Regulation Of Switching Research Articles

Artemisinin attenuates the development of atherosclerotic lesions by the regulation of vascular smooth muscle cell phenotype switching

AimsThe purpose of this study was to investigate the therapeutic effect of artemisinin (ART) on atherosclerosis and explore the molecular mechanisms involved by RNA sequencing (RNA-Seq). Main methodsEight-week-old male ApoE−/- mice were treated with ART for eight weeks. Atherosclerotic lesion sizes were determined by Oil Red O staining, and RNA-Seq was used to detect the profile of differentially expressed genes following the administration of ART. The expressions of contractile phenotypic markers were detected by western blot and qRT-PCR, and the ability of the MOVAS cells to migrate and proliferate were assessed using the wound healing and CCK8 assays. Key findingsArtemisinin treatment significantly reduced plaque area in the ApoE−/- mice and increased the expression of contractile phenotypic markers. RNA-Seq of aorta tissue revealed a distinct change in gene expression patterns after the mice were treated with ART. Our bioinformatics analysis demonstrated that the most prominently enriched pathway was a set of genes involved in vascular smooth muscle contractile function. Using an in vitro cell model, we demonstrated that ART could effectively reverse PDGF-activated MOVAS migration and proliferation, and elevate the level of proteins involved in the contractile phenotype. SignificanceWe provide in vivo and in vitro evidence supporting a role for ART in the suppression of atherosclerosis, partly through the inhibition of vascular smooth muscle cell phenotype switching to a de-differentiated phenotype. These data further advances our understanding for a potential role for ART and suggests that ART is an excellent candidate for the treatment of atherosclerosis.

Regulation of flagellar motor switching by c-di-GMP phosphodiesterases in Pseudomonas aeruginosa

The second messenger cyclic diguanylate (c-di-GMP) plays a prominent role in regulating flagellum-dependent motility in the single-flagellated pathogenic bacterium Pseudomonas aeruginosa The c-di-GMP-mediated signaling pathways and mechanisms that control flagellar output remain to be fully unveiled. Studying surface-tethered and free-swimming P. aeruginosa PAO1 cells, we found that the overexpression of an exogenous diguanylate cyclase (DGC) raises the global cellular c-di-GMP concentration and thereby inhibits flagellar motor switching and decreases motor speed, reducing swimming speed and reversal frequency, respectively. We noted that the inhibiting effect of c-di-GMP on flagellar motor switching, but not motor speed, is exerted through the c-di-GMP-binding adaptor protein MapZ and associated chemotactic pathways. Among the 22 putative c-di-GMP phosphodiesterases, we found that three of them (DipA, NbdA, and RbdA) can significantly inhibit flagellar motor switching and swimming directional reversal in a MapZ-dependent manner. These results disclose a network of c-di-GMP-signaling proteins that regulate chemotactic responses and flagellar motor switching in P. aeruginosa and establish MapZ as a key signaling hub that integrates inputs from different c-di-GMP-signaling pathways to control flagellar output and bacterial motility. We rationalized these experimental findings by invoking a model that postulates the regulation of flagellar motor switching by subcellular c-di-GMP pools.

A novel GCN5b lysine acetyltransferase complex associates with distinct transcription factors in the protozoan parasite Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite that has a tremendous impact on human health and livestock. High seroprevalence among humans and other animals is facilitated by the conversion of rapidly proliferating tachyzoites into latent bradyzoites that are housed in tissue cysts, which allow transmission through predation. Epigenetic mechanisms contribute to the regulation of gene expression events that are crucial in both tachyzoites as well as their development into bradyzoites. Acetylation of histones is one of the critical histone modifications that is linked to active gene transcription. Unlike most early-branching eukaryotes, Toxoplasma possesses two GCN5 homologues, one of which, GCN5b, is essential for parasite viability. Surprisingly, GCN5b does not associate with most of the well-conserved proteins found in the GCN5 complexes of other eukaryotes. Of particular note is that GCN5b interacts with multiple putative transcription factors that have plant-like DNA-binding domains denoted as AP2. To understand the function of GCN5b and its role(s) in epigenetic gene regulation of stage switching, we performed co-immunoprecipitation of GCN5b under normal and bradyzoite induction conditions. We report the greatest resolution of the GCN5b complex to date under these various culture conditions. Moreover, reciprocal co-IPs were performed with distinct GCN5b-interacting AP2 factors (AP2IX-7 and AP2XII-4) to delineate the interactomes of each putative transcription factor. Our findings suggest that GCN5b is associated with at least two distinct complexes that are characterized by two different pairs of AP2 factors, and implicate up to four AP2 proteins to be involved with GCN5b-mediated gene regulation.

