Publications by authors named "Steven Hahn"

79 Publications

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

J Mol Biol 2021 Jan 13:166813. Epub 2021 Jan 13.

Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmb.2021.166813DOI Listing
January 2021

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.

Circ Cardiovasc Qual Outcomes 2020 11 25;13(11):e007303. Epub 2020 Sep 25.

Department of Medicine, NYC Health and Hospitals/Jacobi (J.A.M., M.M., S.R., S.I.S., M.L., S.H., E.L., R.S., I.S., P.G., K.J., V.C., J.M., D.M., D.U., A.F., A.K., N.A., G.R., W.I., W.L., M.G., R.T.F.), Albert Einstein College of Medicine, Bronx.

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; <0.001), were overall shorter in duration (median time of 11 minutes [8.5-26.5] versus 15 minutes [7.0-20.0], =0.001), led to fewer endotracheal intubations (52% versus 85%, <0.001), and had overall worse survival rates (3% versus 13%; =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.
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http://dx.doi.org/10.1161/CIRCOUTCOMES.120.007303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7673640PMC
November 2020

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

Mol Cell 2020 06 15;78(5):890-902.e6. Epub 2020 May 15.

Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2020.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275923PMC
June 2020

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

Elife 2020 Jan 8;9. Epub 2020 Jan 8.

Fred Hutchinson Cancer Research Center, Seattle, United States.

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.
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http://dx.doi.org/10.7554/eLife.50109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6977968PMC
January 2020

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

Mayo Clin Proc 2019 05 4;94(5):842-856. Epub 2019 Apr 4.

Center for Women's Health and Division of General Internal Medicine, Mayo Clinic, Rochester, MN.

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.
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http://dx.doi.org/10.1016/j.mayocp.2019.01.009DOI Listing
May 2019

Phase Separation, Protein Disorder, and Enhancer Function.

Authors:
Steven Hahn

Cell 2018 12;175(7):1723-1725

Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2018.11.034DOI Listing
December 2018

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

Cell Rep 2018 03;22(12):3251-3264

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2018.02.097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908246PMC
March 2018

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

Mol Cell Biol 2018 05 30;38(10). Epub 2018 Apr 30.

The Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

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.
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http://dx.doi.org/10.1128/MCB.00038-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954196PMC
May 2018

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

PLoS One 2017 15;12(11):e0186122. Epub 2017 Nov 15.

Medical Affairs, Americas Astellas Pharma Global Development, Inc., Northbrook, Illinois.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186122PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687746PMC
December 2017

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

Nat Struct Mol Biol 2017 Dec 6;24(12):1139-1145. Epub 2017 Nov 6.

Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.

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.
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http://dx.doi.org/10.1038/nsmb.3500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741190PMC
December 2017

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

Mol Cell Biol 2018 Jan 13;38(1). Epub 2017 Dec 13.

Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

A yeast 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 findings. TFIID depletion can be complemented by additional recombinant TATA binding protein (TBP) at only the TATA-containing promoters. In contrast, high levels of TBP did not complement Taf1 depletion 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.
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http://dx.doi.org/10.1128/MCB.00448-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730718PMC
January 2018

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

Mol Cell 2017 Oct 14;68(1):118-129.e5. Epub 2017 Sep 14.

Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2017.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679267PMC
October 2017

Recommendations for the medical management of chronic venous disease: The role of Micronized Purified Flavanoid Fraction (MPFF).

