PRMT5 epigenetically regulates the E3 ubiquitin ligase ITCH to influence lipid accumulation during mycobacterial infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), triggers enhanced accumulation of lipids to generate foamy macrophages (FMs). This process has been often attributed to the surge in the expression of lipid influx genes with a concomitant decrease in those involved in lipid efflux. Here, we define an Mtb-orchestrated modulation of the ubiquitination of lipid accumulation markers to enhance lipid accretion during infection. We find that Mtb infection represses the expression of the E3 ubiquitin ligase, ITCH, resulting in the sustenance of key lipid accrual molecules viz. ADRP and CD36, that are otherwise targeted by ITCH for proteasomal degradation. In line, overexpressing ITCH in Mtb-infected cells was found to suppress Mtb-induced lipid accumulation. Molecular analyses including loss-of-function and ChIP assays demonstrated a role for the concerted action of the transcription factor YY1 and the arginine methyl transferase PRMT5 in restricting the expression of Itch gene by conferring repressive symmetrical H4R3me2 marks on its promoter. Consequently, siRNA-mediated depletion of YY1 or PRMT5 rescued ITCH expression, thereby compromising the levels of Mtb-induced ADRP and CD36 and limiting FM formation during infection. Accumulation of lipids within the host has been implicated as a pro-mycobacterial process that aids in pathogen persistence and dormancy. In line, we found that perturbation of PRMT5 enzyme activity resulted in compromised lipid levels and reduced mycobacterial survival in mouse peritoneal macrophages (ex vivo) and in a therapeutic mouse model of TB infection (in vivo). These findings provide new insights into the role of PRMT5 and YY1 in augmenting mycobacterial pathogenesis. Thus, we posit that our observations could help design novel adjunct therapies and combinatorial drug regimen for effective anti-TB strategies.

Introduction Mycobacterium tuberculosis (Mtb), the principal etiological agent of the pulmonary ailment, tuberculosis (TB), continues to co-evolve with the human population making itself one of the major infectious diseases afflicting mankind. Globally in 2020, there were an estimated 1.5 million deaths due to TB, a steep increase, following the onset of the COVID-19 pandemic (WHO, Annual TB Report, 2021).
Upon infecting the host, Mtb coordinates the formation of a highly organized immune structure-the granuloma [1]. TB granulomas are constituted by macrophages, neutrophils, monocytes, dendritic cells, B-and T-cells, fibroblasts, and epithelial cells [2,3]. Anatomical dissection into the distinct features of the macrophages within the TB granuloma has revealed accumulated lipids as one of the leading physiological countenances. Also referred to as foamy macrophages (FM), these cells act as a protective niche wherein the bacterium thrives and persists. Mtb deregulates the expression of host molecules to trigger increased lipid intake into the cells with a concomitant decrease in the efflux of lipids-thereby contributing to a lipid-rich environment [4]. While Mtb-mediated regulation of key host molecules that contribute to FM formation has been studied at the transcript level; there are scant reports on their post-translational regulation. Parallel studies have highlighted deregulated autophagy [5] and lipophagy [6] to be instrumental in regulating lipid turnover during Mtb infection. To our interest, ubiquitin-dependent degradation of specific proteins implicated in FM formation forms another mechanism which control the stability of proteins that aid in lipid accumulation [7]. In this context, Nedd4-family ligases (a sub-family of HECT-domain containing E3 ligases), among others, are known to play distinctive roles in maintaining the stability of diverse proteins involved in FM formation. Notably, NEDD4-1 can interact with the ATP-Binding Cassette (ABC) transporters-ABCG1 and ABCG4, two essential regulators of cellular lipid efflux [8]. Besides, another report alluded to the role of AIP4 or ITCH in maintaining the levels of the important FM protein, ADRP [9].
During TB, accumulated host lipids have been associated with mycobacterial survival as they provide essential nutrients, contribute to reduced antigen presentation [10,11,12] and aid in pathogen latency and reactivation [13]. A separate study revealed that knocking down Nedd4 in THP-1 macrophages elicited higher CFU of Mtb and other intracellular pathogens [14]. This encouraged us to investigate NEDD4-family of E3 ligases in the context of Mtbdriven FM formation and consequently pathogen survival.
Pathogen-driven epigenetic changes have been indicated to govern the expression of distinct molecules that contribute to mycobacterial survival [4,15,16,17,18]. Emerging literature have indicated towards epigenetic regulation of NEDD4-family ligases, where HDAC2-dependent downregulation of histone methyltransferase Ehmt2 (G9a) was shown to activate NEDD4 [19]. While a plethora of epigenetic regulators have been associated with the progression of several infectious diseases, including TB [4,16,18,20,21], there is a dearth of reports on the role of arginine methyl transferases such as Protein arginine methyl transferases (PRMTs) during distinct infections and the specific regulation of E3 ligases. Of all the characterized PRMTs, PRMT1, PRMT2, PRMT3, PRMT6, PRMT8 and CARM1 catalyze asymmetric methylation, while PRMT5 and PRMT9 bring about symmetric methylation on specific arginine residues [22]. Available reports have demonstrated the role of PRMT5 in regulating lipid biogenesis. Notably, PRMT5 has been shown to arbitrate seipin-mediated lipid droplet biogenesis in adipocytes [23]. Besides, PRMT5 could enhance lipogenesis in cancer cells by methylating SREBP1a [24]. These reports encouraged us to investigate a PRMT5-mediated regulation of FM formation during Mtb infection.
In this study, we sought to understand the role of Mtb-mediated post-translational regulation of key molecules in the formation of FMs. Employing the use of siRNA and pharmacological inhibitors, we highlight the role of PRMT5 in repressing the expression of the E3 ligase, ITCH, which contributed to lipid accumulation during Mtb infection both in vitro and in vivo.

