DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes.

DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at its adjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.

Anti-Inflammatory Chromatinscape Suggests Alternative Mechanisms of Glucocorticoid Receptor Action.

Despite the widespread use of glucocorticoids (GCs), their anti-inflammatory effects are not understood mechanistically. Numerous investigations have examined the effects of glucocorticoid receptor (GR) activation prior to inflammatory challenges. However, clinical situations are emulated by a GC intervention initiated in the midst of rampant inflammatory responses. To characterize the effects of a late GC treatment, we profiled macrophage transcriptional and chromatinscapes with Dexamethasone (Dex) treatment before or after stimulation by lipopolysaccharide (LPS). The late activation of GR had a similar gene-expression profile as from GR pre-activation, while ameliorating the disruption of metabolic genes. Chromatin occupancy of GR was not predictive of Dex-regulated gene expression, contradicting the "trans-repression by tethering" model. Rather, GR activation resulted in genome-wide blockade of NF-κB interaction with chromatin and directly induced inhibitors of NF-κB and AP-1. Our investigation using GC treatments with clinically relevant timing highlights mechanisms underlying GR actions for modulating the "inflamed epigenome."

The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency.

Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA and a divergent non-coding (DNC) transcript of unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. Using high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements, we identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC transcription. Nascent transcription profiling showed a genome-wide role of Hda1C in repression of DNC transcription. Live-cell imaging of transcription revealed that mutations in the Hda3 subunit increased the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC transcription regions, supporting DNC transcription repression by histone deacetylation. Our data support the interpretation that DNC transcription results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

Transcription factor clusters enable target search but do not contribute to target gene activation.

Many transcription factors (TFs) localize in nuclear clusters of locally increased concentrations, but how TF clustering is regulated and how it influences gene expression is not well understood. Here, we use quantitative microscopy in living cells to study the regulation and function of clustering of the budding yeast TF Gal4 in its endogenous context. Our results show that Gal4 forms clusters that overlap with the GAL loci. Cluster number, density and size are regulated in different growth conditions by the Gal4-inhibitor Gal80 and Gal4 concentration. Gal4 truncation mutants reveal that Gal4 clustering is facilitated by, but does not completely depend on DNA binding and intrinsically disordered regions. Moreover, we discover that clustering acts as a double-edged sword: self-interactions aid TF recruitment to target genes, but recruited Gal4 molecules that are not DNA-bound do not contribute to, and may even inhibit, transcription activation. We propose that cells need to balance the different effects of TF clustering on target search and transcription activation to facilitate proper gene expression.

Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting.

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

Following the tracks: How transcription factor binding dynamics control transcription.

Transcription, the process of copying genetic information from DNA to messenger RNA, is regulated by sequence-specific DNA-binding proteins known as transcription factors (TFs). Recent advances in single-molecule tracking (SMT) technologies have enabled visualization of individual TF molecules as they diffuse and interact with the DNA in the context of living cells. These SMT studies have uncovered multiple populations of DNA-binding events characterized by their distinctive DNA residence times. In this perspective, we review recent insights into how these residence times relate to specific and non-specific DNA binding, as well as the contribution of TF domains on the DNA-binding dynamics. We discuss different models that aim to link transient DNA binding by TFs to bursts of transcription and present an outlook for how future advances in microscopy development may broaden our understanding of the dynamics of the molecular steps that underlie transcription activation.

Benzochalcogendiazole-based conjugated molecules: investigating the effects of substituents and heteroatom juggling.

A convenient and effective synthetic approach for benzochalcogendiazole-based small molecules has been achieved using polyaniline (PANI)-anchored palladium as a heterogeneous catalyst. The photophysical properties of the synthesized benzochalcogendiazole-based small molecules, having different terminal substituents, have been compared. Moreover, the structural aspects, including the packing patterns and non-bonding interactions of the conjugated molecules, have been investigated using the single crystal X-ray diffraction (SCXRD) technique.

HIV Testing, Linkage to HIV Medical Care, and Interviews for Partner Services Among Youths - 61 Health Department Jurisdictions, United States, Puerto Rico, and the U.S. Virgin Islands, 2015.

