# Histone deacetylase 7 mediates lipopolysaccharide-inducible mitochondrial fission in macrophages

Rishika Abrol, Syeda Farhana Afroz, James Curson, Karoline Raven, Kaustav

Das Gupta, Kimberley Gunther, Alun Jones, Robert Reid, Zherui Xiong,

Jennifer Gunter, et al.

► To cite this version:

Rishika Abrol, Syeda Farhana Afroz, James Curson, Karoline Raven, Kaustav Das Gupta, et al. Histone deacetylase 7 mediates lipopolysaccharide-inducible mitochondrial fission in macrophages. Journal of Cell Science, 2025, 138 (19), pp.jcs264376. 10.1242/jcs.264376. hal-05245118

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Histone deacetylase 7 mediates lipopolysaccharide-inducible
mitochondrial fission in macrophages

Rishika Abrol<sup>1,2,‡,¶</sup>, Syeda Farhana Afroz<sup>1,2,§,¶</sup>, James E. B. Curson<sup>1,2</sup>, Karoline D. Raven<sup>1,2</sup>, Kaustav Das Gupta<sup>1,2,3</sup>, Kimberley S. Gunther<sup>1,2</sup>, Alun Jones<sup>1</sup>, Robert C. Reid<sup>1,2</sup>, Zherui Xiong<sup>1</sup>, Jennifer H. Gunter<sup>4</sup>, Jessica A. Engel<sup>5</sup>, Christian R. Engwerda<sup>5</sup>, Antje Blumenthal<sup>6</sup>, David P. Fairlie<sup>1,2,7</sup>, Robert G. Parton<sup>1,8</sup>, Steven Zuryn<sup>9</sup>, Ronan Kapetanovic<sup>1,2,10</sup>, Divya Ramnath<sup>1,2,*</sup>, Matthew J. Sweet<sup>1,2,**</sup>

<footnote n="1"><sup>1</sup>Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia</footnote>

<footnote n="2"><sup>2</sup>Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, 4072, Australia</footnote>

<footnote n="3"><sup>3</sup>Division of Infectious Disease, The Lundquist Institute for Biomedical Innovation at Harbor-University of California, Los Angeles Medical Center, Torrance, California, USA</footnote>

<footnote n="4"><sup>4</sup>Queensland University of Technology (QUT), Australian Prostate Cancer Research Centre - Queensland, Centre for Genomics and Personalised Health, School of Biomedical Sciences, Translational Research Institute, Brisbane, Queensland, 4102, Australia</footnote>

<footnote n="5"><sup>5</sup>QIMR Berghofer Medical Research Institute, Brisbane, Queensland, 4006, Australia</footnote>

<footnote n="6"><sup>6</sup>Frazer Institute, The University of Queensland, Brisbane, Queensland, 4102, Australia</footnote>

<footnote n="7"><sup>7</sup>ARC Centre of Excellence for Innovations in Peptide and Protein Science, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia</footnote>

<footnote n="8"><sup>8</sup>Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Queensland, 4072, Australia</footnote>

<footnote n="9"><sup>9</sup>Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, 4072, Australia</footnote>

<footnote n="10"><sup>10</sup>INRAE, Université de Tours, Infectiologie et Santé Publique (ISP), Nouzilly 37380, France</footnote>

<footnote>*Authors for correspondence: m.sweet@imb.uq.edu.au or d.ramnath@imb.uq.edu.au</footnote>

<footnote>‡Present address: Immunity and Inflammatory Diseases, Garvan Institute of Medical Research, Darlinghurst, and St Vincent's Healthcare Clinical Campus, Faculty of Medicine, UNSW Sydney, Sydney, New South Wales, Australia</footnote>

<footnote>§Present address: Department of Cell and Biological Systems, Penn State College of Medicine, Hershey, Pennsylvania, 17033, USA</footnote>

<footnote>¶These authors contributed equally to this work.</footnote>

<footnote>**Lead Contact</footnote>
## SUMMARY

Histone deacetylase 7 (HDAC7) drives several immunometabolism-related processes in macrophages including lipopolysaccharide (LPS)-inducible glycolysis and inflammatory mediator production. Using an advanced biotin ligase TurboID system in human macrophages, we report 104 candidate HDAC7 interaction partners that may contribute to its immunometabolic functions. One such protein is the mitochondrial fission-promoting GTPase dynamin-related protein 1 (DRP1), which associates with HDAC7 in cells. Using gain- and loss-of-function genetic approaches, we show that HDAC7 promotes LPS-inducible mitochondrial fission in macrophages, as well as DRP1-dependent metabolic and inflammatory responses. HDAC7 enzymatic activity was dispensable for LPS-inducible fission, as previously reported for LPS-inducible glycolysis. However, a pharmacological inhibitor of HDAC7 attenuated fission in primary human and mouse macrophages, implicating its acetyl-lysine docking function in this response. HDAC7 thus drives inducible mitochondrial fission in macrophages. Small molecules targeting the acetyl-lysine docking function of HDAC7 may have applications in preventing pathological processes driven by dysregulated mitochondrial fission.

**Keywords:** dynamin-related protein 1; glycolysis; histone deacetylase; immunometabolism; lysine deacetylase, macrophages; mitochondrial dynamics; mitochondrial fission; post-translational modification; toll-like receptor

## INTRODUCTION

Macrophages detect danger through pattern recognition receptors (PRRs) expressed on the cell surface, endosomal compartments, and within the cytosol¹. Toll-like receptors (TLRs) are among the most widely studied PRRs, sensing and responding to threats by initiating inflammatory and antimicrobial responses, priming adaptive immunity, and controlling cell survival². TLR signalling also reprograms macrophage metabolism, with this required for specific functional responses³,⁴. For example, lipopolysaccharide (LPS)-mediated TLR4 activation increases glycolysis and signals the glycolytic enzyme pyruvate kinase isoform M2 (PKM2) to drive inflammatory gene expression via the transcription factor HIF-1α in macrophages⁵.
Many metabolic enzymes, including PKM2, are regulated by lysine acetylation⁶. Classical histone deacetylases (HDAC1-11) deacetylate target proteins and/or act as scaffolding proteins to control immune cell functions⁷. Class IIa HDACs (HDAC4, 5, 7, 9) are generally considered to be enzymatically inactive because they lack a critical active-site tyrosine residue that is conserved in other classical HDAC enzymes⁸. Nonetheless, enzyme activity of one class IIa HDAC, HDAC7, is regulated in activated macrophages. Signalling via multiple TLRs rapidly increases HDAC7 enzymatic activity in macrophages, as measured by deacetylation of an artificial class IIa HDAC-specific trifluoroacetyl-lysine-containing substrate⁹. Except for TLR3 that signals via TRIF¹⁰, TLR-mediated activation of HDAC7 enzyme activity requires the MyD88 adaptor⁹. Upon TLR activation, HDAC7 drives PKM2-dependent activation of HIF-1α for inducible IL-1β production in macrophages¹¹ and also promotes TLR-inducible glycolysis⁹,¹¹, which has been linked to inflammation¹² and chronic conditions such as rheumatoid arthritis¹³. Interestingly, HDAC7 enzymatic activity is required for LPS-induced IL-1β production⁹ but not inducible glycolysis⁹ in macrophages, suggesting that HDAC7 has both enzyme-dependent and enzyme-independent functions in these cells. Collectively, these studies position HDAC7 as a central player in TLR-regulated immunometabolism in macrophages¹⁴.

Mitochondria play critical roles in macrophage functions¹⁵,¹⁶. These dynamic organelles alternate between a fused mitochondrial network (fusion) and fragmented mitochondria (fission). Mitochondrial dynamics is regulated by several dynamin-related GTPases, including fusion-promoting mitofusin 1 and 2¹⁷ and fission-promoting dynamin-related protein 1 (DRP1) that is recruited to mitochondrial membranes¹⁸ for membrane scission¹⁹. Increasing evidence suggests a connection between mitochondrial fission and LPS-inducible glycolysis. LPS and other inflammatory stimuli that rewire cellular metabolism towards glycolysis²⁰,²¹ also skew mitochondrial dynamics towards fission in macrophages²², and inhibiting mitochondrial fission reduces LPS-induced glycolytic reprogramming in various cell types²³⁻²⁵.

In this study, we explored mechanisms by which HDAC7 controls metabolic functions in macrophages via a proximity-based screen for HDAC7 interaction partners. Using the TurboID advanced biotin ligase system²⁶ coupled with proteomic analysis, we reveal that the HDAC7 macrophage interactome includes DRP1. This led us to discover that HDAC7 promotes LPS-inducible mitochondrial fission in macrophages, as well as DRP1-dependent metabolic and
inflammatory responses. Our studies also revealed a two-stage mechanism of LPS-inducible
mitochondrial fission, with HDAC7 playing a particularly pronounced role in the latter. Our
findings provide mechanistic insight into how TLR activation connects mitochondrial dynamics
and metabolic changes in macrophages, while also revealing HDAC7 as a potential target for
attenuating dysregulated mitochondrial fission in innate immune cells.

RESULTS

TurboID identifies 104 candidate HDAC7 interaction partners

To identify HDAC7 interaction partners, we used the TurboID advanced biotin ligase system²⁷. In this system, cells expressing a HDAC7-TurboID fusion protein biotinylate proximal proteins (Fig. S1A), which can then be characterized (Fig. S1B). We used a doxycycline-inducible system to conditionally express HDAC7-TurboID in THP1 cells²⁸ for identifying candidate HDAC7-interacting partners. As specificity controls, we generated THP1 cells inducibly expressing TurboID alone (empty vector, EV) or Natural Killer Granule Protein 7 (NKG7)-TurboID, as an inflammation-related control protein²⁹. We verified doxycycline-induced expression of FLAG-tagged HDAC7 or V5-tagged NKG7 (Fig. S1C) and, using a class IIa HDAC-specific enzyme assay in which a trifluoroacetyl-lysine-containing substrate is deacetylated⁹, we confirmed that the HDAC7-TurboID fusion protein was functional. As expected, doxycycline-stimulated HDAC7-expressing THP1 cells had elevated basal and LPS-inducible class IIa HDAC activity, compared to control cells (Fig. S1D-E).

To identify candidate HDAC7 interaction partners, we isolated biotinylated proteins by
streptavidin pull-down in THP1 cells expressing TurboID fusion proteins³⁰. As expected, the
HDAC7 and NKG7 bait fusion proteins were themselves biotinylated as indicated by their
detection in the streptavidin pull-down samples (Fig. 1A). Other biotinylated proteins were
identified by mass spectrometry, with the top ten proteins specific to HDAC7 and NKG7
displayed in Fig. 1B and proteins significantly enriched in the HDAC7-TurboID versus EV-
TuboID pull-downs shown in Fig. 1C (124 proteins were significantly enriched, with at least 2
peptides detected at >95% confidence). Known HDAC7 interaction partners, such as PKM2¹¹,
14-3-3 proteins³¹, NCOR1³², and MEF2D³³ were all unique to the HDAC7-TurboID pull-downs
(Fig. 1C), thus validating our approach. After false discovery rate correction, 104 proteins were unique to the HDAC7-TurboID pull-down samples when compared to the EV and NKG7-TurboID controls in each of three independent experiments (Fig. 1D, Table S1). To gain insights into the HDAC7 interactome, GO and KEGG pathway analyses were employed³⁴ (Fig. 1E-F, Fig. S1F-G). HDAC7 partner proteins were significantly enriched in cytoplasmic compartments (Fig. 1E), where HDAC7 is localized in macrophages⁹. GO analysis revealed that these proteins were enriched in several pathways, including actin filament organization and regulation of GTPase activity (Fig. 1F, Fig. S1F). KEGG analysis also revealed the involvement of HDAC7 partners in bacterial infection and phagocytosis (Fig. S1G), consistent with known HDAC7 functions³⁵. These findings broadly validate the candidate HDAC7 interaction partners identified.