Phlebology 2017 Apr;32(1_suppl):3-19

6 Extrapolate LLC, USA.

Scope A systematic review of the clinical literature concerning medical management of chronic venous disease with the venoactive therapy Micronized Purified Flavonoid Fraction was conducted in addition to an investigation of the hemodynamics and mechanism of chronic venous disease. Methods The systematic review of the literature focused on the use of Micronized Purified Flavonoid Fraction (diosmin) which has recently become available in the US, in the management of chronic venous disease. The primary goal was to assess the level of evidence of the role of Micronized Purified Flavonoid Fraction in the healing of ulcers, and secondarily on the improvement of the symptoms of chronic venous disease such as edema. An initial search of Medline, Cochrane Database for Systematic Reviews and Google Scholar databases was conducted. The references of articles obtained in the primary search, including a Cochrane review of phlebotonics for venous insufficiency, were reviewed for additional studies. Studies were included if patients had a diagnosis of chronic venous disease documented with Doppler and Impedance Plethysmography. Studies excluded were those that had patients with arterial insufficiency (Ankle Brachial Index < .6), comorbidity of diabetes, obesity, rheumatological diseases, or if other causes of edema were present (congestive heart failure, renal, hepatic or lymphatic cause), or if the patient population had recent surgery or deep vein thrombosis, or had been using diuretics (in studies of edema). Other elements of the study design were to note specifically the type of compression therapy used in conjunction with Micronized Purified Flavonoid Fraction. Results The literature review yielded 250 abstracts, 65 of which met criteria for further review and 10 papers were selected for consideration in the systematic review. Conclusion In summary, the general level of evidence supports the recommendation that the use of medical therapy with Micronized Purified Flavonoid Fraction has beneficial outcomes without serious adverse events. In the United States, diosmiplex is the only available prescription formulation of Micronized Purified Flavonoid Fraction. It is derived from the rinds of oranges and is categorized as a medical food and not as a drug; and may be a particularly attractive therapy for many chronic venous disease patients because of its favorable safety profile. The Working Group for chronic venous disease concurs with previous guidance by the International European Society for Vascular Surgery in 2015 which recommended the use of Micronized Purified Flavonoid Fraction for the healing of venous ulcers, alone and adjunctive to compression therapy, and for the reduction in symptoms of chronic venous disease such as edema.
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http://dx.doi.org/10.1177/0268355517692221DOI Listing
April 2017

Hypoactive Sexual Desire Disorder: A Review of Epidemiology, Biopsychology, Diagnosis, and Treatment.

Sex Med Rev 2016 04 6;4(2):103-120. Epub 2016 Feb 6.

Albert Einstein College of Medicine/Jacobi Medical Center, Bronx, NY, USA.

Introduction: Hypoactive Sexual Desire Disorder (HSDD) is defined in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Revised (DSM-IV-TR) as persistent deficient sexual fantasies and desire for sexual activity that causes marked distress or interpersonal difficulty. In the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), HSDD has been subsumed by Female Sexual Interest/Arousal Disorder. However, decades of research based on DSM-IV-TR HSDD criteria form the foundation of our understanding of the essential symptom of distressing low sexual desire, its epidemiology, clinical management, and treatment.

Aim: This publication reviews the state of knowledge about HSDD.

Methods: A literature search was performed using terms HSDD and female sexual dysfunction (FSD).

Main Outcome Measures: Physicians acknowledge that FSD is common and distressing; however, they infrequently address it, citing low confidence, time constraints, and lack of treatment as barriers.

Results: HSDD is present in 8.9% of women ages 18 to 44, 12.3% ages 45 to 64, and 7.4% over 65. Although low sexual desire increases with age, distress decreases; so prevalence of HSDD remains relatively constant across age. HSDD is associated with lower health-related quality of life; lower general happiness and satisfaction with partners; and more frequent negative emotional states. HSDD is underdetected and undertreated. Less than half of patients with sexual problems seek help from or initiate discussions with physicians. Patients are inhibited by fear of embarrassing physicians and believe that physicians should initiate discussions. The Decreased Sexual Desire Screener, a tool for detecting and diagnosing HSDD, is validated for use in general practice.

Conclusion: Women can benefit from intervention in primary care, behavioral health and sexual medicine settings. Psychotherapeutic and pharmacological interventions aim to enhance sexual excitatory process and decrease inhibitory processes. Flibanserin, the first centrally acting daily medication for HSDD, was recently approved in the US for premenopausal women.
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http://dx.doi.org/10.1016/j.sxmr.2015.11.009DOI Listing
April 2016

Mediator binding to UASs is broadly uncoupled from transcription and cooperative with TFIID recruitment to promoters.

EMBO J 2016 11 20;35(22):2435-2446. Epub 2016 Oct 20.