Identifying persons living with human immunodeficiency virus (HIV) who are unaware of their infection, linking them to HIV medical care, and reducing health disparities are important national goals (1). Of the 8,841 teens and young adults aged 13-24 years (collectively referred to as youths in this report) who received a diagnosis of HIV in 2014, 70% were young men who have sex with men (MSM) (2). In the same year, an estimated 52% of young MSM living with HIV were unaware of their infection compared with 15% among all persons living with HIV (3). An average of 22% of high school students who have had sexual intercourse and 33% of young adults (persons aged 18-24 years) reported ever receiving an HIV test (4). CDC recommends screening all persons aged 13-64 years, with annual rescreening for persons at high risk for HIV infection (5). Analysis of CDC-funded program data for youths submitted by 61 health departments in 2015 revealed that young MSM, who accounted for 83% of new diagnoses among all youths in non-health care facilities, received 28% of HIV tests.* The 2020 national goal is to link at least 85% of HIV-positive persons to HIV medical care within 30 days of diagnosis. In this analysis, 66% of youths who received positive test results for HIV infection were linked to care within 90 days of diagnosis. Increasing the number of youths at risk for HIV infection who are tested for HIV on a regular basis and ensuring that youths who receive positive test results for HIV are rapidly linked to and retained in appropriate medical care, including early initiation of antiretroviral therapy, are essential steps for reducing HIV infection in this vulnerable population.

Comprehensive coverage on anti-mycobacterial endeavour reported during 2022.

TB being one of the deadliest diseases and second most common infectious cause of deaths, poses the severe threat to global health. The extended duration of therapy owing to resistance and its upsurge in immune-compromised patients have been the driving force for the development of novel of anti-TB scaffolds. Recently, we have compiled the account of anti-mycobacterial scaffolds published during 2015-2020 and updated them in 2021. The present work involves the insights on the anti-mycobacterial scaffolds reported in 2022 with their mechanism of action, structure activity relationships, along with the key perceptions for the design of newer anti-TB agents for the broader interests of medicinal chemists.

Quantifying how post-transcriptional noise and gene copy number variation bias transcriptional parameter inference from mRNA distributions.

Transcriptional rates are often estimated by fitting the distribution of mature mRNA numbers measured using smFISH (single molecule fluorescence in situ hybridization) with the distribution predicted by the telegraph model of gene expression, which defines two promoter states of activity and inactivity. However, fluctuations in mature mRNA numbers are strongly affected by processes downstream of transcription. In addition, the telegraph model assumes one gene copy but in experiments, cells may have two gene copies as cells replicate their genome during the cell cycle. While it is often presumed that post-transcriptional noise and gene copy number variation affect transcriptional parameter estimation, the size of the error introduced remains unclear. To address this issue, here we measure both mature and nascent mRNA distributions of GAL10 in yeast cells using smFISH and classify each cell according to its cell cycle phase. We infer transcriptional parameters from mature and nascent mRNA distributions, with and without accounting for cell cycle phase and compare the results to live-cell transcription measurements of the same gene. We find that: (i) correcting for cell cycle dynamics decreases the promoter switching rates and the initiation rate, and increases the fraction of time spent in the active state, as well as the burst size; (ii) additional correction for post-transcriptional noise leads to further increases in the burst size and to a large reduction in the errors in parameter estimation. Furthermore, we outline how to correctly adjust for measurement noise in smFISH due to uncertainty in transcription site localisation when introns cannot be labelled. Simulations with parameters estimated from nascent smFISH data, which is corrected for cell cycle phases and measurement noise, leads to autocorrelation functions that agree with those obtained from live-cell imaging.

Optimized protocol for single-molecule RNA FISH to visualize gene expression in S. cerevisiae.

Single-molecule RNA fluorescence in situ hybridization (smFISH) allows subcellular visualization, localization, and quantification of endogenous RNA molecules in fixed cells. The spatial and intensity information of each RNA can be used to distinguish mature from nascent transcripts inside each cell, revealing both past and instantaneous transcriptional activity. Here, we describe an optimized protocol for smFISH in Saccharomyces cerevisiae with optimized lyticase digestion time and hybrization steps for more homogenous results. For complete details on the use and execution of this protocol, please refer to Donovan et al. (2019).

