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1.
Development ; 145(9)2018 04 30.
Article in English | MEDLINE | ID: mdl-29650591

ABSTRACT

Precise control of the relative ratio of retinal neurons and glia generated during development is essential for visual function. We show that Lhx2, which encodes a LIM-homeodomain transcription factor essential for specification and differentiation of retinal Müller glia, also plays a crucial role in the development of retinal neurons. Overexpression of Lhx2 with its transcriptional co-activator Ldb1 triggers cell cycle exit and inhibits both Notch signaling and retinal gliogenesis. Lhx2/Ldb1 overexpression also induces the formation of wide-field amacrine cells (wfACs). In contrast, Rnf12, which encodes a negative regulator of LDB1, is necessary for the initiation of retinal gliogenesis. We also show that Lhx2-dependent neurogenesis and wfAC formation requires Ascl1 and Neurog2, and that Lhx2 is necessary for their expression, although overexpression of Lhx2/Ldb1 does not elevate expression of these proneural bHLH factors. Finally, we demonstrate that the relative level of the LHX2-LDB1 complex in the retina decreases in tandem with the onset of gliogenesis. These findings show that control of Lhx2 function by Ldb1 and Rnf12 underpins the coordinated differentiation of neurons and Müller glia in postnatal retina.


Subject(s)
DNA-Binding Proteins/metabolism , Ependymoglial Cells/metabolism , LIM Domain Proteins/metabolism , LIM-Homeodomain Proteins/metabolism , Neurogenesis/physiology , Retinal Neurons/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA-Binding Proteins/genetics , Ependymoglial Cells/cytology , LIM Domain Proteins/genetics , LIM-Homeodomain Proteins/genetics , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Retinal Neurons/cytology , Transcription Factors/genetics , Ubiquitin-Protein Ligases/genetics
2.
Nat Methods ; 15(5): 330-338, 2018 05.
Article in English | MEDLINE | ID: mdl-29638227

ABSTRACT

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.


Subject(s)
Antibodies, Monoclonal/immunology , Protein Array Analysis/methods , Transcription Factors/metabolism , Animals , Cloning, Molecular , Databases, Factual , Female , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Reproducibility of Results
3.
Proc Natl Acad Sci U S A ; 115(45): 11643-11648, 2018 11 06.
Article in English | MEDLINE | ID: mdl-30348778

ABSTRACT

The circadian clock orchestrates 24-h rhythms in physiology in most living organisms. At the molecular level, the dogma is that circadian oscillations are based on a negative transcriptional feedback loop. Recent studies found the NAD+-dependent histone deacetylase, SIRT1, directly regulates acetylation status of clock components and influences circadian amplitude in cells. While Nakahata et al. [Nakahata Y, Kaluzova M (2008) Cell 134:329-340] reported that loss of SIRT1 increases amplitude through BMAL1 acetylation, Asher et al. [Asher G, Gatfield D (2008) Cell 134:317-328] reported that loss of SIRT1 decreases amplitude through an increase in acetylated PER2. To address this SIRT1 paradox, we developed a circadian enzymatic model. Predictions from this model and experimental validation strongly align with the findings of Asher et al., with PER2 as the primary target of SIRT1. Further, the model suggested SIRT1 influences BMAL1 expression through actions on PGC1α. We validated this finding experimentally. Thus, our computational and experimental approaches suggest SIRT1 positively regulates clock function through actions on PER2 and PGC1α.


Subject(s)
Circadian Clocks/genetics , Feedback, Physiological , Models, Biological , Period Circadian Proteins/genetics , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Sirtuin 1/genetics , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Animals , Cell Line , Computer Simulation , Cytokines/genetics , Cytokines/metabolism , Gene Expression Regulation , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/metabolism , Mice , NAD/metabolism , Nicotinamide Phosphoribosyltransferase/genetics , Nicotinamide Phosphoribosyltransferase/metabolism , Nuclear Receptor Subfamily 1, Group D, Member 1/genetics , Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Period Circadian Proteins/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Signal Transduction , Sirtuin 1/metabolism
4.
PLoS Biol ; 12(4): e1001840, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24737000

ABSTRACT

Over the last decades, researchers have characterized a set of "clock genes" that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.


