Biology subjects

Yen, A.

Publications and source records attributed to Yen, A..

8 recordsLinked to original sources

bioRxiv2025

Prior epigenetic status predicts future susceptibility to seizures in mice

Wide variation of responses to identical stimuli presented to genetically inbred mice suggests the hypothesis that stochastic epigenetic variation during neurodevelopment can mediate such phenotypic differences. However , this hypothesis is largely untested since capturing pre-existing molecular states requires non-destructive, longitudinal recording. Therefore, we tested the potential of Calling Cards (CC) to record transient neuronal enhancer activity during postnatal development, and thereby associate epigenetic variation with a subsequent phenotypic presentation - degree of seizure response to the pro-convulsant pentylenetetrazol. We show that recorded differences in epigenetics at 243 loci predict a severe vs. mild response, and that these are enriched near genes associated with human epilepsy. We also validated pharmacologically a seizure -modifying role for two novel genes, Htr1f and Let7c. This proof-of-principle supports using CC broadly to discover predisposition loci for other neuropsychiatric traits and behaviors. Finally, as, human disease is also influenced by non-inherited factors, similar epigenetic predispositions are possible in humans.

genomics↗

bioRxiv2024

FGR Src Family Kinase Causes Signaling and Phenotypic Shift Mimicking Retinoic Acid-Induced Differentiation of Leukemic Cells

Retinoic acid (RA) is an embryonic morphogen used in cancer differentiation-therapy. It causes a plethora of changes in gene expression culminating in cell differentiation. We now find that amongst them, expression of the Src-family-kinase, FGR, by itself causes cell differentiation analogous to RA. The historically dominant/classical paradigm for RA mechanism of action is transcriptional activation via binding to the ligand-activated nuclear receptors, RAR/RXR. In the HL-60 human myelo-monocytic leukemia model, an actively proliferating, phenotypically immature, lineage bi-potent NCI-60 cell line, RA causes election of the myeloid lineage and phenotypic maturation with G1/0 growth inhibition. It thereby converts transformed immature proliferating tumor cells to mature growth retarded cells that bear fidelity to non-transformed mature myeloid cells. The present study finds that expression of the FGR SFK(SRC-family-kinase) alone is sufficient to induce differentiation. Akin to RA, the phenotypic conversion manifests as expression of CD38, CD11b, and ROS, as well as the p27(kip1) CDKI (cyclin-dependent-kinase-inhibitor that retards cells in G1/0) characteristic of mature myeloid cells. To pursue mechanistic insight, signaling attributes known to promote RA-induced differentiation were analyzed to see what FGR affected. RA is known to cause expression of FGR which is incorporated into and activates a putative novel cytosolic macromolecular signaling machine(signalsome) that propels differentiation. RA enhances the abundance of signalsome constituents, their associations, and their phosphorylation. The signalsome contains connected nodes that appear as a spine to which the other components are connected. The apparent "nodes" are RAF, LYN, FGR, SLP-76 and CBL. All of these become enriched in the nucleus after RA-treatment. NUMB and VAV appear to provide further scaffolding functions enhanced by RA. RAF in the nucleus complexes with a RARE (retinoic acid-response-element) in the promoter of the blr1 gene, which encodes a serpentine G-protein-coupled-receptor. blr1 transcriptional activation by RA depends on RAF binding. BLR1 expression is necessary to propel RA-induced differentiation, although by itself is not sufficient to cause phenotypic differentiation. Analyzing this signaling process revealed that expression of FGR mimics RA-induced enhancement of the signalsome nodes, enhancing expression of RAF and its phosphorylation, and causing BLR1 expression. Interestingly, for cd38 and blr1, FGR apparently causes expression of genes targeted by RAR/RXR even without RA. FGR thus appears to cause signaling events and phenotypic shift characteristic of RA. In sum, the data indicate that FGR is the "trigger" for RA-induced differentiation. Given the historical perception of FGR as a pro-proliferation, transforming-viral-oncogene, this is a surprising paradigm shift.

molecular biology↗

bioRxiv2024

Norepinephrine Signals Through Astrocytes To Modulate Synapses

AbstractLocus coeruleus (LC)-derived norepinephrine (NE) drives network and behavioral adaptations to environmental saliencies by reconfiguring circuit connectivity, but the underlying synapse-level mechanisms are elusive. Here, we show that NE remodeling of synaptic function is independent from its binding on neuronal receptors. Instead, astrocytic adrenergic receptors and Ca2+ dynamics fully gate the effect of NE on synapses as the astrocyte-specific deletion of adrenergic receptors and three independent astrocyte-silencing approaches all render synapses insensitive to NE. Additionally, we find that NE suppression of synaptic strength results from an ATP-derived and adenosine A1 receptor-mediated control of presynaptic efficacy. An accompanying study from Chen et al. reveals the existence of an analogous pathway in the larval zebrafish and highlights its importance to behavioral state transitions. Together, these findings fuel a new model wherein astrocytes are a core component of neuromodulatory systems and the circuit effector through which norepinephrine produces network and behavioral adaptations, challenging an 80-year-old status quo.

