In Vitro Studies of Nucleosome Positioning and Stability at the PHO5 and PHO8 Promoters in Saccharomyces cerevisiae

The PHO5 and PHO8 genes in yeast provide typical examples for the role of chromatin in promoter regulation. Both genes are regulated by the same transcriptional activator, Pho4, which initiates nucleosome remodeling and transcriptional activation. In spite of this co-regulation, there are important differences in gene activity and in the way promoter chromatin undergoes chromatin remodeling. First, PHO5 belongs to one of the most strongly induced genes in yeast being 10-fold more active than the PHO8 gene (Oshima, 1997; Barbaric et al., 1992). Second, chromatin remodeling at the PHO5 promoter affects four nucleosomes (Almer et al., 1986), whereas only two nucleosomes are afffected at the PHO8 promoter (Barbaric et al., 1992). Third, neither the histone acetyl transferase Gcn5 nor chromatin remodeling complex Swi/Snf seem to be critically required for chromatin remodeling at the PHO5 promoter (Barbaric et al., 2001; Reinke and Hörz, 2003; Dhasarathy and Kladde, 2005; Neef and Kladde, 2003). At the PHO8 promoter, on the other hand, absence of Swi/Snf results in the complete loss of chromatin remodeling under inducing conditions. Furthermore, Gcn5 is required for full remodeling and transcriptional activation at this promoter (Gregory et al., 1999). Ever since these differences were recognized there have been speculations about the underlying reasons. This work shows that these discrepancies are not a direct consequence of the position or strength of the UASp elements driving the activation of transcription. Instead, these differences result from different stabilities of the two promoter chromatin structures. The basis for these results was the development of a competitive yeast in vitro assembly technique in which differences in nucleosome stability between promoter regions could be directly compared. This technique originated from a yeast in vitro chromatin assembly system that generated the characteristic PHO5 promoter chromatin structre (Korber and Hörz, 2004). As shown here, this system also assembles the native PHO8 promoter nucleosome pattern. Using the competitive assembly system it was shown that the PHO8 promoter has greater nucleosome positioning power, and that the properly positioned nucleosomes are more stable than at the PHO5 promoter. This provided for the first time evidence for the correlation of inherently more stable chromatin with stricter co-factor requirements. Remarkably, the positioning information for the in vitro assembly of the native PHO5 and PHO8 promoter chromatin patterns was specific to the yeast extract. Salt gradient dialysis or Drosophila embryo extract assemblies did not support the proper nucleosome positioning. However, nucleosomes in chromatin generated in these systems could be shifted to their in vivo-like positions by the addition of yeast extract. This indicates that the nucleosome positioning mechanisms in vitro are uncoupled from the nucleosome loading machinery. The nucleosome positioning at the PHO5 and PHO8 promoters was energy dependent suggesting a role of chromatin remodeling machines in generation of the repressed promoter chromatin structure. In spite of this, the chromatin remodeling machines Swi/Snf, Isw1, Isw2 and Chd1 were dispensable nucleosome positioning at both promoters

Longitudinal Effects of Activity-Based Flexible Office Design on Teamwork

This three-wave longitudinal interview study (time lag: 12 and 18 months) investigates the impact of working in an activity-based flexible office (A-FO) on processes within and across teams (i.e., communication, trust, cohesion, and collaboration) and team management. Based on a new theoretical framework on benefits and risks of A-FOs (A-FO-M; Wohlers and Hertel, 2017), we conducted interviews with 25 employees of an in-house training institute who recently switched from single cell or shared offices to an A-FO. The A-FO consisted of a main open-layout environment without assigned workstations and provided additional working zones appropriate for specific work activities. According to the A-FO-M, A-FO features are expected to alter visibility and proximity of employees compared to office environments with assigned workstations. Altered visibility and proximity, in turn, should be related to team processes, such as communication. The interview material was analyzed using qualitative content analysis. This textual analysis procedure revealed that the interviewees reported that inter-team collaboration improved while working in the A-FO. Reasons that were mentioned for this positive effect were more contact, communication, collaboration possibilities (joint project work), and trusting relationships. However, interviewees also reported negative effects, such as that teamwork suffered due to less communication and cooperation. Along with that, especially ensuring team cohesion and communication among team partners were the most often mentioned challenges for management since team members were spatially dispersed within the office building. Theoretical and practical implications, such as assigning additional team areas to support teamwork, as well as recommendations for future research are discussed

Stuck in the Past? Rumination-Related Memory Integration

Memories connected to ruminative concerns repetitively capture attention, even in situations designed to alter them. However, recent research on memory updating suggests that memory for benign substitutes (e.g., reinterpretations) might be facilitated by integration with the ruminative memories. As a first approach, two experiments (Ns = 72) mimicked rumination-related memories with rumination-themed stimuli and an imagery task. College undergraduates screened for ruminative status first studied and imaged ruminative cue-target word pairs, and then in a second phase they studied the same cues re-paired with benign targets (along with new and repeated pairs). On the test of cued recall of benign targets, they judged whether each recalled word had been repeated or changed across the two phases (or was new in the second phase). When target changes were not remembered, recall of benign targets revealed proactive interference that was insensitive to ruminative status. However, when participants remembered change and the ruminative targets, their recall of benign targets was facilitated, particularly if they identified as ruminators (Experiment 1). When the test simply asked for recall of either or both targets (Experiment 2), ruminators recalled both targets more frequently than did others. These outcomes suggest that ruminative memories might provide bridges to remembering associated benign memories, such as reinterpretations, under conditions consistent with everyday ruminative retrieval