An Improved Fundamental Harmonic Approximation to Describe Filter Inductor Influence on Steady-State Performance of Parallel-Type Resonant Converter

The influence of a filter inductor on the steady-state performance of a parallel-type resonant converter is elaborated and analyzed in this paper. By steady-state operation analysis, it is shown that the ripple current of the filter inductor results in an inaccurate prediction of fundamental harmonic approximation (FHA), and such problem would become serious when the filter inductance is small. In order to portray such feature, an improved fundamental harmonic approximation (IFHA) is proposed. Unlike an FHA equivalent circuit, an equivalent inductor is added to the ac resistance parallelly in an IFHA equivalent circuit. Due to that, the equivalent inductor branch takes account of the ripple current of the filter inductor, and the IFHA is expected to own higher accuracy and can be used in a parameter design procedure. In this paper, the equivalent inductor expressions of full-bridge rectifier and current-doubler rectifier are derived as examples. In order to verify the theoretical analysis, a 500 W LCC resonant converter is built as a prototype. The close-loop experiment results show that small filter inductance would lead to the failure of output voltage regulation and hard-switching operation of power switches. And open-loop experiment results show the IFHA gives more accurate predictions than those of FHA.

Chaperone-mediated secretion switching from early to middle substrates in the type III secretion system encoded by Salmonella pathogenicity island 2

The bacterial type III secretion system (T3SS) delivers virulence proteins, called effectors, into eukaryotic cells. T3SS comprises a transmembrane secretion apparatus and a complex network of specialized chaperones that target protein substrates to this secretion apparatus. However, the regulation of secretion switching from early (needle and inner rod) to middle (tip/filament and translocators) substrates is incompletely understood. Here, we investigated chaperone-mediated secretion switching from early to middle substrates in the T3SS encoded by Salmonella pathogenicity island 2 (SPI2), essential for systemic infection. Our findings revealed that the protein encoded by ssaH regulates the secretion of an inner rod and early substrate, SsaI. Structural modeling revealed that SsaH is structurally similar to class III chaperones, known to associate with proteins in various pathogenic bacteria. The SPI2 protein SsaE was identified as a class V chaperone homolog and partner of SsaH. A pulldown analysis disclosed that SsaH and SsaE form a heterodimer, which interacted with another early substrate, the needle protein SsaG. Moreover, SsaE also helped stabilize SsaH and a middle substrate, SseB. We also found that SsaE regulates cellular SsaH levels to translocate the early substrates SsaG and SsaI and then promotes the translocation of SseB by stabilizing it. In summary, our results indicate that the class III chaperone SsaH facilitates SsaI secretion, and a heterodimer of SsaH and the type V chaperone SsaE then switches secretion to SsaG. This is the first report of a chaperone system that regulates both early and middle substrates during substrate switching for T3SS assembly.

Molecular mechanism underlying regulation of vascular smooth muscle phenotype switching by TRPC6

Molecular mechanism underlying regulation of vascular smooth muscle phenotype switching by TRPC6

