scholarly journals Comparative Transcriptomics Highlights the Role of the Activator Protein 1 Transcription Factor in the Host Response to Ebolavirus

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
James W. Wynne ◽  
Shawn Todd ◽  
Victoria Boyd ◽  
Mary Tachedjian ◽  
Reuben Klein ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Human Cells ◽  
Activator Protein 1 ◽  
Host Gene Expression ◽  
Rna Seq ◽  
Content Type ◽  
Molecular Events ◽  
Ebov Infection

ABSTRACT Ebolavirus and Marburgvirus comprise two genera of negative-sense single-stranded RNA viruses that cause severe hemorrhagic fevers in humans. Despite considerable research efforts, the molecular events following Ebola virus (EBOV) infection are poorly understood. With the view of identifying host factors that underpin EBOV pathogenesis, we compared the transcriptomes of EBOV-infected human, pig, and bat kidney cells using a transcriptome sequencing (RNA-seq) approach. Despite a significant difference in viral transcription/replication between the cell lines, all cells responded to EBOV infection through a robust induction of extracellular growth factors. Furthermore, a significant upregulation of activator protein 1 (AP1) transcription factor complex members FOS and JUN was observed in permissive cell lines. Functional studies focusing on human cells showed that EBOV infection induces protein expression, phosphorylation, and nuclear accumulation of JUN and, to a lesser degree, FOS. Using a luciferase-based reporter, we show that EBOV infection induces AP1 transactivation activity within human cells at 48 and 72 h postinfection. Finally, we show that JUN knockdown decreases the expression of EBOV-induced host gene expression. Taken together, our study highlights the role of AP1 in promoting the host gene expression profile that defines EBOV pathogenesis. IMPORTANCE Many questions remain about the molecular events that underpin filovirus pathophysiology. The rational design of new intervention strategies, such as postexposure therapeutics, will be significantly enhanced through an in-depth understanding of these molecular events. We believe that new insights into the molecular pathogenesis of EBOV may be possible by examining the transcriptomic response of taxonomically diverse cell lines (derived from human, pig, and bat). We first identified the responsive pathways using an RNA-seq-based transcriptomics approach. Further functional and computational analysis focusing on human cells highlighted an important role for the AP1 transcription factor in mediating the transcriptional response to EBOV infection. Our study sheds new light on how host transcription factors respond to and promote the transcriptional landscape that follows viral infection.

scholarly journals Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1870
Author(s):  
Klaudia Skrzypek ◽  
Grażyna Adamek ◽  
Marta Kot ◽  
Bogna Badyra ◽  
Marcin Majka
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Migration Activity ◽  
Id Proteins ◽  
Rh30 Cell ◽  
Str Profile ◽  
And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

Transcriptional control of human papillomavirus type 18 oncogene expression in different cell lines: Role of transcription factor YY1

Virus Genes ◽  
1995 ◽  
Vol 11 (1) ◽  
pp. 53-58 ◽  
Author(s):  
Franziska Jundt ◽  
Ingrid Herr ◽  
Peter Angel ◽  
Harald Zur Hausen ◽  
Tobias Bauknecht
Keyword(s):  
Transcription Factor ◽  
Human Papillomavirus ◽  
Cell Lines ◽  
Transcriptional Control ◽  
Oncogene Expression ◽  
Transcription Factor Yy1

scholarly journals The Global Transcription Factor Lrp Controls Virulence Modulation in Xenorhabdus nematophila

2015 ◽  
Vol 197 (18) ◽  
pp. 3015-3025 ◽  
Author(s):  
Elizabeth A. Hussa ◽  
Ángel M. Casanova-Torres ◽  
Heidi Goodrich-Blair
Keyword(s):  
Transcription Factor ◽  
Phenotypic Variation ◽  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Inverse Correlation ◽  
Xenorhabdus Nematophila ◽  
Insect Larvae ◽  
Content Type ◽  
Expression Levels

