ESX1 gene as a potential candidate responsible for male infertility in nonobstructive azoospermia

Infertility is a problem that affects approximately 15% of couples, and male infertility is responsible for 40–50% of these cases. The cause of male infertility is still poorly diagnosed and treated. One of the prominent causes of male infertility is disturbed spermatogenesis, which can lead to nonobstructive azoospermia (NOA). Whole-genome sequencing (WGS) allows us to identify novel rare variants in potentially NOA-associated genes, among others, in the ESX1 gene. The aim of this study was to activate the ESX1 gene using CRISPRa technology in human germ cells (testicular seminoma cells—TCam-2). Successful activation of the ESX1 gene in TCam-2 cells using the CRISPRa system was achieved, and the expression level of the ESX1 gene was significantly higher in modified TCam-2 cells than in WT cells or the negative control with nontargeted gRNA (p < 0.01). Using RNA-seq, a network of over 50 genes potentially regulated by the ESX1 gene was determined. Finally, 6 genes, NANOG, CXCR4, RPS6KA5, CCND1, PDE1C, and LINC00662, participating in cell proliferation and differentiation were verified in azoospermic patients with and without a mutation in the ESX1 gene as well as in men with normal spermatogenesis, where inverse correlations in the expression levels of the observed genes were noted.

but more closer to the end of this region (p.Pro365Arg and p.Leu366Val) and found that the compound variant compromised the stabilizing effect of ESX1 on cyclin A, thereby causing the failure of M phase arrest in cells 12 .
The human ESX1 gene is present on the X chromosome at position Xq22.2 and has 4 exons.It is a homeotic gene that is only expressed in the male gonad of adult males 13,14 .This gene, which encodes a dual-function 65 kDa protein, contains a homeodomain in the N-terminal fragment and a proline-rich region in the C-terminal fragment 15 .It has been reported that the ESX1 transcript in the semen of men with NOA is a suitable molecular marker for predicting the presence of residual foci of spermatogenesis in the testis 16 .Moreover, ESX1 may inhibit cyclin degradation 15 and may be involved in the regulation of cell cycle progression during human spermatogenesis 12 .
Here, we present studies of the ESX1 gene using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activation system in the testicular seminoma cell line TCam-2 as the first step to investigate its potential function regarding gene cluster regulated by ESX1.In this study we also shown that the previously identified variants lead to amino acid sequence changes in the proline-rich repeat region (PRR) of the ESX1 protein which may affect the correct folding of the protein.As PRRs have been observed to bind the major groove of DNA, the identified mutations: c.1040C > G (p.Pro347Arg) and c.1042C > G (p.Leu348Val) are predicted to affect the overall regular helical structure inherent in the PRR of ESX1, thereby potentially affecting sequencespecific interactions.

Sanger validation of identified SNVs (single nucleotide variants) from WGS
In our previous published studies using whole genome sequencing-WGS analysis, we identified two ultrarare variants in the ESX1 gene (c.1040C> G, c.1042C > G) presented as compound mutation in 2 patients with NOA at the postmeiotic arrest phase, as detailed by Malcher et al. 11 .Using Sanger sequencing, we confirmed the obtained SNVs (single nucleotide variants) from WGS analysis of these patients (Supplementary Fig. 1).