## HDAC7 associates with mitochondrial fission protein DRP1

We focused on HDAC7-specific candidates that may contribute to HDAC7-dependent TLR-inducible glycolysis¹¹. Five glycolysis-related proteins (PKM, G6PD, DNM1L/DRP1, HSPA5; GNB2L1/RACK1)²³,³⁶,³⁷ were identified (Fig. S1H). DNM1L/DRP1 is essential for mitochondrial fission²² and glycolysis²³,³⁸,³⁹ in various cell types, so was selected for further analysis. We next employed additional methods to assess the association between HDAC7 and DRP1 in cells. Immunoprecipitation of DRP1 with an antibody against endogenous DRP1 also pulled down ectopically-expressed HDAC7 in stably transduced THP1 cells (Fig. 2A). Using co-immunoprecipitation, we additionally confirmed an association between endogenous HDAC7 and DRP1 in PMA-differentiated THP1 cells (Fig. 2B). To further validate this interaction, proximity ligation assays (PLA) were performed on bone marrow-derived macrophages (BMM) from either mice overexpressing V5-tagged HDAC7 in myeloid cells (MacBlue x UAS-HDAC7 mice, hereafter MacHDAC7 mice)¹¹ or littermate control MacBlue mice. In this system, a tissue-specific promoter restricts expression of the GAL4-VP16 transcriptional activator to myeloid cells in MacBlue mice. Consequently, when MacBlue mice are crossed with UAS-HDAC7 mice, mice that inherit both transgenes (MacHDAC7 mice) express high levels of HDAC7-V5 in macrophages¹¹. These PLA experiments with anti-V5 and anti-DRP1 revealed that HDAC7 and DRP1 are in close proximity in primary macrophages from MacHDAC7 mice, with a PLA signal not detected using the same antibody pair in MacBlue control BMM (Fig. 2C-D). The specificity
of the interaction was also confirmed using antibodies against HDAC7-V5 and p38 as an unrelated cytoplasmic control protein.

**HDAC7 is essential for LPS-inducible mitochondrial fission in macrophages**

We previously showed that LPS promotes DRP1-dependent mitochondrial fission in macrophages, without affecting mitochondrial mass or mitochondrial DNA replication<sup>22</sup>. To investigate the significance of the HDAC7 and DRP1 association, LPS-inducible mitochondrial fission was assessed in *Hdac7*-deficient BMM. Stimulation with sub-maximal (0.5 ng/mL) or maximal stimulatory (100 ng/mL) LPS concentrations for 6 h led to mitochondrial fragmentation and an increase in mitochondria numbers in *Hdac7**<sup>+/+</sup> control BMM (**Fig. 2E-F, Fig. S2A**). This LPS-inducible increase in mitochondrial numbers is dependent on DRP1 in macrophages, indicating increased mitochondrial fission<sup>22</sup>. Consistent with this, LPS treatment reduced mitochondrial branch length, a measure of the average length of connected mitochondrial segments (**Fig. S2B**), as well as form factor, a composite measure of mitochondrial shape complexity and elongation (**Fig. S2C**). Reductions in these parameters indicate a shift toward a more fragmented, punctate mitochondrial network. In contrast, LPS did not promote mitochondrial fission in *Hdac7*-deficient macrophages (**Fig. 2E-F, Fig. S2A-C**). This defect could not be explained by perturbations in mitochondrial mass, which was unaffected by *Hdac7* deficiency in BMM (**Fig. S2D**). Furthermore, the failure of LPS to induce fission in *Hdac7*-deficient BMM was not reflective of a broader defect in LPS responsiveness, as these cells retained the ability to respond to LPS with inducible expression of *Tnf* mRNA (**Fig. S2E**)¹¹. We next utilized gain-of-function MacHDAC7 mice¹¹ that overexpress HDAC7 in macrophages to further test the role of HDAC7 in fission. MacHDAC7 BMM displayed increased basal and LPS-inducible mitochondrial fission by comparison to MacBlue control BMM, with the inducible response being enhanced at both low dose (0.5 ng/mL) and high dose (100 ng/mL) LPS concentrations (**Fig. 2G-H, Fig. S2F-G**). Collectively, these data indicate that HDAC7 mediates LPS-induced fission in macrophages at 6 h post-stimulation.
**HDAC7 enzyme activity is dispensable for LPS-inducible fission**

Acute LPS-inducible glycolysis in macrophages does not require HDAC7 enzyme activity⁹. We therefore predicted that HDAC7 enzyme activity would be dispensable for LPS-inducible mitochondrial fission. To examine this, we used an HDAC7 point mutant that lacks catalytic activity due to the mutation of a key histidine residue to alanine in the active site (H649A), as previously described¹¹. Hereafter, we refer to this mutant as enzyme-dead (ED) HDAC7. Reconstitution of *Hdac7*-deficient BMM with wild type (WT) or ED HDAC7 (Fig. 3A)¹¹, but not the empty vector control (EV), rescued LPS-inducible mitochondrial fission (Fig. 3B-C, Fig. S3A) without affecting DRP1 protein levels (Fig. S3B). Interestingly however, TMP195, a pharmacological inhibitor of class IIa HDACs⁴⁰, attenuated LPS-inducible fission in mouse BMM (Fig. 3D, Fig. S3C) and human monocyte-derived macrophages (HMDM) (Fig. 3E, Fig. S3D). This further supports a requirement for HDAC7 in LPS-inducible fission in macrophages and implies that the TMP195-binding pocket may be required. We further investigated this by examining the interaction between DRP1 and HDAC7-WT versus HDAC7-ED in immunoprecipitation experiments in co-transfected HEK293T cells, with TLR signalling protein SCIMP⁴¹ serving as a negative control (Fig. 3F). Whereas DRP1 associated with wild type HDAC7 in macrophages (Fig. 2A-D), it immunoprecipitated HDAC7-ED but not HDAC7-WT in the HEK293T cell co-transfection system. The reasons for this are unclear but may relate to cell type-specific differences and/or different sensitivities in methods used. Since HDAC7 enzyme activity was not required for LPS-induced fission (Fig. 3B-C), this enhanced interaction with enzyme-dead HDAC7 is unlikely due to increased binding of DRP1 as a substrate. Instead, we presume the enzymatic pocket of HDAC7 facilitates the interaction between HDAC7 and DRP1, with this pocket being more accessible in the enzyme-dead form of HDAC7. Interestingly, DRP1 also interacted with class IIa HDAC5 and class IIb HDAC6 in co-transfected HEK293T cells (Fig. S3E). This suggests that additional factors beyond DRP1 binding alone are required for the non-redundant role of HDAC7 in driving LPS-inducible mitochondrial fission in macrophages (Fig. 2E-F). Collectively, our data suggest that the acetyl-lysine docking function of HDAC7, but not its enzymatic activity, is likely to mediate LPS-inducible mitochondrial fission.
### HDAC7 has different roles in transient versus sustained LPS-inducible mitochondrial fission

To gain further insight into HDAC7-dependent mitochondrial fission, we considered the kinetics of the response. We previously provided evidence of two distinct stages of LPS-induced mitochondrial fission, with LPS triggering transient DRP1 phosphorylation at S635 (numbering based on NP_001346936.1; corresponds to human S616), followed by subsequent DRP1 dephosphorylation at S656 (corresponds to human S637) at 6 h post-LPS stimulation and beyond²². To test the involvement of these post-translational modifications (PTMs), we reconstituted *Drp1*-deficient MEF cells²² with a wild-type (wt) DRP1 construct, a phosphodeficient S635A DRP1 mutant (predicted to be defective in early stage fission) and a phosphomimetic S656E DRP1 mutant (predicted to be defective in late stage fission). As expected, reconstitution of wt DRP1 in *Drp1*⁻/⁻ MEF cells restored the early (3 h) and late (8 h) LPS-inducible fission responses, with effects being comparable to WT MEF cells (Fig. S4A-B). Moreover, DRP1-S635A-expressing MEF cells were defective in the first but not the second stage of inducible fission, whereas the reverse was true for DRP1-S656E-expressing MEF cells. These data support a model in which LPS-induced DRP1-S635 phosphorylation permits a first stage of fission, with subsequent S656 dephosphorylation enabling a second stage of fission.

Next, we used *Hdac7*-deficient BMM to examine the role of HDAC7 in these two stages of fission. HDAC7 was only partially required for inducible fission at 2 h post-LPS stimulation, whereas the response was completely HDAC7-dependent by 6 h (Fig. S5A-B). These defects were not due to reduced DRP1 protein levels or overall mitochondrial content as assessed by TOM20 protein levels, both of which were similar between wild type and *Hdac7*-deficient BMM over an LPS time course (Fig. S5C-E). Assessing DRP1 modifications, HDAC7 was not required for inducible DRP1-S635 phosphorylation (Fig. S5C, F), despite it having a partial involvement in the first stage of LPS-induced fission (Fig. S5A-B). In contrast, the apparent DRP1-S656 dephosphorylation, as detected with a pDRP1 S656 antibody (CST, #4867S), was abrogated in *Hdac7*⁻/⁻ BMM (Fig. S5C, G). This antibody detected a higher molecular weight band of >90 kDa (instead of ~80 kDa for DRP1), which we presumed to be a SUMOylated version of DRP1 corresponding to this size⁴²,⁴³. However, this band was still detected in *Drp1*⁻/⁻ MEF cells when using the pDRP1 S656 antibody, whereas this was not the case for an antibody detecting total DRP1 (Fig. S5H). This suggests that the pDRP1 S656 antibody (CST, #4867S) detects a cross-reactive protein but not DRP1 itself (hereafter referred to as fission-related
protein, FRP). Given that DRP1-S656 dephosphorylation was required for the second stage of fission (Fig. S4A-B) and that HDAC7 was required for both the second stage of LPS-induced fission (Fig. S5A-B) and the corresponding loss of FRP (Fig. S5C, G), we conclude that the LPS-induced decrease in FRP correlates with DRP1-S656 dephosphorylation.

To further probe how HDAC7 may connect to the second wave of LPS-inducible fission, we explored other signalling components. As expected, TLR4 was required for both the first and second stages of inducible fission (Fig. S6A), as well as acute DRP1-S635 phosphorylation and the subsequent loss of FRP (Fig. S6B). We next assessed MyD88 involvement, since this adaptor is required for inducible fission at 6 h post-LPS stimulation²². Here we observed that acute LPS-inducible fission did not require MyD88 (Fig. S6C-D). This presumably reflects the involvement of other TLR adaptors, since TAK-242/resatorvid that antagonizes MAL/TIRAP- and TRAM-mediated signalling⁴⁴,⁴⁵ attenuated both stages of inducible fission (Fig. S6E) and acute DRP1-S635 phosphorylation (Fig. S6F). We conclude that HDAC7 partially contributes to the first stage of TLR4-induced fission independently of DRP1-S635 phosphorylation, whereas it is essential for the subsequent sustained second stage of fission, likely by connecting MyD88 to DRP1-S656 dephosphorylation.

### HDAC7 promotes LPS-inducible glycolysis and a subset of inflammatory responses via DRP1

To investigate the potential biological significance of HDAC7-dependent TLR-inducible fission, we examined the effect of HDAC7 over-expression on metabolic and inflammatory responses in macrophages lacking DRP1. To do so, we generated *Drp1*-deficient RAW264.7 cells by CRISPR (Fig. 4A) and confirmed that DRP1 reconstitution restored inducible fission in these cells (Fig. 4B). To determine whether HDAC7 promotes LPS-inducible glycolysis via DRP1, we transfected parent and *Drp1*-deficient RAW264.7 cells with mRNAs encoding HDAC7 or GFP as a control (Fig. 4C), then measured extracellular acidification rate (ECAR) as a proxy for glycolytic activity (Fig. 4D-E). We noted that unstimulated *Drp1*-deficient RAW264.7 cells exhibited a higher ECAR baseline than parental RAW264.7 cells, highlighting metabolic heterogeneity between clonal populations of this cell line. For this reason, we focused on the effects of HDAC7 on metabolic and inflammatory responses in each cell population individually. In parental RAW264.7 cells, LPS increased ECAR in a dose-dependent manner, and this effect
was further enhanced by HDAC7 overexpression (Fig. 4D, right). In contrast, LPS only modestly increased ECAR in Drp1-deficient RAW264.7 cells and HDAC7 overexpression had no additional effect (Fig. 4E, right). Thus, DRP1 is required for HDAC7 to promote LPS-inducible glycolysis in macrophages.

Finally, we assessed whether HDAC7 enhances inflammatory gene expression via DRP1 in macrophages. Here we found that ectopic HDAC7 expression in parental but not Drp1-deficient RAW264.7 cells modestly but significantly increased LPS-inducible mRNA levels of two previously reported HDAC7 inflammatory gene targets, *Ccl2*¹¹ and *Edn1*⁴⁶, that encode the monocyte-recruiting chemokine CCL2 and the potent vasoconstrictive and inflammatory peptide endothelin-1, respectively (Fig. 4F-G). These effects were selective as LPS-inducible *Tnf* mRNA levels were not affected by HDAC7 in either cell line (Fig. 4H). Collectively, these data indicate that HDAC7 requires DRP1 to both promote LPS-inducible glycolysis and to fine-tune inflammatory responses in macrophages.

DISCUSSION

HDAC7 has key roles in regulating metabolic, inflammatory and antimicrobial responses in macrophages⁹,¹¹,³⁵,⁴⁶. In this study, we provide mechanistic insights into immunometabolic functions of HDAC7 in these cells. We show that this class IIa HDAC interacts with the GTPase DRP1, that it is essential for LPS-inducible mitochondrial fission, and that it promotes LPS-inducible glycolysis and fine tunes inflammatory responses via DRP1. As far as we are aware, this is the first published evidence linking HDAC7 to DRP1 functions and/or mitochondrial dynamics in any biological system. There are, however, a small number of studies implicating HDAC7 in mitochondrial biology. For example, HDAC7 localised to mitochondria in prostate epithelial cells and was redistributed to the cytoplasm during cellular apoptosis⁴⁷. HDAC7 also negatively regulated genes encoding TCA cycle enzymes in the context of renal cell carcinoma⁴⁸, but this was not related to direct effects on mitochondria. In contrast, we introduce HDAC7 as a direct regulator of mitochondrial dynamics in macrophages.

Mitochondrial fission is generally indicative of cells being skewed towards glycolysis²⁵, with evidence supporting a functional connection between mitochondrial fission and glycolysis. LPS
promotes mitochondrial fission in several cell types²²,⁴⁹,⁵⁰, with inhibition of fission reducing glycolytic reprogramming²⁴,²⁵. In T cells, glycolytic gene expression and glycolysis required DRP1, with *Drp1*-deficient T cells being skewed towards OXPHOS⁵¹. Similarly, DRP1 was required for the metabolic shift towards glycolysis during somatic cell reprogramming⁵². Given these functional connections between mitochondrial fission and metabolic reprogramming, it seems likely that HDAC7 promotes LPS-inducible glycolysis in macrophages⁹,¹¹, at least in part, via inducible mitochondrial fission. This is consistent with our observations that HDAC7 enzyme activity was not required for either LPS-inducible mitochondrial fission (Fig. 3B-C) or acute inducible glycolysis⁹ in macrophages, and that HDAC7 failed to enhance LPS-inducible glycolysis in *Drp1*-deficient macrophages (Fig. 4D-E).

Since HDAC7 enzyme activity was not required for LPS-inducible fission, HDAC7 probably mediates these effects independently of protein deacetylation. Indeed, a conserved active site tyrosine present in most classical HDACs is replaced with a histidine in HDAC7 and other class IIa HDACs, resulting in this sub-class of HDACs having very weak lysine deacetylase activity in vitro⁸. Although the N-terminal domain of HDAC7 interacts with a range of proteins to control cell functions¹⁴, it seems more likely that HDAC7 promotes LPS-inducible fission by docking an acetyl-lysine-containing protein into the C-terminal enzymatic pocket, given that TMP195⁵³ inhibited LPS-inducible fission (Fig. 3D-E). However, TMP195 inhibits all class IIa HDACs⁵³ and we cannot exclude the possibility that other class IIa HDACs may also be essential for LPS-inducible fission, particularly given that multiple HDACs can interact with DRP1 in cells (Fig. S3E).

Past studies on regulated mitochondrial dynamics have primarily focused on DRP1 PTMs, including phosphorylation¹⁶. For example, phosphorylation of rat DRP1 at S585 (mouse S635) was associated with DRP1 recruitment to mitochondria and mitochondrial fission⁵⁴. In line with the transient kinetics of LPS-inducible DRP1-S635 phosphorylation in macrophages²², we confirmed that this PTM mediates acute LPS-induced mitochondrial fission. Furthermore, this early phosphorylation of DRP1-S635 is independent of both MyD88²² and HDAC7 (Fig. S5C, F). In contrast, the later dephosphorylation of DRP1 enhances DRP1 GTPase activity⁵⁵,⁵⁶. DRP1 reconstitution experiments implicate DRP1-S656 dephosphorylation in the late second stage (8 h) of LPS-induced fission (Fig. S4A-B). Both HDAC7 (Fig. S5A-B) and MyD88²² were required
for the late fission response in macrophages, so it seems likely that a MyD88-HDAC7 axis
coordinates DRP1 activation, potentially via DRP1 dephosphorylation at S656, for sustained
LPS-inducible fission.

HDAC7 has been widely implicated in the development and/or pathology of certain cancers, metabolic conditions and inflammatory diseases¹⁴. It likely contributes to inflammation-related pathology through multiple mechanisms, for example reinforcing Th17 cell transcriptional programs in T cells⁵⁷ and promoting immunometabolism-related pro-inflammatory cytokines in macrophages¹¹. The connection between HDAC7 and mitochondrial fission identified here may provide further insights into how this protein drives dysregulated inflammation. Activated microglia produce inflammatory mediators that contribute to neuron destruction in Parkinson's disease and Alzheimer's disease⁵⁸, with TLR signaling implicated in these processes⁵⁹. Pharmacological and genetic approaches link excessive mitochondrial fission to inflammation¹⁶ and neurodegeneration⁶⁰. Although HDAC7 has neuroprotective functions in neurons⁶¹,⁶², our findings suggest that it might also drive fission-mediated pathology in activated microglia. For example, HDAC7 amplified LPS-inducible expression of genes encoding the inflammatory mediators CCL2 and endothelin-1 via DRP1 (Fig. 4F-G), with both of these mediators implicated in various neuroinflammatory conditions⁶³,⁶⁴. Excessive mitochondrial fission also occurs in metabolic conditions, such as type II diabetes (T2D)⁶⁵. Elevated HDAC7 mRNA levels, along with reduced insulin secretion, were observed in pancreatic islet cells from people with T2D compared to non-diabetic controls⁶⁶. A similar reduction in insulin secretion was observed in HDAC7-overexpressing rat islet and clonal β-cells, along with a reduction in mitochondrial respiration in those cells⁶⁷. Whether the HDAC7-DRP1-fission axis contributes to this dysregulation remains an open question.

## Study limitations and future directions

While this study has revealed that HDAC7 interacts with DRP1 and drives TLR-inducible mitochondrial fission in macrophages, the precise mechanisms by which it controls DRP1 functions are unknown. The significance of HDAC7-mediated fission in macrophages to different pathological processes, for example neuroinflammatory and metabolic conditions, also remains to be determined. Finally, HDAC7 is localised to the cytoplasm in macrophages⁹ and
subcellular localisation of HDAC7 dictates its functions in different cell types¹⁴, so it is unclear
whether HDAC7 promotes fission in cell types other than macrophages. Thus, future studies
should determine whether HDAC7 promotes mitochondrial fission in other cell types in which
this protein is cytoplasmic, the precise mechanisms by which it does so, and the contribution of
HDAC7-mediated fission to physiological and pathophysiological processes.

ACKNOWLEDGMENTS

This work was supported by a National Health and Medical Research Council of Australia
(NHMRC) Ideas grant (1184885 to M.J.S. and D.R.), NHMRC Investigator grants (1194406 to
M.J.S. and 2009551 to D.P.F), an Australian Research Council (ARC) Discovery Project
(DP230102124 to M.J.S. and S.Z.), an ARC Laureate Fellowship (R.G.P), and an Australian
Infectious Disease Research Centre seed grant (C.E., M.J.S., J.E. and D.R.). We acknowledge
the IMB Mass Spectrometry Facility, the ARC Centre of Excellences in Advanced Molecular
Imaging (CE140100011) and Innovations in Peptide and Protein Science (CE200100012), the
Transgenic Animal Facility of Queensland (a division of The University of Queensland
Biological Resources department), the Biological Research Facility at the Translational Research
Institute and the University of Queensland BASE Facility. We thank Prof Eric Olson and Prof
Rhonda Bassel-Duby (UT Southwestern Medical Centre, United States) for kindly providing
*Hdac7* flo/flo mice used in this study, Prof Mike Ryan (Monash Biomedicine Discovery Institute,
Australia) for providing *Drp1*⁻/⁻ MEF cells, Jordan Atkinson for generating *Hdac7* mRNA, and
Ben Matthews (Queensland Brain Institute) for help with proteomic analyses. Figures S1A-B
and the Graphical Abstract were created using BioRender.com.

AUTHOR CONTRIBUTIONS

Conceptualisation, R.A., S.F.A., S.Z., R.K., D.R., M.J.S.; Methodology, R.A., S.F.A., A.J., R.G.P., R.K., D.R., M.J.S.; Investigation, R.A., S.F.A., J.E.B.C., K.D.R., K.D.G., K.S.G., A.J., Z.X., J.H.G., D.R.; Resources, R.C.R., J.H.G., J.A.E., C.R.E., A.B., D.P.F.; Writing- original draft, R.A., S.F.A., D.R., M.J.S.; Writing- Review/Writing, R.A., S.F.A., J.E.B.C., K.D.R., K.D.G., K.S.G., A.J., R.C.R., Z.X., J.H.G., J.A.E., C.R.E., A.B., D.P.F., R.G.P., S.Z., R.K., D.R.,
M.J.S.; Supervision, D.P.F., R.G.P., R.K., D.R., M.J.S.; Funding acquisition, J.A.E., C.R.E., D.P.F., S.Z., D.R., M.J.S.

