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Principal Investigator: SHU CHIEN
Organization: UNIVERSITY OF SOUTHERN CALIFORNIA
Fiscal Year: 2024
Award: $583,656
Funding agency: National Heart Lung and Blood Institute
Endothelial cells (ECs) play a critical role in regulating vascular functions. We and others have demonstrated
that, through epigenetic and transcriptional regulations, laminar pulsatile shear stress (PS) induces athero-
protective genes to maintain EC homeostasis, whereas disturbed flow with oscillatory shear (OS) elevates
athero-prone genes to cause EC dysfunctions. We have performed single-cell RNA sequencing (scRNA-seq)
analyses to demonstrate that the transcriptomic effects of PS are distinct from those of OS. In addition, we have
shown that PS caused enrichments of histone active mark (H3K27ac) at genes related to EC homeostasis and
histone repressing mark (H3K9me3) at genes related to inflammation. We also demonstrated that the PS-
induced H3K9me3 is dependent on the nuclear envelop proteins lamin/emerin. These findings have led to our
hypothesis that PS and OS modulate EC functions through the coupling of lamin/emerin and chromatin to recruit
histone modifiers, thus leading to differential changes in histone epigenetics and the associated genomic and
transcriptomic regulations, and hence the opposite functional outcomes. The couplings between lamin/emerin
and chromatin/genome can transduce the mechanical signals from physical space into genome space for gene
and cell fate regulations. In order to test our hypothesis, we will conduct ChIP-seq to identify the lamin/emerin
associated genome regions (LEAGRs) under PS and OS, and determine the LEAGR-associated histone
modifications (i.e., epigenome). To visualize the differential flow-modulations of the dynamic interaction between
LEAGRs and lamin/emerin in single live cells, we will employ endonuclease-deficient Cas9 (dCas9) together
with small guide RNAs (sgRNAs) and engineered biosensors to track the dynamics of the histone profiles of
these genomic loci, particularly those related to EC homeostasis or inflammation. We will then determine the
roles of the locus-specific epigenetic profiles in regulating the transcriptome and cellular functions under different
flows. We will conduct studies in vivo on aorta arch (OS) and thoracic aorta (PS) in mice to validate our in vitro
results, and assess their impacts on atherogenesis by using atherosclerotic mouse models. Specifically, the MR
(magnetic resonance)-guided FUS (focused ultrasound) (MRg-FUS) system will be used to remotely and
noninvasively activate the inducible shRNA and CRISPRa/i (CRISPR activation or interference) systems to
manipulate lamin/emerin and locus-specific histone epigenetics at local tissue areas of mouse with partially
ligated carotid arteries to examine their functional roles in vivo. Accordingly, three specific aims are proposed: 1)
In vitro investigation of lamin/emerin and EC epigenome/transcriptome under different flows, 2) Imaging of locus-
specific epigenetic and chromatin remodeling in single live ECs, 3) In vivo examination and validation of the
epigenome/transcriptome regulation in mouse atherosclerosis models. With the integrated multi-omics, single-
cell imaging, and noninvasive locus-specific modulation, we will be able to identify and mitigate the key molecules
to develop mechanomedicine for vascular diseases.
Terms: <Area><Atherosclerosis><Atherosclerotic Cardiovascular Disease><Autoregulation><Biosensor><Blood Vessels><Blood flow><Body Tissues><CRISPR><CRISPR activation><CRISPR activator><CRISPR based activation><CRISPR gene activation><CRISPR interference><CRISPR transcription activation><CRISPR transcriptional activation><CRISPR-Cas-9-mediated gene activation><CRISPR-based gene activation><CRISPR-dCAS9 Activator><CRISPR-dCas9-mediated repression><CRISPR-mediated transcriptional activation><CRISPR/CAS9 activation><CRISPR/CAS9 gene activation><CRISPR/Cas system><CRISPR/dCas9 activation><CRISPR/dCas9 interference><CRISPR/dCas9-based transcriptional activation><CRISPR/dCas9-mediated transcriptional inhibition><CRISPRa><CRISPRi><Carotid Arteries><Cell Body><Cell Fate Control><Cell Fate Regulation><Cell Function><Cell Physiology><Cell Process><Cells><Cellular Function><Cellular Mechanotransduction><Cellular Physiology><Cellular Process><ChIP Sequencing><ChIP-seq><ChIPseq><Chemicals><Chromatin><Closure by Ligation><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats interference><Coupling><Dysfunction><Endothelial Cells><Engineering><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><FRET><Fluorescence Resonance Energy Transfer><Focused Ultrasound><Functional disorder><Förster Resonance Energy Transfer><Gene Targeting><Genes><Genetic><Genome><Genomic Segment><Genomics><Guide RNA><Histones><Homeostasis><Image><In Vitro><Individual><Inflammation><Investigation><Knowledge><Laboratories><Lamins><Ligation><Link><Location><Magnetic Resonance><Mechanical Signal Transduction><Mechanosensory Transduction><Mice><Mice Mammals><Modeling><Monitor><Murine><Mus><Nature><Nuclear><Nuclear Envelope><Nuclear Membrane><Pattern><Physiological Homeostasis><Physiopathology><Play><Prevention><Proteins><Regulation><Repression><Role><STA protein><Series><Site><Subcellular Process><System><Testing><Therapeutic><Thoracic aorta><Time><Tissues><Transcription Regulation><Transcriptional Control><Transcriptional Regulation><Type V IF Protein><Validation><Vascular Diseases><Vascular Disorder><Vascular Endothelial Cell><Viral><Visualization><activating CRISPR technology><aortic arch><atherogenesis><atheromatosis><atheroprotection><atheroprotective><atherosclerotic disease><atherosclerotic vascular disease><biological sensor><blood vessel disorder><cell imaging><cellular imaging><chromatin immunoprecipitation-sequencing><chromatin remodeling><emerin><endonuclease><epigenetic regulation><epigenetically><epigenome><experiment><experimental research><experimental study><experiments><functional outcomes><gRNA><gain of function><gene locus><genetic locus><genome segment><genomic location><genomic locus><genomic profiles><genomic region><global gene expression><global transcription profile><histone modification><imaging><in vivo><inhibitor><intravenous administration><loss of function><mechanosensing><mechanotransduction><mouse model><multiomics><multiple omics><murine model><novel><panomics><pathophysiology><recruit><repressing CRISPR-dCas9 system><scRNA-seq><shRNA><shear stress><short hairpin RNA><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><small hairpin RNA><social role><tool><transcriptome><transcriptomics><validations><vascular><vascular dysfunction><vasculopathy>