Subject(s)
ARNTL Transcription Factors/metabolism , Circadian Clocks/physiology , Circadian Rhythm Signaling Peptides and Proteins/metabolism , Histone Deacetylases/metabolism , Repressor Proteins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Artificial Intelligence , Cell Line , Circadian Clocks/genetics , Circadian Rhythm/genetics , Circadian Rhythm/physiology , Circadian Rhythm Signaling Peptides and Proteins/biosynthesis , Circadian Rhythm Signaling Peptides and Proteins/genetics , Cryptochromes/genetics , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Nuclear Receptor Subfamily 1, Group D, Member 1/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Glucocorticoid/metabolism , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Sequence Alignment , Transcription, Genetic/genetics
7.
Photochem Photobiol Sci ; 13(3): 531-40, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24407555

ABSTRACT

Grp1-associated scaffold protein (Grasp), the product of a retinoic acid-induced gene in P19 embryonal carcinoma cells, is expressed primarily in brain, heart, and lung of the mouse. We report herein that Grasp transcripts are also found in mouse skin in which the Grasp gene is robustly induced following acute ultraviolet-B (UVB) exposure. Grasp(-/-) mice were found to exhibit delayed epidermal proliferation and a blunted apoptotic response after acute UVB exposure. Immunohistochemical analyses revealed that the nuclear residence time of the tumor suppressor protein p53 was reduced in Grasp(-/-) mice after UVB exposure. Taken together, our results suggest that a physiological role of Grasp may be to regulate skin homeostasis after UVB exposure, potentially by influencing p53-mediated apoptotic responses in skin.


Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Skin Physiological Phenomena/radiation effects , Skin/radiation effects , Animals , Apoptosis/physiology , Apoptosis/radiation effects , Carrier Proteins/genetics , Cell Nucleus/physiology , Cell Nucleus/radiation effects , Cell Proliferation/radiation effects , Cells, Cultured , Dermis/physiology , Dermis/radiation effects , Epidermis/pathology , Epidermis/physiology , Epidermis/radiation effects , Fibroblasts/physiology , Fibroblasts/radiation effects , Homeostasis/physiology , Homeostasis/radiation effects , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Tumor Suppressor Protein p53/metabolism , Ultraviolet Rays
8.
J Cardiovasc Imaging ; 31(3): 125-132, 2023 Jul.
Article in English | MEDLINE | ID: mdl-37488916

ABSTRACT

BACKGROUND: There is limited data on the residual echocardiographic findings including strain analysis among post-coronavirus disease (COVID) patients. The aim of our study is to prospectively phenotype post-COVID patients. METHODS: All patients discharged following acute COVID infection were systematically followed in the post-COVID-19 Recovery Clinic at Vancouver General Hospital and St. Paul's Hospital. At 4-18 weeks post diagnosis, patients underwent comprehensive echocardiographic assessment. Left ventricular ejection fraction (LVEF) was assessed by 3D, 2D Biplane Simpson's, or visual estimate. LV global longitudinal strain (GLS) was measured using a vendor-independent 2D speckle-tracking software (TomTec). RESULTS: A total of 127 patients (53% female, mean age 58 years) were included in our analyses. At baseline, cardiac conditions were present in 58% of the patients (15% coronary artery disease, 4% heart failure, 44% hypertension, 10% atrial fibrillation) while the remainder were free of cardiac conditions. COVID-19 serious complications were present in 79% of the patients (76% pneumonia, 37% intensive care unit admission, 21% intubation, 1% myocarditis). Normal LVEF was seen in 96% of the cohort and 97% had normal right ventricular systolic function. A high proportion (53%) had abnormal LV GLS defined as < 18%. Average LV GLS of septal and inferior segments were lower compared to that of other segments. Among patients without pre-existing cardiac conditions, LVEF was abnormal in only 1.9%, but LV GLS was abnormal in 46% of the patients. CONCLUSIONS: Most post-COVID patients had normal LVEF at 4-18 weeks post diagnosis, but over half had abnormal LV GLS.