## DECLARATION OF INTERESTS

The authors have no conflicts of interests to declare.

## METHODS

Detailed methods are provided underneath and include the following:

* RESOURCES TABLE

* LEAD CONTACT AND MATERIALS AVAILABILITY

* EXPERIMENTAL MODELS

* Animals

* Primary cell culture

* Cell lines

* Ethics statement

* METHOD DETAILS

* Generation of lentiviral constructs

* Gene overexpression by lentiviral transduction in THP1 cells

* Construction of ΔDrp1 RAW264.7 cells

* Class IIa HDAC enzyme activity assay

* Strepavidin pulldown

* Mass spectrometry

* Proteomic analysis

* Protein lysates and immunoblotting

* Mammalian expression constructs

* Co-immunoprecipitation

* Gene overexpression by retroviral transduction in BMM

* Transient transfection of MEF cells

* Proximity ligation assay

* mRNA synthesis and Neon transfection of RAW264.7 cells
* Real time analysis of extracellular acidification rate (ECAR)

* RNA extraction and qPCR

* Mitotracker staining and quantification of mitochondrial numbers

* QUANTIFICATION AND STATISTICAL ANALYSIS

* DATA AND CODE AVAILABILITY

## LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Matthew J. Sweet (m.sweet@imb.uq.edu.au).

All unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement and reasonable compensation by the requestor for their processing and shipping.

## EXPERIMENTAL MODELS

### Animals

Mice aged 8-12 weeks were used for the study. Wild-type (C57BL/6) mice were acquired from an in-house breeding colony. *Hdac7<sup>+/+</sup>*(*Hdac7<sup>fl/fl</sup>* ) were kindly provided by Prof Eric Olson and Prof Rhonda Bassel-Duby (UT Southwestern Medical Centre, United States), which were further crossed with LysM<sup>Cre</sup> mice to obtain myeloid specific *Hdac7*-deficient mice ( *Hdac7<sup>fl/fl</sup>*/*Lysm<sup>Cre</sup>)<sup>11</sup>. MacBlue mice express cyan fluorescent protein (CFP) in monocytes and macrophages due to the presence of two transgenes including the *c-fms/Csf1r* promoter that drives expression of the transcriptional activator Gal4-VP16 and UAS-CFP<sup>68</sup>. MacBlue mice were then crossed with C57BL/6J expressing the UAS-HDAC7-u-V5 transgene to generate progeny that expresses HDAC7-u<sup>69</sup> specifically in the myeloid compartment (MacHDAC7)<sup>11</sup>. *Tlr4<sup>-/-</sup>* and *MyD88<sup>-/-</sup>* mice on a C57BL/6 background were used as previously described<sup>22</sup>.

### Primary cell culture

To generate murine BMM, bone marrow cells were isolated from the femurs and tibias of mice. Post-euthanasia, the bones were sterilized using 70% ethanol and then flushed using a 27-gauge needle containing PBS to isolate the cells. The obtained cells were then cultured in RPMI1640
media (Invitrogen) with 2 mM L-glutamine (Gibco), 10% fetal bovine serum (FBS) (Gibco), 50 U/mL penicillin (Invitrogen), 50 µg/mL streptomycin (Invitrogen), and 150 ng/mL human colony stimulating factor-1 (CSF-1). Cells were harvested on day 6 of BMM differentiation and plated as required for experiments.

HMDM were generated by positive selection of CD14+ monocytes from buffy coats obtained from the Australian Red Cross Lifeblood. In brief, buffy coat samples were subjected to Ficoll (Ficoll-Paque Plus, GE Healthcare Life Sciences, USA) density gradient centrifugation to isolate peripheral blood mononuclear cells. Subsequently, the cells were incubated with CD14+ Microbeads (Miltenyi Biotech, Germany) and transferred into LS columns (Miltenyi Biotech, Germany). The eluted CD14+ monocytes were then cultured in IMDM media (Gibco) with 10% FBS, 50 U/mL penicillin, 50 µg/mL streptomycin and 150 ng/mL CSF-1 for 6 days, before plating for experiments as required.

## Cell lines

THP1 cells were cultured in biotin-containing RPMI1640 media with 2 mM L-glutamine, 10% FBS, 50 U/mL penicillin, 50 µg/mL streptomycin, 10 mM HEPES (Gibco) and 1 mM sodium pyruvate (Gibco). These cells were stimulated with 30 ng/mL PMA (Sigma Aldrich) for 48 h, followed by PMA removal for 6 h before all functional experiments. PlatE, MEF, HEK293 and HEK293T cells were cultured in DMEM (Invitrogen) with 10% FBS, 50 U/mL penicillin and 50 µg/mL streptomycin. When plating for experiments, MEF cells were serum starved overnight (~16 h) in the presence of DMEM (Invitrogen) with 0.5% FBS, 50 U/mL penicillin and 50 µg/mL streptomycin. The murine macrophage-like cell line RAW264.7 (ATCC) was cultured in RPMI1640 media containing 2 mM Glutamax, 5% FBS, 50 U/mL penicillin and 50 µg/mL streptomycin.

## Ethics statement

All animal studies had approval from the appropriate University of Queensland Animal Ethics Committee. All work involving human primary cells was approved by the University of
Queensland Institutional Human Research Ethics Committee. Human blood was collected from heathy donors following informed consent and deidentified buffy coats were obtained from Australian Red Cross Lifeblood for this study.

# METHOD DETAILS

## Generation of lentiviral constructs

TurboID-Empty vector was generated by inserting the TurboID gene block in the doxycycline-inducible lentiviral vector-pF_TRE3G_PGK_puro⁷⁰,⁷¹. HDAC7-FLAG-TurboID plasmid was generated by using restriction enzymes to insert HDAC7-FLAG into the TurboID-Empty vector. NKG7-V5-TurboID by using restriction enzymes to insert NKG7-V5 into the TurboID-Empty vector. All plasmids were sequence-verified prior to use.

## Gene overexpression by lentiviral transduction in THP1 cells

2 x 10⁶ HEK293T cells per transfection were plated and transfected with 0.8 µg/mL of lentiviral packaging and envelope plasmids including pCMV-dR8.2dvpr and pCMV-VSV-G (Addgene), and 1.2/mL µg of plasmid DNA along with 60 µL lipofectamine 2000 (Invitrogen) in 1.5 mL OptiMEM media (Invitrogen). At 6 h post-transfection, the media was replaced with complete THP1 media, and the cells were incubated for 24 h. The viral supernatants were collected, and filtered using PVDF syringe filters (0.45 mm) (Millipore) and supplemented with 6 µL polybrene (Merck). This mix was then added to 2 wells of 2 x 10⁶ THP1 cells and spinfected at 1000 g at 35°C for 2 h. At 48 h post-transduction, the cells were washed with media thrice and cultured in 1 µg/mL puromycin (Sigma-Aldrich) containing media. The differentiated cells were then stimulated with 100 ng/mL doxycycline (Sigma-Aldrich) to confirm inducible gene expression. For experiments, THP1 stable cell lines were differentiated with 30 ng/mL PMA for 48 h, after which cells were stimulated ± doxycycline (100 ng/mL) for 16 h for proteomic studies or for 6 h for Class IIa HDAC enzyme activity assays, prior to subsequent analyses.
## Construction of ΔDrp1 RAW264.7 cells

A combined protocol from Lipofectamine CRISPRMAX (Invitrogen) and Integrated DNA technologies (IDT) was used to construct ΔDrp1 RAW264.7 cell lines. Three distinct guide RNA (gRNA) duplexes (1 µM) were generated by annealing three different crRNAs (IDT, sequence details provided in the Resources Table) with a tracrRNA. A mixture of three different gRNA duplexes were used for transfection to maximize the chances of gRNA-dependent Drp1 targeting and subsequent Cas9-mediated deletion. 1 x 10⁵ RAW264.7 cells per 500 µL per well were plated in a 24-well plate on the day before transfection. A transfection mix was prepared containing 7 µL of the gRNA mix, 0.9 µL of Cas9 (8 µM), 0.25 µg blasticidin-resistant selection vector pEF6/V5-His TOPO, 125 µL lipofectamine Cas9 plus (Invitrogen) and 31.25 µL OptiMEM media. This was added to 31.25 µL of OptiMEM media containing 1.9 µL CRISPRMAX lipofectamine reagent (Invitrogen) and left at RT for 10 min, followed by dropwise addition to the previously plated RAW264.7 cells. The cells were then incubated for 4 h at 37°C, and then washed once before replenishing with fresh media, supplemented with 2 µg/mL blasticidin. After overnight incubation and 24 h of transfection, media was replaced with fresh blasticidin-containing media. After 48 h of selection, cells were transferred to a 10 cm TC dish without blasticidin. The cells were then left undisturbed at 37°C until they formed visible colonies. Single colonies were then picked using pipette tips and transferred into individual wells of a 12-well plate. Once confluent, cells were split and again transferred to sterilin plates. At this stage, whole cell lysates were harvested and screened for absence of DRP1 by western blotting.

## Class IIa HDAC enzyme activity assay

1 x 10⁵ cells were plated in a black 96-well plate (Corning) and incubated at 37°C overnight. After appropriate treatments, cells were lysed in 10 µL mild lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP40, pH 7.4) on ice for 15 min. Class IIa HDAC assay buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, pH 8) containing 200 µM class IIa HDAC substrate (BOC-Lys(trifluoroacetyl)-AMC)⁵³ was added to the lysates and then incubated at 37°C for 30 min. After incubation, the reaction was stopped by adding 20 mM SAHA⁷² (manufactured in-house) and 1 mg/mL trypsin (Invitrogen) diluted in class IIa HDAC assay buffer. The fluorescence of the resulting solution was measured using a plate reader (Infinite M
Plex, Tecan) at the excitation wavelength and emission wavelength of 350 nm and 460 nm, respectively.

## Streptavidin pulldown

The streptavidin pulldown protocol was modified from a previous study³⁰. 30 x 10⁶ PMA-differentiated THP1 cells were washed using PBS and lysed using RIPA buffer supplemented with phosphatase inhibitor (PhosStop™, Roche) and protease inhibitor cocktail (cOmplete™, Roche). 2 µL benzonase (Sigma-Aldrich) was added per sample and incubated for 20 min room temperature to remove nucleic acids. The samples were then subjected to centrifugation at maximum speed and the one-fourth of the lysate was stored as input. The lysates were passed through a PD-10 desalting column (GE Healthcare) to eliminate the presence of free biotin. Dynabeads MyOne streptavidin C1 (Thermo Fisher Scientific) were washed three times using RIPA buffer and 400 µL of the beads was added to each sample. These samples were incubated overnight at 4°C. Following incubation, the samples were placed on a magnetic rack and beads were washed twice with RIPA buffer, once in SDS wash buffer (2% SDS in 50 mM Tris-HCl pH 7) and once in urea buffer (2 M urea in 10 mM Tris-HCl pH 8). Finally, the beads were washed and resuspended in 50 µL 0.1 M ammonium bicarbonate for downstream applications.

