Important Regulatory Modules Research Articles

Adapting cytoskeleton-mitochondria patterning with myocyte differentiation by promyogenic PRR33.

Coordinated cytoskeleton-mitochondria organization during myogenesis is crucial for muscle development and function. Our understanding of the underlying regulatory mechanisms remains inadequate. Here, we identified a novel muscle-enriched protein, PRR33, which is upregulated during myogenesis and acts as a promyogenic factor. Depletion of Prr33 in C2C12 represses myoblast differentiation. Genetic deletion of Prr33 in mice reduces myofiber size and decreases muscle strength. The Prr33 mutant mice also exhibit impaired myogenesis and defects in muscle regeneration in response to injury. Interactome and transcriptome analyses reveal that PRR33 regulates cytoskeleton and mitochondrial function. Remarkably, PRR33 interacts with DESMIN, a key regulator of cytoskeleton-mitochondria organization in muscle cells. Abrogation of PRR33 in myocytes substantially abolishes the interaction of DESMIN filaments with mitochondria, leading to abnormal intracellular accumulation of DESMIN and mitochondrial disorganization/dysfunction in myofibers. Together, our findings demonstrate that PRR33 and DESMIN constitute an important regulatory module coordinating mitochondrial organization with muscle differentiation.

Simultaneous knockout of cytosolic and plastidial disproportionating enzymes disrupts grain setting and filling in rice.

Rice (Oryza sativa) plants contain plastidial and cytosolic disproportionating enzymes (DPE1 and DPE2). Our previous studies showed that DPE2 acts on maltose, the major product of starch degradation in pollens, releasing one glucose to fuel pollen tube growth and fertilization, whereas DPE1 participates in endosperm starch synthesis by transferring maltooligosyl groups from amylose to amylopectin, and removing excess short maltooligosaccharides. However, little is known about their integrated function. Here, we report that the coordinated actions of DPE1 and DPE2 contribute to grain setting and filling in rice. The dpe1dpe2 mutants could not be isolated from the progeny of heterozygous parental plants but were obtained via anther culture. Unlike that reported in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), the dpe1dpe2 rice plants grew normally but only yielded a small number of empty, unfilled seeds. In the dpe1dpe2 seeds, nutrient accumulation was substantially reduced, and dorsal vascular bundles were also severely malnourished. Zymogram analyses showed that changes in activities of the major starch-synthesizing enzymes matched well with various endosperm phenotypes of mutant seeds. Mechanistically, DPE1 deficiency allowed normal starch mobilization in leaves and pollens but affected starch synthesis in endosperm, while DPE2 deficiency blocked starch degradation, resulting in substantially decreased levels of the sugars available for pollen tube growth and grain filling. Overall, our results demonstrate the great potential of DPE1-DPE2 as an important regulatory module to realize higher crop yields and present a promising target for regulating nutrient accumulation in cereal crop endosperm.

Auxin homeostasis is maintained by sly-miR167-SlARF8A/B-SlGH3.4 feedback module in the development of locular and placental tissues of tomato fruits.

The locular gel, produced by the placenta, is important for fruit flavor and seed development in tomato. However, the mechanism underlying locule and placenta development is not fully understood yet. Here, we show that two SlARF transcription factors, SlARF8B and SlARF8A (SlARF8A/B), promote the development of locular and placenta tissues. The expression of both SlARF8A and SlARF8B is repressed by sly-microRNA167 (sly-miR167), allowing for the activation of auxin downstream genes. In slarf8a, slarf8b, and slarf8a/b mutants, the auxin (IAA) levels are decreased, whereas the levels of inactive IAA conjugates including IAA-Ala, IAA-Asp, and IAA-Glu are increased. We further find that SlARF8B directly inhibits the expression of SlGH3.4, an acyl acid amino synthetase that conjugates the amino acids to IAA. Disruption of such auxin balance by the increased expression of SlGH3.4 or SlGH3.2 results in defective locular and placental tissues. Taken together, our findings reveal an important regulatory module constituted by sly-miR167-SlARF8A/B-SlGH3.4 during the development of locular and placenta tissues of tomato fruits.