A Survivor-Derived Approach to Addressing Trafficking in the Pediatric ED.

OBJECTIVES: Our objective was to elicit the perspectives of survivors of child trafficking on addressing trafficking in the pediatric emergency department (ED) and, secondarily, to provide a survivor-derived framework to help pediatric emergency medicine (PEM) providers discuss trafficking with their patients. METHODS: We conducted in-depth, semistructured interviews with young adults who experienced trafficking as children and/or as adolescents. In the interviews, we employed a novel video-elicitation method designed by the research team to elicit detailed participant feedback and recommendations on the pediatric ED through an interactive, immersive discussion with the interviewer. A grounded theory approach was employed. RESULTS: Seventeen interviews were conducted revealing the following themes, which we present in an integrated framework for PEM providers: (1) fear is a significant barrier; (2) participants do want PEM providers to ask about trafficking, and it is not harmful to do so; (3) PEM providers should address fear through emphasizing confidentiality and privacy and encouraging agency; (4) PEM providers should approach the patient in a direct, sensitive, and nonjudgmental manner; and (5) changes to the ED environment may facilitate the conversation. Suggested wordings and tips from survivors were compiled. CONCLUSIONS: Trafficking survivors feel that the pediatric ED can be a place where they can be asked about trafficking, and that when done in private, it is not harmful or retraumatizing. Fear is a major barrier to disclosure in the pediatric ED setting, and PEM providers can mitigate this by emphasizing privacy and confidentiality and increasing agency by providing choices. PEM providers should be direct, sensitive, and nonjudgmental in their approach to discussing trafficking.

Single-Molecule Fluorescence Imaging in Living Saccharomyces cerevisiae Cells.

This protocol describes how to image fluorescently tagged proteins, RNA, or DNA inside living Saccharomyces cerevisiae cells at the single-molecule level. Imaging inside living cells, as opposed to fixed materials, gives access to real-time kinetic information. Although various single-molecule imaging applications are discussed, we focus on imaging of gene transcription at the single-RNA level. To obtain the best possible results, it is important that both imaging parameters and yeast culture conditions are optimized. Here, both aspects are described. For complete details on the use and execution of this protocol, please refer to Lenstra et al. (2015) and Donovan et al. (2019).

Integrative analysis suggests cell type-specific decoding of NF-κB dynamics.

The complex signaling dynamics of transcription factors can encode both qualitative and quantitative information about the extracellular environment, which increases the information transfer capacity and potentially supports accurate cellular decision-making. An important question is how these signaling dynamics patterns are translated into functionally appropriate gene regulation programs. To address this question for transcription factors of the nuclear factor κB (NF-κB) family, we profiled the single-cell dynamics of two major NF-κB subunits, RelA and c-Rel, induced by a panel of pathogen-derived stimuli in immune and nonimmune cellular contexts. Diverse NF-κB-activating ligands produced different patterns of RelA and c-Rel signaling dynamic features, such as variations in duration or time-integrated activity. Analysis of nascent transcripts delineated putative direct targets of NF-κB as compared to genes controlled by other transcriptional and posttranscriptional mechanisms and showed that the transcription of more than half of the induced genes was tightly linked to specific dynamic features of NF-κB signaling. Fibroblast and macrophage cell lines shared a cluster of such "NF-κB dynamics-decoding" genes, as well as cell type-specific decoding genes. Dissecting the subunit specificity of dynamics-decoding genes suggested that target genes were most often linked to both RelA and c-Rel or to RelA alone. Thus, our analysis reveals the cell type-specific interpretation of pathogenic information through the signaling dynamics of NF-κB.

TUBB4A mutations result in both glial and neuronal degeneration in an H-ABC leukodystrophy mouse model.