## Mass spectrometry

200 µL of sequencing grade Trypsin/Lys-C (Promega) (20 ng/µL in 50 mM ammonium bicarbonate pH 8) was added to each sample and incubated overnight at 37°C. After incubation, the beads were separated and the sample was dried using vacuum centrifugation and reconstituted in 0.1% formic acid. The sample was analysed using LC-MS/MS on a Triple TOF 6600 mass spectrometer (SCIEX).

## Proteomic analysis

The initial MS peak area analysis was performed using Skyline software (MacCoss Lab). Subsequently, protein and peptide identification from the final MS/MS spectra was performed using ProteinPilot™ software (SCIEX). The MS/MS spectra were mapped against Uniprot
database (*Homo sapiens*) and the search parameter was set to conduct False Discovery Rate (FDR) analysis with a 1% global FDR cut-off for protein identification. The resulting data was then analysed to identify the unique and overlapping proteins in each sample. Pathway analysis was performed using the ClusterProfiler package in R⁷³. 104 HDAC7 candidate proteins were subjected to GO Cellular component, Biological process and Molecular function analyses and KEGG pathway analysis in ClusterProfiler package in R. Furthermore, the interaction between glycolysis-related proteins was analysed using STRING⁷⁴.

## Protein lysates and immunoblotting

Cells were lysed using RIPA buffer supplemented with phosphatase inhibitor (PhosStop™) and protease inhibitor cocktail (cOmplete™) and homogenized using a 21-gauge needle. 1X loading buffer (Bolt™ LDS sample buffer, Invitrogen) and 1X reducing agent (NuPAGE sample reducing agent, Invitrogen) was added to the lysates and then heat denatured at 100°C for 10 min. Samples were then loaded on 4-20% precast gels (Bio-Rad) and subjected to electrophoresis at 200 V for 30 min. The samples were transferred onto a nitrocellulose membrane (Bio-Rad) using a semi-dry Turbo-transfer system (Bio-Rad) at 25 V for 9 min. Membrane were blocked in 5% skimmed milk made in tris buffered saline (10 mM Tris-HCl, 150 mM NaCl) with 0.05% Tween-20 (TBST) for 1 h and then incubated with primary antibody (diluted in 5% milk or 5% BSA in TBST) overnight. The membranes were washed with TBST and incubated with secondary antibody (diluted in 5% milk or 5% BSA in TBST) along with Rhodamine antibodies (Tubulin or GAPDH) for 1 h before being visualized using Clarity ECL (Bio-Rad) on the Bio-Rad Chemidoc-MP system (Bio-Rad).

## Mammalian expression constructs

The pEF6-HDAC7-V5, pEF6-HDAC7-ED-V5 (pEF6-HDAC7H649A-V5), pEF6-HDAC5-V5, pEF6-HDAC6-V5 and pEF6-SCIMP-V5 constructs have previously been described¹¹,⁴¹,⁴⁶,⁵⁴. The pMIGR-HDAC7 and pMIGR-HDAC7-ED constructs used for retroviral-mediated gene expression have been previously described¹¹. The full-length mouse DRP1 transcript variant 2 (mouse isoform b, NCBI Reference Sequence: NM_001025947.2) (without stop codon) was amplified by PCR from a pool of BMM cDNA (Forward Primer:
GTCATGGAGGCGCTGATCC, Reverse Primer: GAAATCCGAGAGACTCATCTTTGG). The amplified construct was then cloned into the pEF6/V5-His TOPO TA expression vector (Invitrogen) to generate the mouse pEF6-DRP1 construct. The DRP1 phosphomutant constructs, pEF6-DRP1-S635A and pEF6-DRP1-S656E (pEF6/V5-His TOPO TA backbone), were purchased from Gene Universal (USA). Endotoxin-free plasmid maxi-prep kits (Qiagen) were used to generate plasmid DNA for transfection.

## Co-immunoprecipitation

For assessing interactions of endogenous proteins, 10 x 10⁶ THP1 cells were PMA-differentiated for 48 h, following which they were cross-linked with 1% formaldehyde (Thermo Fisher Scientific) for 10 min. Reactions were then quenched using 0.1 M Glycine. Cells were washed twice with ice-cold PBS and then lysed in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor. One-tenth of the lysates were stored as input. For assessing interactions of ectopically-expressed proteins, HEK293T cells were transfected with indicated constructs for 24 h after which cells were lysed in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor. 2 µL benzonase was added per sample and incubated for 20 min at room temperature to remove nucleic acids. The samples were then subjected to centrifugation at maximum speed and one-fourth of the lysate was stored as input. 1 µg of Mouse anti-DRP1 or Mouse IgG1 isotype control antibody (see Resources Table) was added to each sample and incubated overnight at 4°C. 25 µL Dynabeads protein G magnetic beads (Invitrogen) were then added to each sample and incubated on a rotor for 2 h at 4°C. The beads were then washed thrice with RIPA buffer and eluted using 1X loading buffer (Bolt™ LDS sample buffer, Invitrogen) and 1X reducing agent (NuPAGE sample reducing agent, Invitrogen). The samples were immunoblotted as described above.

## Gene overexpression by retroviral transduction in BMM

2 x 10⁶ PlatE cells per transfection were plated on 10 cm dishes and were transfected with 24 µg of Empty Vector plasmid DNA or molar equivalent of indicated constructs along with 30 µL lipofectamine 2000 in 1.5 mL OptiMEM media. At 24 h post-transfection, the media was replaced with complete BMM media, and the cells were incubated at 32°C for another 48 h. The
viral supernatants were collected, filtered using a PVDF syringe filter (0.45 µm) and supplemented with 1 M HEPES, 10 µg/mL polybrene, and 150 ng/mL CSF1 and incubated at room temperature for 20 min. The viral supernatants were then added to day 2 BMM and spinfected at 1000 g at 35°C for 2 h. On day 6 of BMM differentiation, cells were harvested and plated for experiments.

Transient transfection of MEF cells

*Drp1*-deficient MEF cells have previously been described⁷⁵. 4.5 x 10⁴ wild type or *Drp1*-deficient MEF cells were plated in a 24-well plate with serum-starved MEF media and incubated overnight at 37°C. On the following day, 0.5 µg of the indicated plasmids were transfected into MEF cells using lipofectamine 3000 (Thermo Fisher Scientific), as per the manufacturer's protocol, after which the cells were incubated at 37°C. At 6 h post-transfection, the media was replaced with complete MEF media, with further analyses performed at 24 h post-transfection.

Proximity ligation assay

5 x 10⁴ BMM were plated over the coverslips in a 24-well plate and fixed in 4% paraformaldehyde for 15 min. Cells were then washed 3 times with PBS and incubated with blocking buffer for 1 h. The blocking buffer was then replaced with the primary antibody diluted in the blocking buffer and kept at 4°C overnight. After washing with PBS, cells were incubated with Duolink In Situ Reagents Orange (Sigma) reagents according to the manufacturer's protocol. Cells were again washed with PBS and stained with wheat germ agglutinin -Alexa Fluor 647 (Invitrogen) and 4',6-diamidino-2-phenylidole (DAPI) for 20 min. Cells were washed 3 times with PBS, and the coverslips were mounted using IMBIOL mounting media (Gelvatol mounting media, IMB). Subsequently, slides were imaged using Zeiss Axiovert 200 Inverted Microscope (Zeiss). Cells were defined using wheat germ agglutinin and DAPI, and the PLA puncta were identified using ImageJ software. The threshold for the identification of puncta-positive cells was set to ≥ 3 puncta/cell. PLA-positive cells were counted for each sample, and the percentage of PLA-positive cells was plotted.
## mRNA synthesis and Neon transfection of RAW264.7 cells

Mouse *Hdac7* and *Gfp* mRNA sequences were acquired from NCBI. Codon optimization was performed using mRNAId<sup>76</sup>, and the sequences were inserted between incomplete 5' and 3' UTRs designed for expression by the BASE Facility (The University of Queensland). DNA templates (synthesized by GenScript) were used in PCR reactions, from which mRNAs with complete UTRs were *in vitro* transcribed using the HiScribe™ T7 mRNA kit with CleanCap reagent AG (New England Bioscience) as per the manufacturers instructions, except standard UTP was substituted with 5-methoxy UTP (Thermo Fisher Scientific Milwaukee) to reduce immunogenicity<sup>77</sup>. mRNA was purified using a Monarch RNA clean up kit (NEB), as per manufacturers instructions and transfected into RAW264.7 cells with the Neon NxT Electroporation System (100 µL kit, Thermo Fisher Scientific) using 1400 V, 20 ms and 2 pulses. Transfected cells were immediately transferred into antibiotic-free media and plated as required for the different assays.

## Real time analysis of extracellular acidification rate (ECAR)

For real time analysis of ECAR, transfected RAW264.7 cells were plated at 5 x 10<sup>4</sup> cells/mL in XF96 cell culture plates overnight. The following day, cells were washed and left for 1 h in unbuffered seahorse XF DMEM media supplemented with glucose (10 mM), glutamine (2 mM), and pyruvate (1 mM). ECAR measurements were performed using a Seahorse XF96 Extracellular Flux Analyser (Agilent). Three baseline readings were taken before LPS (0.5 or 100 ng/mL) was added to the cells. Changes in ECAR were measured post-LPS stimulation for a period of up to 3 h. MTT assays (Sigma-Aldrich) were performed in parallel and ECAR values were normalized to MTT data to account for any variations in plating densities.

## RNA extraction and qPCR

5 x 10<sup>5</sup> cells were lysed in 350 µL of RLT buffer (Qiagen), after which total RNA was extracted using QIAGEN RNAesy extraction kits, as per manufacturers guidelines. cDNA was synthesised from 1 µg of RNA using oligo dT priming and Superscript III reverse transcriptase (Invitrogen), as per the manufacturers instructions. A no RT control was generated from RNA pooled from all samples in the preparation and treated as above but without the addition of reverse transcriptase.
The synthesized cDNA was diluted 10-fold in ultrapure DNase/RNase free water (Gibco) and used for qPCR analysis. mRNA levels of *Ccl2*, *Edn1* and *Tnf*, relative to the housekeeping gene hypoxanthine phosphoribosyltransferase (*Hprt*), were determined by qPCR using the delta Ct method, SYBR Green PCR MasterMix (Applied BioSystems) and a Viia7 RT-PCR system (Applied BioSystems).