Structural protein analysis of ESX1
The three-dimensional structural model of the homeobox protein ESX1 generated by the machine-learning-based AlphaFold (Deepmind Technologies, Ltd.) 17 indicates several regions of disorder surrounding the confidently modelled homeobox domain (data not shown).The residues of interest fall within a C-terminal proline-rich region consisting of a nine-residue P-P-x-x-P-x-P-P-x motif repeated fifteen times 15,18 , spanning amino acids 244-378 (Fig. 1A).We used ColabFold AlphaFold2 and ColabFold AlphaFold2_advanced_beta to model this region 17,19 .As expected, both programs generated polyproline II-type structures (Fig. 1B,C and Supplementary Fig. 2).This secondary structure has three amino acids per left-hand turn, creating a fairly rigid structural element 20,21 .Proline-rich regions foster protein-protein interactions, including the recruitment, stabilization, and scaffolding of protein complexes 20,21 .Consistent with these findings, ColabFold AlphaFold2 predicted regions of disorder surrounding the homeobox domain of ESX1 17 , although disordered regions are difficult to predict accurately.
The p.Pro347Arg/p.Leu348Val compound mutation could affect several aspects of the larger structure.First, it inverts the surface potential of the region from negative to positive (Fig. 1C).The atomic shape of this area of the polyproline helix would also change; the bulky side chain of proline is topologically linked to the polypeptide backbone, limiting the rotational space that the residue can occupy.Thus, these mutations would be expected to affect its ability to interact with binding partners.

Localization of the ESX1 gene in human testis
Immunofluorescence analysis of control testicular tissue showed that ESX1 protein is produced mostly in the nucleus of spermatogonia in normal adult human testis (Fig. 2A-D).In the co-staining of ESX1 with UTF1, most of the nuclear UTF1-positive undifferentiated spermatogonia presented strongly stained ESX1 protein, however, there were also cells strongly positive only for ESX1 or exclusively for UTF1 (Fig. 2B).In the case of ESX1 costained with MLH1, we observed mostly the same positive-stained nuclear spermatogonia for both proteins and only some were strongly positive exclusively for ESX1 or for MLH1 protein (Fig. 2C).Double staining of the ESX1 and c-KIT proteins presented cytoplasmatic localization for c-KIT and nucleus for ESX1 in differentiated spermatogonia, but also there were some cells positive only for ESX1 protein (Fig. 2D).The c-KIT also showed a strong expression in Leydig cells.

Activation of the ESX1 gene by transfection of TCam-2 cells with the CRISPRa system
The transfection efficiency was estimated after antibiotic selection and induction with doxycycline (after 72 h) and was approximately 40% of GFP-positive signal for both samples-TCam-2 with the activated ESX1 gene-35% and the negative control-TCam-2 with nonspecific gRNAs (guide RNAs) for the human genome-42% (Fig. 3A).ESX1 gene activation was determined after 72 h at the mRNA level using real-time PCR (Fig. 3B) and at the protein level using Western blot and immunofluorescence staining (Fig. 3C,D).We observed significant upregulation (p < 0.001) of the ESX1 gene in TCam-2 samples with the activated ESX1 gene (marked as ESX1) in comparison to both control groups-TCam-2 wild type (WT) and the negative control, which was TCam-2 with nonspecific gRNAs for the human genome (NC) (Fig. 3B).We also confirmed ESX activation at the protein level in Western blot analysis, where we observed the presence of the ESX1 protein, while in wild-type and negative controls, the protein was absent (Fig. 3C).Additionally, by using immunofluorescent staining, we observed the presence of ESX1 protein with very strong (47%) and weak signals (53%) in the nuclei of modified cells with gRNA for the ESX1 gene, while in both applied controls, no protein signal was detected (Fig. 3D).

Verification of gene expression levels in NOA patients
We also verified the 13 selected differentially expressed genes (CCND1, HMGA2, KDR, L1TD1, LGALS3, MKRN1, NANOG, PDE1C, WNT11, CXCR4, FGF4, LINC00662, and RPS6KA5) in NOA patients by real-time PCR.CCND1, HMGA2, PDE1C, and LINC00662 were downregulated (p < 0.05), and LGALS3, NANOG, WNT11, CXCR4 and RPS6KA5 were upregulated (p < 0.05) in the male gonad tissue from an azoospermic patient with a mutation in the ESX1 gene in comparison to an azoospermic patient without mutation in the ESX1 gene as well as to control samples with normal spermatogenesis (Fig. 6).The KDR, MKRN1, and FGF4 genes showed no significant changes in NOA patient with mutations in the ESX1 gene compared to the other samples (Fig. 6).