Nucleotide Modification Alters MicroRNA-Dependent Silencing of MicroRNA Switches

mRNA therapeutics hold great promise for the treatment of human diseases. While incorporating naturally occurring modified nucleotides during synthesis has greatly increased their potency and safety, challenges in selective expression have hindered clinical applications. MicroRNA (miRNA)-regulated in vitro-transcribed mRNAs, called miRNA switches, have been used to control the expression of exogenous mRNA in a cell-selective manner. However, the effect of nucleotide modifications on miRNA-dependent silencing has not been examined. Here we show that the incorporation of pseudouridine, N1-methylpseudourdine, or pseudouridine and 5-methylcytidine, which increases translation, tends to decrease the regulation of miRNA switches. Moreover, pseudouridine and 5-methylcytidine modification enables one miRNA target site at the 3′ UTR to be as effective as four target sites. We also demonstrate that the effects of pseudouridine, pseudouridine and 5-methylcytidine, and N1-methylpseudourdine modification are miRNA switch specific and do not correlate with the proportion of modified nucleotides in the miRNA target site. Furthermore, modified miRNA switches containing seed-complementary target sites are poorly regulated by miRNA. We also show that placing the miRNA target site in the 5′ UTR of the miRNA switch abolishes the effect of nucleotide modification on miRNA-dependent silencing. This work provides insights into the influence of nucleotide modifications on miRNA-dependent silencing and informs the design of optimal miRNA switches.

Control of Human Hemoglobin Switching By LIN28B-Mediated Regulation of BCL11A Translation

Both sickle cell disease and β-thalassemia are major sources of morbidity and mortality worldwide. Continued production of the β-like γ-globin genes that form fetal hemoglobin after infancy has been shown to ameliorate the severity of these disorders. As a result, there has been considerable interest in understanding the underlying regulation of the physiologic fetal-to-adult hemoglobin switch in humans to be able to better manipulate this process for therapeutic purposes. To date, only a single factor, BCL11A, has been identified as being involved in the developmental regulation of human hemoglobin switching. BCL11A is a direct transcriptional repressor of the γ-globin genes. Moreover, BCL11A is expressed in a developmental stage-specific manner to regulate human hemoglobin switching. However, despite extensive studies, the mechanisms that act upstream to regulate BCL11A expression and thereby control hemoglobin switching have remained elusive. To gain further insights, we have directly explored the developmental regulation of BCL11A expression at various stages of human erythropoiesis. We find that BCL11A is regulated at the level of mRNA translation during development. While BCL11A mRNA is comparably expressed in a similar manner at all developmental stages in erythroid cells, robust protein expression only occurs in adult erythroid cells. Importantly, we demonstrate that at the earlier stages of development, the observed reduction in protein expression is attributable to decreased synthesis and not increased degradation of BCL11A through direct assessment of both protein synthesis and degradation rates in primary erythroid cells from various stages of human development. Interestingly, while BCL11A protein is not well synthesized at these earlier stages of development, we find that its mRNA curiously continues to be associated with ribosomes in a comparable manner between newborn and adult erythroid precursors using polysome fractionation of stage-matched erythroid cells. Through use of an unbiased proteomic analysis approach involving RNA affinity purification of the 18S ribosomal RNA in erythroid cells, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a reciprocal pattern to BCL11A, directly interacts with ribosomes. We additionally show that the observed suppression of BCL11A protein translation is mediated by LIN28B. Using a newly developed RNA isolation and crosslinking immunoprecipitation approach coupled to massively parallel sequencing we mapped the direct interaction of LIN28B with BCL11A mRNA at nucleotide resolution. Through a number of functional assays in both newborn and adult erythroid cells, we additionally demonstrate that this alteration of BCL11A translation is entirely independent of the role of LIN28B in the biogenesis of let-7 microRNAs. Finally, we show that BCL11A is the major functional target in LIN28B-mediated fetal hemoglobin induction through functional complementation experiments in primary human erythroid cells. Our results reveal a previously unappreciated regulatory mechanism underlying human hemoglobin switching. Moreover, our findings highlight opportunities for developing improved treatments for sickle cell disease and β-thalassemia by understanding the upstream regulation of human hemoglobin switching. DisclosuresNo relevant conflicts of interest to declare.

Thalamic regulation of switching between cortical representations enables cognitive flexibility.

Interactions between the prefrontal cortex (PFC) and mediodorsal thalamus (MD) are critical for cognitive flexibility, yet the underlying computations are unknown. To investigate fronto-thalamic substrates of cognitive flexibility, we developed a behavioral task, where mice switched between different sets of learned cues that guided attention towards either visual or auditory targets. We found that PFC responses reflected both the individual cues and their meaning as task rules, indicating a hierarchical cue-to-rule transformation. Conversely, MD responses reflected the statistical regularity of cue presentation, and were required for switching between such experimentally-specified cueing contexts. A subset of these thalamic responses sustained context-relevant PFC representations, while another suppressed the context-irrelevant ones. Through modeling and experimental validation, we find that thalamic-mediated suppression may not only reduce PFC representational interference but could also preserve unused cortical traces for future use. Overall, our study provides a computational foundation for thalamic engagement in cognitive flexibility.

The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

Form confers function: Case of the 3'X region of the hepatitis C virus genome.

At the 3’ end of genomic hepatitis C virus (HCV) RNA there is a highly conserved untranslated region, the 3’X-tail, which forms part of the 3’UTR. This region plays key functions in regulation of critical processes of the viral life cycle. The 3’X region is essential for viral replication and infectivity. It is also responsible for regulation of switching between translation and transcription of the viral RNA. There is some evidence indicating the contribution of the 3’X region to the translation efficiency of the viral polyprotein and to the encapsidation process. Several different secondary structure models of the 3’X region, based on computer predictions and experimental structure probing, have been proposed. It is likely that the 3’X region adopts more than one structural form in infected cells and that a specific equilibrium between the various forms regulates several aspects of the viral life cycle. The most intriguing explanations of the structural heterogeneity problem of the 3’X region came with the discovery of its involvement in long-range RNA-RNA interactions and the potential for homodimer formation. This article summarizes current knowledge on the structure and function of the 3’X region of hepatitis C genomic RNA, reviews previous opinions, presents new hypotheses and summarizes the questions that still remain unanswered.

IRF4 regulates the rate of cell cycle during B cell differentiation.

Abstract Cell division is required for the initiation of B cell differentiation, the regulation of isotype class switching, and ultimately entry of cells into the plasma cell (PC) lineage. Division coupled changes in the expression of transcription factors, such as interferon regulatory factor-4 (IRF4), that coordinate the PC transcriptional program also occur; however, little is known regarding how these factors coordinate cell division and exit from the cell cycle after differentiation. To begin to address this gap in knowledge, we assessed the cell division kinetics of wildtype (WT) B cells responding to the T cell independent antigen lipopolysaccharide (LPS). Interestingly, we found that WT B cells undergo at least 8 divisions before differentiating into PCs. Computational modeling of division rates over time defined a proliferative burst between 48 and 60 hours after LPS injection that is characterized by a rapid increase in the rate of cell division. IRF4-deficient B cells divided but failed to undergo the proliferative burst resulting in fewer activated B cells. Indeed, IRF4 is expressed at low levels during these divisions before being upregulated in post-mitotic CD138+ PCs after 8 divisions. Notably, while more than 40% of WT B cells reached division 8+ by 72 hours, IRF4-null B cells appeared to be stalled at divisions 2–5. Taken together, these data suggest that IRF4 regulates the rate of cellular proliferation during B cell differentiation that occurs around divisions 2–5 and that this role may be concentration-dependent.

Transcriptional Regulation of Telomeric Expression Sites and Antigenic Variation in Trypanosomes.

Introduction: Trypanosoma brucei uses antigenic variation to evade the host antibody clearance by periodically changing its Variant Surface Glycoprotein (VSGs) coat. T. brucei encode over 2,500 VSG genes and pseudogenes, however they transcribe only one VSG gene at time from one of the 20 telomeric Expression Sites (ESs). VSGs are transcribed in a monoallelic fashion by RNA polymerase I from an extranucleolar site named ES body. VSG antigenic switching occurs by transcriptional switching between telomeric ESs or by recombination of the VSG gene expressed. VSG expression is developmentally regulated and its transcription is controlled by epigenetic mechanisms and influenced by a telomere position effect.Conclusion: Here, we discuss 1) the molecular basis underlying transcription of telomeric ESs and VSG antigenic switching; 2) the current knowledge of VSG monoallelic expression; 3) the role of inositol phosphate pathway in the regulation of VSG expression and switching; and 4) the developmental regulation of Pol I transcription of procyclin and VSG genes.

Precise theranostic nanomedicines for inhibiting vulnerable atherosclerotic plaque progression through regulation of vascular smooth muscle cell phenotype switching.

