Essential Regulatory Elements Research Articles

Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs.

The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.

Single Nuclei Multiomics Analysis of Transcriptional and Chromatin Accessibility of Tumor Cells Uncovers Molecular Signatures and Regulatory Elements of Malignant Clonal Evolution in Multiple Myeloma

Category Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational Introduction Multiple myeloma (MM) is a hematological cancer resulting from abnormal proliferation of plasma cells (PC) in bone marrow. The progression of the disease from Monoclonal Gammopathy of Undetermined Significance (MGUS), Smoldering Multiple Myeloma (SMM), to MM involves multiple drivers and dynamic interactions of premalignant/malignant cells. In the advancement of single-cell technologies, the same-cell readouts of nucleus RNA sequencing (snRNA-seq) and Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) provide a less biased measurement of gene expressions and chromatin accessible regions. Profiling and analyzing both omics data at different stages of MM may reveal essential molecular signatures and regulatory elements for microenvironmental modification and evolution. These insights could benefit MM patients as novel targets for early therapeutic intervention at different stages of progression. Methods Bone marrow aspirates were collected from 33 individuals: Healthy donors (n=3), patients with MGUS (n=2), SMM (n=5), MM (n=21), and Plasma Cell Leukemia (PCL; n=2). Viable mononuclear cells underwent CD138+ sorting and nuclei isolation prior to library preparation. Libraries were prepared with the Chromium Multiome kit (10X Genomics) and sequenced on NovaSeq S4 flow cells at UAMS. The clonal V(D)J arrangements of each sample and expression level from HD samples were used to distinguish malignant (MM) from normal plasma cells (PC). The MM data from snATAC-seq and snRNA-seq were integrated using Signac with the weighted nearest neighbor methods from Seurat. All inter- and intra-tumor clusters were evaluated for markers of high-risk disease, differential gene expression, differentially accessible regions, and transcription factor activities. Results We performed snRNA-seq and snATAC-seq sequencing to generate multi-stage, molecular classified-specific chromatin accessibility and transcriptional profiles of plasma cell neoplasms comprising 439,134 cells across 33 samples. The malignant plasma cells for each sample were extracted by the unique clonotypic signature of the immunoglobulin gene rearrangement. Of these, we detected the highest clonality in MGUS samples, suggesting clonal competition at the premalignant stage. Based on our primary microarray data, we detected the overexpression of molecular signatures for most cells in each sample, confirming the intratumor cell heterogeneity. Furthermore, we used the ratio of 70-gene signature of aggressive disease and proliferation signatures to investigate the clonal composition of MM cells corresponding to the risk of relapse. Surprisingly, the proportion of high-risk cells increased respectively from low to high-risk samples. The expansion of high-risk clonality might exhibit competition among malignant cell subpopulations regarding microenvironmental adaptation. The multiomics integration generated less distinct clusters between MGUS and SMM, indicating the gradual complex alterations of subclonal gene expression and chromatin accessibility in malignant transformation. Interestingly, most accessible regions were found in intronic/distal intergenic regions implying the long-range gene regulation from cis-regulatory elements. Based on the positive/negative correlation of binding motifs within differentially accessible regions, we identified unique enriched transcription factors for each disease stage, suggesting the mechanism patterns in the evolutionary process of malignant cells. Conclusion In the largest multiomics study to date of CD138+ cells, we demonstrate the malignant clonal evolution using gene expression and chromatin accessibility profiling from patients with early-stage disease to highly aggressive form. Throughout the time of disease progression, we found an increase in the proportion of malignant cells with high-risk signatures. Also, we uncover unique signatures of chromatin accessibility for each molecular classification that can further refine our understanding of functional heterogeneity in multiple myeloma. Collectively, our joint single nuclei profiling reveals additional insights into the malignant clonal evolution and investigates novel regulatory elements involved in disease progression.

