CRISPRa Analysis of Phosphoinositide Phosphatases Shows That TMEM55A Is a Positive Regulator of Autophagy

Abstract

Transcriptional activation, based on Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) and known as CRISPR activation (CRISPRa), is a specific and safe tool to upregulate endogenous genes. Therefore, CRISPRa is valuable not only for analysis of molecular mechanisms of cellular events, but also for treatment of various diseases. Regulating autophagy has been proposed to enhance effects of some therapies. In this study, we upregulated genes for phosphoinositide phosphatases, SACM1L, PIP4P1, and PIP4P2, using CRISPRa, and their effects on autophagy were examined. Our results suggested that TMEM55A/PIP4P2, a phosphatidylinositol-4,5-bisphosphate 4-phosphatase, positively regulates basal autophagy in 293A cells. Furthermore, it was also suggested that SAC1, a phosphatidylinositol 4-phosphatase, negatively regulates basal autophagic degradation.

INTRODUCTION

Epigenomic regulation using the Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system can activate or inhibit transcription of guide RNA (gRNA)-targeted genes. In this system, an endonuclease-dead mutant of spyCas9 (dCas9) is fused to transcriptional activation or repression domains to target promoter regions of genes of interest (GOI) with specific gRNAs. The transcriptional activation system, known as CRISPR activation (CRISPRa), is expected to have more desirable effects than overexpression of exogenous genes, since it activates endogenous transcriptional machinery to promote expression of gene variants specific for target cells. Recently, therapeutic applications of CRISPRa have been proposed for retinitis pigmentosa, Parkinson’s disease, obesity, and cancer.^1–4)

Autophagy is one of the catabolic pathways in which double-membranous autophagosomes encapsulate cytosolic components, such as cytosolic molecules and organelles and then fuse with lysosomes to generate autolysosomes. Cellular components in autolysosomes are degraded by lysosomal hydrolases. Autophagy is needed to maintain organismal homeostasis.⁵⁾ Defects in autophagy may have carcinogenic consequences, inducing neurodegenerative diseases.^6,7) Bacteria entered host cells are eliminated by antibacterial autophagy, termed xenophagy.⁸⁾ Some viruses, such as Middle East respiratory syndrome–related coronavirus and severe acute respiratory syndrome coronavirus 2, suppress autophagic activity to escape lysosomal degradation in host cells.^9,10) Advanced cancer cells upregulate autophagic activity to resist chemotherapy. On the other hand, autophagy enhances the effect of immunotherapy by promoting immune responses.^6,11) Consequently, proper regulation of autophagic activity is valuable for therapies of many diseases.

Phosphoinositides regulate many steps of autophagy.¹²⁾ At the onset of autophagy, phosphatidylinositol 3-phosphate (PI(3)P) is produced on the cytosolic surface of endoplasmic reticulum (ER) membranes, mainly by the class III PI 3-kinase complex.¹³⁾ PI(3)P recruits effector proteins to induce elongation of small template membranes, termed isolation membranes or phagophores, scission of omegasomes, and closure of phagophores to generate autophagosomes.^14,15) It has been suggested that PI(4)P, controlled by PI 4-kinases, 4-phosphatase, and lipid transfer proteins, regulates autophagosome-lysosome fusion.^16,17) To promote autophagy flux, lysosome reformation is also important. This process is thought to require PI(4,5)P2, produced by PIP5Kγ.¹⁸⁾

Previous reports showed that PI(4,5)P2 4-phosphatase, TMEM55B (PIP4P1), but not its isoform TMEM55A (PIP4P2), accelerates clustering of lysosomes to increase lysosomal degradation.^19,20) However, it was also suggested that overexpressed TMEM55B suppresses phagosome-lysosome fusion.²⁰⁾ In this study, we upregulated expression of endogenous TMEM55s gene using the CRISPRa system in 293A cells. As a positive control, we also examined effects of transcriptional activation of the gene for the PI(4)P phosphatase, SAC1, using CRISPRa, as it was reported to be a regulator of autophagy.¹⁷⁾ We upregulated expression of these genes with single-guide RNAs (sgRNAs), effectiveness of which was evaluated by in vitro cleavage assays in advance. Unexpectedly, our results suggest that TMEM55A, but not TMEM55B, is a positive regulator of basal autophagy.