Department of Biology, Indiana University, Bloomington, IN, USA

Mediator is a conserved, essential transcriptional coactivator complex, but its in vivo functions have remained unclear due to conflicting data regarding its genome-wide binding pattern obtained by genome-wide ChIP Here, we used ChEC-seq, a method orthogonal to ChIP, to generate a high-resolution map of Mediator binding to the yeast genome. We find that Mediator associates with upstream activating sequences (UASs) rather than the core promoter or gene body under all conditions tested. Mediator occupancy is surprisingly correlated with transcription levels at only a small fraction of genes. Using the same approach to map TFIID, we find that TFIID is associated with both TFIID- and SAGA-dependent genes and that TFIID and Mediator occupancy is cooperative. Our results clarify Mediator recruitment and binding to the genome, showing that Mediator binding to UASs is widespread, partially uncoupled from transcription, and mediated in part by TFIID.
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http://dx.doi.org/10.15252/embj.201695020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5109241PMC
November 2016

Function of Conserved Topological Regions within the Saccharomyces cerevisiae Basal Transcription Factor TFIIH.

Mol Cell Biol 2016 10 12;36(19):2464-75. Epub 2016 Sep 12.

Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

TFIIH is a 10-subunit RNA polymerase II basal transcription factor with a dual role in DNA repair. TFIIH contains three enzymatic functions and over 30 conserved subdomains and topological regions. We systematically tested the function of these regions in three TFIIH core module subunits, i.e., Ssl1, Tfb4, and Tfb2, in the DNA translocase subunit Ssl2, and in the kinase module subunit Tfb3. Our results are consistent with previously predicted roles for the Tfb2 Hub, Ssl2 Lock, and Tfb3 Latch regions, with mutations in these elements typically having severe defects in TFIIH subunit association. We also found unexpected roles for other domains whose function had not previously been defined. First, the Ssl1-Tfb4 Ring domains are important for TFIIH assembly. Second, the Tfb2 Hub and HEAT domains have an unexpected role in association with Tfb3. Third, the Tfb3 Ring domain is important for association with many other TFIIH subunits. Fourth, a partial deletion of the Ssl1 N-terminal extension (NTE) domain inhibits TFIIH function without affecting subunit association. Finally, we used site-specific cross-linking to localize the Tfb3-binding surface on the Rad3 Arch domain. Our cross-linking results suggest that Tfb3 and Rad3 have an unusual interface, with Tfb3 binding on two opposite faces of the Arch.
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http://dx.doi.org/10.1128/MCB.00182-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021372PMC
October 2016

Two-stage screening for early dementia in primary care.

J Clin Exp Neuropsychol 2016 11 7;38(9):1038-49. Epub 2016 Jun 7.

a Department of Neurology , Albert Einstein College of Medicine and Montefiore Medical Center , Bronx , NY , USA.

Objective: The objective was to compare two screening strategies for dementia in an urban primary care clinic, serving a low-education, minority community composed largely of Latino and African American patients.

Method: Two hundred and fifty-seven patients underwent two-stage patient-based screening (PBS) and informant-based screening (IBS) followed by a diagnostic evaluation. In the first stage, PBS included brief tests of episodic memory (Memory Impairment Screen), semantic memory (Animal Fluency), and executive function (Reciting Months Backwards). For IBS, the first stage consisted of the short Informant Questionnaire on Cognitive Decline in the Elderly, administered to a family member or friend. Patients who screened positive in the first stage of either strategy underwent testing with the picture version of the Free and Cued Selective Reminding Test with Immediate Recall to identify memory impairment. Sensitivity, specificity, and positive and negative predictive values were computed for various cutoffs of each test and combination of tests. Dementia was diagnosed using Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition (DSM-IV) criteria without access to the screening test results.

Results: We identified 66 patients (25.7%) with previously undiagnosed dementia. Sensitivity was the same (77%) for both strategies but specificity was higher for IBS than for PBS (92% versus 83%). IBS's higher specificity makes it the preferred strategy if a knowledgeable informant is available.

Conclusion: Unrecognized dementia is common in primary care. Case-finding can be improved using either PBS or IBS two-stage screening strategies.
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http://dx.doi.org/10.1080/13803395.2016.1187117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790123PMC
November 2016

Structural biology: Snapshots of transcription initiation.

Nature 2016 05 11;533(7603):331-2. Epub 2016 May 11.

Department of Biochemical Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

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http://dx.doi.org/10.1038/nature18437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490667PMC
May 2016

Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II.

J Biol Chem 2016 Jun 17;291(25):13040-7. Epub 2016 Apr 17.

From the Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 and

Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.
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http://dx.doi.org/10.1074/jbc.M116.724583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933221PMC
June 2016

Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH.

Mol Cell 2015 Sep;59(5):794-806

Institute for Systems Biology, 401 Terry Avenue North, Seattle, WA 98109, USA. Electronic address:

TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit.
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http://dx.doi.org/10.1016/j.molcel.2015.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4560838PMC
September 2015

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

Proc Natl Acad Sci U S A 2015 Mar 16;112(13):3961-6. Epub 2015 Mar 16.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.
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http://dx.doi.org/10.1073/pnas.1417709112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4386358PMC
March 2015

Ellis Englesberg and the discovery of positive control in gene regulation.

Authors:
Steven Hahn

Genetics 2014 Oct;198(2):455-60

Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.

Based on his work with the Escherichia coli l-arabinose operon, Ellis Englesberg proposed in 1965 that the regulatory gene araC was an "activator gene" required for positive control of the ara operon. This challenged the widely held belief in a universal mechanism of negative regulation proposed earlier by Jacob and Monod. For years, Englesberg's model was met with deep skepticism. Despite much frustration with complex ad hoc explanations used to challenge his model, Englesberg persisted until the evidence for positive control in ara and other systems became overwhelming. Englesberg's pioneering work enriched the original operon model and had a lasting impact in opening new and exciting ways of thinking about transcriptional regulation.
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http://dx.doi.org/10.1534/genetics.114.167361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196604PMC
October 2014

Architecture of the Saccharomyces cerevisiae SAGA transcription coactivator complex.

EMBO J 2014 Nov 12;33(21):2534-46. Epub 2014 Sep 12.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

The conserved transcription coactivator SAGA is comprised of several modules that are involved in activator binding, TBP binding, histone acetylation (HAT) and deubiquitination (DUB). Crosslinking and mass spectrometry, together with genetic and biochemical analyses, were used to determine the molecular architecture of the SAGA-TBP complex. We find that the SAGA Taf and Taf-like subunits form a TFIID-like core complex at the center of SAGA that makes extensive interactions with all other SAGA modules. SAGA-TBP binding involves a network of interactions between subunits Spt3, Spt8, Spt20, and Spt7. The HAT and DUB modules are in close proximity, and the DUB module modestly stimulates HAT function. The large activator-binding subunit Tra1 primarily connects to the TFIID-like core via its FAT domain. These combined results were used to derive a model for the arrangement of the SAGA subunits and its interactions with TBP. Our results provide new insight into SAGA function in gene regulation, its structural similarity with TFIID, and functional interactions between the SAGA modules.
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http://dx.doi.org/10.15252/embj.201488638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4283410PMC
November 2014

Architecture of the Saccharomyces cerevisiae RNA polymerase I Core Factor complex.

Nat Struct Mol Biol 2014 Sep 17;21(9):810-6. Epub 2014 Aug 17.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Core Factor (CF) is a conserved RNA polymerase (Pol) I general transcription factor comprising Rrn6, Rrn11 and the TFIIB-related subunit Rrn7. CF binds TATA-binding protein (TBP), Pol I and the regulatory factors Rrn3 and upstream activation factor. We used chemical cross-linking-MS to determine the molecular architecture of CF and its interactions with TBP. The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Pol I factor SL1. We validated the cross-linking-derived model through a series of genetic and biochemical assays. Our combined results show the architecture of CF and the functions of the CF subunits in assembly of the complex. We extend these findings to model how CF assembles into the Pol I preinitiation complex, providing new insight into the roles of CF, TBP and Rrn3.
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http://dx.doi.org/10.1038/nsmb.2873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219626PMC
September 2014

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface.

Proc Natl Acad Sci U S A 2014 Aug 13;111(34):E3506-13. Epub 2014 Aug 13.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and

Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.
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http://dx.doi.org/10.1073/pnas.1412088111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151740PMC
August 2014

Screening older Latinos for dementia in the primary care setting.

J Int Neuropsychol Soc 2014 Sep 14;20(8):848-55. Epub 2014 Aug 14.

1Department of Neurology,Albert Einstein College of Medicine and Montefiore Medical Center,Bronx,New York.

The purpose was to compare the Spanish language picture version of the Free and Cued Selective Reminding Test with Immediate Recall (pFCSRT+IR) and the Mini Mental State Exam (MMSE) in identifying very mild dementia among Spanish speaking Latino patients. The tests and an independent diagnostic assessment were administered to 112 Latino patients free of medically diagnosed dementia from an urban primary care clinic. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) were used to examine differences in the operating characteristics of the pFCSRT+IR and the MMSE. Cut scores were manipulated to equate sensitivities (specificities) at clinically relevant values to compare differences in specificities (sensitivities) using the Pearson Chi Square test. Youden's index was used to select the optimal cut scores. Twenty-four of the 112 primary care patients (21%) received a research dementia diagnosis, indicating a substantial burden of unrecognized dementia. MMSE scores but not free recall scores were associated with years of education in patients free of dementia. AUC was significantly higher for free recall than for MMSE. Free recall performed significantly better than the MMSE in sensitivity and in specificity. Using optimal cut scores, patients with impaired free recall were 10 times more likely to have dementia than patients with intact recall, and patients with impaired MMSE scores were 4.5 times more likely to have dementia than patients with intact scores. These results suggest that the Spanish language pFCSRT+IR may be an effective tool for dementia screening in educationally diverse Latino primary care populations.
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http://dx.doi.org/10.1017/S1355617714000708DOI Listing
September 2014

Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

Mol Cell Biol 2014 Aug 27;34(15):2929-43. Epub 2014 May 27.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription.
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http://dx.doi.org/10.1128/MCB.01685-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135567PMC
August 2014

Distinct dendritic cell subsets dictate the fate decision between effector and memory CD8(+) T cell differentiation by a CD24-dependent mechanism.

Immunity 2014 Mar 13;40(3):400-13. Epub 2014 Mar 13.

Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, VA 22908, USA; Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA; Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

The contribution of different DC subsets to effector and memory CD8(+) T cell generation during infection and the mechanism by which DCs controls these fate decisions is unclear. Here we demonstrated that the CD103(+) and CD11b(hi) migratory respiratory DC (RDC) subsets after influenza virus infection activated naive virus-specific CD8(+) T cells differentially. CD103(+) RDCs supported the generation of CD8(+) T effector (Teff) cells, which migrate from lymph nodes to the infected lungs. In contrast, migrant CD11b(hi) RDCs activated CD8(+) T cells characteristic of central memory CD8(+) T (CD8(+) Tcm) cells including retention within the draining lymph nodes. CD103(+) RDCs expressed CD24 at an elevated level, contributing to the propensity of this DC subpopulation to support CD8(+) Teff cell differentiation. Mechanistically, CD24 was shown to regulate CD8(+) T cell activation through HMGB1-mediated engagement of T cell RAGE. Thus, there is distribution of labor among DC subsets in regulating CD8(+) T cell differentiation.
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http://dx.doi.org/10.1016/j.immuni.2014.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4017923PMC
March 2014

Teaching and assessing residents' skills in managing heroin addiction with objective structured clinical examinations (OSCEs).

Subst Abus 2013 ;34(4):350-5

a Division of General Internal Medicine, Department of Medicine , Albert Einstein College of Medicine and Montefiore Medical Center , Bronx , New York , USA.

Background: Heroin-abusing patients present a significant challenge. Objective Structured Clinical Examinations (OSCEs) allow evaluation of residents' clinical skills. The objective of this study was to examine residents' OSCE performance assessing and managing heroin abuse.

Methods: Evaluation and comparison of heroin-specific communication, assessment, and management skills in a 5-station postgraduate year 3 (PGY3) substance abuse OSCE. Faculty used a 4-point Likert scale to assess residents' skills; standardized patients provided written comments.

Results: Two hundred sixty-five internal and family medicine residents in an urban university hospital participated over 5 years. In the heroin station, residents' skills were better (P < .001 for both comparisons) in communication (mean overall score: 316 ± 0.51) than in either assessment (mean overall score: 2.66 ± 0.60) or management (mean overall score: 2.50 ± 0.73). The mean score for assessing specific high-risk behaviors was lower than the mean overall assessment score (222 ± 1.01 vs. 2.74 ± .59; P < .0001), and the mean score for recommending appropriate harm reduction management strategies was lower than the mean overall management score (2.39 ± .89 vs. 2.54 ± .74; P < .005). Standardized patients' comments reflected similar weaknessess in residents' skills.

Conclusions: Assessment and management of heroin abuse were more challenging for residents than general communication. Additional training is required for residents to assess and counsel patients about high-risk behaviors.
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http://dx.doi.org/10.1080/08897077.2013.776658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811017PMC
September 2014