MRT-ModSeq – Rapid Detection of RNA Modifications with MarathonRT

Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we introduce MRT-ModSeq for rapid, simultaneous detection of multiple RNA modifications using MarathonRT. MRT-ModSeq employs distinct divalent cofactors to generate 2-D mutational profiles that are highly dependent on nucleotide identity and modification type. As a proof of concept, we use the MRT fingerprints of well-studied rRNAs to implement a general workflow for detecting RNA modifications. MRT-ModSeq rapidly detects positions of diverse modifications across a RNA transcript, enabling assignment of m1acp3Y, m1A, m3U, m7G and 2′-OMe locations through mutation-rate filtering and machine learning. m1A sites in sparsely modified targets, such as MALAT1 and PRUNE1 could also be detected. MRT-ModSeq can be trained on natural and synthetic transcripts to expedite detection of diverse RNA modification subtypes across targets of interest.

DeepCIP: A multimodal deep learning method for the prediction of internal ribosome entry sites of circRNAs

Circular RNAs (circRNAs) have been found to have the ability to encode proteins through internal ribosome entry sites (IRESs), which are essential RNA regulatory elements for cap-independent translation. Identification of IRES elements in circRNA is crucial for understanding its function. Previous studies have presented IRES predictors based on machine learning techniques, but they were mainly designed for linear RNA IRES. In this study, we proposed DeepCIP (Deep learning method for CircRNA IRES Prediction), a multimodal deep learning approach that employs both sequence and structural information for circRNA IRES prediction. Our results demonstrate the effectiveness of the sequence and structure models used by DeepCIP in feature extraction and suggest that integrating sequence and structural information efficiently improves the accuracy of prediction. The comparison studies indicate that DeepCIP outperforms other comparative methods on the test set and real circRNA IRES dataset. Furthermore, through the integration of an interpretable analysis mechanism, we elucidate the sequence patterns learned by our model, which align with the previous discovery of motifs that facilitate circRNA translation. Thus, DeepCIP has the potential to enhance the study of the coding potential of circRNAs and contribute to the design of circRNA-based drugs. DeepCIP as a standalone program is freely available at https://github.org/zjupgx/DeepCIP.

Enhancer in cancer pathogenesis and treatment.

Enhancers are essential cis-acting regulatory elements that determine cell identity and tumor progression. Enhancer function is dependent on the physical interaction between the enhancer and its target promoter inside its local chromatin environment. Enhancer reprogramming is an important mechanism in cancer pathogenesis and can be driven by both cis and trans factors. Super enhancers are acquired at oncogenes in numerous cancer types and represent potential targets for cancer treatment. BET and CDK inhibitors act through mechanisms of enhancer function and have shown promising results in therapy for various types of cancer. Genome editing is another way to reprogram enhancers in cancer treatment. The relationship between enhancers and cancer has been revised by several authors in the past few years, which mainly focuses on the mechanisms by which enhancers can impact cancer. Here, we emphasize SE's role in cancer pathogenesis and the new therapies involving epigenetic regulators (BETi and CDKi). We suggest that understanding mechanisms of activity would aid clinical success for these anti-cancer agents.

Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes.

Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in biology. Despite the development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches to capture interactions driven by specific post-translational modifications (PTMs). Myristoylation is a lipid PTM added to more than 200 human proteins, where it may regulate membrane localization, stability or activity. Here we report the design and synthesis of a panel of novel photocrosslinkable and clickable myristic acid analog probes, and their characterization as efficient substrates for human N-myristoyltransferases NMT1 and NMT2, both biochemically and through X-ray crystallography. We demonstrate metabolic incorporation of probes to label NMT substrates in cell culture and in situ intracellular photoactivation to form a covalent crosslink between modified proteins and their interactors, capturing a snapshot of interactions in the presence of the lipid PTM. Proteomic analyses revealed both known and multiple novel interactors of a series of myristoylated proteins, including ferroptosis suppressor protein 1 (FSP1) and spliceosome-associated RNA helicase DDX46. The concept exemplified by these probes offers an efficient approach for exploring the PTM-specific interactome without the requirement for genetic modification, which may prove broadly applicable to other PTMs.