MATERIALS AND METHODS

Cell Culture and Transfection

293A cells (Thermo Fisher Scientific, Waltham, MA, U.S.A.) were cultured in D-MEM medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum and penicillin/streptomycin in a 37 °C incubator in a humidified, 5% CO₂ atmosphere. Four micrograms of polyethylenimine (PEI)-MAX (Polysciences, PA, U.S.A.) were diluted with 50 µL of Opti-MEM (Thermo Fisher Scientific). Zero point seven micrograms of pcDNA3-3xNLS-FLAG-dCas9-VPR with or without 0.4 µg of sgRNAs were also diluted with an equal amount of Opti-MEM. Diluted PEI and DNA solutions were mixed and incubated for 15 min at room temperature. 293A cells were grown on a 24-well plate, and culture medium was replaced with 150 µL of antibiotic-free medium. Then 100 µL of the PEI-DNA mixture was added. After 1.5 h, medium was replaced with fresh complete medium, and these cells were cultured for 48 h. For purine starvation, medium was replaced with fresh medium containing 10 µM mycophenolate (MPA, Tokyo Kasei, Tokyo, Japan) 48 h after transfection, and cells were then cultured for 24 h.

RNA Extraction and RT-Quantitative PCR (qPCR)

Total RNA was extracted from 293A cells with Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan). First-strand cDNA was synthesized from total RNA by reverse transcription using ReverTra Ace reverse transcriptase (TOYOBO, Osaka, Japan) and random primers. RT-qPCR analysis of the resulting cDNA was conducted using KOD SYBR qPCR Mix (TOYOBO) and primers listed in Supplementary Table S3 with a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Relative mRNA expression levels were determined using the delta–delta Ct method.

Western Blotting

293A cells grown on a 24-well plate were washed with phosphate-buffered saline (PBS), and solubilized in NP-40 lysis buffer (25 mM Tris–HCl (pH 7.6), 100 mM NaCl, 1 mM ethylenediaminetetra-acetic acid (EDTA), 1% Nonidet P-40, 30 mM sodium fluoride, 1 mM sodium orthovanadate, 200 µM phenylmethylsulfonyl fluoride, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, U.S.A.). Then they were mixed with 5× sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer, as described previously.²¹⁾ Proteins were separated by SDS-PAGE using NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific) and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, U.S.A.). Membranes were treated with 5% skim milk, and incubated with the indicated antibodies for LC3 (LC3B Antibody, NB100-2220, Novus Biologicals, CO, U.S.A.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (anti-GAPDH monoclonal antibody (mAb), M171-3, MBL, Tokyo, Japan). After washing, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using the Clarity Western ECL Substrate (BIO-RAD, Hercules, CA, U.S.A.).

Immunofluorescence Analysis

293A cells in glass-bottom dishes were fixed with 4% paraformaldehyde-contained PBS. After washing with PBS, cells were incubated with TBS containing 3% BSA and 0.02% Triron X-100 for 30 min and further incubated with anti-LC3B mouse mAB (Cosmo Bio, Tokyo, Japan) and anti-flag rabbit polyclonal antibody (MBL) overnight. After washing with TBS containing 0.1% Tween 20, cells were incubated with a AlexaFluor488-labeled anti-mouse immunoglobulin G (IgG), AlexaFluor647-labeled anti-rabbit IgG (Cell Signaling, Danvers, MA, U.S.A.) and 4′,6-diamidino-2-phenylindole (DAPI) for 1 h. Microscopic analysis was performed using a Keyence BZ-9000 equipped with a CFI Plan Apo VC60xH lens (Keyence, Osaka, Japan).