ABSTRACTThe bacteriumXenorhabdus nematophilaengages in phenotypic variation with respect to pathogenicity against insect larvae, yielding both virulent and attenuated subpopulations of cells from an isogenic culture. The global regulatory protein Lrp is necessary forX. nematophilavirulence and immunosuppression in insects, as well as colonization of the mutualistic host nematodeSteinernema carpocapsae, and mediates expression of numerous genes implicated in each of these phenotypes. Given the central role of Lrp inX. nematophilahost associations, as well as its involvement in regulating phenotypic variation pathways in other bacteria, we assessed its function in virulence modulation. We discovered that expression oflrpvaries within an isogenic population, in a manner that correlates with modulation of virulence. Unexpectedly, although Lrp is necessary for optimal virulence and immunosuppression, cells expressing high levels oflrpwere attenuated in these processes relative to those with low to intermediatelrpexpression. Furthermore, fixed expression oflrpat high and low levels resulted in attenuated and normal virulence and immunosuppression, respectively, and eliminated population variability of these phenotypes. These data suggest that fluctuatinglrpexpression levels are sufficient to drive phenotypic variation inX. nematophila.IMPORTANCEMany bacteria use cell-to-cell phenotypic variation, characterized by distinct phenotypic subpopulations within an isogenic population, to cope with environmental change. Pathogenic bacteria utilize this strategy to vary antigen or virulence factor expression. Our work establishes that the global transcription factor Lrp regulates phenotypic variation in the insect pathogenXenorhabdus nematophila, leading to attenuation of virulence and immunosuppression in insect hosts. Unexpectedly, we found an inverse correlation between Lrp expression levels and virulence: high levels of expression of Lrp-dependent putative virulence genes are detrimental for virulence but may have an adaptive advantage in other aspects of the life cycle. Investigation ofX. nematophilaphenotypic variation facilitates dissection of this phenomenon in the context of a naturally occurring symbiosis.

A Novel Long Non-Coding RNA, SNHG4 Complex With Eukaryotic Initiation Factor-4E and Regulate Aberrant Protein Translation In Mantle Cell Lymphoma: Implications For Novel Biomarker

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 81-81
Author(s):  
Guangzhen Hu ◽  
Thomas E Witzig ◽  
Mamta Gupta
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Rna Binding ◽  
Cell Lymphoma ◽  
Protein Translation ◽  
Rna Seq ◽  
Lymphoma Cells ◽  
Binding Motifs ◽  
Mantle Cell