neuroscience↗

bioRxiv2024

MYT1L deficiency impairs excitatory neuron trajectory during cortical development

Mutations that reduce the function of MYT1L, a neuron-specific transcription factor, are associated with a syndromic neurodevelopmental disorder. Furthermore, MYT1L is routinely used as a proneural factor in fibroblast-to-neuron transdifferentiation. MYT1L has been hypothesized to play a role in the trajectory of neuronal specification and subtype specific maturation, but this hypothesis has not been directly tested, nor is it clear which neuron types are most impacted by MYT1L loss. In this study, we profiled 313,335 nuclei from the forebrains of wild-type and MYT1L-deficient mice at two developmental stages: E14 at the peak of neurogenesis and P21, when neurogenesis is complete, to examine the role of MYT1L levels in the trajectory of neuronal development. We found that MYT1L deficiency significantly disrupted the relative proportion of cortical excitatory neurons at E14 and P21. Significant changes in gene expression were largely concentrated in excitatory neurons, suggesting that transcriptional effects of MYT1L deficiency are largely due to disruption of neuronal maturation programs. Most effects on gene expression were cell autonomous and persistent through development. In addition, while MYT1L can both activate and repress gene expression, the repressive effects were most sensitive to haploinsufficiency, and thus more likely mediate MYT1L syndrome. These findings illuminate the intricate role of MYT1L in orchestrating gene expression dynamics during neuronal development, providing insights into the molecular underpinnings of MYT1L syndrome.

neuroscience↗

bioRxiv2023

Calling Cards: a customizable platform to longitudinally record protein-DNA interactions over time in cells and tissues

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon (SRT) "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile specific transcription factor binding with custom transcription factor (TF)-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Card reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 1-2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. Basic Protocol 1: Preparation and delivery of Calling Cards reagents Basic Protocol 2: Sample preparation Basic Protocol 3: Sequencing library preparation Basic Protocol 4: Library pooling and sequencing Basic Protocol 5: Data analysis

genomics↗

bioRxiv2023

CDK7 is a Novel Therapeutic Vulnerability in Fibrolamellar Carcinoma

Fibrolamellar carcinoma (FLC) is a rare and lethal cancer that afflicts young individuals. The tumor arises in the background of a healthy liver, and patients typically present with advanced cancer at the time of diagnosis. Unfortunately, for these patients with advanced or recurrent cancer, no proven systemic therapies exist resulting in only 30-45% of patients surviving to 5 years. Investigations into the molecular underpinning of FLC have revealed a unique gene fusion between heat shock protein 40 (DNAJB1) and the catalytic subunit alpha of protein kinase A (PRKACA), leading to the formation of an oncoprotein (DNAJ-PKAc) that retains kinase activity and is a proven tumor-causing event in FLC. To uncover potential therapeutic targets, we engineered an FLC cell line by introducing the DNAJB1-PRKACA oncogene rearrangement into human hepatocellular cells using CRISPR/Cas9. We identified aberrant cell cycle progression, and follow-up molecular analysis revealed evidence of enhanced cyclin dependent kinase 7 (CDK7) activation in the DNAJB1-PRKACA expressing FLC cells. These findings were confirmed in human samples of FLC. In turn, targeting CDK7 with selective inhibitors demonstrated efficacy in several patient-derived models of FLC, with minimal toxicity to normal liver. Collectively, this work uncovers a novel candidate therapeutic vulnerability in FLC.

cancer biology↗

bioRxiv2021

Mecp2 deletion results in profound alterations of developmental and adult functional connectivity

As a regressive neurodevelopmental disorder with a well-established genetic cause, Rett Syndrome and its Mecp2 loss-of-function mouse model provide an excellent opportunity to define potentially translatable functional signatures of disease progression, as well as offer insight into Mecp2s role in functional circuit development. Thus, we applied optical fluorescence imaging to assess mesoscale calcium functional connectivity (FC) in the Mecp2 cortex prior to symptom onset as well as during decline. We found that FC was profoundly disrupted in Mecp2 males both in juvenile development and early adulthood. Female Mecp2 mice displayed a subtle homotopic contralateral increase in motor cortex as juveniles but not in adulthood, where instead parietal regions were implicated. Additionally, conditional rescue studies indicated FC phenotypes are driven by excitatory neurons. Altogether, the female results identify subtle candidate translatable biomarkers of disease progression, while the male results indicate MeCP2 protein is needed in a circuit-specific manner for FC.

neuroscience↗

bioRxiv2017

An Effective Model Of The Retinoic Acid Induced HL-60 Differentiation Program

In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPAR$[gamma] were critical to the ATRA-induced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.

systems biology↗