Statistical Analysis

The statistical significance of CRISPRa effects in Western blotting was determined by two-tailed Student’s t-tests for each data set with the corresponding controls without sgRNA. Data flagged with one asterisk or two asterisks have values of * p < 0.05 or ** p < 0.01, respectively.

The statistical analysis on formation of LC3-positive structures was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test in GraphPad Prism. Data flagged with two asterisks have values of ** p < 0.01.

RESULTS

In Vitro Evaluation of sgRNA Activity

It is important to use specific, effective gRNAs to recruit the transcription activation domain on the promoter region of a target gene. We selected three to four gRNA candidates for each promoter region of genes of interest using a web program, CRISPOR (http://crispor.tefor.net/).²²⁾ Since the predicted efficiency of gRNA in the program is not often reflected in the actual output, which may depend on the sequence contexts of target sites, gRNA efficacy was evaluated by in vitro DNA digestion using designed sgRNA and Cas9 complexes.²³⁾ Measured cleaving activity appeared consistent with the affinity of the sgRNA-Cas9 complex for the target site. We succeeded in identifying multiple effective sgRNAs for each promoter region (Supplementary Fig. S1).

Transcriptional Activation by CRISPRa with the Most Effective sgRNAs

We next evaluated transcriptional activation of each gene induced by CRISPRa using the identified sgRNAs in 293A cells. dCas9-VPR, which consisted of dCas9 and three transcriptional activation domains, VP64-p65-Rta, was employed for CRISPRa.^24,25) Levels of mRNA of target genes in cells transfected with plasmids encoding dCas9-VPR and/or sgRNAs were evaluated. To evaluate multiplexed gRNA effects, combinations of identified gRNAs for each gene were also tested. To activate the SAC1 gene (SACM1L), sgRNA2, sgRNA3 and their combination were tested, and sgRNA2 alone was most effective (Fig. 1A). Activations of the TMEM55A gene (PIP4P2) and the TMEM55B gene (PIP4P1) were also performed utilizing four and three identified sgRNAs, respectively. TMEM55A activation was most effective when sgRNA2 and sgRNA3 were used in combination (Fig. 1B). In case of TMEM55B, a combination of sgRNA1 + sgRNA4 show comparable activation with a combination of all gRNA (1–4) (Fig. 1C). The most effective gRNA or combinations were utilized to examine effects of gene activation on autophagy.

Fig. 1. Transcriptional Activation by CRISPRa with Effective sgRNAs for SAC1 (A), TMEM55A (B), and TMEM55B (C)

293A cells were transfected with pcDNA3-flag-dCas9-VPR and indicated sgRNA expression vectors using PEI-MAX reagent. After 48 h, total RNA was extracted, and cDNA was synthesized. Relative mRNA expression levels were analyzed with real-time qPCR using GOI-specific primers. Data are shown as means ± standard deviation (S.D.) (n = 4), except for gRNA1 + 4 and gRNA1 + 2 + 3 + 4 in PIP4P1, which show average of duplicated experiments.

Effect of Transcriptional Activation of Phosphoinositide Phosphatase Genes on LC3-II Levels