Discussion
The genetic causes of nonobstructive azoospermia are not yet well known; therefore, there is still a need to study the human genome to identify the gene variants that could be responsible for NOA.In recent years, due to the development of new NGS and genome editing technologies, new variants of genes that are potentially responsible for NOA have been identified.One such finding is the ESX1 gene, of which novel, ultrarare variants (c.1040C > G, c.1042C > G) manifesting as a compound mutation were discovered by our group in whole-genome sequencing analysis in men with nonobstructive azoospermia at postmeiotic arrest 11 .Using Sanger sequencing, we confirmed the SNVs obtained from WGS analysis for these patients (Supplementary Fig. 1).
Commonly found in cytoskeletal proteins, HOX proteins and zinc-finger transcription factors, the polyproline-II helix binds to specific protein folds, including SH3, WW, GYF, EUV, profilin, and EVH1 domains [20][21][22][23] .Examination of several proteomic databases for ESX1-interacting proteins revealed one SH3-domain-containing candidate protein, Dlg4 [24][25][26] .This cytoplasmic protein plays a role in neuronal signalling; however, this interaction has limited relevance here.It is possible that other ESX1 interactors could recruit other factors with affinity for the polyproline region of ESX1.The C-terminal end of the polyproline region of ESX1 has two types of proline-rich repeats (Fig. 1A): one containing a proline and leucine and one containing an arginine and valine.The compound mutation of p.Pro347Arg/p.Leu348Val substitutes the former type of repeat for the latter one.This substitution could potentially change the global structure of the region.These repeats could form molecular barriers, recruiting some binding partners and eliminating others.Therefore, mutation of the core residues of the mutated repeat likely shifts this putative molecular barrier for aligning molecules in three-dimensional space, which would be expected to destabilize or even eliminate functional complexes.
Summarizing the co-staining of ESX1 protein with each specific spermatogonia marker, such as: UTF1, MLH1 and c-KIT, we observed that ESX1 protein is presented in state 1 through 4 spermatogonia (Fig. 2).The co-staining of ESX1 with UTF1, which is a specific marker in state 0 and 1 undifferentiated spermatogonia, demonstrated the positive signal for both proteins in state 1.The spermatogonia in state 0 were positive only for UTF1 protein and negative for ESX1, which suggests that the expression of ESX1 protein starts in state 1 spermatogonia.The positive signals of ESX1 in MLH1-and c-KIT-positive cells were a strong evidence that ESX1 is also presented during further states of spermatogonia.The multiplex immunofluorescence staining of testis from the Human Protein Atlas database shows nuclear localization in state 1 and 4 spermatogonia as well as preleptotene spermatocytes for MLH1 protein and cytoplasmic localization in state 2-3 spermatogonia for c-KIT protein.The double staining of ESX1 and MLH1 proteins showed mostly positive signals for the same cells, suggesting that ESX1 is presented in state 1 and 4 spermatogonia, but not at the spermatocytes stage, where only www.nature.com/scientificreports/MLH1-signal was strong.Whereas, the co-staining of ESX1 with c-KIT-a specific marker of spermatogonia in state 2-3, showed cytoplasmic staining for c-KIT and nucleus for ESX1 in the same cells, which indicates that ESX1 is also localized in state 2 and 3 spermatogonia.It was previously noted that Esx1 hemizygous mutant males were fertile, demonstrating that Esx1 is not essential for mouse spermatogenesis 27 .However, it was later proven that human ESX1 and mouse Esx1 proteins exhibit an overall high level of sequence divergence (34%), which concentrates mostly in the C-terminal domain implicated in the cell cycle control 28,29 .Because of these data, we decided to use a human germ cell  www.nature.com/scientificreports/model-TCam-2 cells, which do not express the ESX1 gene.Activation of the ESX1 gene in TCam-2 cells was successfully achieved at the mRNA and protein levels (Fig. 3), which allowed us to analyse by RNA-seq the general expression profile of TCam-2 cells with the activated ESX1 gene in comparison to controls-TCam-2 wild type and negative control (TCam-2 cells with nonspecific gRNAs for the human genome) (Fig. 4).We selected over 50 genes connected with spermatogenesis and/or characterized by high expression levels in the testis that were differentially expressed in ESX1-activated TCam-2 cells compared to both controls.Using STRING gene database, we checked ESX1 gene interactions, and we found that ESX1 gene directly interacts with ELF3 and NANOG.It also interacts indirectly with CLDN4, VIM, CCND1, FGF4, SQSTM1, LGALS3, FGF13, CXCR4, KDR, PTBRB, SPP1, L1TD1, ZSCAN10, HMGA2, TIMP3, OGFBP5, ADAMTS6; due to their interaction with the aforementioned ELF3 and NANOG (Supplementary Fig. 3D).Interestingly, upon observation of RNA sequencing results, we noticed that the genes taking part in the mitotic and proliferation processes, or responsible for maintaining the spermatogonia population (NANOG, FGF4, CXCR4, FGF13, VIM, L1TD1, HMGA2, ZSCAN10, ADAMTS9) were downregulated in the ESX1-activated TCam-2 cells.On the other hand, genes participating in the differentiation or occurring at later stages of spermatogenesis (ELF3, SPP1, TIMP3, KDR, WNT11, IGFBP5, PTPRB, SQSTM1, CLDN4, CCND1, CX3CL1, LGALS3) showed higher expression in TCam-2 cells with ESX1 activation in comparison to both applied controls.These results may suggest