Coronary heart disease is a prevalent and fatal killer caused by vulnerable atherosclerotic plaques (VASPs). However, the precise detection and treatment of VASPs remains a difficult challenge. Here, we present the development of noninvasive human serum albumin (HSA)-based theranostic nanomedicines (NMs) for the specific diagnosis and effective therapy of VASPs.Methods: The ICG/SRT@HSA-pept NMs were formulated to contain payloads of the near-infrared (NIR) fluorescent dye indocyanine green (ICG) and the sirtuin 1 (Sirt1) activator SRT1720, and modified with a peptide moiety targeting osteopontin (OPN). The in vivo atherosclerotic mouse model was established with the high-fat diet (HFD). The in vitro vascular smooth muscle cells (VSMCs) phenotypic switching was induced using the ox-LDL stimulation.Results: Due to the overexpression of OPN in activated VSMCs and VASPs, the targeted NMs specifically accumulated within the VASPs region after intravenous injection into the atherosclerotic mice, achieving the precise detection of VASPs. In addition, in the presence of SRT1720, the NMs could activate intracellular Sirt1 and activate an antiatherogenesis effect by inhibiting the phenotypic switching of VSMCs, which is an essential contributor to the vulnerability and progression of atherosclerotic plaques. After therapeutic administration of the ICG/SRT@HSA-pept NMs for two weeks, the physiological sizes and plaque compositions of VASPs were markedly improved. Furthermore, ICG/SRT@HSA-pept NMs-treated mice presented a more favorable plaque phenotype than that was observed in free SRT1720-treated mice, suggesting the enhanced delivery of pharmaceutical agents to the atherosclerotic lesions and improved therapeutic efficacy of NMs compared with free SRT1720.Conclusions: The theranostic ICG/SRT@HSA-pept NMs showed great potential for the precise identification and targeted treatment of atherosclerotic diseases.

Alkaline Phosphatase Controls Lineage Switching of Mesenchymal Stem Cells by Regulating the LRP6/GSK3β Complex in Hypophosphatasia.

Lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs) is the key to bone-fat reciprocity in bone marrow. To date, the regulators of BMMSC lineage switching have all been identified to be transcription factors, and researchers have not determined whether other genes control this process. This study aims to reveal a previously unknown role of tissue-nonspecific alkaline phosphatase (TNSALP) in controlling BMMSC lineage selection.Methods: We compared the characteristics of cultured BMMSCs from patients with hypophosphatasia (HPP), which is caused by mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene, and an ALPL knockout (ko) mouse model. We performed ALPL downregulation and overexpression experiments to investigate the regulatory role of ALPL in BMMSC lineage switching. Using the PathScan array, coimmunoprecipitation experiments and pathway-guided small molecule treatments, we explored the possible mechanism underlying the regulatory effects of ALPL on cell differentiation and evaluated its therapeutic effect on ALPL ko mice.Results: BMMSCs from both patients with HPP and ALPL ko mice exhibited defective lineage differentiation, including a decrease in osteogenic differentiation and a parallel increase in adipogenic differentiation. Mechanistically, TNSALP directly interacted with LRP6 and regulated the phosphorylation of GSK3β, subsequently resulting in lineage switching of BMMSCs. Re-phosphorylation of GSK3β induced by LiCl treatment restored differentiation of BMMSCs and attenuated skeletal deformities in Alpl+/- mice.Conclusion: Based on our findings, TNSALP acts as a signal regulator to control lineage switching of BMMSCs by regulating the LRP6/GSK3β cascade.

Regulation of mating type switching by the mating type genes and RME1 in Ogataea polymorpha

Saccharomyces cerevisiae and its closely related yeasts undergo mating type switching by replacing DNA sequences at the active mating type locus (MAT) with one of two silent mating type cassettes. Recently, a novel mode of mating type switching was reported in methylotrophic yeast, including Ogataea polymorpha, which utilizes chromosomal recombination between inverted-repeat sequences flanking two MAT loci. The inversion is highly regulated and occurs only when two requirements are met: haploidy and nutritional starvation. However, links between this information and the mechanism associated with mating type switching are not understood. Here we investigated the roles of transcription factors involved in yeast sexual development, such as mating type genes and the conserved zinc finger protein Rme1. We found that co-presence of mating type a1 and α2 genes was sufficient to prevent mating type switching, suggesting that ploidy information resides solely in the mating type locus. Additionally, RME1 deletion resulted in a reduced rate of switching, and ectopic expression of O. polymorpha RME1 overrode the requirement for starvation to induce MAT inversion. These results suggested that mating type switching in O. polymorpha is likely regulated by two distinct transcriptional programs that are linked to the ploidy and transmission of the starvation signal.