Transcriptome-wide association study coupled with eQTL analysis reveals the genetic connection between gene expression and flowering time in Arabidopsis.

Genome-wide association study (GWAS) has improved our understanding of complex traits, but challenges exist in distinguishing causation versus association caused by linkage disequilibrium. Instead, transcriptome-wide association studies (TWAS) detect direct associations between expression levels and phenotypic variations, providing an opportunity to better prioritize candidate genes. To assess the feasibility of TWAS, we investigated the association between transcriptomes, genomes, and various traits in Arabidopsis, including flowering time. The associated genes formerly known to regulate growth allometry or metabolite production were first identified by TWAS. Next, for flowering time, six TWAS-newly identified genes were functionally validated. Analysis of the expression quantitative trait locus (eQTL) further revealed a trans-regulatory hotspot affecting the expression of several TWAS-identified genes. The hotspot covers the FRIGIDA (FRI) gene body, which possesses multiple haplotypes differentially affecting the expression of downstream genes, such as FLOWERING LOCUS C (FLC) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1). We also revealed multiple independent paths towards the loss of function of FRI in natural accessions. Altogether, this study demonstrates the potential of combining TWAS with eQTL analysis to identify important regulatory modules of FRI-FLC-SOC1 for quantitative traits in natural populations.

Growth-regulating factor 15-mediated gene regulatory network enhances salt tolerance in poplar.

Soil salinity is an important determinant of crop productivity and triggers salt stress response pathways in plants. The salt stress response is controlled by transcriptional regulatory networks that maintain regulatory homeostasis through combinations of transcription factor (TF)-DNA and TF-TF interactions. We investigated the transcriptome of poplar 84 K (Populus alba × Populus glandulosa) under salt stress using samples collected at 4- or 6-h intervals within 2 days of salt stress treatment. We detected 24,973 differentially expressed genes, including 2,231 TFs that might be responsive to salt stress. To explore these interactions and targets of TFs in perennial woody plants, we combined gene regulatory networks, DNA affinity purification sequencing, yeast two-hybrid-sequencing, and multi-gene association approaches. Growth-regulating factor 15 (PagGRF15) and its target, high-affinity K+ transporter 6 (PagHAK6), were identified as an important regulatory module in the salt stress response. Overexpression of PagGRF15 and PagHAK6 in transgenic lines improved salt tolerance by enhancing Na+ transport and modulating H2O2 accumulation in poplar. Yeast two-hybrid assays identified more than 420 PagGRF15-interacting proteins, including ETHYLENE RESPONSE FACTOR TFs and a zinc finger protein (C2H2) that are produced in response to a variety of phytohormones and environmental signals and are likely involved in abiotic stress. Therefore, our findings demonstrate that PagGRF15 is a multifunctional TF involved in growth, development, and salt stress tolerance, highlighting the capability of a multifaceted approach in identifying regulatory nodes in plants.

Activation of TnSmu1, an integrative and conjugative element, by an ImmR-like transcriptional regulator in Streptococcus mutans.

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immR Smu (immunity repressor) and a metalloprotease immA Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of immR Smu and repression of immR Smu with CRISPRi (CRISPR interference) causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immR Smu mutant. Deletion of immR Smu also leads to production of a circular intermediate form of TnSmu1, which is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology, we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems and a type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRA Smu module, and that activation is deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host.

New Insights into Phase Separation Processes and Membraneless Condensates of EIN2.

Recent technological advances allow us to resolve molecular processes in living cells with high spatial and temporal resolution. Based on these technological advances, membraneless intracellular condensates formed by reversible functional aggregation and phase separation have been identified as important regulatory modules in diverse biological processes. Here, we present bioinformatic and cellular studies highlighting the possibility of the involvement of the central activator of ethylene responses EIN2 in such cellular condensates and phase separation processes. Our work provides insight into the molecular type (identity) of the observed EIN2 condensates and on potential intrinsic elements and sequence motifs in EIN2-C that may regulate condensate formation and dynamics.