IPromoter-Seqvec: identifying promoters using bidirectional long short-term memory and sequence-embedded features

BackgroundPromoters, non-coding DNA sequences located at upstream regions of the transcription start site of genes/gene clusters, are essential regulatory elements for the initiation and regulation of transcriptional processes. Furthermore, identifying promoters in DNA sequences and genomes significantly contributes to discovering entire structures of genes of interest. Therefore, exploration of promoter regions is one of the most imperative topics in molecular genetics and biology. Besides experimental techniques, computational methods have been developed to predict promoters. In this study, we propose iPromoter-Seqvec – an efficient computational model to predict TATA and non-TATA promoters in human and mouse genomes using bidirectional long short-term memory neural networks in combination with sequence-embedded features extracted from input sequences. The promoter and non-promoter sequences were retrieved from the Eukaryotic Promoter database and then were refined to create four benchmark datasets.ResultsThe area under the receiver operating characteristic curve (AUCROC) and the area under the precision-recall curve (AUCPR) were used as two key metrics to evaluate model performance. Results on independent test sets showed that iPromoter-Seqvec outperformed other state-of-the-art methods with AUCROC values ranging from 0.85 to 0.99 and AUCPR values ranging from 0.86 to 0.99. Models predicting TATA promoters in both species had slightly higher predictive power compared to those predicting non-TATA promoters. With a novel idea of constructing artificial non-promoter sequences based on promoter sequences, our models were able to learn highly specific characteristics discriminating promoters from non-promoters to improve predictive efficiency.ConclusionsiPromoter-Seqvec is a stable and robust model for predicting both TATA and non-TATA promoters in human and mouse genomes. Our proposed method was also deployed as an online web server with a user-friendly interface to support research communities. Links to our source codes and web server are available at https://github.com/mldlproject/2022-iPromoter-Seqvec.

Lineage-specific insertions in T-box riboswitches modulate antibiotic binding and action.

T-box riboswitches (T-boxes) are essential RNA regulatory elements with a remarkable structural diversity, especially among bacterial pathogens. In staphylococci, all glyS T-boxes synchronize glycine supply during synthesis of nascent polypeptides and cell wall formation and are characterized by a conserved and unique insertion in their antiterminator/terminator domain, termed stem Sa. Interestingly, in Staphylococcus aureus the stem Sa can accommodate binding of specific antibiotics, which in turn induce robust and diverse effects on T-box-mediated transcription. In the present study, domain swap mutagenesis and probing analysis were performed to decipher the role of stem Sa. Deletion of stem Sa significantly reduces both the S. aureus glyS T-box-mediated transcription readthrough levels and the ability to discriminate among tRNAGly isoacceptors, both in vitro and in vivo. Moreover, the deletion inverted the previously reported stimulatory effects of specific antibiotics. Interestingly, stem Sa insertion in the terminator/antiterminator domain of Geobacillus kaustophilus glyS T-box, which lacks this domain, resulted in elevated transcription in the presence of tigecycline and facilitated discrimination among proteinogenic and nonproteinogenic tRNAGly isoacceptors. Overall, stem Sa represents a lineage-specific structural feature required for efficient staphylococcal glyS T-box-mediated transcription and it could serve as a species-selective druggable target through its ability to modulate antibiotic binding.