Exercise induces muscle fiber type switching via transient receptor potential melastatin 2-dependent Ca2+ signaling.

The aim of the present study was to examine whether transient receptor potential melastatin 2 (TRPM2) plays a role in muscle fiber-type transition during exercise. Mice were trained at a speed of 12 m/min at a slope of 0° for 60 min for 5 consecutive days/wk for 4 wk. Exhaustion tests were performed on the treadmill (the speed was set at 6 m/min at a slope of 0° and increased at a rate of 1 m/min every 6 min). Isolated primary skeletal muscle cells from TRPM2-knockout (KO) mice showed lower amplitudes of electrical stimuli (ES)-induced Ca2+ signals when compared with wild-type (WT) mice due to a defect in Ca2+ influx. Moreover, TRPM2-KO mice had a higher proportion of fast-twitch skeletal muscle fibers and a lower proportion of slow-twitch muscle fibers before exercise than WT mice. After exercise, the expression of slow-twitch skeletal muscle fibers was increased only in WT mice but not in TRPM2-KO mice. ES-induced nuclear translocation of the Ca2+-dependent transcription factor NFATc1 was significantly lower in TRPM2-KO mice than in WT mice. TRPM2-KO mice also showed decreased mitochondrial Ca2+ and membrane potential. Lactate levels were higher in the skeletal muscle cells of TRPM2-KO mice before and after ES compared with WT mice. Collectively, these data indicate that TRPM2-mediated Ca2+ signaling plays a critical role in the regulation of fiber-type switching and mitochondrial function in skeletal muscle. NEW & NOTEWORTHY TRPM2 has been shown to play an important role in a variety of cellular functions. However, the role of TRPM2 in skeletal muscle remains poorly understood. Here, we provide evidence that TRPM2-mediated Ca2+ signaling is required for training-induced improvement in skeletal muscle mitochondrial function and fiber type transition.

Asymmetrical hybrid‐controlled half‐bridge LCC resonant converter with low conduction loss and wide ZVS operation range

An asymmetrical hybrid-controlled half-bridge LCC resonant converter which integrated a buck–boost converter and a half-bridge LCC resonant converter is proposed. It benefits with wide soft switching range, low conduction loss and high power density. By utilising an asymmetrical hybrid control strategy, its switching frequency and duty ratio can be adjusted simultaneously. The proposed LCC resonant converter maintains output voltage regulation and zero-voltage switching of switches in wide input voltage and load variation range. Experiment results of a 500 W prototype are provided to verify the theoretical analysis results.

Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na+-Driven Flagella

The flagellar motor protein complex consists of rotor and stator proteins. Their interaction generates torque of flagellum, which rotates bidirectionally, clockwise (CW) and counterclockwise. FliG, one of the rotor proteins, consists of three domains: N-terminal (FliGN), middle (FliGM), and C-terminal (FliGC). We have identified point mutations in FliGC from Vibrio alginolyticus, which affect the flagellar motility. To understand the molecular mechanisms, we explored the structural and dynamic properties of FliGC from both wild-type and motility-defective mutants. From nuclear magnetic resonance analysis, changes in signal intensities and chemical shifts between wild-type and the CW-biased mutant FliGC are observed in the Cα1-6 domain. Molecular dynamics simulations indicated the conformational dynamics of FliGC at sub-microsecond timescale, but not in the CW-biased mutant. Accordingly, we infer that the dynamic properties of atomic interactions around helix α1 in the Cα1-6 domain of FliGC contribute to ensure the precise regulation of the motor switching.

CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.

ABSTRACTPlasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family.