Protein levels of LC3-II, a phosphatidylethanolamine-conjugated form of LC3, is correlated with the number of autophagosomes.²⁶⁾ Autophagosome synthesis in a certain period can be estimated by LC3-II level in the presence of the vacuolar H⁺-ATPase inhibitor, Bafilomycin A1 (BafA1).²⁷⁾ By utilizing BafA1, effects on autophagic degradation, not on autophagosome formation, in autophagic flux can be addressed. When the LC3-II levels are increased without BafA1, but they are not changed in the presence of BafA1, it could indicate that the autophagic degradation is suppressed. The basal level of LC3-II in SAC1-upregulated cells was higher than that in control cells (Fig. 2A). Autophagosome synthesis in SAC1-upregulated cells under treatment with BafA1 also tended to be higher than in control cells, but not significantly (Fig. 2A). The levels of p62/SQSTM1, which is an autophagy receptor,²⁸⁾ were also examined to address autophagic degradation. The amount of p62/SQSTM1 was increased when SAC1 gene was upregulated, but it remained unchanged in the presence of BafA1 (Supplementary Fig. S2A). These results imply that excess SAC1 suppresses autophagic degradation under basal conditions. Unexpectedly, in TMEM55A-upregulated cells, autophagosome synthesis was significantly higher than in control cells. In addition, the basal level of LC3-II tended to be higher than in control cells (Fig. 2B). The results indicate that TMEM55A is a positive regulator of autophagy. The level of p62/SQSTM1 in the TMEM55A-upregulated cells tended to be higher than in control cells when treated with BafA1, but did not change in the absence of BafA1 (Supplementary Fig. S2B). The result may indicate that autophagic degradation is not suppressed by upregulation of TMEM55A. On the contrary, transcriptional activation of TMEM55B, which has been thought to regulate autophagic degradation, did not influence the basal level of LC3-II and autophagosome synthesis (Fig. 2C). Purine starvation, attributed to inhibition of de novo synthesis, induces inactivation of mTORC1 to promote autophagy.^29,30) We also examined the effect of upregulation of phophoinositide phosphatases on purine starvation-induced autophagy. Upon treatment with mycophenolate (MPA), an inhibitor of inosine monophosphate dehydrogenase,³¹⁾ LC3-II levels tended to increase in the presence or absence of BafA1 (Figs. 2A–C). Autophagosome formation in the presence of MPA tended to decrease in both SAC1- and TMEM55A-upregulated cells, but not significantly (Figs. 2A, B). The aforementioned data imply that these phosphatases may inhibit MPA-elicited signals that promote autophagy. When treated with MPA, TMEM55B-upregulated cells showed slight decrease of the LC3-II and the p62/SQSTM1 levels in the absence of BafA1, but did not in the presence of BafA1 (Fig. 2C, Supplementary Fig. S2C). The results may indicate that TMEM55B slightly enhances autophagic degradation because the result in the presence of BafA1 indicate that TMEM55B upregulation does not affect to autophagosome synthesis induced by MPA.

Fig. 2. Effect of Transcriptional Activation of Phosphoinositide Phosphatase Genes, SAC1 (A), TMEM55A (B), and TMEM55B (C), on LC3-II Levels

293A cells were transfected with pcDNA3-flag-dCas9-VPR and indicated sgRNA expression vectors using PEI-MAX reagent. After 48 h, cells were incubated in the presence or absence of 10 µM mycophenolate (MPA) for 22 h. Then, they were further incubated in the presence or absence of 0.1 µM BafA1 for 2 h. Cell lysates were analyzed by Western blotting. Representative blots from 3 experiments are shown. LC3-II protein levels were normalized against GAPDH. Values relative to those of control cells treated with Bafilomycin A1 are shown as means ± S.D. (n = 3).

Effect of Transcriptional Activation of Phosphoinositide Phosphatase Genes on LC3-Positive Structure Formation

Since basal autophagy was affected by upregulation of SAC1 and TMEM55A genes, LC3-positive structure (autophagosome) formation was further examined. When the SAC1 gene was upregulated, the number of LC3-positive structures tended to increase in untreated cells, but not in BafA1-treated cells. The result may also support inhibitory effect of excess SAC1 on autophagic degradation (Fig. 3). In TMEM55A-upregulated cells, LC3-positive structures clearly increased regardless of the presence or absence of BafA1 (Fig. 3). This result is consistent with the above results (Fig. 2), and indicates that TMEM55A is a positive regulator of basal autophagy flux in 293A cells.