Mammary epithelial cell transcriptome reveals potential roles of lncRNAs in regulating milk synthesis pathways in Jersey and Kashmiri cattle

BackgroundLong noncoding RNAs (lncRNAs) are now proven as essential regulatory elements, playing diverse roles in many biological processes including mammary gland development. However, little is known about their roles in the bovine lactation process.ResultsTo identify and characterize the roles of lncRNAs in bovine lactation, high throughput RNA sequencing data from Jersey (high milk yield producer), and Kashmiri cattle (low milk yield producer) were utilized. Transcriptome data from three Kashmiri and three Jersey cattle throughout their lactation stages were utilized for differential expression analysis. At each stage (early, mid and late) three samples were taken from each breed. A total of 45 differentially expressed lncRNAs were identified between the three stages of lactation. The differentially expressed lncRNAs were found co-expressed with genes involved in the milk synthesis processes such as GPAM, LPL, and ABCG2 indicating their potential regulatory effects on milk quality genes. KEGG pathways analysis of potential cis and trans target genes of differentially expressed lncRNAs indicated that 27 and 48 pathways were significantly enriched between the three stages of lactation in Kashmiri and Jersey respectively, including mTOR signaling, PI3K-Akt signaling, and RAP1 signaling pathways. These pathways are known to play key roles in lactation biology and mammary gland development.ConclusionsExpression profiles of lncRNAs across different lactation stages in Jersey and Kashmiri cattle provide a valuable resource for the study of the regulatory mechanisms involved in the lactation process as well as facilitate understanding of the role of lncRNAs in bovine lactation biology.

Dynamic Interactions of Transcription Factors and Enhancer Reprogramming in Cancer Progression.

While improved tumor treatment has significantly reduced the overall mortality rates, invasive progression including recurrence, therapy resistance and metastasis contributes to the majority of deaths caused by cancer. Enhancers are essential distal DNA regulatory elements that control temporal- or spatial-specific gene expression patterns during development and other biological processes. Genome-wide sequencing has revealed frequent alterations of enhancers in cancers and reprogramming of distal enhancers has emerged as one of the important features for tumors. In this review, we will discuss tumor progression-associated enhancer dynamics, its transcription factor (TF) drivers and how enhancer reprogramming modulates gene expression during cancer invasive progression. Additionally, we will explore recent advancements in contemporary technology including single-cell sequencing, spatial transcriptomics and CUT&RUN, which have permitted integrated studies of enhancer reprogramming in vivo. Given the essential roles of enhancer dynamics and its drivers in controlling cancer progression and treatment outcome, understanding these changes will be paramount in mitigating invasive events and discovering novel therapeutic targets.

High Expression of miR-483-5p Predicts Chemotherapy Resistance in Epithelial Ovarian Cancer.

Ovarian cancer is the most deadly cancer that requires novel diagnostics and therapeutics. MicroRNAs are viewed as essential gene regulatory elements involved in different pathobiological mechanisms of many cancers, including ovarian cancer. This study examined the relationship between microRNA (miRNA) expression and response to platinum-based chemotherapy. Genome-wide miRNA expression analysis was conducted using Epithelial Ovarian Cancer (EOC) tissues from 25 patients with 17 malignant tumors and eight benign ovarian tumors. Candidate miRNAs that respond to platinum-based chemotherapy were selected for validation by quantitative RT-PCR. Among 2,578 mature human miRNAs, high expression of miR-483-5p correlated with poor responses to platinum-based chemotherapy in EOC patients. Furthermore, high levels of miR-483-5p in the resistant group suppressed expression of the apoptotic regulator TAOK-1. A possible marker for the prediction of chemotherapy response and resistance in patients may be miR-483-5p. Choosing the right treatment for each patient with EOC can avoid the risk of developing chemotherapy resistance.

