD function in yeast and trained a deep neural network (ADpred) on the AD-positive and -negative sequences. ADpred identifies known acidic ADs within transcription factors and accurately predicts the consequences of mutations. Our work reveals that strong acidic ADs contain multiple clusters of hydrophobic residues near acidic side chains, explaining why ADs often have a biased amino acid composition. ADs likely use a binding mechanism similar to avidity where a minimum number of weak dynamic interactions are required between activator and target to generate biologically relevant affinity and in vivo function. This mechanism explains the basis for fuzzy binding observed between acidic ADs and targets.

Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA.

Deletions within genes coding for subunits of the transcription coactivator SAGA caused strong genome-wide defects in transcription and SAGA-mediated chromatin modifications. In contrast, rapid SAGA depletion produced only modest transcription defects at 13% of protein-coding genes - genes that are generally more sensitive to rapid TFIID depletion. However, transcription of these 'coactivator-redundant' genes is strongly affected by rapid depletion of both factors, showing the overlapping functions of TFIID and SAGA at this gene set. We suggest that this overlapping function is linked to TBP-DNA recruitment. The remaining 87% of expressed genes that we term 'TFIID-dependent' are highly sensitive to rapid TFIID depletion and insensitive to rapid SAGA depletion. Genome-wide mapping of SAGA and TFIID found binding of both factors at many genes independent of gene class. Promoter analysis suggests that the distinction between the gene classes is due to multiple components rather than any single regulatory factor or promoter sequence motif.

The International Society for the Study of Women's Sexual Health Process of Care for the Identification of Sexual Concerns and Problems in Women.

Sexual problems are common in women of all ages. Despite their frequency and impact, female sexual dysfunctions (FSDs) are often unrecognized and untreated in clinical settings. In response, the International Society for the Study of Women's Sexual Health convened a multidisciplinary, international expert panel to develop a process of care (POC) that outlines recommendations for identification of sexual problems in women. This POC describes core and advanced competencies in FSD for clinicians who are not sexual medicine specialists and serve as caregivers of women and, therefore, is useful for clinicians with any level of competence in sexual medicine. The POC begins with the expectation of universal screening for sexual concerns, proceeds with a 4-step model (eliciting the story, naming/reframing attention to the problem, empathic witnessing of the patient's distress and the problem's impact, and referral or assessment and treatment) that accommodates all levels of engagement, and delineates a process for referral when patients' needs exceed clinician expertise. Distressing problems related to desire, arousal, and orgasm affect 12% of women across the lifespan. Low desire is the most common sexual problem, but sexual pain and other less common disorders of arousal and orgasm are also seen in clinical practice. Screening is best initiated by a ubiquity statement that assures the patient that sexual concerns are common and can be revealed. Patient-centered communication skills facilitate and optimize the discussion. The goal of the POC is to provide guidance to clinicians regarding screening, education, management, and referral for women with sexual problems.

Phase Separation, Protein Disorder, and Enhancer Function.

New findings suggest that transcription enhancers work by recruitment of a large dynamic network of coactivators and other factors responsible for gene activation. Formation of these condensates is driven by DNA-bound transcription factors, their intrinsically disordered activation domains, and dynamic low-specificity interactions within the complex.

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements.

Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator.

Eukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While the AD function of both factors depended on hydrophobic residues, Ino2 further required key conserved acidic and polar residues for optimal function. Binding studies showed that the ADs bound multiple Med15 activator-binding domains (ABDs) with similar orders of micromolar affinity and similar but distinct thermodynamic properties. Protein cross-linking data show that no unique complex was formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

Mechanistic Differences in Transcription Initiation at TATA-Less and TATA-Containing Promoters.

A yeast in vitro system was developed that is active for transcription at both TATA-containing and TATA-less promoters. Transcription with extracts made from cells depleted of TFIID subunit Taf1 demonstrated that promoters of both classes are TFIID dependent, in agreement with recent in vivo findings. TFIID depletion can be complemented in vitro by additional recombinant TATA binding protein (TBP) at only the TATA-containing promoters. In contrast, high levels of TBP did not complement Taf1 depletion in vivo and instead repressed transcription from both promoter types. We also demonstrate the importance of the TATA-like sequence found at many TATA-less promoters and describe how the presence or absence of the TATA element is likely not the only feature that distinguishes these two types of promoters.

TFIIH generates a six-base-pair open complex during RNAP II transcription initiation and start-site scanning.

Eukaryotic mRNA transcription initiation is directed by the formation of the megadalton-sized preinitiation complex (PIC). After PIC formation, double-stranded DNA (dsDNA) is unwound to form a single-stranded DNA bubble, and the template strand is loaded into the polymerase active site. DNA opening is catalyzed by Ssl2 (XPB), the dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, transcription initiation proceeds through a scanning phase during which downstream DNA is searched for optimal start sites. Here, to test models for initial DNA opening and start-site scanning, we measure the DNA-bubble sizes generated by Saccharomyces cerevisiae PICs in real time using single-molecule magnetic tweezers. We show that ATP hydrolysis by Ssl2 opens a 6-base-pair (bp) bubble that grows to 13 bp in the presence of NTPs. These observations support a two-step model wherein ATP-dependent Ssl2 translocation leads to a 6-bp open complex that RNA polymerase II expands via NTP-dependent RNA transcription.

Physician-patient communication about overactive bladder: Results of an observational sociolinguistic study.

INTRODUCTION: Overactive bladder (OAB) and urinary incontinence are common problems that have significant impact on quality of life (QOL). Less than half of sufferers seek help from their physicians; many who do are dissatisfied with treatment and their physicians' understanding of their problems. Little is known about the sociolinguistic characteristics of physician-patient communication about OAB in community practice. METHODS: An IRB-approved observational sociolinguistic study of dialogues between patients with OAB and treating physicians was conducted. Study design included semi-structured post-visit interviews, post-visit questionnaires, and follow-up phone calls. Conversations were analyzed using techniques from interactional sociolinguistics. RESULTS: Communication was physician- rather than patient-centered. Physicians spoke the majority of words and 83% of questions were closed-ended. The impact of OAB on QOL and concerns about and adherence to treatment were infrequently addressed by physicians, who were poorly aligned with patients in their understanding. These topics were addressed more frequently when open-ended questions successfully eliciting elaborated responses were used in ask-tell-ask or ask-tell sequences. DISCUSSION: Clinical dialogue around OAB is physician-centered; topics critical to managing OAB are infrequently and inadequately addressed. The use of patient-centered communication is correlated with more discussion of critical topics, and thus, more effective management of OAB.

Transcription of Nearly All Yeast RNA Polymerase II-Transcribed Genes Is Dependent on Transcription Factor TFIID.

Previous studies suggested that expression of most yeast mRNAs is dominated by either transcription factor TFIID or SAGA. We re-examined the role of TFIID by rapid depletion of S. cerevisiae TFIID subunits and measurement of changes in nascent transcription. We find that transcription of nearly all mRNAs is strongly dependent on TFIID function. Degron-dependent depletion of Taf1, Taf2, Taf7, Taf11, and Taf13 showed similar transcription decreases for genes in the Taf1-depleted, Taf1-enriched, TATA-containing, and TATA-less gene classes. The magnitude of TFIID dependence varies with growth conditions, although this variation is similar genome-wide. Many studies have suggested differences in gene-regulatory mechanisms between TATA and TATA-less genes, and these differences have been attributed in part to differential dependence on SAGA or TFIID. Our work indicates that TFIID participates in expression of nearly all yeast mRNAs and that differences in regulation between these two gene categories is due to other properties.