that ESX1 gene regulates the genes participating in the differentiation process and preparing the spermatogonia to transit into the next stage of spermatogenesis.As for the remaining of the genes, which were either down or upregulated by the activation of ESX1 gene but had no shown interaction in the STRING gene database, further studies should be made in order to test their possible interaction with ESX1 gene.Thus we can conclude that ESX1 gene may take part in the promotion of cell differentiation in spermatocytes and/or regulate the balance between cell proliferation and differentiation, through direct interaction with NANOG, involved in the proliferation process, and ELF3, involved in the differentiation of the cells.Functional studies are required to justify this hypothesis.We verified 13 key genes by real-time PCR due to their involvement in the cell cycle, proliferation and apoptosis and with high expression in the testes (CCND1, HMGA2, KDR, L1TD1, LGALS3, MKRN1, NANOG, PDE1C, WNT11, CXCR4, FGF4, LINC00662, and RPS6KA5) (Fig. 5).These genes were differentially expressed in cells with the activated ESX1 gene compared to controls applied, and except for the LINC00662 gene, we confirmed the results obtained from RNA-seq analysis.Therefore, as expected, CCND1, KDR, LGALS3, PDE1C, and WNT11 were upregulated (p < 0.05), and HMGA2, L1TD1, MKRN1, NANOG, CXCR4, FGF4, and RPS6KA5 were downregulated (p < 0.05) in TCam-2 cells with ESX1 activation compared to controls (Fig. 5).However, it is worth highlighting the results obtained in the male gonad tissues from a patient with azoospermia with a detected mutation in the ESX1 gene, where we noted an inverse correlation.Hence, the genes that were downregulated (NANOG, CXCR4 and RPS6KA5) in ESX1-activated TCam-2 cells in comparison to the control cells (Figs. 4, 5) were characterized by increasing expression in the patient with mutation in the ESX1 gene in comparison to the control samples (Fig. 6).Similar to genes that were upregulated (CCND1, PDE1C, and LINC00662) in the ESX1-activated cells, a decreased expression of these genes was observed in the patient with ESX1 mutation in comparison to men with normal spermatogenesis and NOA patient without ESX1 mutation.
Ultimately, 6 genes, NANOG, CXCR4, RPS6KA5, CCND1, PDE1C, and LINC00662 were identified as participants in cell proliferation and differentiation, and together with the analysis of their expression levels, provided an interesting interpretation of the ESX1 gene as promoting the process of cell differentiation in spermatocytes.The NANOG gene is a homeobox transcription factor and plays a key role in the pluripotency process.Normally, the NANOG gene is expressed in pluripotent cell lines, including embryonic stem (ES) and embryonic germ (EG) cells, and loss of NANOG causes a tendency for ES cells to differentiate 30 .In our study, activation of the ESX1 gene led to the downregulation of NANOG gene expression in TCam-2 cells (Figs. 4, 5), whereas the mutation (with compound variants) in the ESX1 gene in azoospermic humans was characterized by increased NANOG gene expression (Fig. 6).It can be suggested that the ESX1 gene impacts NANOG gene expression, causing its decrease, and dividing cells may undergo differentiation towards spermatocytes.In turn, when there is a mutation in the ESX1 gene, this process may be disturbed by maintaining increased NANOG gene expression, and thus, the balance between the division and differentiation of gametogenic cells may be disturbed.Another interesting gene that was also highlighted CCND1, which is responsible for controlling the cell cycle at the G1/S phase.In 293T cells, it was shown that the truncated CCND1 isoform did indeed promote cell proliferation and accelerated cell cycle progression 31 .In our case, the balance between the division and differentiation of gametogenic cells may also be destabilized since the mutation in the ESX1 gene affects the downregulation of the CCND1 gene (Fig. 6).The same applies to the RPS6KA5 proliferation-promoting gene 32 , which in our study showed increased expression levels in the patient with a mutation in the ESX1 gene and decreased expression in TCam-2 cells with an activated ESX1 gene.Here, it also seems that under normal conditions, the ESX1 gene potentially regulates the expression level of the RPS6KA5 gene and consequently inhibits cell proliferation, leading to the differentiation process.Our hypothesis regarding the role of the ESX1 gene in the promotion of cell differentiation in spermatocytes may also be supported by its influence on the PDE1C gene, which is expressed in germ cells from early meiotic prophase through the meiotic and postmeiotic stages 33 .In our studies, the PDE1C gene was characterized by increased expression levels in ESX1-activated TCam-2 cells (Figs. 4, 5), whereas in the NOA patient with ESX1 gene mutation, PDEC1 gene expression was decreased (Fig. 6).In turn, the CXCR4 gene, which is a crucial receptor for spermatogonial stem cell maintenance 34 , was characterized in our studies by decreased expression in TCam-2 cells with an activated ESX1 gene (Figs.4, 5), while in the patient with ESX1 gene mutation, upregulation of the CXCR4 gene was observed (Fig. 6).This finding also suggests that the ESX1 gene may regulate the process of spermatogonia proliferation and directs cells to differentiate into spermatocytes.The last selected gene is LINC00662, which is a spermatogenesis-associated lncRNA (long noncoding RNA); however, its function in spermatogenesis is still not clarified 35 .It was previously reported that LINC00662 was upregulated