Whole Aegilops tauschii Transcriptome Investigation Revealed Nine Novel miRNAs Involved in Stress Response

Background: Aegilops tauschii is a wild relative of bread wheat. This species has been reported as the donor of bread wheat D genome. There are also several reports that mentioned the importance of Ae. tauschii in biotic and abiotic stress tolerance. On the other hands, miRNAs have been reported as the essential regulatory elements in stress response. Objective: Therefore, it is important to discover novel miRNAs involved in stress tolerance in this species. The aim of the current study was to predict novel miRNAs in Ae. tauschii and also uncover their potential role in stress response. Methods: For this purpose, ESTs, TSAs, and miRBase databases were obtained and used to predict new miRNAs. Results: Our results discovered nine novel stem-loop miRNAs. These predicted miRNAs could be introduced as the new members of previously identified miRNA families in Ae. tauschii, including miR156, miR168, miR169, and miR319. The result indicating that miR397 and miR530 are novel families in this species. Furthermore, several novel stem-loop miRNAs predicted for T. aestivum showed remarkable similarities to novel Ae. tauschii stem-loops. Conclusion: Our results demonstrated that predicted novel miRNAs could play a significant role in stress response.

Transcription of the WUSCHEL-RELATED HOMEOBOX 4 gene in Arabidopsis thaliana

Phylogenetic shadowing and chromatin accessibility data suggested that essential regulatory elements are absent in the 2.9 kb immediate upstream region of the published WOX4pro::YFP cambium marker. Inclusion of an additional 6.3 kb of upstream promoter sequence and confocal imaging with different fluorophores in transgenic Arabidopsis lines revealed a much wider cell-type-specific expression pattern in parenchymous cells of the aerial plant body. The previously demonstrated activity of the WOX4pro::YFP marker in the cambium of vascular strands in the young Arabidopsis inflorescence stem depicts only sectors of a circular subcortical layer of parenchymous AtWOX4-positive cells. Transcription starts in subepidermal cells within the inflorescence apex in a phyllotactic pattern and extends into successively branching lateral organs, which are connected via small tube-like domains of AtWOX4-expressing cells with the circular subcortical parenchymal layer that extends basipetally down the stem. AtWOX4 expression is most dynamic in leaves, where promoter activity is observed transiently at the adaxial side of the lamina and remains detectable later in the palisade parenchyma, although at a weaker level than in the vasculature. In the root the extended AtWOX4 promoter is active through the proximal root meristem, i.e. in the quiescent centre (QC) and its surrounding initials, a pattern that is broader than transcription of its stem cell promoting relative AtWOX5 in the QC. Outside the proximal meristem AtWOX4 transcription is observed in upper cell layers of the columella root cap beneath or above within the stele in proto- and metaxylem cells, in a ribbon-type pattern which divides the central cylinder in two equal halves. This xylem-specific expression it the root stele relates to established AtWOX4 activity in xylem parenchyma specificity within vascular bundles of the stem.

Regulatory genome variants in human susceptibility to infection.

Genome studies have accelerated the discovery of common and rare genetic variants associated with susceptibility to infection and with disease severity. Genome-wide association studies identified many common genetic variants associated with modest risk for infection. Over 80% of these common variants map to the non-coding genome and are thought to modulate the regulatory networks. Exome sequencing has rapidly expanded the number of recognized primary immunodeficiencies through the identification of rare coding variants. In contrast, less than 29 primary immunodeficiencies have causative rare variation mapped outside protein-coding regions. In the future, whole genome sequencing will accelerate the identification of rare variants of substantial phenotypic impact that disrupt essential regulatory elements and the three-dimensional structure of chromatin.

The C4 Ppc promoters of many C4 grass species share a common regulatory mechanism for gene expression in the mesophyll cell.

C4 photosynthetic plants have evolved from C3 ancestors and are characterized by differential expression of several hundred genes. Strict compartmentalization of key C4 enzymes either to mesophyll (M) or bundle sheath cells is considered a crucial step towards the evolution of C4 photosynthesis. In this study, we demonstrate that the 5'-flanking sequences of the C4 type phosphoenolpyruvate carboxylase (Ppc) gene from three C4 grass species could drive M-cell-specific expression of a reporter gene in rice. In addition to that, we identified about 450bp (upstream of their transcription start site) of the analyzed C4 Ppc promoters contain all the essential regulatory elements for driving M-cell-specific expression in rice leaves. Importantly, four motifs of conserved nucleotide sequences (CNSs) were also determined, which are essential for the activity of the promoter. A putative interaction between the CNSs and an unknown upstream element(s) is required for driving M-cell-specific expression. This work identifies the evolutionary conservation of C4 Ppc regulatory mechanisms of multiple closely related C4 grass species.