9.
Cell Biol Int ; 36(12): 1115-28, 2012.
Article in English | MEDLINE | ID: mdl-22931251

ABSTRACT

GRASP interacts with Grp1 (general receptor for phosphoinositides 1; cytohesin 3), which catalyses nucleotide exchange on and activation of Arf6 (ADP-ribosylation factor-6). Arf6 is a low-molecular-mass GTPase that regulates key aspects of endocytic recycling pathways. Overexpressed GRASP accumulated in the juxtanuclear ERC (endocytic recycling compartment). GRASP co-localized with a constitutively inactive mutant of Arf6 in the ERC such that it was reversed by expression of wild-type Grp1. Co-expression of GRASP and Grp1 promoted membrane ruffling, a cellular hallmark of Arf6 activation. GRASP accumulation in ERC was found to block recycling of the MHC-I (major histocompatibility complex-I), which is trafficked by the Arf6-dependent pathway. In contrast, overexpression of GRASP had no effect on the recycling of transferrin receptors, which are trafficked by a clathrin-dependent pathway. The findings suggest that GRASP regulates the non-clathrin/Arf6-dependent, plasma membrane recycling and signalling pathways.


Subject(s)
ADP-Ribosylation Factors/metabolism , Carrier Proteins/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/analysis , ADP-Ribosylation Factors/genetics , Carrier Proteins/analysis , Carrier Proteins/genetics , Endosomes/metabolism , Gene Expression , HeLa Cells , Humans , Membrane Proteins/analysis , Membrane Proteins/genetics , Point Mutation , Protein Transport , Receptors, Cytoplasmic and Nuclear/analysis , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Up-Regulation , rab GTP-Binding Proteins/analysis , rab GTP-Binding Proteins/metabolism
10.
Biochem Biophys Res Commun ; 398(1): 7-12, 2010 Jul 16.
Article in English | MEDLINE | ID: mdl-20510202

ABSTRACT

Multiple drug resistance protein 1 (MDR1) is composed of two homologous halves separated by an intracellular linker region. The linker has been reported to bind myosin regulatory light chain (RLC), but it is not clear how this can occur in the context of a myosin II complex. We characterized MDR1-RLC interactions and determined that binding occurs via the amino terminal of the RLC, a domain that typically binds myosin heavy chain. MDR1-RLC interactions were sensitive to the phosphorylation state of the light chain in that phosphorylation by myosin light chain kinase (MLCK) resulted in a loss of binding in vitro. We used ML-7, a specific inhibitor of MLCK, to study the functional consequences of disrupting RLC phosphorylation in intact cells. Pretreatment of polarized Madin-Darby canine kidney cells stably expressing MDR1 with ML-7 produced a significant increase in apical to basal permeability and a corresponding decrease in the efflux ratio (threefold; p<0.01) of [(3)H]-digoxin, a classic MDR1 substrate. Together these data show that MDR1-mediated transport of [(3)H]-digoxin can be modulated by pharmacological manipulation of myosin RLC, but direct MDR1-RLC interactions are atypical and not explained by the structure of the myosin II holoenzyme.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Myosin Light Chains/metabolism , ATP Binding Cassette Transporter, Subfamily B , Animals , Azepines/pharmacology , Cell Line , Digoxin/metabolism , Dogs , Holoenzymes/metabolism , Humans , Myosin Type II/metabolism , Myosin-Light-Chain Kinase/antagonists & inhibitors , Naphthalenes/pharmacology , Phosphorylation
11.
Nat Commun ; 11(1): 137, 2020 01 09.
Article in English | MEDLINE | ID: mdl-31919425

ABSTRACT

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here, we present ASCOT, a resource that uses annotation-free methods to rapidly analyze and visualize splice variants across tens of thousands of bulk and single-cell data sets in the public archive. To demonstrate the utility of ASCOT, we identify novel cell type-specific alternative exons across the nervous system and leverage ENCODE and GTEx data sets to study the unique splicing of photoreceptors. We find that PTBP1 knockdown and MSI1 and PCBP2 overexpression are sufficient to activate many photoreceptor-specific exons in HepG2 liver cancer cells. This work demonstrates how large-scale analysis of public RNA-Seq data sets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events.