Subjects
In this study, we used blood samples from 2 out of 39 NOA patients (marked as 10L and 33P).They were characterized by the compound mutation p.Pro347Arg/p.Leu348Val in the ESX1 gene, detected in our previous research using whole-genome sequencing (WGS) 11 .Moreover, for the real-time PCR verification we used RNA extracted from the testicular tissue of a NOA patient with postmeiotic arrest without mutation in ESX1 (marked as 2L) and a patient with NOA with compound mutation p.Pro347Arg/p.Leu348Val in the ESX1 gene (marked as 10L) 11 .All the details concerning patients characterization were previously described by Malcher et al. 11 .As control sample, we used commercial RNA obtained from normal testicular tissue with preserved spermatogenesis pooled from 39 Caucasians aged between 16 and 64 years (Clontech Laboratories, Mountain View, CA, USA); whereas for the immunofluorescence staining we used the commercial human testis paraffin sections (Zyagen, San Diego, CA, USA).This study was approved by the Local Bioethics Committee of Poznan University of Medical Sciences (Permission No. 1003/18), and all participants provided informed consent.All experiments were performed in accordance with relevant guidelines and regulations.

Extraction and sequencing of DNA from patient blood samples
Blood samples from 2 NOA patients with postmeiotic arrest caused by the compound mutation p.Pro347Arg/p.Leu348Val] in the ESX1 gene, which was identified by WGS analysis 11 , were used to extract DNA.We performed Sanger sequencing of the ESX1 gene for both studied patients to confirm the previously identified variants.The reaction mixture for sequencing contained 15-30 ng of DNA, 1 µl of primer for the ESX1 gene (20 µM), 2 µl of BigDye (5x) buffer, and BigDye Terminator v3.1 (Applied Biosystems Life Technologies, Carlsbad, CA, USA) in a final volume of 20 µl.The primer pairs used for the sequencing reaction are listed in Supplementary Table 2.The products were separated on an ABI Prism 310 (Applied Biosystems, Life Technologies, Carlsbad, CA, USA).Changes in the patients' sequenced ESX1 fragments were identified with respect to the reference DNA (made available in the NCBI database) using the program CLC Workbench 6.0.The ESX1 sequence of each patient was compared to the NCBI Reference Sequence GRCh37.p13:NC_000023.10103,494,719.103499614.