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

Initial steps in the sex determination of the (human) testis depend on SRY regulating SOX9, but the exact mechanism that controls SOX9 expression remains unknown. These authors discovered four overlapping copy number variations (CNVs) upstream of SOX9 as the causes of sex reversal in two 46,XX DSD (with duplications) and two 46,XY DSD patients (with deletions). Prompted by this, they performed studies of these CNVs in cell systems and in mice, and found three essential regulatory elements for normal testis and male sex development, namely eSR-A, eSR-B and eALDI.

Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

A Messenger Ribonucleic Acid: A Platform for Protein Synthesis

The messenger Ribonucleic Acid (mRNA) has come to play as potential regulators of gene expression that is translates in the ribosome by the RNA polymerase and yields amino acid and protein. This plays a tremendous role in the protein synthesis and is not just mere template in translation but also contains essential regulatory elements as protein supplement, for therapy vaccine production and generation of pluripotent stem cells. This review highlights the new approaches and insights in the role of mRNA as a template in protein synthesis, functional approach, clinical uses and in vivo usage which is still a challenge underway. Keywords: MRNA; Translation; Protein synthesis; DNA; Gene regulation.

Impact of Autophagy and Aging on Iron Load and Ferritin in Drosophila Brain.

Biometals such as iron, copper, potassium, and zinc are essential regulatory elements of several biological processes. The homeostasis of biometals is often affected in age-related pathologies. Notably, impaired iron metabolism has been linked to several neurodegenerative disorders. Autophagy, an intracellular degradative process dependent on the lysosomes, is involved in the regulation of ferritin and iron levels. Impaired autophagy has been associated with normal pathological aging, and neurodegeneration. Non-mammalian model organisms such as Drosophila have proven to be appropriate for the investigation of age-related pathologies. Here, we show that ferritin is expressed in adult Drosophila brain and that iron and holoferritin accumulate with aging. At whole-brain level we found no direct relationship between the accumulation of holoferritin and a deficit in autophagy in aged Drosophila brain. However, synchrotron X-ray spectromicroscopy revealed an additional spectral feature in the iron-richest region of autophagy-deficient fly brains, consistent with iron–sulfur. This potentially arises from iron–sulfur clusters associated with altered mitochondrial iron homeostasis.

CRE and SRE mediate LPA-induced CCN1 transcription in mouse aortic smooth muscle cells

Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein (ox-LDL), is a potent bioactive phospholipid. Our recent data reveal that LPA induces matricellular protein CCN1 (also known as Cyr61) expression in aortic smooth muscle cells (SMCs) and that CCN1 bridges LPA and integrin signaling pathways leading to SMC migration. Whether and how LPA regulates the transcriptional machinery of the CCN1 gene are unknown. In this study, we found that LPA markedly induces CCN1 mRNA expression in SMCs. Using deleting mutation and reporter gene strategies, we demonstrated regions from -2038 to -1787 and from -101 to +63 of the CCN1 promoter contain the essential regulatory elements. The serum response element (SRE) and cyclic AMP-response element (CRE) are located in these regions. LPA induced time-dependent phosphorylation of serum response factor (SRF) and CRE-binding protein (CREB) in mouse SMCs. Luciferase assays of a series of deleted, mutated CCN1 promoter-reporter gene constructs and dominant negative construct revealed the distal SRE and the proximal CRE in the CCN1 promoter are required for LPA-induced CCN1 gene expression. Our results imply that elevated LPA levels may trigger SMC migration and exacerbate restenosis and atherosclerotic lesions through the induced CCN1, which communicates with a set of plasma membrane proteins and intracellular kinases.