Abstract Long noncoding RNAs (lncRNAs) are defined as RNA-like transcripts that are over 200 nucleotides and lack significant open reading frames. Some lncRNAs such as HOTAIR, MALAT1 and H19 have been found to be associated with clinical prognosis and are potential drivers of cancer progression in cancers of the breast, lung, and liver respectively. The role of lncRNAs in lymphoma is unknown. Dysregulation of eIF4E (a key component of the translation initiation complex eIF4F) influences global protein translation, especially the translation of “weak” mRNAs that can be malignancy-related. We and others have found that eIF4E is dysregulated in B-cell lymphoma. The aim of this study is to identify eIF4E-associated lncRNAs through next generation RNA-Sequencing (NGS RNA-Seq) and delineate their role in protein translation in lymphoma. RNA-immunoprecipitation (RNA-IP) was used to pull down eIF4E-bound lncRNA in lymphoma cells. eIF4E-bound lncRNAs were immunoprecipitated with eIF4E antibody or IgG control in Jeko, a mantle cell lymphoma (MCL) cell line and sent for microarray analysis and NGS-RNA-Seq for identification of lncRNAs. The microarray analysis showed that several lncRNAs were enriched with eIF4E antibody compared to IgG control. These included SNHG4 (13.6 fold), SNHG12 (4.8 fold), NCRNA00171 (4.8 fold) and IPW (4.6 fold), GNASAS (3.5 fold), SNHG7 (3.3 fold), NCRNA00182 (2.7 fold), NCRNA00094 (2.6 fold), NCRNA00188 (2.4 fold) and NCRNA00201 (2.1 fold). The binding of these lncRNAs to eIF4E was further confirmed by RT-PCR in Jeko, Mino and Granta MCL cell lines. Next, we looked the expression of these lncRNAs by qRT- PCR in the MCL cell lines and normal controls. We found SNHG4 and IPW to be overexpressed in all the MCL cell lines, while SNHG12 and NCRNA00201 were overexpressed in the selected cell lines. No significant difference was found for the expression of NCRNA00171 and NCRNA00182 in any of the MCL cell lines compared to controls. Overall, these data suggest that several lncRNA have altered expression in malignant B-cells. Considering that the microarray assay only covered a limited number of lncRNAs, we further confirmed eIF4E bound lncRNA by NGS RNA-Seq in Jeko MCL and normal control. The binding of 10/13 lncRNA mentioned above with eIF4E were found upregulated by NGS-RNA-Seq. In addition several novel lncRNAs such as SNHG1 (161.6), AC091814.2 (98.8) and RP11-304L19.5 (64.2) showed up in NGS-RNA-Seq data. These data suggest that lncRNAs, such as SNHG12, SNHG4, and SNHG1 bind to eIF4E with high affinity in malignant B-cells and might play a role in protein translation. We knocked down the expression of SNHG4 through siRNA and demonstrated that cell proliferation and global protein translation was inhibited in lymphoma cells. To further confirm the role of SNHG4 in translation regulation, a plasmid, which contains a renilla luciferase driven by SV40 promoter, was co-transfected with SNHG4 siRNA into Mino cells. The luciferase signal, decreased compared with the cells transfected with nontargeting siRNA. These data suggest that SNHG4 is involved in the regulation of protein translation. In order to clarify the mechanism of lncRNAs bound to eIF4E we searched for RNA binding sites or motifs in eIF4E protein using the web-based tools, BindN and PPRInt. Interestingly two RNA binding motifs, KNKRGGRWLITLNKQQRRS and SHADTATKSGSTTKNR, were found in eIF4E based on the prediction. To examine whether lncRNAs bind with eIF4E through these RNA binding motifs, an eIF4E mutant plasmid with both RNA binding motifs deleted (eIF4EDel), was constructed and transfected transiently into HEK-293T cells along with eIF4EWT plasmid. RNA-IP data showed that the lncRNAs SNHG12, SNHG4 and SNHG1 were not able to bind with eIF4E in eIF4EDel-transfected cells compared with that of eIF4EWT, suggesting that these lncRNAs complex with eIF4E through RNA-binding motifs within the eIF4E. Overall, our results show that the lncRNAs, SNHG1 and SNHG4 are able to bind with eIF4E and regulate protein translation. Since lncRNAs had been found to play roles in the regulation of gene expression, including transcription, splicing and mRNA stability, our results may broaden the view of the functional role of lncRNAs in translation in lymphoma cells and in other cancers. Furthermore, our results also suggested that SNHG4 lncRNAs might be served as potential biomarkers for MCL and other B cell lymphomas for translation therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The β-Arrestin-Like Protein Rim8 Is Hyperphosphorylated and Complexes with Rim21 and Rim101 To Promote Adaptation to Neutral-Alkaline pH

2012 ◽  
Vol 11 (5) ◽  
pp. 683-693 ◽  
Author(s):  
Jonathan Gomez-Raja ◽  
Dana A. Davis
Keyword(s):  
Signal Transduction ◽  
Transcription Factor ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Multivesicular Bodies ◽  
Ph Sensing ◽  
Content Type ◽  
Protein Levels ◽  
G Protein Coupled

ABSTRACTβ-Arrestin proteins are critical for G-protein-coupled receptor desensitization and turnover. However, β-arrestins have recently been shown to play direct roles in nonheterotrimeric G-protein signal transduction. TheCandida albicansβ-arrestin-like protein Rim8 is required for activation of the Rim101 pH-sensing pathway and for pathogenesis. We have found thatC. albicansRim8 is posttranslationally modified by phosphorylation and specific phosphorylation states are associated with activation of the pH-sensing pathway. Rim8 associated with both the receptor Rim21 and the transcription factor Rim101, suggesting that Rim8 bridges the signaling and activation steps of the pathway. Finally, upon activation of the Rim101 transcription factor,C. albicansRim8 was transcriptionally repressed and Rim8 protein levels were rapidly reduced. Our studies suggest that Rim8 is taken up into multivesicular bodies and degraded within the vacuole. In total, our results reveal a novel mechanism for tightly regulating the activity of a signal transduction pathway. Although the role of β-arrestin proteins in mammalian signal transduction pathways has been demonstrated, relatively little is known about how β-arrestins contribute to signal transduction. Our analyses provide some insights into potential roles.

scholarly journals The Pit-1/Pou1f1 transcription factor regulates and correlates with prolactin expression in human breast cell lines and tumors

2010 ◽  
Vol 17 (1) ◽  
pp. 73-85 ◽  
Author(s):  
I Ben-Batalla ◽  
S Seoane ◽  
M Macia ◽  
T Garcia-Caballero ◽  
L O Gonzalez ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Transcription Factor ◽  
Cell Lines ◽  
Independent Effect ◽  
Breast Cancer Cell Lines ◽  
Transcriptional Level ◽  
Human Breast ◽  
Small Interference Rna ◽  
Breast Invasive Ductal Carcinomas

The transcription factor Pit-1/Pou1f1 regulates GH and prolactin (PRL) secretion in the pituitary gland. Pit-1 expression and GH regulation by Pit-1 have also been demonstrated in mammary gland. However, no data are available on the role of Pit-1 on breast PRL. To evaluate this role, several human breast cancer cell lines were transfected with either the Pit-1 expression vector or a Pit-1 small interference RNA construct, followed by PRL mRNA and protein evaluation. In addition, transient transfection of MCF-7 cells by a reporter construct containing the proximal PRL promoter, and ChIP assays were performed. Our data indicate that Pit-1 regulates mammary PRL at transcriptional level by binding to the proximal PRL promoter. We also found that Pit-1 raises cyclin D1 expression before increasing PRL levels, suggesting a PRL-independent effect of Pit-1 on cell proliferation. By using immunohistochemistry, we found a significant correlation between Pit-1 and PRL expression in 94 human breast invasive ductal carcinomas. Considering the possible role of PRL in breast cancer disorders, the function of Pit-1 in breast should be the focus of further research.

scholarly journals Single cell RNA-seq uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19

2021 ◽  
Author(s):  
Marina Aznaourova ◽  
Nils Schmerer ◽  
Harshavardhan Janga ◽  
Zhenhua Zhang ◽  
Kim Pauck ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Noncoding Rna ◽  
Rna Seq ◽  
Blood Leukocytes ◽  
Antiviral Responses ◽  
Dead End ◽  
Key Drivers ◽  
Feedback Regulator

The systemic immune response to viral infection is shaped by master transcription factors such as NFκB or PU.1. Although long non-coding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA-seq approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9 - key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling characterized PIRAT as a nuclear decoy RNA, diverting the PU.1 transcription factor from alarmin promoters to dead-end pseudogenes in naive monocytes. NFκB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Our results suggest a major role of nuclear noncoding RNA circuits in systemic antiviral responses to SARS-CoV-2 in humans.

scholarly journals Regulation of the Later Stages of Nodulation Stimulated by IPD3/CYCLOPS Transcription Factor and Cytokinin in Pea Pisum sativum L.

Plants ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 56
Author(s):  
Elizaveta S. Rudaya ◽  
Polina Yu. Kozyulina ◽  
Olga A. Pavlova ◽  
Alexandra V. Dolgikh ◽  
Alexandra N. Ivanova ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Comparative Analysis ◽  
Pisum Sativum ◽  
Nodule Development ◽  
Rna Seq ◽  
Wild Type ◽  
Development Stages ◽  
Pisum Sativum L ◽  
Pea Mutant

The IPD3/CYCLOPS transcription factor was shown to be involved in the regulation of nodule primordia development and subsequent stages of nodule differentiation. In contrast to early stages, the stages related to nodule differentiation remain less studied. Recently, we have shown that the accumulation of cytokinin at later stages may significantly impact nodule development. This conclusion was based on a comparative analysis of cytokinin localization between pea wild type and ipd3/cyclops mutants. However, the role of cytokinin at these later stages of nodulation is still far from understood. To determine a set of genes involved in the regulation of later stages of nodule development connected with infection progress, intracellular accommodation, as well as plant tissue and bacteroid differentiation, the RNA-seq analysis of pea mutant SGEFix--2 (sym33) nodules impaired in these processes compared to wild type SGE nodules was performed. To verify cytokinin’s influence on late nodule development stages, the comparative RNA-seq analysis of SGEFix--2 (sym33) mutant plants treated with cytokinin was also conducted. Findings suggest a significant role of cytokinin in the regulation of later stages of nodule development.

