Biology subjects

Kugel, J.

Publications and source records attributed to Kugel, J..

6 recordsLinked to original sources

bioRxiv2022

Bayesian Accounts of Perceptual Decisions in the Nonclinical Continuum of Psychosis: Greater Imprecision in Both Top-down and Bottom-up Processes

Neurocomputational accounts of psychosis propose mechanisms for how information is integrated into a predictive model of the world, in attempts to understand the occurrence of altered perceptual experiences. Conflicting Bayesian theories postulate aberrations in either top-down or bottom-up processing. The top-down theory predicts an overreliance on prior beliefs or expectations resulting in aberrant perceptual experiences, whereas the bottom-up theory predicts an overreliance on current sensory information, as aberrant salience is directed towards objectively uninformative stimuli. This study empirically adjudicates between these models. We use a perceptual decision-making task in a neurotypical population with varying degrees of psychotic-like experiences. Bayesian modelling was used to compute individuals reliance on prior relative to sensory information. Across two datasets (discovery dataset n=363; independent replication in validation dataset n=782) we showed that psychotic-like experiences were associated with an overweighting of sensory information relative to prior expectations, which seem to be driven by decreased precision afforded to prior information. However, when prior information was more uncertain, participants with greater psychotic-like experiences encoded sensory information with greater noise. Greater psychotic-like experiences were also associated with generally heightened perceptions of task instability, which we suggest may be the cause for the observed aberrant precision in the encoding both prior and likelihood information. Our study lends empirical support to notions of both weaker bottom-up and weaker (rather than stronger) top-down perceptual processes, as well as aberrancies in belief updating that extend into the non-clinical continuum of psychosis.

neuroscience↗

bioRxiv2022

p53 binds more dynamically to response elements that are closer to the consensus sequence

The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Critical for understanding how p53 regulates gene expression is unraveling the fundamental mechanisms by which it binds to REs. Toward this goal we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of tetrameric p53 to five native REs in real time under equilibrium conditions. We found little evidence of dimer/DNA complexes as intermediates to the formation or dissociation of p53 tetramer/DNA complexes; however, tetramer/DNA complexes can exchange dimers at some REs. Determining rate constants for association and dissociation revealed two kinetically distinguishable populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing the more favorable binding sequences form the least kinetically stable complexes. Together our real time measurements provide insight into mechanisms with which tetrameric p53 forms complexes on different native REs.

biochemistry↗

bioRxiv2021

Epigenetic remodeling during monolayer cell expansion reduces therapeutic potential

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies require two-dimensional (2D) cell expansion processes to achieve large cell populations critical for the repair of damaged (e.g. connective and musculoskeletal) tissues. However, the influence of mechanical memory on cell fate following expansion is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we show that the organization of histone H3 trimethylated at lysine 9 (H3K9me3) and expression of tissue-identifying genes in primary cartilage cells (chondrocytes) transferred to three-dimensional (3D) hydrogels depends on the number of previous population doublings on tissue culture plastic during 2D cell expansion. Decreased levels of H3K9me3 occupying promoters of dedifferentiation genes after the 2D culture were also retained in 3D culture. Suppression of H3K9me3 during expansion of cells isolated from a murine model similarly resulted in the loss of the chondrocyte phenotype and global remodeling of nuclear architecture. In contrast, increasing levels of H3K9me3 through inhibiting H3K9 demethylases partially rescued the chondrogenic nuclear architecture and gene expression, which has important implications for tissue repair therapies, where expansion of large numbers of phenotypically-suitable cells is required. Overall, our findings indicate mechanical memory in primary cells is encoded in the chromatin architecture, which impacts cell fate and the phenotype of expanded cells. SIGNIFICANCE STATEMENTTissue regeneration procedures, such as cartilage defect repair (e.g. Matrix-induced Autologous Chondrocyte Implantation) often require cell expansion processes to achieve sufficient cells to transplant into an in vivo environment. However, the chondrocyte cell expansion on 2D stiff substrates induces epigenetic changes that persist even when the chondrocytes are transferred to a different (e.g. 3D) or in vivo environment. Treatments to alter epigenetic gene regulation may be a viable strategy to improve existing cartilage defect repair procedures and other tissue engineering procedures that involve cell expansion.

bioengineering↗

bioRxiv2021

The herpes simplex virus 1 protein ICP4 acts as both an activator and repressor of host genome transcription during infection

Infection by Herpes simplex virus 1 (HSV-1) impacts nearly all steps of gene expression in the host cell. The regulatory mechanisms by which this occurs, and the interplay between host and viral factors, have yet to be fully elucidated. Here we investigated how the occupancy of RNA polymerase II (Pol II) on the host genome changes during HSV-1 infection and is impacted by the viral immediate early protein ICP4. Pol II ChIP-seq experiments revealed a reduction of Pol II occupancy across the bodies of hundreds of host genes that was dependent upon ICP4. Concomitantly, Pol II levels increased across the bodies of several hundred genes, the majority of which also depended on ICP4 for activation. Our data suggest ICP4 regulates repression of Pol II at host genes by inhibiting recruitment of Pol II, while it regulates activation by promoting release of Pol II from promoter proximal pausing into productive elongation. Consistent with this, relative levels of the pausing factors NELF-A and Spt5 were reduced on an HSV-1 activated gene in an ICP4 dependent manner. Exogenous expression of ICP4 revealed that ICP4 can activate, but not repress, transcription of some genes in the absence of infection in a manner that correlates with the chromatin state of the gene. Together our data support the model that ICP4 decreases promoter proximal pausing on host genes activated by infection, and ICP4 is necessary, but not sufficient, to repress transcription from host genes during viral infection.

genomics↗

bioRxiv2019

Release of human TFIIB from actively transcribing complexes is triggered upon synthesis of 7 nt and 9 nt RNAs

RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important to maintain the transcriptional activity of Pol II as initiating complexes transition into elongation complexes.

biochemistry↗

bioRxiv2019

Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

The tumor suppressor protein p53 is at the nexus of cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds to DNA response elements to regulate transcription of its target genes, a process activated by cellular stress. p53 response elements consist of two decameric half-sites, and most data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding to DNA, unanswered questions remain, due in part to the need for more quantitative and structural studies with the full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding to DNA in real time. The data reveal a dynamic interaction with many p53 binding and dissociation events occurring on single DNA molecules over minutes. We found that p53 tetramers bound to response elements containing only a single half site. The kinetic stability of tetramer/DNA complexes depended on the number of half sites and the helical phasing between them, with the most stable complexes forming on DNA containing two adjacent half sites. The forward rate of binding was not strongly impacted when one half site was mutated. These studies provide real time kinetic measurements of full length p53 tetramers binding to single molecules of DNA, and reveal new insight into the mechanisms by which this nucleoprotein complex forms.

biochemistry↗