Response to comment on 'AIRE-deficient patients harbor unique high-affinity disease-ameliorating autoantibodies'

In 2016, we reported four substantial observations of APECED/APS1 patients, who are deficient in AIRE, a major regulator of central T cell tolerance (Meyer et al., 2016). Two of those observations have been challenged. Specifically, 'private' autoantibody reactivities shared by only a few patients but collectively targeting >1000 autoantigens have been attributed to false positives (Landegren, 2019). While acknowledging this risk, our study-design included follow-up validation, permitting us to adopt statistical approaches to also limit false negatives. Importantly, many such private specificities have now been validated by multiple, independent means including the autoantibodies ' molecular cloning and expression. Second, a significant correlation of antibody-mediated IFN a neutralization with an absence of disease in patients highly disposed to Type I diabetes has been challenged because of a claimed failure to replicate our findings (Landegren, 2019). However, flaws in design and implementation invalidate this challenge. Thus, our results present robust, insightful, independently validated depictions of APECED/APS1, that have spawned productive follow-up studies.Non peer reviewe

Autoantibody Repertoire in APECED Patients Targets Two Distinct Subgroups of Protiens

High titer autoantibodies produced by B lymphocytes are clinically important features of many common autoimmune diseases. APECED patients with deficient autoimmune regulator (AIRE) gene collectively display a broad repertoire of high titer autoantibodies, including some which are pathognomonic for major autoimmune diseases. AIRE deficiency severely reduces thymic expression of gene-products ordinarily restricted to discrete peripheral tissues, and developing T cells reactive to those gene-products are not inactivated during their development. However, the extent of the autoantibody repertoire in APECED and its relation to thymic expression of self-antigens are unclear. We here undertook a broad protein array approach to assess autoantibody repertoire in APECED patients. Our results show that in addition to shared autoantigen reactivities, APECED patients display high inter-individual variation in their autoantigen profiles, which collectively are enriched in evolutionarily conserved, cytosolic and nuclear phosphoproteins. The APECED autoantigens have two major origins; proteins expressed in thymic medullary epithelial cells and proteins expressed in lymphoid cells. These findings support the hypothesis that specific protein properties strongly contribute to the etiology of B cell autoimmunity.Peer reviewe

AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies

APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.Peer reviewe

Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

Human cytomegalovirus (hCMV) is a highly prevalent pathogen that, upon primary infection, establishes life-long persistence in all infected individuals. Acute hCMV infections cause a variety of diseases in humans with developmental or acquired immune deficits. In addition, persistent hCMV infection may contribute to various chronic disease conditions even in immunologically normal people. The pathogenesis of hCMV disease has been frequently linked to inflammatory host immune responses triggered by virus-infected cells. Moreover, hCMV infection activates numerous host genes many of which encode pro-inflammatory proteins. However, little is known about the relative contributions of individual viral gene products to these changes in cellular transcription. We systematically analyzed the effects of the hCMV 72-kDa immediate-early 1 (IE1) protein, a major transcriptional activator and antagonist of type I interferon (IFN) signaling, on the human transcriptome. Following expression under conditions closely mimicking the situation during productive infection, IE1 elicits a global type II IFN-like host cell response. This response is dominated by the selective up-regulation of immune stimulatory genes normally controlled by IFN-γ and includes the synthesis and secretion of pro-inflammatory chemokines. IE1-mediated induction of IFN-stimulated genes strictly depends on tyrosine-phosphorylated signal transducer and activator of transcription 1 (STAT1) and correlates with the nuclear accumulation and sequence-specific binding of STAT1 to IFN-γ-responsive promoters. However, neither synthesis nor secretion of IFN-γ or other IFNs seems to be required for the IE1-dependent effects on cellular gene expression. Our results demonstrate that a single hCMV protein can trigger a pro-inflammatory host transcriptional response via an unexpected STAT1-dependent but IFN-independent mechanism and identify IE1 as a candidate determinant of hCMV pathogenicity

Getting more from many & mdash;A framework of community resourcefulness in new venture creation

In this study, we move beyond the predominant focus entrepreneurship researchers have put on the acquisition of financial capital from professional investors by exploring how, and with what effects, entrepreneurs can mobilize all required resources?financial, human, physical, and social?from local communities. Our temporal analysis of the resource mobilization processes of seven cases of community-based enterprises (CBEs) reveals four sets of activities with distinct goals and effects, which explain how entrepreneurs can meet or even exceed their resource mobilization goals by mobilizing a greater variety of resources from a broader base of resource providers. Importantly, the findings show how entrepreneurs can achieve a multiplier effect meaning that they can perpetuate the inflow of significant amounts of unsolicited resources by continuously engaging in activities targeted at creating a sense of identification and ownership, which require comparatively little extra effort and resource inputs. We synthesize our findings in a framework of community resourcefulness in new venture creation. This framework adds a new perspective of resourcefulness as ?getting more from many,? and demonstrates that resourceful behavior is not necessarily about individuals? ability to respond to situational constraints but also about their ability to recognize and seize situational resource potentials. Our findings have important implications for our understanding of resourcefulness in entrepreneurship and the nascent body of literature on community-based enterprises