Meet the President: Russell B. Melchert, PhD, RPh

<h3>Abstract</h3> Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the <i>Streptococcus mutans</i> chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain <i>immR</i>_Smu (immunity repressor) and a metalloprotease <i>immA</i>_Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of <i>immR</i>_Smu and repression of <i>immR</i>_Smu with CRISPRi causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an <i>immR</i>_Smu mutant. Deletion of <i>immR</i>_Smu also leads to production of a circular intermediate form of TnSmu1, that is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmR_Smu and broader effects on <i>S. mutans</i> UA159 physiology we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems, and a Type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across <i>S. mutans</i> isolates. Taken together, our results show that activation of TnSmu1 is controlled by the <i>immRA</i>_Smu module, and that activation is deleterious to <i>S. mutans</i>, highlighting the complex interplay between mobile elements and their host.

SIGNET: single-cell RNA-seq-based gene regulatory network prediction using multiple-layer perceptron bagging.

High-throughput single-cell RNA-seq data have provided unprecedented opportunities for deciphering the regulatory interactions among genes. However, such interactions are complex and often nonlinear or nonmonotonic, which makes their inference using linear models challenging. We present SIGNET, a deep learning-based framework for capturing complex regulatory relationships between genes under the assumption that the expression levels of transcription factors participating in gene regulation are strong predictors of the expression of their target genes. Evaluations based on a variety of real and simulated scRNA-seq datasets showed that SIGNET is more sensitive to ChIP-seq validated regulatory interactions in different types of cells, particularly rare cells. Therefore, this process is more effective for various downstream analyses, such as cell clustering and gene regulatory network inference. We demonstrated that SIGNET is a useful tool for identifying important regulatory modules driving various biological processes.

The Core Promoter Is a Regulatory Hub for Developmental Gene Expression.

The development of multicellular organisms and the uniqueness of each cell are achieved by distinct transcriptional programs. Multiple processes that regulate gene expression converge at the core promoter region, an 80 bp region that directs accurate transcription initiation by RNA polymerase II (Pol II). In recent years, it has become apparent that the core promoter region is not a passive DNA component, but rather an active regulatory module of transcriptional programs. Distinct core promoter compositions were demonstrated to result in different transcriptional outputs. In this mini-review, we focus on the role of the core promoter, particularly its downstream region, as the regulatory hub for developmental genes. The downstream core promoter element (DPE) was implicated in the control of evolutionarily conserved developmental gene regulatory networks (GRNs) governing body plan in both the anterior-posterior and dorsal-ventral axes. Notably, the composition of the basal transcription machinery is not universal, but rather promoter-dependent, highlighting the importance of specialized transcription complexes and their core promoter target sequences as key hubs that drive embryonic development, differentiation and morphogenesis across metazoan species. The extent of transcriptional activation by a specific enhancer is dependent on its compatibility with the relevant core promoter. The core promoter content also regulates transcription burst size. Overall, while for many years it was thought that the specificity of gene expression is primarily determined by enhancers, it is now clear that the core promoter region comprises an important regulatory module in the intricate networks of developmental gene expression.

Identification of Key Regulators of Hepatitis C Virus-Induced Hepatocellular Carcinoma by Integrating Whole-Genome and Transcriptome Sequencing Data.

Background: Hepatitis C virus (HCV) infection is a major cause of cirrhosis and hepatocellular carcinoma (HCC). Despite recent advances in the understanding of the biological basis of HCC development, the molecular mechanisms underlying HCV-induced HCC (HCC-HCV) remain unclear. The carcinogenic potential of HCV varies according to the genotype and mutation in its viral sequence. Moreover, regulatory pathways play important roles in many pathogenic processes. Therefore, identifying the pathways by which HCV induces HCC may enable improved HCC diagnosis and treatment.Methods: We employed a systematic approach to identify an important regulatory module in the process of HCV-HCC development to find the important regulators. First, an HCV-related HCC subnetwork was constructed based on the gene expression in HCC-HCV patients and HCC patients. A priority algorithm was then used to extract the module from the subnetworks, and all the regulatory relationships of the core genes of the network were extracted. Integrating the significantly highly mutated genes involved in the HCC-HCV patients, core regulatory modules and key regulators related to disease prognosis and progression were identified.Result: The key regulatory genes including EXO1, VCAN, KIT, and hsa-miR-200c-5p were found to play vital roles in HCV-HCC development. Based on the statistics analysis, EXO1, VCAN, and KIT mutations are potential biomarkers for HCV–HCC prognosis at the genomic level, whereas has-miR-200c-5P is a potential biomarker for HCV–HCC prognosis at the expression level.Conclusion: We identified three significantly mutated genes and one differentially expressed miRNA, all related to HCC prognosis. As potential pathogenic factors of HCC, these genes and the miRNA could be new biomarkers for HCV-HCC diagnosis.