Structural protein modelling
The sequence of human ESX1 (Q8N693) was obtained from UniProt 18 , and residues 244-378 were searched in software representing iterative improvements of AlphaFold2, ColabFold2 AlphaFold2 and ColabFold_Alpha-Fold2_advanced_beta without relaxation 17,19 .Alignments were performed using LSQ-based alignment in Coot over the mainchain atoms 37 .Structure-based figures were made in PyMol (Version 2.0, Schrödinger, LLC).

Plasmid preparation and transfection
Three gRNAs were designed for the ESX1 gene using the CRISPOR tool (http:// crisp or.tefor.net/).Candidate sequences for CRISPRa design were selected from specific regions of the genomic sequence relative to the gene's transcription start site (TSS) at positions from − 300 to 0 bases from the TSS region.The gRNA sequences are listed in the 5′-3′ direction: gRNA_1-AAG GCA CCG TAC CGC TAT AT; gRNA_2-GAC ACG AAT GCG TCC CCC www.nature.com/scientificreports/GG; and gRNA_3-AAG GCA CCG TAC CGC TAT AT.The selected gRNAs were cloned into the vector px335_G2P, which also includes GFP sequence.We performed co-transfection with 2.5 µg of DNA mix, which consisted of the 3 different px335_G2P vectors with cloned gRNAs specific for the ESX1 gene and the PB-TRE-dCas9-VPR vector, using 3.75 µl of Lipofectamine 3000 in TCam-2 cells (passages 35-37).As a negative control we used px335_G2P vector with nonspecific gRNAs for the human genome.The vector pX335_G2P was a kind gift from Boris Greber (Max Planck Institute for Molecular Biomedicine, Münster) to the Dept.Herrmann at the Max Planck Institute for Molecular Genetics in Berlin, and PB-TRE-dCas9-VPR (Addgene #63,800, Addgene, Watertown, MA, USA) was purchased by the Max Planck Institute for Molecular Genetics in Berlin (Dept.Herrmann), which has the right to use the construct.The cells were selected with puro and doxycycline for 72 h.After 72 h, the cells were collected, and ESX1 gene expression was analysed.The experiment was done in 3 biological replicates.The transfection efficiency of the modified TCam-2 cells was confirmed on a Juli FL fluorescence microscope (NanoEnTek, Seoul, South Korea) and the percentage of positively transfected cells (GFP-positive cells) was counted with multi-point tool using ImageJ2 38 .

RNA, DNA and protein extraction
The cell or the testicular samples were lysed in 350 µl of RLT buffer with β-mercaptoethanol, and RNA, DNA and protein were extracted according to the manufacturer's protocol of the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany).The RNA samples were further purified according to the manufacturer's protocol of the Turbo DNA-free Kit (Thermo Fisher Scientific, MT, USA).At the end of the procedure, the protein was dissolved in a buffer consisting of 8 M urea, 50 mM Tris-HCl, pH 8.0, 1% SDS (1:1) and a protease inhibitor cocktail (Roche, Basel, Switzerland).

Real-time PCR
The cDNA was synthesized from 1 μg of total RNA using iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in a 20 μL reaction volume in a C1000 Touch thermocycler (Bio-Rad Laboratories, Hercules, CA, USA).Real-time PCR was performed using specific primers for the studied genes (detailed in Supplementary Table 2) with SsoAdvanced ™ SYBR ® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's protocol.The threshold cycle (Ct) values of each transcript studied were analysed with a CFX384 Touch™ Real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) using standard cycling parameters.All TCam2 cell samples-WT (n = 3 biological replicates), NC (n = 3 biological replicates), ESX1 (n = 3 biological replicates) as well as the standard curve were run in duplicate.The relative expression level of each studied transcript was normalized with reference to two housekeeping genes (β-actin and GAPDH) according to CFX Maestro software (Bio-Rad Laboratories, Hercules, CA, USA).

Figure 1 .
Figure 1.The proline-rich region of ESX1 likely forms polyproline (II) helices.(A) Alignment of the last seven repeats of the proline-rich region of ESX1.(B) Alignment of the ColabFold AlphaFold2-generated structures of the proline-rich regions of the wild-type and P347R/L348V ESX1 variants shown in ribbon representation.The N-and C-termini are labelled.Residues at positions 347 and 348 are shown as spheres.(C) Zoomed view of the surface representation of the region surrounding residues 347 and 348 modelled by ColabFold AlphaFold2.The surface is coloured by electrostatic potential as calculated in PyMOL; blue, positive; red, negative (~ − 30-30 eV).The compound mutation of p.Pro347Arg/p.Leu348Val would be expected to affect its ability to interact with binding partners.

Figure 3 .
Figure 3. Activation of the ESX1 gene using the CRISPRa system.(A) TCam-2 cells at 72 h after transfection under a Juli FL fluorescence microscope; scale bar: 100 μm.(B) Real-time PCR analysis of ESX1 gene expression.(C) Representative Western blot analysis of ESX1 protein isolated from TCam-2 cells, exposure time 300 s; and its graph of the relative quantity of the ESX1 protein normalized with reference to HPRT1 analysed by Image Lab 6.1 tools.(D) Immunofluorescence analysis of TCam-2 cells.A Leica DMi8 with a proper filter set (DAPI/TxR/Triple) was used; objectives: 40x, scale bar: 50 μm; software: LASX.Arrows indicate examples of ESX1-positive staining.The transfection efficiency after antibiotic selection and induction with doxycycline was estimated for approximately 40% for both TCam-2-cells with the activated ESX1 gene and the negative control-cells with nonspecific gRNAs (guide RNAs) for the human genome.Activation of the ESX1 gene in TCam-2 cells was successfully achieved at the mRNA level with significant upregulation (p < 0.001) of the ESX1 gene in comparison to both applied control samples-WT and NC; and that was also observed at the protein level, where the presence of the ESX1 protein was observed in TCam-2 cells with active ESX1, while in WT and NC, the protein was absent.WT wild type, NC negative control with nonspecific gRNAs for the human genome, ESX1 cells with the activated ESX1 gene using specific gRNAs for the ESX1 sequence, BR brightfield, FLU fluorescence.

Figure 4 .
Figure 4. Box-plot graphs of 52 selected genes analysed in TCam-2 cells with activated ESX1 gene and controls analysed by RNA-seq.The 33 out of the selected genes presented upregulation and 19 downregulation with a minimum of a twofold change (p < 0.05) in cells with activated ESX1 in comparison to both applied controls.WT wild type, NC negative control with nonspecific gRNAs for the human genome, ESX1 cells with the activated ESX1 gene using specific gRNAs for the ESX1 sequence.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.