Fig. 3. Effect of Transcriptional Activation of Phosphoinositide Phosphatase Genes on Formation of LC3-Positive Structures

293A cells were transfected with pcDNA3-flag-dCas9-VPR and indicated sgRNA expression vectors using PEI-MAX reagent. After 48 h, cells were incubated in the presence or absence of 0.1 µM BafA1 for 2 h. Cells were fixed and immunostained with indicated primary antibodies and corresponding fluorescent secondary antibodies, and counterstained with DAPI. (A) Representative images from 3 experiments are shown. Scale bars show 10 µm. (B) Quantification of LC3 in cells. Intensities of green fluorescence in flag-dCas9-VPR expressed cells were quantified and are shown relative to those of control cells. For each experimental condition, 15 cells were analyzed. Data are shown as means ± S.D. (n = 15).

DISCUSSION

CRISPRa is a powerful and safe tool to upregulate endogenous genes in a specific manner without altering genes.³²⁾ Furthermore, it can target even dormant genes in cells of interest. Therefore, transcriptional activation of endogenous genes using CRISPRa is valuable for analysis of molecular mechanisms of cellular events and should be useful to treat various diseases.^1–4,33) We upregulated genes of three phosphoinositide phosphatases, and found that two, SAC1 and TMEM55A, are involved in basal autophagy flux.

It has been reported that SAC1 deficiency prevents autolysosome formation in yeast and HeLa cells.¹⁷⁾ Although we predicted that upregulation of SAC1 would accelerate autolysosome formation, excess SAC1 slightly suppressed autophagic degradation (Fig. 3). One of the PI 4-kinase isoforms, PI4K2A, produces PI(4)P on autophagosomes and is required for autolysosome formation.^18,34) These previous results indicate that a proper level of PI(4)P balanced by PI4K2A and SAC1 is probably important for autolysosome formation. As shown in this study, excess SAC1 may associated with reduction of PI(4)P levels and resulted in suppression of autolysosome formation.

TMEM55A and TMEM55B, also known as PIP4P2 and PIP4P1, respectively, are PI(4,5)P2 4-phosphatases. Their amino acid sequences are 51% identical in humans. TMEM55B facilitates lysosome clustering to enhance lysosomal degradation, whereas TMEM55A does not.¹⁹⁾ In this study, upregulation of TMEM55B did not affect basal autophagy flux. On the other hand, TMEM55A gene upregulation enhanced basal autophagy flux (Figs. 2, 3), indicating TMEM55A has a role in inducing autophagy rather than autophagic degradation. These results in the present study may have been obtained due to increased expression of TMEM55s by endogenous transcriptional activation using CRISPRa. It has been suggested that PI(5)P promotes autophagosome formation via PI(3)P-interacting proteins.³⁵⁾ PI(4,5)P2 phosphatase activity of TMEM55A was previously suggested to be higher than that of TMEM55B in cells.³⁶⁾ The result may indicate that TMEM55A promotes autophagosome formation by producing PI(5)P. In order to confirm these mechanisms, further investigations examining the dependence of TMEM55A mRNA and protein levels on the amounts of autophagosome and PI(5)P levels are necessary.

Autophagy defects or enhancement occurs in various diseases.^6,7,9,10) It has also been proposed that regulation of autophagy potentiates effects of some therapies.^6,11) Thus, this study suggests that CRISPRa could be a future therapeutic choice for autophagy-related diseases.

Acknowledgments

We thank Dr. Steven D. Aird for English language editing. This work was supported in part by the New Energy and Industrial Technique Development Organization (NEDO) of Japan, the Japan Society for the Promotion of Science (JSPS) KAKENHI (JP22H02201, JP19H02827 and JP20K21253 to WN, JP21K14740 to DM), and funding from Core Research for Organelle Diseases at Hiroshima University (the MEXT program to enhance Japanese research universities), HIRAKU-Global Program which is funded by MEXT’s “Strategic Professional Development Program for Young Researchers” (to DM), funding from Takeda Science Foundation, the Naito Foundation, the Uehara Memorial Foundation, the Mochida Memorial Foundation, and the Suzuken Memorial Foundation (to WN).

Author Contributions

KN and WN conceived and designed the study. KN, YF, CS, DM conducted experiments and data analysis. WN supervised the project. KN and WN wrote the manuscript and all authors reviewed the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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