Synonymous mutation in Growth Regulating Factor 15 of miR396a target sites enhances photosynthetic efficiency and heat tolerance in poplar.

Heat stress damages plant tissues and induces multiple adaptive responses. Complex and spatiotemporally specific interactions among transcription factors (TFs), microRNAs (miRNAs), and their targets play crucial roles in regulating stress responses. To explore these interactions and to identify regulatory networks in perennial woody plants subjected to heat stress, we integrated time-course RNA-seq, small RNA-seq, degradome sequencing, weighted gene correlation network analysis, and multi-gene association approaches in poplar. Results from Populus trichocarpa enabled us to construct a three-layer, highly interwoven regulatory network involving 15 TFs, 45 miRNAs, and 77 photosynthetic genes. Candidate gene association studies in a population of P. tomentosa identified 114 significant associations and 696 epistatic SNP-SNP pairs that were linked to 29 photosynthetic and growth traits (P<0.0001, q<0.05). We also identified miR396a and its target, Growth-Regulating Factor 15 (GRF15) as an important regulatory module in the heat-stress response. Transgenic plants of hybrid poplar (P. alba × P. glandulosa) overexpressing a GRF15 mRNA lacking the miR396a target sites exhibited enhanced heat tolerance and photosynthetic efficiency compared to wild-type plants. Together, our observations demonstrate that GRF15 plays a crucial role in responding to heat stress, and they highlight the power of this new, multifaceted approach for identifying regulatory nodes in plants.

Cold-Induced CBF–PIF3 Interaction Enhances Freezing Tolerance by Stabilizing the phyB Thermosensor in Arabidopsis

Growth inhibition and cold-acclimation strategies help plants withstand cold stress, which adversely affects growth and survival. PHYTOCHROME B (phyB) regulates plant growth through perceiving both light and ambient temperature signals. However, the mechanism by which phyB mediates the plant response to cold stress remains elusive. Here, we show that the key transcription factors mediating cold acclimation, C-REPEAT BINDING FACTORs (CBFs), interact with PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) under cold stress, thus attenuating the mutually assured destruction of PIF3–phyB. Cold-stabilized phyB acts downstream of CBFs to positively regulate freezing tolerance by modulating the expression of stress-responsive and growth-related genes. Consistent with this, phyB mutants exhibited a freezing-sensitive phenotype, whereas phyB-overexpression transgenic plants displayed enhanced freezing tolerance. Further analysis showed that the PIF1, PIF4, and PIF5 proteins, all of which negatively regulate plant freezing tolerance, were destabilized by cold stress in a phytochrome-dependent manner. Collectively, our study reveals that CBFs–PIF3–phyB serves as an important regulatory module for modulating plant response to cold stress.

Weighted gene co‑expression network analysis to identify key modules and hub genes associated with atrial fibrillation.

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia and significantly increases the risks of morbidity, mortality and health care expenditure; however, treatment for AF remains unsatisfactory due to the complicated and incompletely understood underlying mechanisms. In the present study, weighted gene co-expression network analysis (WGCNA) was conducted to identify key modules and hub genes to determine their potential associations with AF. WGCNA was performed in an AF dataset GSE79768 obtained from the Gene Expression Omnibus, which contained data from paired left and right atria in cardiac patients with persistent AF or sinus rhythm. Differentially expressed gene (DEG) analysis was used to supplement and validate the results of WGCNA. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were also performed. Green and magenta modules were identified as the most critical modules associated with AF, from which 6 hub genes, acetyl-CoA Acetyltransferase 1, death domain-containing protein CRADD, gypsy retrotransposon integrase 1, FTX transcript, XIST regulator, transcription elongation factor A like 2 and minichromosome maintenance complex component 3 associated protein, were hypothesized to serve key roles in the pathophysiology of AF due to their increased intramodular connectivity. Functional enrichment analysis results demonstrated that the green module was associated with energy metabolism, and the magenta module may be associated with the Hippo pathway and contain multiple interactive pathways associated with apoptosis and inflammation. In addition, the blue module was identified to be an important regulatory module in AF with a higher specificity for the left atria, the genes of which were primarily correlated with complement, coagulation and extracellular matrix formation. These results suggest that may improve understanding of the underlying mechanisms of AF, and assist in identifying biomarkers and potential therapeutic targets for treating patients with AF.

