Biology subjects

Martin, R. D.

Publications and source records attributed to Martin, R. D..

9 recordsLinked to original sources

bioRxiv2022

Spt5 C-terminal repeat domain phosphorylation and length negatively regulate heterochromatin through distinct mechanisms

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomcyes pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that Spt5 CTD repeat length and phosphorylation have distinct regulatory effects on transcription.

genetics↗

bioRxiv2022

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. Post-mortem tissue or biopsies from diseased patients, which remain scarcely available, rendered it possible to study end-stage heart disease yet the inclusion of healthy human cardiac materials for basic science research was beyond reach. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human inducible stem cell derived cardiomyocytes presented a renewed opportunity. We think it necessary to conduct a comparative study, assessing cellular signalling through cardiac G protein-coupled receptors and bulk transcriptomics of traditional rat neonatal cardiomyocytes and human iPSC-CMs. Genetically-encoded biosensors were used to interrogate nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) in rat and human-derived cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted for an in-depth examination of existing differences between both in vitro cardiomyocyte models. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human CMs. In line with this, bulk RNA-seq demonstrated key differences regarding the expression patterns of GPCRs, G proteins and effectors. Overall, our study demonstrates that human stem cell derived models of the cardiomyocyte do provide significant advantages and should be taken advantage of.

biochemistry↗

bioRxiv2021

Dopamine D1 receptor activation and cAMP/PKA signalling mediate Brd4 recruitment to chromatin to regulate gene expression in rat striatal neurons

The activity of striatal medium-spiny projection neurons is regulated by dopamine acting principally at D1 and D2 dopamine receptors. The dopamine D1 receptor (D1R) is a Gs/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Transcriptional regulation downstream of the D1R involves the activation of PKA, which can translocate to the nucleus to phosphorylate various targets. The chromatin reader Brd4 regulates transcription induced by neurotrophic factors in cortical neurons and has also been implicated in dopamine-dependent striatal functions. Brd4 is activated by phosphorylation; this facilitates its binding to acetylated histones at promoters and enhancers. In non-neuronal cells, Brd4 is recruited to chromatin in response to PKA signalling. However, it is unknown whether Brd4 is involved in transcriptional activation by the D1R in striatal neurons. Here, we demonstrate that cAMP/PKA signalling increases Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown or inhibition of Brd4 modulated D1R-induced gene expression. Specifically, inhibition of Brd4 with the bromodomain inhibitor JQ1 suppressed the expression of [~]25% of D1R-upregulated genes, while increasing the expression of a subset of immediate-early genes, including Fos and Jun. This pro-transcriptional effect of JQ1 was P-TEFb-dependent, and mediated through inhibition of the BD1 bromodomain of Brd4. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify Brd4 as a novel regulator of D1R-dependent transcription and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.

neuroscience↗

bioRxiv2021

Benchmark of data processing methods and machine learning models for gut microbiome-based diagnosis of inflammatory bowel disease

BackgroundInflammatory bowel disease (IBD) patients wait months and undergo numerous invasive procedures between the initial appearance of symptoms and receiving a diagnosis. In order to reduce time until diagnosis and improve patient wellbeing, machine learning algorithms capable of diagnosing IBD from the gut microbiomes composition are currently being explored. To date, these models have had limited clinical application due to decreased performance when applied to a new cohort of patient samples. Various methods have been developed to analyze microbiome data which may improve the generalizability of machine learning IBD diagnostic tests. With an abundance of methods, there is a need to benchmark the performance and generalizability of various machine learning pipelines (from data processing to training a machine learning model) for microbiome-based IBD diagnostic tools. ResultsWe collected fifteen 16S rRNA microbiome datasets (7707 samples) from North America to benchmark combinations of gut microbiome features, data normalization methods, batch effect reduction methods, and machine learning models. Pipeline generalizability to new cohorts of patients was evaluated with four binary classification metrics following leave-one dataset-out cross validation, where all samples from one study were left out of the training set and tested upon. We demonstrate that taxonomic features obtained from QIIME2 lead to better classification of samples from IBD patients than inferred functional features obtained from PICRUSt2. In addition, machine learning models that identify non-linear decision boundaries between labels are more generalizable than those that are linearly constrained. Prior to training a non-linear machine learning model on taxonomic features, it is important to apply a compositional normalization method and remove batch effects with the naive zero-centering method. Lastly, we illustrate the importance of generating a curated training dataset to ensure similar performance across patient demographics. ConclusionsThese findings will help improve the generalizability of machine learning models as we move towards non-invasive diagnostic and disease management tools for patients with IBD.