## Mitotracker staining and quantification of mitochondrial numbers

1 x 10⁵ cells were plated over the coverslips in a 24-well plate and incubated at 37°C overnight. After indicated treatments, the media was replaced with fresh media containing 150 nM (macrophages) or 200 nM (MEF cells) MitoTracker™ Deep Red FM (Invitrogen) for 20 min. Cells were then washed 3 times with PBS and fixed in 4% paraformaldehyde for 15 min. Cells were again washed 3 times with PBS and stained with 20 ng/mL DAPI for 20 min. After washing, the coverslips were mounted using IMBiol mounting media and imaged using Zeiss Axiovert 200 Inverted Microscope with 40 to 63x magnification (mitochondria: red, nuclei: blue). For mitochondrial quantification, 15-30 cells were randomly selected from a minimum of 5 independent fields of view to quantify mitochondrial numbers by identifying maxima of intensity using ImageJ, as previously described⁷⁸. Data from multiple experiments were combined for statistical analysis, and violin plots of representative experiments were used to show variability in mitochondria numbers across cell populations within each experiment. An open-source tool, Mitochondrial Analyzer, integrated as a plugin within ImageJ, was used to quantify mitochondrial branch length and form factor (https://github.com/AhsenChaudhry/Mitochondria-Analyzer)⁷⁹. Image thresholding was applied using a block size of 2.25 and C-value of 5 for all images. The mitochondrial network was analysed for each image using the default settings within the plugin.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed using Prism 10 (GraphPad). Data from 3 or more independent experiments were combined and displayed as mean with standard error of the mean (SEM), with different symbols used to indicate data from individual experiments. Figure legends show details of specific statistical analysis for different data sets, with “n” indicating number of independent
experiments that were performed (either combined data where statistical analysis was performed or representative experiments for immunoblots and microscopy images).

## DATA AND CODE AVAILABILITY

This study did not generate any unique datasets or code.

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<figure><img src="image_2.png" /><figcaption>B</figcaption></figure>

<table><caption>C</caption><thead><tr><th colspan="2">HDAC7</th><th colspan="2">NKG7</th></tr><tr><th>Protein Name</th><th>% Coverage</th><th>Protein Name</th><th>% Coverage</th></tr></thead><tbody><tr><td>ZYX</td><td>64.86</td><td>LAMTOR1</td><td>57.76</td></tr><tr><td>INPP5D</td><td>22.54</td><td>AP3B1</td><td>9.32</td></tr><tr><td>HCLS1</td><td>28.19</td><td>STARD3</td><td>10.79</td></tr><tr><td>TRIM25</td><td>25.24</td><td>ITGB2</td><td>3.90</td></tr><tr><td>DPYSL2</td><td>39.69</td><td>STIM1</td><td>3.50</td></tr><tr><td>GSN</td><td>26.47</td><td>PLEKHO2</td><td>4.69</td></tr><tr><td>PLIN3</td><td>40.09</td><td>LBR</td><td>3.09</td></tr><tr><td>LCP1</td><td>24.08</td><td>RPL8</td><td>6.23</td></tr><tr><td>PTPN6</td><td>23.87</td><td>TMPO</td><td>2.64</td></tr><tr><td>MYO1E</td><td>15.79</td><td>TGFB1</td><td>5.13</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Gene Ontology: Biological Pathway</figcaption></figure>
Fig. 1. TurboID tagging identifies 104 candidate HDAC7 interaction partners.

PMA-differentiated THP1 cell lines expressing HDAC7-FLAG-TurboID, NKG7-V5-TurboID or TurboID alone (EV) were treated ± doxycycline, lysed, and biotinylated proteins were isolated using streptavidin pulldown. The samples were then trypsin-digested and proteins identified by mass spectrometry.

(A) Proteins in input and pulldown samples were detected by immunoblotting with anti-FLAG, anti-V5, or anti-Tubulin antibodies (n=3).

(B) Table shows proteins with the highest peptide coverage identified in the HDAC7-TurboID and NKG7-TurboID samples (ProteinPilot, 1% global false-discovery rate).

(C) Scatter dot plot comparing MS peak area of proteins identified in empty vector-TurboID and HDAC7-TurboID (HDAC7) samples (Skyline). The x-axis indicates the fold change across the two conditions and the y-axis indicates statistical significance for differentially-expressed proteins (dark blue dots: proteins with p< 0.05; orange dots: proteins of interest). Statistical analysis was performed using an unpaired two-tailed t-test (n=3).

(D) Venn diagram representing numbers of proteins identified by mass spectrometry across all experiments, as determined using ProteinPilot (104 proteins unique to HDAC7 in each of three experiments).

(E-F) The 104 HDAC7-TurboID-specific proteins were subjected to Gene Ontology (E) Cellular component and (F) Biological Pathway enrichment analysis (ClusterProfiler). Dot plots represent pathways enriched in proteins identified from cells expressing HDAC7-TurboID. Dot sizes indicate the protein count and colours indicate p-values (n=3).

(See also figure S1)
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** HDAC7 associates with mitochondrial fission protein DRP1 and drives LPS-inducible fission in macrophages.</figcaption></figure>

(A) Indicated PMA-differentiated THP1 stable cell lines were treated ± doxycycline, lysed, and proteins were immunoprecipitated with an anti-DRP1 antibody. Immunoblotting was used to
visualise HDAC7 (anti-FLAG), DRP1 (anti-DRP1) and NKG7 (anti-V5) and Tubulin (anti-Tubulin) (n=3).

(B) PMA-differentiated THP1 cells were crosslinked using formaldehyde then lysed, after which proteins were immunoprecipitated with an anti-DRP1 antibody or an isotype control (IC). Immunoblotting was used to visualise DRP1, HDAC7 and Tubulin in inputs and immunoprecipitates (n=2).

(C-D) MacHDAC7 and MacBlue BMM were fixed, and PLA was performed using antibodies against the V5 tag (HDAC7-V5) and either endogenous DRP1 or MAPK p38 (control). (B) Cells were stained with DAPI (blue) and WGA (red), then visualized by confocal microscopy (scale bar=5 µm, n=3). (C) % PLA-positive cells in each condition were quantified using a threshold of ≥ 3 puncta/cell (mean ± SEM, * p< 0.05, RM two-way ANOVA, n=3).

(E) *Hdac7<sup>+/+</sup> and *Hdac7<sup>-/-</sup> BMM were stimulated with LPS (0.5, 100 ng/mL) for 6 h, stained with MitoTracker (red) and DAPI (blue), then visualized by confocal microscopy (scale bar=5 µm, n=5).

(F) Mitochondrial numbers per cell from images in E were quantified (mean ± SEM, * p< 0.05, ** p< 0.01, *** p< 0.001, RM two-way ANOVA, Dunnett's multiple comparisons test, n=5, data normalized to unstimulated *Hdac7<sup>+/+</sup> controls).

(G) MacBlue and MacHDAC7 BMM were stimulated with LPS (100 ng/mL) for 6 h. Cells were stained with MitoTracker (red) and DAPI (blue), then visualized by confocal microscopy (scale bar=5 µm, n=3).

(H) Mitochondrial numbers per cell from images in G were quantified (mean ± SEM, * p< 0.05, RM two-way ANOVA, Tukey's multiple comparisons test, n=3, normalized to unstimulated MacBlue controls).

(See also figure S2 and S3)
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** HDAC7 enzyme activity is dispensable for LPS-inducible fission.</figcaption></figure>

(A-C) *Hdac7*⁻ BMM were transduced with empty vector (EV), wild type (WT) HDAC7 or enzyme-dead HDAC7 (ED), with *Hdac7*⁺/BMM used as a positive control.

(A) HDAC7 protein expression in transduced *Hdac7*⁻ BMMs (anti-V5).

(B) Indicated cells were stimulated with LPS (100 ng/mL) for 6 h, stained with MitoTracker (red) and DAPI (blue), then visualized by confocal microscopy (scale bar=5 µm).

(C) Mitochondrial numbers per cell from images in **B** were quantified (mean ± SEM, **p**<0.01, RM two-way ANOVA, Sidak's multiple comparisons test, n=3, data normalized to WT BMM unstimulated control).

(D) BMM were pre-treated with DMSO or TMP195 (0.5 µM) for 1 h, then LPS-stimulated (100 ng/mL) for 6 h. Cells were stained with MitoTracker and DAPI, visualized by confocal microscopy, and mitochondrial numbers per cell quantified (mean ± SEM, **p**<0.01, RM one-
way ANOVA, Dunnett's multiple comparisons test, n=3, normalized to the unstimulated DMSO-treated control).

(E) HMDM were pre-treated with DMSO or TMP195 (10 µM) for 1 h, then LPS-stimulated (100 ng/mL) for 6 h. Cells were stained with MitoTracker and DAPI, visualized by confocal microscopy, and mitochondrial numbers per cell quantified (mean ± SEM, ns = not significant, ** p<0.01, RM one-way ANOVA, Dunn's multiple comparisons test, n=5, normalized to the unstimulated DMSO-treated control).

(F) HEK293T cells were co-transfected with V5-tagged DRP1 and either HDAC7, enzyme-dead (HDAC7-ED), or SCIMP (unrelated control). Cells were lysed, samples were immunoprecipitated (anti-endogenous DRP1), and immunoblotting was used to visualise the V5 tag (detecting DRP1 and candidate interactors) and Tubulin (n=3).

(See also figure S4, S5 and S6)
<figure><img src="image_9.png" /><figcaption>Fig. 4. HDAC7 requires DRP1 to promote LPS-inducible glycolysis and to fine-tune inflammatory gene expression in macrophages.</figcaption></figure>

(A) Immunoblotting was used to assess DRP1 protein levels in wild-type (WT) and *Drp1*-deficient (*ΔDrp1*) RAW264.7 cells generated by CRISPR-Cas9.

(B) Left: Δ*Drp1* RAW264.7 cells were transfected with empty vector (EV) or DRP1 plasmids, stimulated with LPS (100 ng/mL) for 6 h, stained with MitoTracker (red) and DAPI (blue), then visualized by confocal microscopy (scale bar=10 µm). Right: Mitochondrial numbers per cell
were quantified (mean ± SEM, * p < 0.05, ns = not significant, RM two-way ANOVA, Sidak's multiple comparisons test, n=3, data normalized to DRP1-transfected unstimulated control).

(C) Wild-type and ΔDrp1 RAW264.7 cells were transfected with mRNAs encoding either HDAC7 or GFP (control), after which levels of HDAC7 (V5), DRP1 and Tubulin were assessed by immunoblotting.

(D) Wild-type and (E) ΔDrp1 RAW264.7 cells were transfected with mRNAs encoding either HDAC7 or GFP (control), after which ECAR levels were measured following LPS treatment, as indicated. Left: ECAR plots from a representative experiment, right: area under the curve (AUC) analysis (mean ± SEM, *p < 0.05, ns = not significant, two-way ANOVA with Sidak's post-test, n=3, normalized to the GFP control).

(F-H) Wild-type and ΔDrp1 RAW264.7 cells were transfected with mRNAs encoding either HDAC7 or GFP (control). After 24 h, cells were stimulated with LPS (100 ng/ml) for 4 h, after which mRNA levels of (F) Ccl2, (G) Edn1 and (H) Tnf (relative to Hprt) were quantified by qPCR (mean ± SEM, *p < 0.05, ****p < 0.0001, ns = not significant, two-way ANOVA with Sidak's post-test, n=3, normalized to the GFP control).
<figure><img src="image_10.png" /><figcaption>Fig. S1. Generation and functional validation of THP1 stable cell lines expressing TurboID fusion proteins.</figcaption></figure>

(A) A schematic diagram of the TurboID system.

(B) Lentiviral vectors expressing HDAC7-FLAG-TurboID, NKG7-V5-TurboID fusion proteins or TurboID alone were transduced into THP1 cells. THP1 stable cell lines were
differentiated with PMA for 48 h, then stimulated ± doxycycline (100 ng/mL) for 16 h. Cells
were then lysed, and the biotinylated proteins were isolated using streptavidin pulldown. The
samples were then digested using trypsin and proteins identified by mass spectrometry analysis.

(C) Lentiviral vectors encoding HDAC7-FLAG-TurboID and NKG7-V5-TurboID fusion proteins were transduced in THP1 cells. Two sets of stable cell lines (generated in independent experiments) were selected using 1 µg/mL puromycin for a week, PMA-differentiated for 48 h, then stimulated ± doxycycline (100 ng/mL) for 16 h. Immunoblotting for epitope tags were used to visualise HDAC7 (anti-FLAG), NKG7 (anti-V5), and GAPDH as a loading control. Displayed immunoblots are representative of three independent experiments.

(D) Empty Vector (EV), HDAC7-TurboID, and NKG7-TurboID THP1 stable cell lines were PMA-differentiated for 48 h, then treated with doxycycline (100 ng/mL) for 16 h, after which cells were lysed, and class IIa HDAC enzyme activity was determined. Data are combined from three independent experiments (mean ± SEM, * p<0.05, RM one-way ANOVA followed by Dunnett's correction).

(E) Empty vector (EV) and HDAC7-TurboID THP1 stable cell lines were PMA-differentiated for 48 h, then stimulated ± doxycycline (100 ng/mL) for 6 h. Cells were then washed, left for a further 6 h in fresh media, then stimulated ± LPS (10 ng/mL) for 1 h. Cells were then lysed, and class IIa HDAC enzyme activity was determined. Data are combined from three independent experiments (mean ± SEM, ns – not significant, ** p<0.01; *** p<0.001, **** p<0.0001, RM two-way ANOVA followed by Sidak's multiple comparison test).

(F-G) The 104 proteins identified as being unique to the HDAC7-TurboID pulldown (candidate HDAC7 partners, **Figure 1**) were subjected to (E) Gene Ontology-Molecular Function and (F) KEGG Pathway enrichment analysis using the ClusterProfiler package. The dot plots above represent the pathways enriched in proteins identified from cells expressing HDAC7-FLAG-TurboID. The size of the dot indicates the protein count, and the colour indicates the *p*-values. *p*-values in (G) are nominal, without post-corrections, and only "Pathogenic E. coli infection" and "Yersinia infection" pathways remained significant post-FDR correction. Data are combined from three independent experiments.

(H) Protein-protein interaction network of the glycolysis-related proteins identified in the candidate HDAC7 partners. The protein interaction analysis was performed using STRING.
<figure><img src="image_11.png" /><figcaption>Fig. S2. Effects of HDAC7 on mitochondrial fission, mitochondrial mass, and inflammatory responses in macrophages.</figcaption></figure>

(A) BMM from *Hdac7+/+* and *Hdac7-/-* mice were stimulated with LPS (0.5 ng/mL or 100 ng/mL) for 6 h. Cells were stained with 100 nM MitoTracker, then visualized by confocal microscopy. The numbers of mitochondria per cell from images were quantified by identifying maxima of intensity using ImageJ. 15 cells were counted per condition across five independent fields of view.

(B-C) BMM from *Hdac7+/+* and *Hdac7-/-* mice were stimulated with LPS (0.5 ng/mL or 100 ng/mL) for 6 h. Cells were stained with 100 nM MitoTracker and mitochondrial branch length (B) and form factor (C) were determined by Mitochondria Analyzer plugin in ImageJ (mean ± SEM, n=4, * p< 0.05, **** p< 0.0001, ns = not significant, RM two-way ANOVA, Sidak's multiple comparisons test).

(D) BMM from *Hdac7+/+* and *Hdac7-/-* mice were stimulated with LPS (100 ng/mL) for 2 or 6 h. Cells were stained with 100 nM MitoTracker and relative mitochondrial mass was determined by flow cytometry (mean ± SEM, data normalized to unstimulated *Hdac7+/+* controls, n=3).
(E) BMM from *Hdac7+/+* and *Hdac7/-* mice were stimulated with LPS (10 ng/mL) for 4 or 8 h, after which mRNA levels of *Tnf* (relative to *Hprt*) were quantified by qPCR (mean ± SEM, n=3).

(F) BMM from MacBlue and MacHDAC7 mice were stimulated with LPS (100 ng/mL) for 6 h. Cells were stained with 150 nM MitoTracker, then visualized by confocal microscopy. Numbers of mitochondria per cell were quantified by identifying maxima of intensity using ImageJ. 18 cells were counted per condition across five independent fields of view.

(G) BMM from MacBlue and MacHDAC7 mice were stimulated with 0.5 ng/mL LPS for 6 h. Cells were stained with 150 nM MitoTracker, then visualized by confocal microscopy. Numbers of mitochondria per cell were quantified by identifying maxima of intensity using ImageJ (mean ± SEM, * p< 0.05, RM two-way ANOVA, Sidak's multiple comparisons test, n=3).
<figure><img src="image_12.png" /><figcaption>Fig. S3. Role of HDAC7 enzyme activity in LPS-inducible mitochondrial fission.</figcaption></figure>

(A) BMM from myeloid *Hdac7*-/- mice were retrovirally transduced with empty vector (EV), a wild-type (WT) HDAC7 construct or an enzyme-dead HDAC7 mutant (ED) construct. BMM from wild-type C57BL/6 mice (WT BMM) were used as a control population. Cells from the indicated populations were stimulated with LPS (100 ng/mL) for 6 h and then stained with 150 nM MitoTracker (mitochondria in red) and 20 ng/mL DAPI (nuclei in blue) and visualized by confocal microscopy. Numbers of mitochondria per cell were quantified using ImageJ by
identifying maxima of intensity. 20 cells were counted per condition across five independent
fields of view.

(B) Retrovirally transduced BMM from myeloid *Hdac7*-/- mice were transduced with constructs as described in (A), after which DRP1, HDAC7 (V5) and Tubulin levels were assessed by immunoblotting. Representative immunoblots are displayed (top), with total DRP1 levels relative to Tubulin being quantified (bottom, mean ± SEM, n=3).

(C) BMM from C57BL/6 mice were pre-treated with either DMSO or TMP195 (0.5 µM) for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Cells were stained with 150 nM MitoTracker, then visualized by confocal microscopy. Numbers of mitochondria per cell were quantified using ImageJ by identifying maxima of intensity. 20 cells were counted per condition across five independent fields of view.

(D) HMDM were pre-treated with either DMSO or TMP195 (10 µM) for 1 h, then stimulated with LPS (100 ng/mL) for 6 h. Cells were stained with 150 nM MitoTracker, then visualized by confocal microscopy. The numbers of mitochondria per cell were quantified using ImageJ by identifying maxima of intensity. In the violin plots, the middle dark dotted line in the violin plot indicates median, while the light dotted line indicates the quartiles. A minimum of 15 cells were counted per condition across five independent fields of view.

(E) HEK293T cells were co-transfected with V5-tagged DRP1 and either empty vector (EV), HDAC7-V5 (H7), ED-HDAC7-V5 (ED-H7), HDAC5-V5 (H5) or HDAC6-V5 (H6). Cells were lysed, samples were immunoprecipitated (anti-DRP1), and immunoblotting was used to visualise the V5 tag (detecting both DRP1 and candidate interactors) and Tubulin (n=3).
<figure><img src="image_13.png" /><figcaption>Fig. S4. Reconstitution of DRP1 in Drp1⁻/⁻ MEF cells restores both stages of LPS-inducible mitochondrial fission.</figcaption></figure>

(A-B) Drp1⁻/⁻ MEF cells were transfected with an empty vector control (EV) or indicated DRP1 constructs, with EV-transfected wild type (WT) MEF cells included as a positive control. After 24 h, cells were stimulated with LPS (100 ng/mL) for 3 or 8 h, stained with MitoTracker (red) and DAPI (blue), visualized by confocal microscopy (A), and mitochondrial numbers per cell quantified (B) (scale = 10 µm, mean ± SEM, ** p< 0.01, *** p< 0.001, **** p< 0.0001, RM two-way ANOVA, Tukey's multiple comparisons test, n=4, data normalized to the unstimulated WT MEF-EV control).
<figure><img src="image_14.png" /><figcaption>Fig. S5. Roles of HDAC7 in the two stages of LPS-inducible mitochondrial fission.</figcaption></figure>

(A) *Hdac7+/+* and *Hdac7/-* BMM were stimulated with LPS (100 ng/mL) for 2 or 6 h. Cells were stained with MitoTracker (red) and DAPI (blue), visualized by confocal microscopy, and mitochondrial numbers per cell quantified (mean ± SEM, *** p< 0.001, **** p< 0.0001, RM two-way ANOVA, Dunnett's multiple comparisons test, n=4, normalized to the unstimulated control cells).

(B) A representative experiment from the compiled data in (A) showing numbers of mitochondria per cell, quantified by identifying maxima of intensity using ImageJ. In the violin plots, the middle dark dotted line in the violin plot indicates median, while the light dotted line indicates the quartiles. 20 cells were counted per condition across five independent fields of view.

(C) *Hdac7+/+* and *Hdac7/-* BMM were stimulated with LPS (100 ng/mL), with whole cell extracts analysed by immunoblotting for pDRP1 (S635), FRP (detected by pDRP1 S656 antibody), total DRP1, TOM20 and Histone H3 (n=4).

(D-G) Quantification of data from (C) above. Intensity of total DRP1 relative to loading control (D), TOM20 relative to loading control (E), pDRP1 (S635) relative to total DRP1 levels (F) and FRP relative to total DRP1 levels (G) were quantified using Bio-Rad ImageLab software,
with all data plotted relative to the *Hdac7<sup>+/+</sup>* unstimulated control cells (mean ± SEM, *p<0.05*, **p<0.01*, RM two-way ANOVA, Dunnett's multiple comparisons test, n=4).

(H) Whole cell extracts from WT and *Drp1<sup>-/-</sup>* MEF cells were collected and analysed by western blot for FRP (detected by pDRP1 S656 antibody), total DRP1 and Tubulin (n=3).
<figure><img src="image_15.png" /><figcaption>Fig. S6. TLR4 and TLR adaptors are essential for both stages of LPS-inducible fission in macrophages.</figcaption></figure>

(A) BMM from *Tlr4<sup>+/+</sup>* and *Tlr4<sup>-/-</sup>* mice were stimulated with LPS (100 ng/mL) for 2 or 6 h. Cells were stained with 150 nM MitoTracker (mitochondria in red) and 20 ng/mL DAPI (nuclei in blue), then visualized by confocal microscopy. Numbers of mitochondria per cell were quantified by identifying maxima of intensity using ImageJ. Data (mean ± SEM, ** p<0.01, *** p<0.001, ns = not significant, RM two-way ANOVA, Sidak's multiple comparisons test, n=3, normalized to the *Tlr4<sup>+/+</sup>* unstimulated control cells).

(B) BMM from *Tlr4<sup>+/+</sup>* and *Tlr4<sup>-/-</sup>* mice were stimulated with LPS (100 ng/mL) for the indicated time points, after which whole cell extracts were collected and analysed by western blot for
pDRP1 (S635), FRP (detected by pDRP1 S656 antibody), total DRP1 and Tubulin as a loading control (n=4).

(C) *MyD88<sup>+/+</sup>* and *MyD88<sup>-/-</sup>* BMM were stimulated with LPS (100 ng/mL) for 2 or 6 h. Cells were stained with MitoTracker (red) and DAPI (blue), then visualized by confocal microscopy (scale bar=5 µm).

(D) Numbers of mitochondria per cell in images from C were quantified (mean ± SEM, * p<0.05, ** p<0.01, RM two-way ANOVA, Sidak multiple comparisons test, n=3, normalized to the *MyD88<sup>+/+</sup>* unstimulated control cells).

(E) BMM from C57BL/6 mice were pre-treated with either DMSO or TAK242 (1 µM) for 1 h and then stimulated with LPS (100 ng/mL) for 2 or 6 h. Cells were stained with 150 nM MitoTracker and 20 ng/mL DAPI, then visualized by confocal microscopy. Numbers of mitochondria per cell were quantified using ImageJ by identifying maxima of intensity (mean ± SEM, * p<0.05, ** p<0.01, RM one-way ANOVA, Dunnett's multiple comparisons test, n=3, normalized to the unstimulated DMSO treated control).

(F) BMM from C57BL/6 mice were pre-treated with either DMSO or TAK242 (1 µM) for 1 h and then stimulated with LPS (100 ng/mL) for the indicated time points, after which whole cell extracts were collected and analysed by western blot for pDRP1 (S635), total DRP1, p-ERK1/2 (positive control), and Tubulin (n=3).

**Table S1.** List of 104 proteins that were specifically identified in streptavidin pulldowns for HDAC7-TurboID (versus NKG7-TurboID and empty vector-TurboID) in all three biological replicate experiments.

Available for download at
https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.264376#supplementary-data
RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>V5 (Mouse)</td><td>Bio-Rad</td><td>MCA1360</td></tr><tr><td>FLAG (Rabbit)</td><td>Sigma-Aldrich</td><td>F7425</td></tr><tr><td>DRP1 (Mouse)</td><td>Cell Signalling Technology</td><td>14647S</td></tr><tr><td>DRP1 (Rabbit)</td><td>Cell Signalling Technology</td><td>8570S</td></tr><tr><td>HDAC7 (E708V) (Rabbit)</td><td>Cell Signalling Technology</td><td>10831</td></tr><tr><td>p38 MAPK (Rabbit)</td><td>Cell Signalling Technology</td><td>9212</td></tr><tr><td>phospho-DRP1 (S616) (Rabbit)</td><td>Cell Signalling Technology</td><td>3455S</td></tr><tr><td>phospho-DRP1 (S637) (Rabbit)</td><td>Cell Signalling Technology</td><td>4867S</td></tr><tr><td>phospho-ERK1/2 (Thr202/Tyr204) (Rabbit)</td><td>Cell Signalling Technology</td><td>9101S</td></tr><tr><td>Tom20 (Rabbit)</td><td>Cell Signalling Technology</td><td>42406S</td></tr><tr><td>Histone H3 (Mouse)</td><td>Cell Signalling Technology</td><td>14269</td></tr><tr><td>Mouse (MOPC-21) IgG1 Isotype Control</td><td>Cell Signalling Technology</td><td>4097S</td></tr><tr><td>Tubulin (rhodamine conjugated)</td><td>Bio-Rad</td><td>12004165</td></tr><tr><td>GAPDH (rhodamine conjugated)</td><td>Bio-Rad</td><td>12004167</td></tr><tr><td>anti-mouse IgG HRP-linked antibody</td><td>Cell Signalling Technology</td><td>7076</td></tr><tr><td>anti-rabbit IgG HRP-linked antibody</td><td>Cell Signalling Technology</td><td>7074</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>lipopolysaccharide (LPS)</td><td>Sigma-Aldrich</td><td>L2137</td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P1585</td></tr><tr><td>lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668019</td></tr><tr><td>lipofectamine 3000</td><td>Thermo Fisher Scientific</td><td>L3000015</td></tr><tr><td>polybrene</td><td>Merck</td><td>TR-1003-G</td></tr><tr><td>TMP195</td><td>Fairlie Laboratory, IMB</td><td>N/A</td></tr><tr><td>puromycin</td><td>Sigma-Aldrich</td><td>P7255</td></tr><tr><td>doxycycline</td><td>Sigma-Aldrich</td><td>D 9891</td></tr><tr><td>cOmplete Protease inhibitor cocktail</td><td>Roche</td><td>11697498001</td></tr><tr><td>PhosStop (phosphatase inhibitor)</td><td>Sigma-Aldrich</td><td>4906845001</td></tr><tr><td>trypsin/Lys-C</td><td>Promega</td><td>V5073</td></tr><tr><td>trypsin</td><td>Invitrogen</td><td>15090046</td></tr></tbody></table>
<table><tr><td>SAHA</td><td>Fairlie Laboratory, IMB</td><td>N/A</td></tr><tr><td>MitoTracker™ Deep Red FM</td><td>Invitrogen</td><td>M22426</td></tr><tr><td>wheat germ agglutinin</td><td>Life Technologies</td><td>W32466</td></tr><tr><td>4',6-diamidino-2-phenylidole (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td></tr><tr><td>Class IIa HDAC substrate (BOC-Lys(trifluoroacetyl)-AMC)</td><td>Fairlie Laboratory, IMB</td><td>N/A</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>Duolink In Situ Reagents Orange</td><td>Sigma-Aldrich</td><td>DUO92007</td></tr><tr><td>Duolink In Situ Probe Anti-Mouse Plus</td><td>Sigma-Aldrich</td><td>DUO92001</td></tr><tr><td>Duolink In Situ Probe Anti-Rabbit Plus</td><td>Sigma-Aldrich</td><td>DUO92005</td></tr><tr><td>Qiagen Endofree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td></tr><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>HEK293T</td><td>ATCC</td><td>CRL-3216</td></tr><tr><td>PlatE</td><td>CELL BIOLABS, INC</td><td>RV-101</td></tr><tr><td>THP1</td><td>ATCC</td><td>TIB-202</td></tr><tr><td>WT MEF</td><td>Prof Mike Ryan (Monash Biomedicine Discovery Institute, Australia)</td><td>N/A</td></tr><tr><td>Drp1<sup>-/-</sup> MEF</td><td>Prof Mike Ryan (Monash Biomedicine Discovery Institute, Australia)</td><td>N/A</td></tr><tr><td>WT RAW264.7</td><td>ATCC</td><td>TIB-71</td></tr><tr><td>DDrp1 RAW264.7</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>C57BL/6J</td><td>In-house breeding</td><td>N/A</td></tr><tr><td>MacBlue</td><td>In-house breeding</td><td>N/A</td></tr><tr><td>MacHDAC7</td><td>In-house breeding</td><td>N/A</td></tr><tr><td>Hdac7<sup>+/+</sup> (Hdac7<sup>fl/fl</sup>)</td><td>Prof Eric Olson and Dr Rhonda Bassel-Duby (UT Southwestern Medical Centre, United States)</td><td>N/A</td></tr><tr><td>Hdac7<sup>-/-</sup> (Hdac7<sup>fl/fl</sup>/Lysm<sup>Cre</sup>)</td><td>In-house breeding</td><td>N/A</td></tr><tr><td>Tlr4<sup>+/+</sup></td><td>In-house breeding</td><td>N/A</td></tr><tr><td>Tlr4<sup>-/-</sup></td><td>In-house breeding</td><td>N/A</td></tr><tr><td>MyD88<sup>+/+</sup></td><td>Blumenthal laboratory, TRI</td><td>N/A</td></tr><tr><td>MyD88<sup>-/-</sup></td><td>Blumenthal laboratory, TRI</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>DRP1 cloning primer</td><td>This paper</td><td>N/A</td></tr><tr><td>Fwd: GTCATGGAGGCGCTGATCC</td><td></td><td></td></tr></table>
<table><tr><td>DRP1 cloning primer<br>Rev: GAAATCCGAGAGACTCATCTTTGG</td><td>This paper</td><td>N/A</td></tr><tr><td>mRNA T7 complete primer Fwd:<br>TAATACGACTCACTATAAGGACTCTTCTGGT<br>CCCCACA</td><td>This paper</td><td>N/A</td></tr><tr><td>mRNA PolyA complete primer Rev:<br>T125GCTAGCTCCAGGGTGTGGC</td><td>This paper</td><td>N/A</td></tr><tr><td>Ccl2 qPCR primer<br>Fwd: GCTTCTTTGGGACACCTGCTG</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Ccl2 qPCR primer<br>Rev: CCCACTCACCTGCTGCTACTCA</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Edn1 qPCR primer<br>Fwd: GGAAGGAAGGAAACTACGAAGG</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Edn1 qPCR primer<br>Rev: GTGCGTCAACTTCTGGTCTCT</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Tnf qPCR primer<br>Fwd: CATCTTCTCAAAATTCGAGTGACAA</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Tnf qPCR primer<br>Rev: TGGGAGTAGACAAGGTACAACCC</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Hprt qPCR primer<br>Fwd: GCAGTACAGCCCCAAAATGG</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Hprt qPCR primer<br>Rev: AACAAAGTCTGGCCTGTATCCAA</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td colspan="3"><strong>crRNAs</strong></td></tr><tr><td>GACACTTGTGGATTTACCGG</td><td>This paper</td><td>N/A</td></tr><tr><td>GAGGAACTGGTGTGGTCACC</td><td>This paper</td><td>N/A</td></tr><tr><td>CAGGACGTCTTCAACACAGT</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>Lenti-TurboID-Empty vector</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Lenti-HDAC7-FLAG-TurboID</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>Lenti-NKG7-V5-TurboID</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>pCMV-dR8.2dvpr</td><td>Addgene</td><td>N/A</td></tr><tr><td>pCMV-VSV-G</td><td>Addgene</td><td>N/A</td></tr><tr><td>pEF6-DRP1-V5</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>pEF6-DRP1-S635A</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>pEF6-DRP1-S656E</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>pEF6-HDAC7-V5</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>pEF6-HDAC7-ED-V5</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr><tr><td>pEF6-SCIMP-V5</td><td>Sweet Laboratory,<br>IMB</td><td>N/A</td></tr></table>
<table><tr><td>pEF6-HDAC5-V5</td><td>Sweet Laboratory, IMB</td><td>N/A</td></tr><tr><td>pEF6-HDAC6-V5</td><td>Sweet Laboratory, IMB</td><td>N/A</td></tr><tr><td>pMIGR-HDAC7-V5</td><td>Sweet Laboratory, IMB</td><td>N/A</td></tr><tr><td>pMIGR-HDAC7-ED-V5</td><td>Sweet Laboratory, IMB</td><td>N/A</td></tr><tr><td colspan="3"><strong>Software</strong></td></tr><tr><td>ImageJ</td><td>FIJI</td><td>N/A</td></tr><tr><td>Image Lab</td><td>Bio-Rad</td><td>N/A</td></tr><tr><td>Prism 10</td><td>GraphPad</td><td>N/A</td></tr><tr><td>ProteinPilot™</td><td>SCIEX</td><td>N/A</td></tr><tr><td>Skyline</td><td>MacCoss Lab</td><td>N/A</td></tr><tr><td>R package-clusterProfiler</td><td>Bioconductor</td><td>N/A</td></tr></table>