The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases.

Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling.

The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants. DOI:http://dx.doi.org/10.7554/eLife.01340.001.

RRAS: A key regulator and an important prognostic biomarker in biliary atresia

To characterize the differentially expressed gene profiles in livers from biliary atresia (BA) patients including, ascertain genes, functional categories and pathways that play a central role in the pathogenesis of BA, and identify the novel prognostic markers for BA. Liver tissue samples from control patients, neonatal cholestasis patients, and BA patients at the age of < 60 d, 60-90 d, and > 90 d were pooled for DNA microarray analysis. Bioinformatics analysis was performed using, series test cluster of gene ontology, and Pathway-Finder software. Reverse-transcription polymerase chain reaction was performed to confirm changes in selected genes. Relation between RRAS gene expression and prognosis of 40 BA patients was analyzed in a 2-year follow-up study. The 4 identified significant gene expression profiles could confidently separate BA liver tissue from normal and other diseased liver tissues. The included genes were mainly involved in inflammation response and reconstruction of cellular matrix. The significant pathways associated with BA were primarily involved in autoimmune response, activation of T lymphocytes and its related cytokines. The RRAS, POMC, SLC26A6 and STX3 genes were important regulatory modules in pathogenesis of BA. The expression of RRAS was negatively correlated with the elimination rate of jaundice and positively correlated with the occurrence rate of cholangitis. Autoimmune response mediated by T lymphocytes may play a vital role in the pathogenesis of BA. The RRAS gene is an important regulatory module in the pathogenesis of BA, which may serve as a novel prognostic marker for BA.

Stochastic analysis of the SOS response in Escherichia coli.

BackgroundDNA damage in Escherichia coli evokes a response mechanism called the SOS response. The genetic circuit of this mechanism includes the genes recA and lexA, which regulate each other via a mixed feedback loop involving transcriptional regulation and protein-protein interaction. Under normal conditions, recA is transcriptionally repressed by LexA, which also functions as an auto-repressor. In presence of DNA damage, RecA proteins recognize stalled replication forks and participate in the DNA repair process. Under these conditions, RecA marks LexA for fast degradation. Generally, such mixed feedback loops are known to exhibit either bi-stability or a single steady state. However, when the dynamics of the SOS system following DNA damage was recently studied in single cells, ordered peaks were observed in the promoter activity of both genes (Friedman et al., 2005, PLoS Biol. 3(7):e238). This surprising phenomenon was masked in previous studies of cell populations. Previous attempts to explain these results harnessed additional genes to the system and deployed complex deterministic mathematical models that were only partially successful in explaining the results.Methodology/Principal FindingsHere we apply stochastic methods, which are better suited for dynamic simulations of single cells. We show that a simple model, involving only the basic components of the circuit, is sufficient to explain the peaks in the promoter activities of recA and lexA. Notably, deterministic simulations of the same model do not produce peaks in the promoter activities.Conclusion/SignificanceWe conclude that the double negative mixed feedback loop with auto-repression accounts for the experimentally observed peaks in the promoter activities. In addition to explaining the experimental results, this result shows that including additional regulations in a mixed feedback loop may dramatically change the dynamic functionality of this regulatory module. Furthermore, our results suggests that stochastic fluctuations strongly affect the qualitative behavior of important regulatory modules even under biologically relevant conditions, thus emphasizing the importance of stochastic analysis of regulatory circuits.

Phospholipase C-ϵ Augments Epidermal Growth Factor-dependent Cell Growth by Inhibiting Epidermal Growth Factor Receptor Down-regulation

The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.