key: cord-0334752-0z1tnbzo authors: Borbora, Salik Miskat; Rajmani, R.S.; Balaji, Kithiganahalli Narayanaswamy title: PRMT5 epigenetically regulates the E3 ubiquitin ligase ITCH to influence lipid accumulation during mycobacterial infection date: 2021-11-09 journal: bioRxiv DOI: 10.1101/2021.11.09.467864 sha: 9e454128ca37aedd54acad630191dc5f65258240 doc_id: 334752 cord_uid: 0z1tnbzo 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 genes. Here, we define an Mtb-orchestrated modulation of the ubiquitination mechanism 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 our study, perturbation of PRMT5 enzyme activity resulted in compromised lipid levels and reduced mycobacterial survival in primary murine macrophages (ex vivo) and in a therapeutic mouse model of TB infection (in vivo). These findings provide new insights on 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. Author Summary Mycobacterium tuberculosis generates lipid-laden cells (foamy macrophages-FMs) that offer a favorable shelter for its persistence. During infection, we observe a significant reduction in the expression of the E3 ubiquitin ligase, ITCH. This repression allows the sustenance of key lipid accretion molecules (ADRP and CD36), by curbing their proteasomal degradation. Further, we show the repression of ITCH to be dependent on the concerted action of the bifunctional transcription factor, YY1 and the arginine methyl transferase, PRMT5. NOTCH signaling pathway was identified as a master-regulator of YY1 expression. In vitro and in vivo analyses revealed the significance of PRMT5 in regulating FM formation and consequently mycobacterial burden. Ubiquitin-dependent degradation of specific proteins implicated during FM formation is 87 yet another mechanism that can control the stability of proteins that aid in lipid 88 accumulation, thereby securing the integrity of lipids (7). In this context, Nedd4-family augmenting TB pathology and draws attention towards specific cellular nodes that could 135 be targeted for the development of host-directed therapeutics against TB. Mycobacterial infection elicits differential expression of the E3 ubiquitin ligase, 138 ITCH, to enhance lipid accumulation in cells 139 As distinct bona fide cellular organelles, lipid droplets are formed by the coordinated 140 action of diverse processes which include its biogenesis, maturation, and turnover (25). As introduced, ubiquitination has been implicated in the regulation of proteins that (Fig. 1E, F) . Furthermore, ITCH over expression compromised the 162 elevated levels of key lipid accumulation molecules, ADRP and CD36 (Fig. 1G) . These 163 observations led us to assess the possible role of ITCH in the proteasomal degradation 164 of the said proteins. We found a significant degree of interaction of ITCH with ADRP and 165 CD36 in the uninfected scenario (Fig. 1H) Mtb infection. Next, we compromised Yy1 levels in murine macrophages with targeted 185 siRNA and found a marked rescue in the Mtb-driven repression of ITCH (Fig. 2D) . This 186 was further confirmed by ChIP assay, that indicated an enhanced recruitment of YY1 at 187 its respective binding sites on the promoter of ITCH (Fig. 2E) . 188 With the premise of YY1-dependent downregulation of ITCH, we verified the contribution Here, we sought to examine such upstream regulatory events that can potentially 204 contribute to the differential expression of YY1 during mycobacterial infection. Using by transcript analysis (Fig. S3B ) as well as luciferase reporter assay (Fig. S3C) . 215 To verify the role of NOTCH pathway activation in augmenting YY1 levels, we With these lines of evidence, we highlight the crucial contribution of NOTCH-YY1-PRMT5 307 axis in the pathogenesis of TB disease (Fig. 7) . The enormous success of Mtb in continuing to affect human lives stems from its ability to Emerging trends in the formation 662 and function of tuberculosis granulomas Revisiting the role of the granuloma in tuberculosis Who puts the tubercle in tuberculosis? 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