Mycobacterial infection suppresses the expression of the E3 ubiquitin ligase, ITCH, to enhance lipid accumulation in cells
Lipid droplets are formed by the coordinated action of diverse processes which include its biogenesis, maturation, and turnover [25]. As introduced, ubiquitination has been implicated in the regulation of proteins that contribute to lipid turnover within cells [26], thereby providing distinct regulatory potential to the entire process of lipid accumulation [27]. Since Nedd4-family ligases were reported to be responsible for ubiquitination and degradation of lipid-associated proteins such as ADRP [9], we screened for Nedd4-family ligases that are differentially expressed during Mtb infection. Interestingly, we found the transcript levels of the E3 ligase Itch to be significantly downregulated upon mycobacterial infection both in vitro (S1A Fig) and in vivo (S1B Fig), prompting us to investigate the molecule further. This was corroborated at the protein level as Mtb-infected mouse peritoneal macrophages displayed diminished levels of ITCH protein (Fig 1A), while homogenates from the lungs of Mtb-infected mice exhibited a similar downregulated expression of ITCH ( Fig 1B). Furthermore, immunofluorescence analysis revealed enhanced accumulation of lipids with a concomitant decrease in the levels of ITCH expression upon Mtb infection in murine peritoneal macrophages (Fig 1C and 1D). Also, transient over expression of ITCH in RAW 264.7 macrophages compromised the Mtbinduced FM formation, thereby emphasizing on the role of ITCH in lipid accumulation ( Fig  1E and 1F).
Previously, the role of ADRP and CD36 in Mtb-triggered FM formation was underscored as siRNA-mediated knock down of ADRP and/ or CD36 resulted in diminished lipid pools within Mtb-infected macrophages [4]. Over expression of ITCH compromised the elevated levels of ADRP and CD36 (Fig 1G). This led us to assess the possible role of ITCH in the proteasomal degradation of the key lipid accumulation molecules. We found a significant degree of interaction of ITCH with ADRP and CD36 in the uninfected scenario (Fig 1H), suggesting the contribution of ITCH in maintaining lipid homeostasis in macrophages under basal conditions. However, in the event of ITCH repression during Mtb infection, ADRP and CD36 levels are sustained, thereby aiding in lipid accumulation. Together, these observations indicate a role for ITCH in regulating FM formation during Mtb infection.

Transcription regulator, YY1, aids in Mtb-mediated repression of ITCH
Next, we analyzed the promoter region of Itch gene to identify the potential regulators of ITCH expression. Bioinformatic assessment of the 2kb upstream sequence revealed two putative binding sites of the transcriptional regulator, YY1. A previous report suggests an enhanced expression of YY1 in human TB samples, where it plays a cardinal role in the regulation of specific cytokines [28]. Essentially, YY1 is a zinc finger domain containing protein that can activate or repress genes based on its interacting partners [29]. Mouse peritoneal macrophages infected with Mtb displayed an enhanced expression of YY1 in a time-dependent manner (Fig 2A). A similar trend was observed in vivo as elevated YY1 expression was observed in the lung homogenates of Mtb-infected mice (Fig 2B). Analysis of the subcellular localization of YY1 in murine macrophages revealed that Mtb infection enhanced the nuclear localization of YY1 (Fig 2C), underscoring the possible role of YY1 in mediating transcriptional processes upon Mtb infection. Next, we compromised Yy1 levels in murine macrophages with targeted siRNA and found a marked rescue in the Mtb-driven repression of ITCH ( Fig 2D). This was further confirmed by ChIP assay, that indicated an enhanced recruitment of YY1 at its respective binding sites on the promoter of Itch (Fig 2E).
With the premise of YY1-dependent downregulation of ITCH, we verified the contribution of YY1 in FM generation during Mtb infection. We found the expression of the FM markers, ADRP and CD36, to be compromised in Mtb-infected macrophages transfected with Yy1 siRNA (Fig 2F). Furthermore, depletion of Yy1 in mouse peritoneal macrophages also reduced lipid accumulation in Mtb-infected macrophages as assessed by BODIPY 493/503 staining (Fig 2G and 2H).

PLOS PATHOGENS
Epigenetic regulation of foamy macrophage formation

NOTCH signaling pathway contributes to mycobacteria-induced lipid accumulation through YY1
Perturbation of host signaling pathways has been shown to result in FM formation during Mtb infection. Notably, TNF receptor signaling through the downstream activation of the caspase cascade and the mammalian target of rapamycin complex 1 (mTORC1) is central to triglyceride accumulation in human macrophages infected with Mtb [30]. Besides, NOTCH, EGFR, and PI3K pathways have been separately implicated in Mtb-driven FM formation [4,6].
With this premise, using specific pharmacological interventions against distinct cellular pathways (IWP-2: WNT pathway inhibitor; LY294002: PI3K pathway inhibitor; Gefitinib: EGFR pathway inhibitor; GSI, Gamma secretase inhibitor (GSI): NOTCH pathway inhibitor), we found the possible role of NOTCH signaling in the expression of YY1 as inhibition of the pathway using GSI compromised the ability of Mtb to induce the expression of YY1 in macrophages ( Fig 3A). NOTCH pathway activation is characterized by the cleavage of the intracellular domain of the NOTCH receptor (NICD) by the enzyme Gamma secretase. GSI blocks the activity of Gamma secretase and thereby inhibits the activation of the NOTCH pathway. In line with previous reports [31,32], we found an activation of NOTCH signaling pathway (elevated NICD expression) in mouse peritoneal macrophages infected with Mtb (S3A Fig). Corroborating this observation, the transcription factor, HES1, a bona fide target of NOTCH To verify the role of NOTCH pathway activation in augmenting YY1 levels, we overexpressed NICD in RAW 264.7 macrophages and found enhanced levels of YY1 transcript even in the absence of Mtb infection, underscoring the role of NOTCH in the elevated expression of YY1 ( Fig 3B). Further, Mtb-mediated expression of YY1 was compromised in macrophages expressing Notch1 siRNA ( Fig 3C). Additionally, perturbation of the NOTCH pathway in murine peritoneal macrophages using GSI alleviated the Mtb-mediated diminished expression of ITCH ( Fig 3D); while NICD overexpressing macrophages displayed compromised expression of ITCH (Fig 3E), validating our observations on the role of the NOTCH pathway in regulating the levels of YY1 and consequently the E3 ligase, ITCH. Furthermore, inhibition of the NOTCH pathway hindered the accumulation of lipids during Mtb infection (Fig 3F and 3G) thereby endorsing the significant role of NOTCH-YY1-ITCH axis in Mtb-induced FM formation.
Both NOTCH signaling and lipid accumulation was shown to aid mycobacterial survival within the host [31,4]. In this context, using in vitro CFU analysis, we found that perturbation of NOTCH pathway (using GSI) severely compromised mycobacterial survival ( Fig 3H), without any considerable effect on the bacterial uptake (S4A Fig).