Subject(s)
Alternative Splicing/genetics , Computational Biology/methods , Data Analysis , Photoreceptor Cells/cytology , RNA Splice Sites/genetics , Animals , Cell Line, Tumor , Gene Expression/genetics , Hep G2 Cells , Heterogeneous-Nuclear Ribonucleoproteins/genetics , High-Throughput Nucleotide Sequencing , Humans , Liver Neoplasms/genetics , Mice , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Neurons/cytology , Polypyrimidine Tract-Binding Protein/genetics , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics , Retina/cytology , Sequence Analysis, RNA/methods
12.
Cell Metab ; 25(4): 961-974.e4, 2017 Apr 04.
Article in English | MEDLINE | ID: mdl-28380384

ABSTRACT

The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, ∼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism.


Subject(s)
Circadian Clocks/genetics , Metabolome/genetics , Transcription, Genetic , Animals , Cell Line, Tumor , Cells, Cultured , Circadian Rhythm/genetics , Creatine/metabolism , Cryptochromes/metabolism , Gene Regulatory Networks , Hepatocytes/metabolism , Humans , Liver/metabolism , Mice , Nitrogen/metabolism , Time Factors
13.
Metabolites ; 6(3)2016 Jul 27.
Article in English | MEDLINE | ID: mdl-27472375

ABSTRACT

Oscillations in circadian metabolism are crucial to the well being of organism. Our understanding of metabolic rhythms has been greatly enhanced by recent advances in high-throughput systems biology experimental techniques and data analysis. In an in vitro setting, metabolite rhythms can be measured by time-dependent sampling over an experimental period spanning one or more days at sufficent resolution to elucidate rhythms. We hypothesized that cellular metabolic effects over such a time course would be influenced by both oscillatory and circadian-independent cell metabolic effects. Here we use nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling of mammalian cell culture media of synchronized U2 OS cells containing an intact transcriptional clock. The experiment was conducted over 48 h, typical for circadian biology studies, and samples collected at 2 h resolution to unravel such non-oscillatory effects. Our data suggest specific metabolic activities exist that change continuously over time in this settting and we demonstrate that the non-oscillatory effects are generally monotonic and possible to model with multivariate regression. Deconvolution of such non-circadian persistent changes are of paramount importance to consider while studying circadian metabolic oscillations.

14.
Cell Metab ; 22(6): 1009-19, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26387865

ABSTRACT

The MYC oncogene encodes MYC, a transcription factor that binds the genome through sites termed E-boxes (5'-CACGTG-3'), which are identical to the binding sites of the heterodimeric CLOCK-BMAL1 master circadian transcription factor. Hence, we hypothesized that ectopic MYC expression perturbs the clock by deregulating E-box-driven components of the circadian network in cancer cells. We report here that deregulated expression of MYC or N-MYC disrupts the molecular clock in vitro by directly inducing REV-ERBα to dampen expression and oscillation of BMAL1, and this could be rescued by knockdown of REV-ERB. REV-ERBα expression predicts poor clinical outcome for N-MYC-driven human neuroblastomas that have diminished BMAL1 expression, and re-expression of ectopic BMAL1 in neuroblastoma cell lines suppresses their clonogenicity. Further, ectopic MYC profoundly alters oscillation of glucose metabolism and perturbs glutaminolysis. Our results demonstrate an unsuspected link between oncogenic transformation and circadian and metabolic dysrhythmia, which we surmise to be advantageous for cancer.


Subject(s)
ARNTL Transcription Factors/metabolism , CLOCK Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , ARNTL Transcription Factors/chemistry , ARNTL Transcription Factors/genetics , Base Sequence , Binding Sites , CLOCK Proteins/chemistry , CLOCK Proteins/genetics , Cell Line, Tumor , Circadian Rhythm , Dimerization , Genes, Reporter , Glucose/metabolism , Glutamine/metabolism , Humans , Nuclear Receptor Subfamily 1, Group D, Member 1/antagonists & inhibitors , Nuclear Receptor Subfamily 1, Group D, Member 1/genetics , Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Repressor Proteins/metabolism
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