microbiology↗

bioRxiv2021

Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons

Genetically-encoded biosensors are used to track biochemical activities in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. In the present article, we describe the application of genetically-encoded FRET biosensors with high content microscopy to image the signaling responses of thousands of neurons in response to drug treatments. We applied this approach to reveal intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. The striatum is largely composed of medium-spiny GABAergic neurons which are divided into two broad sub-types based in part on their expression of dopamine D1 vs. D2 receptors. Using high content FRET imaging and immunofluorescence, we identified neuronal sub-populations with unique responses to pharmacological manipulation. Focusing on dopamine- and glutamate-regulated PKA and ERK1/2 signaling in both the cytoplasm and nucleus, we identified pronounced intercellular differences, in both the magnitude and kinetics of signaling responses to drug application. Importantly, we found that a conventional "bulk" analysis that included all cells in culture yielded a different rank order of drug potency than that revealed by our single-cell analysis. The high degree of heterogeneity that we observed at the single cell level would not have been detectable using common population-level analyses, derived for example from western blotting or plate reader-based measurements. In conclusion, our single-cell analytical approach highlights the limitations of population-level analyses, and provides a novel way to study signaling biology.

neuroscience↗

bioRxiv2020

Membrane-tethered peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as allosteric biased ligands

Over the last decade, the urotensinergic system has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases and also cancer. Significant investment toward the development of clinically relevant UT ligands for therapeutic intervention has been made but have met little to no success to date. The UT system, which has yet to be effectively targeted, therefore remains to be therapeutically exploited. The discovery of allosteric sites that allow modulation of receptor activity will increase the searchable chemical space against a disease-relevant target. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics. Therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study UT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of UT, respectively, have been synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and to a lesser extent, IP1 production, stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate hUII- and URP-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue to design allosteric ligands selectively targeting UT signaling that could prove to be useful for the treatment of hUT-associated diseases.

pharmacology and toxicology↗

bioRxiv2020

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

Like many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell-type specific, and can change in response to disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinsons disease (PD), alterations in this pathway lead to activation of extracellular regulated kinases (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Forster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

neuroscience↗

bioRxiv2020

Differential activation of P-TEFb complexes in the development of cardiomyocyte hypertrophy following activation of distinct GPCRs

Pathological cardiac hypertrophy is driven by neurohormonal activation of specific G protein-coupled receptors (GPCRs) in cardiomyocytes and is accompanied by large-scale changes in cardiomyocyte gene expression. These transcriptional changes require activity of positive transcription elongation factor b (P-TEFb), which is recruited to target genes by the bromodomain protein Brd4 or the Super Elongation Complex (SEC). Here we describe GPCR-specific regulation of these P-TEFb complexes and a novel mechanism for activating Brd4 in primary neonatal rat cardiomyocytes. The SEC was required for the hypertrophic response downstream of either the 1-adrenergic receptor (1-AR) or the endothelin receptor (ETR). In contrast, Brd4 inhibition selectively impaired the 1-AR response. This was corroborated by the finding that activation of 1-AR, but not ETR, increased Brd4 occupancy at promoters and super enhancers of hypertrophic genes. Transcriptome analysis demonstrated that activation of both receptors initiated similar gene expression programs, but that Brd4 inhibition attenuated hypertrophic genes more robustly following 1-AR activation. Finally, we show that protein kinase A (PKA) is required for 1-AR stimulation of Brd4 chromatin occupancy. The differential role of the Brd4/P-TEFb complex in response to distinct GPCR pathways has potential clinical implications as therapies targeting this complex are currently being explored for heart failure.

molecular biology↗

bioRxiv2018

A novel interaction between Gβγ and RNA polymerase II regulates cardiac fibrosis.

G{beta}{gamma} subunits are involved in many different signalling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear G{beta}{gamma}, we investigated the functional role of G{beta}{gamma} signalling in regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. Following activation of the angiotensin II type I receptor in these cells, G{beta}{gamma} dimers interact with RNA polymerase II (RNAPII). Our findings suggest that G{beta}1 recruitment to RNAPII negatively regulates the fibrotic transcriptional response, which can be overcome by strong fibrotic stimuli. The interaction between G{beta}{gamma} subunits and RNAPII expands the role for G{beta}{gamma} signalling in cardiac fibrosis. The G{beta}{gamma}-RNAPII interaction was regulated by signaling pathways in HEK 293 cells that diverged from those operating in cardiac fibroblasts. Thus, the interaction may be a conserved feature of transcriptional regulation although such regulation may be cell-specific.

biochemistry↗