PLOS PATHOGENS
Epigenetic regulation of foamy macrophage formation

PRMT5 imparts repressive methylation signature on the promoter of ITCH during mycobacterial infection
As the expression of ITCH was repressed upon mycobacterial infection, we sought to delineate the possible way in which YY1 could accomplish the same. Ample evidence has highlighted the association of YY1 with the Polycomb-group (PcG) proteins to bring about repression of target genes. In this context, upon binding to the DNA, YY1 could initiate PcG protein recruitment that results in concomitant histone deacetylation and methylation [32]. Thus, we surmised if YY1 could bring about repression of ITCH by recruiting members of the PcG proteins. A ChIP assay was performed to assess for the recruitment of the PcG group methyl transferase, EZH2 at the YY1 binding site on the promoter of Itch. It was observed that Mtb infection did not elicit an appreciable recruitment of EZH2 or its cognate methylation signature H3K27me3 at the YY1 binding sites on the promoter of Itch (Fig 4A). Based on this result, we construed that YY1 might interact with distinct epigenetic molecules other than EZH2 to bring about the repression of ITCH. Extensive review of literature revealed that PRMT1, an arginine methyl transferase could interact with YY1 to elicit the enhanced expression of genes [33]. It is important to note that PRMT1 effectuates asymmetric dimethylation in the H4R3 residue (H4R3me2a) to initiate the expression of genes. Interestingly, symmetric dimethylation at the same arginine residue of histone 4 (H4R3me2s) by PRMT5 brings about gene repression [34]. Thus, we conjectured if YY1 could interact with PRMT5 and catalyze the symmetric dimethylation (H4R3me2s) signature on the promoter region of Itch and arbitrate its repression. A ChIP assay confirmed the occupancy of PRMT5 and its cognate repressive

PLOS PATHOGENS
Epigenetic regulation of foamy macrophage formation methylation mark at the YY1-binding sites on the promoter of Itch (Fig 4B). We also observed that upon the perturbation of PRMT5 enzymatic activity with a specific inhibitor EPZ015666, Mtb-mediated diminished expression of ITCH was rescued in mouse peritoneal macrophages ( Fig 4C). Besides, gene-specific knockdown of Prmt5 in mouse peritoneal macrophages alleviated Mtb-mediated repression of ITCH during infection ( Fig 4D). Further, immunoprecipitation analysis in murine macrophages revealed enhanced interaction between YY1 and PRMT5 upon mycobacterial infection (Fig 4E), implying the interaction between YY1 and PRMT5 on ITCH promoter. To conclusively implicate the significance of the association of YY1 and PRMT5 for the recruitment of the repressive methylation signature on the promoter of Itch, siRNA against Yy1 was employed to selectively decrease the levels of Yy1 in mouse peritoneal macrophages. Subsequently, it was observed that cells which were depleted of Yy1 showed compromised recruitment of PRMT5 as well as the repressive methylation mark H4R3me2s on the Itch promoter ( Fig 4F).
Together, these results suggest that Mtb infection mediates the repression of the concerned E3 ligase through the concerted action of YY1 and PRMT5.

PRMT5 activity contributes to sustained lipid accumulation in Mtbinfected macrophages
With the premise that YY1 and PRMT5 orchestrate the Mtb-mediated repression of ITCH, we assessed the role of PRMT5 in FM formation. We found that loss of PRMT5 restricted the Mtb-induced expression of ADRP and CD36, and consequently lipid accumulation (Fig 5A,  5C and 5D). In line, inhibition of PRMT5 enzymatic activity compromised the levels of CD36

PLOS PATHOGENS
Epigenetic regulation of foamy macrophage formation and ADRP and the resultant FMs, thereby validating the essential role of PRMT5 in lipid accrual during mycobacterial infection (Fig 5B, 5E and 5F). Since accumulated lipids provide a favorable niche to the internalized mycobacteria [35], we evaluated mycobacterial survival in PRMT5 inhibitor-treated mouse peritoneal macrophages. Perturbation of PRMT5 enzyme activity in infected macrophages revealed a significant reduction in mycobacterial burden ( Fig  5G), thereby indicating a compelling role of the PRMT5 inhibitor, EPZ015666, in reducing Mtb survival. Similarly, we observe compromised mycobacterial survival in cells knocked down for Prmt5 or Yy1 gene (S4C Fig). Notably, inhibition of PRMT5 did not affect bacterial uptake into host cells (S4A and S4B Fig). Further, it was our interest to investigate if deregulation in lipid levels contributed to the observed differences in mycobacterial survival upon NOTCH pathway or PRMT5 enzyme perturbation. In this context, oleic acid supplementation to mouse peritoneal macrophages resulted in enhanced mycobacterial survival in cells with compromised NOTCH pathway activation or PRMT5 enzymatic activity (S6 Fig), thereby substantiating our inferences on the specific role of PRMT5-mediated sustenance of lipids in mycobacterial survival within macrophages.

Perturbation of PRMT5 activity aids in enhanced mycobacterial killing and resolution of granuloma-like lesions during infection
Having established the role of YY1-PRMT5 axis in mediating ITCH repression and consequently FM formation during Mtb infection, we employed an in vivo mouse model of TB ( Fig  6A) to understand the effect of perturbing the signaling axis on mycobacterial burden and pulmonary pathology. Utilizing the specific pharmacological inhibitor, EPZ015666, we found that perturbation of PRMT5 enzyme activity compromised FM generation in the lungs of infected mice (Fig 6B). Further, the formation of hallmark TB granuloma-like lesions within the lungs

PLOS PATHOGENS
Epigenetic regulation of foamy macrophage formation of treated mice was significantly reduced as indicated by the H and E-stained lung sections and assessment of granuloma-like fraction within the infected lungs (Fig 6C and 6D). Corroborating our histological findings, we observed an appreciable decrease in mycobacterial burden in the lungs of Mtb-infected mice treated with PRMT5 inhibitor.
Thus, we first report the YY1-PRMT5-dependent repression of the E3 ligase, ITCH, during Mtb infection. The reduction in the expression of ITCH contributed to enhanced lipid levels in infected cells, thereby aiding mycobacterial survival, both in vitro and in vivo. With these lines of evidence, we highlight the crucial contribution of NOTCH-YY1-PRMT5 axis in the pathogenesis of TB disease (Fig 7).

Discussion
The enormous success of Mtb in continuing to affect human lives stems from its ability to persist within the body of the infected individual and attain adequate resistance against definitive drugs. FM formation has been attributed as a major development that aids pathogen survival within the hypoxic environment of the host cell. Mtb-induced FMs have been shown to harbor viable mycobacteria wherein it slowly acquires a dormant phenotype [36,37]. Furthermore, FMs displayed enhanced protection from cell death [38] whilst also demonstrating a change to a more favorable inflammatory milieu for mycobacterial survival [4,38]. Since several proteins have been implicated in Mtb-driven FM formation, it becomes pertinent to explore the regulatory mechanisms that contribute to the expression and stability of these proteins within the cells.
Ubiquitination is implicated in diverse cellular processes including protein degradation by the proteasome, cell cycle progression, transcriptional regulation, DNA repair and signal transduction [39]. Pathogenic species' such as Salmonella, Shigella and Legionella secrete ubiquitin ligase-like effectors which are involved in the modulation of critical events of the host that subsequently aid in pathogen survival [40,41]. Besides, mice deficient in the host E3 ligase gene, Hectd3 could enhance host immune responses against bacteria such as Francisella novicida, Mycobacterium bovis (BCG), and Listeria and limit their dissemination [42]. In the current study, analysis of the expression of the NEDD4-family of E3 ligases revealed an appreciable decline in the expression of ITCH. While we established the role of ITCH repression in contributing to pro-mycobacterial FM formation, its role in modulating other cellular processes require further investigation. As mentioned earlier, the role of EGFR pathway in

PLOS PATHOGENS
Epigenetic regulation of foamy macrophage formation augmenting Mtb survival has been underscored previously [6,43]. Besides, a separate report indicated a direct effect of ITCH on the stability of EGF tyrosine kinase receptor [44]. Thus, repression of ITCH could be yet another mechanism that contributes towards EGFR signaling activation during Mtb infection and consequently pathogen survival. Also, ITCH has been shown to regulate the turnover of distinct proteins involved in coordinating T cell immunity [45]. Altogether, additional consequences of Mtb-mediated downregulation of ITCH at the systemic scale other than its role in FM generation, would be promising avenues for future research.
Congruent to earlier reports [28], we found the expression of YY1 to be elevated upon virulent mycobacterial infection. In parallel studies, targeted knockdown of YY1 was shown to decrease the expression of several key effectors of lipid metabolism [46]. Besides, YY1has been reported to promote lipid accumulation in zebrafish liver [47]. Furthermore, YY1 has been implicated in different infectious scenarios. Notably, YY1 could regulate IFN-1 production [48], contribute to viral gene expression [49] and aid the integration of viral DNA into the host chromosomes [50] during discrete viral infections. Thus, it was of our interest to evaluate the role of YY1 in regulating events during mycobacterial infection, where we found its specific relevance in the formation of FMs, among other yet unexplored immune functions. Besides, our study designated a role for the NOTCH signaling pathway in the regulation of YY1.Available literature indicated the association between NOTCH pathway and YY1 upregulation as CBF-1 independent NOTCH signaling could modulate the gene expression of YY1 target genes [51]. An ancillary support for the role of NOTCH pathway could be derived from two reports wherein the juxtacrine signaling pathway was instrumental in M. bovis-mediated SOCS3 upregulation in macrophages on one hand [52], while on the other, YY1 was important for the elevated expression of SOCS3 in neuroinflammation and neuropathic pain [53].
Regulated histone methylation has a diverse role to play during several viral and bacterial infections, including TB [4,18,54,55]. Here, we uncover the role of PRMT5 in mediating lipid accumulation through its close association with the transcriptional regulator, YY1. Histone methyl transferases, including those belonging to the arginine methyl transferase family (PRMTs) have been shown to associate with YY1. Precisely, PRMT1 and PRMT7 interact with YY1 to regulate the expression of specific genes [33,56]. Besides, YY1 could transcriptionally activate PRMT5 and aid in proliferation and invasion in laryngeal cancer cells [57]. With our observation on the close association between PRMT5 and YY1, it would be worthwhile in future to study the association of YY1 with other distinct methyl transferases during mycobacterial infection.
In this study, we show that controlling the turnover of proteins associated with FMs via proteasomal degradation is important to aggregate lipids and that the downregulation of the E3 ubiquitin ligase ITCH by YY1-PRMT5 axis plays a primary role in this process. Besides, the lipids can also undergo turnover by distinct regulated processes such as enzymatic degradation (by lipases) and lipid-specific autophagy. Uncovering the contribution of each of these mechanisms would be useful in designing the most effective combination of drug targets against Mtb infection. Additionally, the effect of the use of the PRMT5 inhibitor in reducing Mtb burden requires further investigation. PRMT5 has been reported to negatively regulate cGAS-mediated antiviral responses [58]. It must be noted that available reports have alluded to the role of PRMT5 in regulating the alternative splicing of genes [59]. In this respect, Mtb infection has been shown to modulate alternate splicing events to program macrophage responses [60,61], The reduced mycobacterial burden and granuloma-like lesions within the lungs of infected mice upon PRMT5 inhibitor administration is indicative of the beneficial effect of PRMT5 inhibition. This might be a result from a combination of LD reduction and the yet-unexplored modulation of alternative splicing and immune mechanisms. Thus, it would be imperative to

PLOS PATHOGENS
Epigenetic regulation of foamy macrophage formation evaluate the role of PRMT5 inhibitor in deregulating alternative splicing events and other discrete immune mechanisms during mycobacterial infection in future. Taken together, the current study unravels the implication of the interplay of specific signaling components and host epigenetic machinery in governing the pathogenesis of Mtb. Future studies involving genespecific knockout animals would further add on to our knowledge of TB pathogenesis.

Ethics statement
Experiments involving mice were carried out after the approval from Institutional Ethics Committee for animal experimentation, Indian Institute of Science (IISc), Bangalore, India

Plasmids and constructs
NICD overexpression (OE) plasmid and β-galactosidase plasmids were received as kind gifts from Prof. Kumaralvel Somasundaram, (IISc, Bangalore). ITCH OE plasmid was procured from Addgene. HES1-luciferase plasmid was a kind gift from Prof. Ryoichiro Kageyama, Institute for Virus Research, Kyoto University.

Transient transfection studies
RAW 264.7 macrophages were transfected with the indicated constructs (NICD OE, ITCH OE, HES1-luc.); or mouse peritoneal macrophages were transfected with 100 nM each of siGLO Lamin A/C, non-targeting siRNA or specific siRNAs with the help of Lipofectamine 3000 for 6 h; followed by 24 h recovery. 70-80% transfection efficiency was observed by counting the number of siGLO Lamin A/C positive cells in a microscopic field using fluorescence microscopy. Transfected cells were subjected to the required infections/ treatments for the indicated time points and processed for analyses.

Luciferase assay
RAW 264.7 cells were transfected with HES1-luciferase and β-galactosidase plasmids using Lipofectamine 3000 for 6 h, followed by 24 h of recovery. subjected to the required infections/ treatments for the indicated time points and processed for analyses. Briefly, cells were harvested and lysed in reporter lysis buffer (Promega) and luciferase activity was assayed using luciferase assay reagent (Promega). The results were normalized for transfection efficiencies by assay of β-galactosidase activity.

RNA isolation and quantitative real time PCR (qRT-PCR)
Treated samples were harvested in TRIzol (Sigma-Aldrich) and incubated with chloroform for phase separation. Total RNA was precipitated from the aqueous layer. Equal amount of RNA was converted into cDNA using First Strand cDNA synthesis kit (Applied Biological Materials Inc.). The cDNA thus obtained was used for SYBR Green (Thermo Fisher Scientific) based quantitative real time PCR analysis for the concerned genes. Gapdh was used as internal control gene. Primer pairs used for expression analyses are provided below (Table 1): Primers were synthesized and obtained from Eurofins Genomics Pvt. Ltd. (India).

Nuclear-cytoplasmic extraction
Cells were treated as indicated, harvested by centrifugation, and gently resuspended in icecold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After incubation on ice for 15 min, cell membranes were disrupted with 10% NP-40 and the nuclear pellets were recovered by centrifugation at 13,226 × g for 15 min at 4˚C. The supernatants from this step were used as cytosolic extracts. Nuclear pellets were lysed with ice-cold Buffer C (20 mM HEPES pH7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF) and nuclear extracts were collected after centrifugation at 13,226 × g for 20 min at 4˚C.

Immunoblotting
Cells post treatment and/or infection were washed with 1X PBS. Whole cell lysate was prepared by lysing in RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/mL each of aprotinin, leupeptin, pepstatin, 1 mM Na 3 VO 4 , 1 mM NaF] on ice for 30min. Total protein from whole cell lysates was estimated by Bradford reagent. Equal amount of protein was resolved on 12% SDS-PAGE and transferred onto PVDF membranes (Millipore) by semi-dry immunoblotting method (Bio-Rad). 5% non-fat dry milk powder in TBST [20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20] was used for blocking nonspecific binding for 60 min. After washing with TBST, the blots were incubated overnight at 4˚C with primary antibody diluted in TBST with 5% BSA. After washing with TBST, blots were incubated with anti-rabbit IgG secondary antibody conjugated to HRP antibody (111-035-045, Jackson ImmunoResearch) for 4h at 4˚C. The immunoblots were developed with enhanced chemiluminescence detection system (Perkin Elmer) as per manufacturer's instructions. For developing more than one protein at a particular molecular weight range, the blots were stripped off the first antibody at 60˚C for 5 min using stripping buffer (62.5 mM Tris-HCl, with 2% SDS 100 mM 2-Mercaptoethanol), washed with 1X TBST, blocked; followed by probing with the subsequent antibody following the described procedure. β-ACTIN was used as loading control.

Immunoprecipitation assay
Immunoprecipitation assays were carried out following a modified version of the protocol provided by Millipore, USA. Treated samples were washed in ice cold PBS and gently lysed in RIPA buffer. The cell lysates obtained were subjected to pre-clearing with BSA-blocked Table 1. Primer sets used for gene expression analysis.

PLOS PATHOGENS
Epigenetic regulation of foamy macrophage formation Protein A beads (Bangalore Genei, India) for 30 min at 4˚C and slow rotation. The amount of protein in the supernatant was quantified and equal amount of protein was used for pull down from each treatment condition; using Protein A beads pre-conjugated with the antibody of interest or isotype control IgG antibody. After incubation of the whole cell lysates with the antibody-complexed beads for 4 h at 4˚C on slow rotation, the pellet containing the beadbound immune complexes were washed with RIPA buffer twice. The complexes were eluted by boiling the beads in Laemmli buffer for 10 min. The bead free samples were resolved by SDS-PAGE and the target interacting partners were identified by immunoblotting. Clean-Blot™ IP Detection Reagent (21230) was obtained from Thermo Scientific.

Chromatin Immunoprecipitation (ChIP) assay
ChIP assays were carried out using a protocol provided by Upstate Biotechnology and Sigma-Aldrich with certain modifications. Briefly, treated samples were washed with ice cold 1X PBS and fixed with 3.6% formaldehyde for 15 min at room temperature followed by inactivation of formaldehyde with 125 mM glycine.

In vitro CFU analysis
Mouse peritoneal macrophages were infected with Mtb H37Rv at MOI 5 for 4 h. Post 4 h, the cells were thoroughly washed with PBS to remove any surface adhered bacteria and medium containing amikacin (0.2 mg/ml) was added for 2 h to deplete any extracellular mycobacteria. After amikacin treatment the cells thoroughly washed with PBS were taken for 0 h time point, and a duplicate set was maintained in antibiotic free medium for next 48 h along with respective inhibitors GSI and EPZ015666. Intracellular mycobacterial burden was enumerated by

In vivo mouse model for TB and treatment with pharmacological inhibitor
BALB/c mice (n = 30) were infected with mid-log phase Mtb H37Rv, using a Madison chamber aerosol generation instrument calibrated to 100 CFU/animal. Aerosolized animals were maintained in securely commissioned BSL3 facility. Post 28 days of established infection, mice were administered eight intra-peritoneal doses (modified from [62]) of EPZ015666 (4mg/kg) every alternate day over 28 days. On 56 th day post inhibitor treatment, mice were sacrificed, the left lung lobe was homogenized in sterile PBS, serially diluted, and plated on 7H11 agar containing OADC to quantify CFU. Upper right lung lobes were fixed in formalin, and processed for hematoxylin and eosin staining, or immunofluorescence analyses. Also, specific lobes from the lungs of mice were homogenized for the extraction of RNA and protein.

Hematoxylin and Eosin staining
Microtome sections (5 μm) were obtained from formalin-fixed, paraffin-embedded mouse lung tissue samples using Leica RM2245 microtome. Deparaffinized and rehydrated sections were subjected to Hematoxylin staining followed by Eosin staining as per manufacturer instructions. After dehydrating, sections were mounted using permount. Sections were kept for drying overnight and handed over to consultant pathologist for blinded analyses. The pathologists have performed the analysis based on the article by Palanisamy et al. Tuberculosis (Edinb). 2008 [63].

Cryosection preparation
The excised lung tissue portions were fixed in 4% paraformaldehyde solution. Subsequently, the tissues were kept in 30% sucrose solution. The fixed lung pieces were placed in the optimal cutting temperature (OCT) media (Jung, Leica). Cryosections of 10 μm were prepared using Leica CM 1510 S or Leica CM 3050 S cryostat and then stored at -80˚C.

MTT assay
Mouse peritoneal macrophages were transfected or treated with the desired siRNA or inhibitors and cultured for 48 h. Thereafter, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; 5 mg/mL) was added to the medium. MTT is a tetrazolium salt that is converted by living cells into blue formazan crystals. After incubating for 3h, the medium is removed from the wells and 200 μL dimethyl sulfoxide was added to dissolve formazan crystals. Thereafter, the absorbance was measured at 570 nm in an enzyme-linked immunosorbent assay reader. For all cytotoxicity assays, the viability of control cells (treated with DMSO or transfected with non-targeting, NT siRNA) was made to 100% and relative viability of test conditions was calculated as a percentage.

Statistical analysis
Levels of significance for comparison between samples were determined by the student's t-test and one-way ANOVA followed by Tukey's multiple-comparisons. The data in the graphs are expressed as the mean ± S.E. for the values from at least 3 or more independent experiments and P values < 0.05 were defined as significant. GraphPad Prism software (6.0, 9.0 versions, GraphPad Software, USA) was used for all the statistical analyses.