scholarly journals Recurrent Non-Coding Mutations in BCL7A May Have Significant Functional Consequence in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 857-857
Author(s):  
Chandraditya Chakraborty ◽  
Eugenio Morelli ◽  
María Linares ◽  
Kenneth C. Anderson ◽  
Mehmet Kemal Samur ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Viability ◽  
Cell Lines ◽  
Chromatin Remodeling ◽  
Colony Formation ◽  
Ectopic Expression ◽  
Advisory Board ◽  
Mass Spectrometry Analysis ◽  
Rna Seq

Multiple myeloma (MM) is a complex hematological malignancy characterized by gene pathway deregulations. Initial sequencing approaches have failed to identify any single frequent (>25%) mutation in the coding genome. We, therefore performed a deep (average coverage > 80X) whole genome sequencing (WGS) on 260 MM samples (208 newly diagnosed and 52 first relapse after uniform treatment) to comprehensively identify recurrent somatic alterations in non-coding regions. We have identified the most frequently involved genes affected by perturbation in neighboring non-coding region and integrate their expression using our matching deep RNA-seq data from the same patients. One of the most prominent examples is mutations in the 5' untranslated region and intron 1 of the BCL7A gene in 76% of myeloma patients. Integration of WGS with RNA-seq data confirmed significant downregulation of its expression (p values < 1e-5) in the MM cells as compared to normal plasma cells (PC). This led us to investigate the consequences of BCL-7A loss in MM. To evaluate the role of BCL7A in MM, using gain of- (GOF) and loss-of-function (LOF) approaches, we have utilized a large panel of MM cell lines with differential expression of BCL7A at the RNA and protein levels. Ectopic expression of BCL7A in a panel of 3 MM cell lines with low basal levels of BCL7a significantly reduced cell viability and colony formation over time. Inhibition of cell viability was associated with induction of apoptotic cell death in the BCL7A overexpressing cells compared to control cells. LOF studies in 3 MM cell lines with relatively higher expression of BCL7a using 3 BCL7A-specific shRNA constructs showed a more proliferative phenotype, with increased growth and viability and enhanced colony formation. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7a-KO cells had higher proliferative rate compared to WT cells and add back of lentiviral BCL7a plasmid reversed this effect. BCL7A is part of the SWI/SNF chromatin remodeling complex. Mutations in the genes encoding m-SWI/SNF subunits are found in more than 20% of human cancers, with subunit- and complex-specific functions. We confirmed that when expressed, BCL7A interacts with BCL11A into the SWI/SNF complex in MM cells. Comparative, mass spectrometry analysis in fact revealed SMARCC2 (BAF170), an integral subunit of SWI/SNF complex, to bind with BCL7A-BCL11A complex. However, BCL7A loss causes decreased SMARCC2 incorporation into SWI/SNF, thus suggesting that presence of BCL7A is crucial in the formation of SWI/SNF complex in MM cells and might play an important role in chromatin remodeling. Interestingly, oncogenes DEK (DNA binding oncogene) and TPD52 (tumor protein D52) involved in cancer cell proliferation and chromatin remodeling formed complex with BCL11A in BCL7A KO MM cells. Additionally, several anti-apoptotic proteins such as ANXA-1 and BCL2 are in complex with BCL11A when BCL7A is lost, suggesting the formation of an anti-apoptotic complex with consequences on MM cell survival. Currently ongoing studies are investigating the molecular mechanism of non-coding mutations impacting BCL7A expression and pathways affected by its downregulation with impact on MM cell growth and survival. In conclusion, we report biological consequences of a frequent (>75% patients) non-coding mutation in MM with cellular and molecular effects of BCL7A loss in which implicates a functional role of the m-SWI/SNF complex in driving a MM cell proliferative phenotype. Disclosures Anderson: Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder . Munshi:Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

scholarly journals MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Mayara S. Bertolini ◽  
Miguel A. Chiurillo ◽  
Noelia Lander ◽  
Anibal E. Vercesi ◽  
Roberto Docampo
Keyword(s):  
Trypanosoma Cruzi ◽  
Cell Lines ◽  
Citrate Synthase ◽  
Host Cells ◽  
Mitochondrial Calcium ◽  
Content Type ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Eukaryotic Supergroup

ABSTRACT The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles. IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.

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