Mutations in TUBB4A result in a spectrum of leukodystrophy including Hypomyelination with Atrophy of Basal Ganglia and Cerebellum (H-ABC), a rare hypomyelinating leukodystrophy, often associated with a recurring variant p.Asp249Asn (D249N). We have developed a novel knock-in mouse model harboring heterozygous (Tubb4aD249N/+) and the homozygous (Tubb4aD249N/D249N) mutation that recapitulate the progressive motor dysfunction with tremor, dystonia and ataxia seen in H-ABC. Tubb4aD249N/D249N mice have myelination deficits along with dramatic decrease in mature oligodendrocytes and their progenitor cells. Additionally, a significant loss occurs in the cerebellar granular neurons and striatal neurons in Tubb4aD249N/D249N mice. In vitro studies show decreased survival and dysfunction in microtubule dynamics in neurons from Tubb4aD249N/D249N mice. Thus Tubb4aD249N/D249N mice demonstrate the complex cellular physiology of H-ABC, likely due to independent effects on oligodendrocytes, striatal neurons, and cerebellar granule cells in the context of altered microtubule dynamics, with profound neurodevelopmental deficits.

Inside human and other animal cells, filaments known as microtubules help support the shape of the cell and move proteins to where they need to be. Defects in microtubules may lead to disease. For example, genetic mutations affecting a microtubule component called TUBB4A cause a rare brain disease in humans known as H-ABC. Individuals with H-ABC display many symptoms including abnormal walking, speech defects, impaired swallowing, and several cognitive defects. Abnormalities in several areas of the brain, including the cerebellum and striatum contribute to these defects. . In these structures, the neurons that carry messages around the brain and their supporting cells, known as oligodendrocytes, die, which causes these parts of the brain to gradually waste away. At this time, there are no therapies available to treat H-ABC. Furthermore, research into the disease has been hampered by the lack of a suitable "model" in mice or other laboratory animals. To address this issue, Sase, Almad et al. generated mice carrying a mutation in a gene which codes for the mouse equivalent of the human protein TUBB4A. Experiments showed that the mutant mice had similar physical symptoms to humans with H-ABC, including an abnormal walking gait, poor coordination and involuntary movements such as twitching and reduced reflexes. H-ABC mice had smaller cerebellums than normal mice, which was consistent with the wasting away of the cerebellum observed in individuals with H-ABC. The mice also lost neurons in the striatum and cerebellum, and oligodendrocytes in the brain and spinal cord. Furthermore, the mutant TUBB4A protein affected the behavior and formation of microtubules in H-ABC mice. The findings of Sase, Almad et al. provide the first mouse model that shares many features of H-ABC disease in humans. This model provides a useful tool to study the disease and develop potential new therapies.

Cadherin-Mediated Cell Coupling Coordinates Chemokine Sensing across Collectively Migrating Cells.

The directed migration of cells sculpts the embryo, contributes to homeostasis in the adult, and, when dysregulated, underlies many diseases [1, 2]. During these processes, cells move singly or as a collective. In both cases, they follow guidance cues, which direct them to their destination [3-6]. In contrast to single cells, collectively migrating cells need to coordinate with their neighbors to move together in the same direction. Recent studies suggest that leader cells in the front sense the guidance cue, relay the directional information to the follower cells in the back, and can pull the follower cells along [7-19]. In this manner, leader cells steer the collective and set the collective's overall speed. However, whether follower cells also participate in steering and speed setting of the collective is largely unclear. Using chimeras, we analyzed the role of leader and follower cells in the collectively migrating zebrafish posterior lateral line primordium. This tissue expresses the chemokine receptor Cxcr4 and is guided by the chemokine Cxcl12a [20-23]. We find that leader and follower cells need to sense the attractant Cxcl12a for efficient migration, are coupled to each other through cadherins, and require coupling to pull Cxcl12a-insensitive cells along. Analysis of cell dynamics in chimeric and protein-depleted primordia shows that Cxcl12a-sensing and cadherin-mediated adhesion contribute jointly to direct migration at both single-cell and tissue levels. These results suggest that all cells in the primordium need to sense the attractant and adhere to each other to coordinate their movements and migrate with robust directionality.

From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting.

Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from "closed" to "open" chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription-the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms.