Long noncoding RNA Malat1 protects against osteoporosis and bone metastasis

MALAT1, one of the few highly conserved nuclear long noncoding RNAs (lncRNAs), is abundantly expressed in normal tissues. Previously, targeted inactivation and genetic rescue experiments identified MALAT1 as a suppressor of breast cancer lung metastasis. On the other hand, Malat1-knockout mice are viable and develop normally. On a quest to discover the fundamental roles of MALAT1 in physiological and pathological processes, we find that this lncRNA is downregulated during osteoclastogenesis in humans and mice. Remarkably, Malat1 deficiency in mice promotes osteoporosis and bone metastasis of melanoma and mammary tumor cells, which can be rescued by genetic add-back of Malat1. Mechanistically, Malat1 binds to Tead3 protein, a macrophage-osteoclast–specific Tead family member, blocking Tead3 from binding and activating Nfatc1, a master regulator of osteoclastogenesis, which results in the inhibition of Nfatc1-mediated gene transcription and osteoclast differentiation. Notably, single-cell transcriptome analysis of clinical bone samples reveals that reduced MALAT1 expression in pre-osteoclasts and osteoclasts is associated with osteoporosis and metastatic bone lesions. Altogether, these findings identify Malat1 as a lncRNA that protects against osteoporosis and bone metastasis.

(CTSK) and acid phosphatase 5 (ACP5, also known as TRAP), followed by maturation of osteoclast precursors and cell-cell fusion 10 .As a master regulator of osteoclastogenesis, the nuclear factor of activated T cells 1 (NFATC1) is induced by RANKL, which in turn forms a complex with other transcription factors 11 and activates the transcription of its own coding gene as well as other genes involved in osteoclast adhesion, cell fusion, and bone resorption [12][13][14] .
Long noncoding RNAs (lncRNAs), transcripts that are longer than 200 nucleotides and are not translated into proteins, function through binding to DNA, other RNA, and proteins 15,16 .LncRNAs usually have low evolutionary conservation.One of the few exceptions, MALAT1, is a highly conserved nuclear lncRNA that is abundantly expressed in many tissues 17 .MALAT1 has been shown to modulate alternative pre-mRNA splicing based on siRNA knockdown results from cultured cell lines 18 .In 2012, three groups reported that Malat1-knockout mice showed no obvious phenotypic differences compared with wild-type mice under physiological conditions, and loss of Malat1 in mice did not affect alternative pre-mRNA splicing [19][20][21] .On the other hand, recent animal studies suggested that Malat1 has important functions under pathological conditions.For instance, through targeted inactivation, restoration (genetic rescue), and overexpression of Malat1 in mouse models, we found that Malat1 suppresses breast cancer lung metastasis through binding and inactivation of the Tead family of transcription factors 22 .Moreover, Malat1-null mice exhibited enhanced antiviral responses, suggesting that Malat1 may suppress antiviral innate immunity 23 .In addition, when fed a high-fat diet, Apoe −/− mice transplanted with Apoe −/− ;Malat1 −/− bone marrow showed higher atherosclerotic plaque burden in the aorta and increased hematopoietic progenitor cells and their progeny, suggesting that Malat1 may regulate hematopoietic cells 24 .
Recent genome-wide association studies (GWAS) showed that single-nucleotide polymorphisms (SNPs) are associated with osteoporosis 1,25 .Interestingly, one such analysis identified an SNP (rs202070768) at the MALAT1 locus that was associated with low BMD 26 .However, functional evidence of MALAT1 alterations having a role in low BMD and osteoporosis is lacking.In the present study, by using genetically engineered mouse models, we identify Malat1 as a negative regulator of osteoporosis and bone metastasis.Mechanistically, Malat1 binds and sequesters Tead3, blocking Tead3 from interacting with and activating Nfatc1.Consequently, loss of Malat1 derepresses Tead3, which in turn promotes Nfatc1-mediated osteoclast differentiation.Notably, single-cell RNA-seq analysis demonstrates an association of reduced MALAT1 expression in the osteoclast lineage with osteoporosis and bone metastasis.

MALAT1 is downregulated during osteoclast differentiation in humans and mice
Hematopoietic stem cells (HSCs) undergo self-renewal and differentiation in the bone marrow.During a hierarchical differentiation process, HSCs turn into multipotent progenitors (MPPs), which then differentiate into oligopotent progenitors, including common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) 27 .Recent reports of Malat1 having a role in regulating hematopoietic cells under pathological conditions 23,24 prompted us to analyze MALAT1 expression during differentiation of HSCs by using publicly available high-throughput sequencing datasets.Interestingly, in both humans and mice, MALAT1 was expressed at higher levels in HSCs than in MPPs or CMPs (Supplementary Fig. 1a-d).
CMPs can differentiate into monocytes and macrophages, which are the precursors of osteoclasts 28 .We analyzed gene expression during the differentiation of human placental CD14 + macrophages into MGCs 29 (Fig. 1a), in which osteoclasts are the major cell population 8 .Compared with CD14 + macrophages, MGCs showed elevated expression of osteoclast markers, including NFATC1, CTSK, DCSTAMP, ATP6V0D2, ATP6V0E2, and ATP6V0A1 (Fig. 1b, c).In contrast, MALAT1 was significantly downregulated in MGCs relative to CD14 + macrophages (Fig. 1a-c).Consistent with the functions of osteoclasts, gene set enrichment analysis (GSEA) indicated that the gene sets enriched in MGCs compared with CD14 + macrophages were related to collagen organization, extracellular structure remodeling, and skeletal development (Fig. 1d and Supplementary Data 1).To further validate the downregulation of Malat1 during the differentiation of macrophages into osteoclasts, we treated a mouse macrophage/pre-osteoclast cell line, RAW264.7, with soluble RANKL to induce osteoclast differentiation 30 .After this treatment, markers of osteoclasts, including Nfatc1, Ctsk, and Trap5, were upregulated in a time-dependent manner (Fig. 1e-g), whereas Malat1 expression levels were markedly decreased (Fig. 1h).Taken together, these results reveal MALAT1 as a lncRNA that is downregulated during osteoclastogenesis in humans and mice.

Genetic models reveal that Malat1 protects against osteoporosis and bone metastasis
To study the role of Malat1 in osteoclastogenesis and osteoporosis in vivo, we used a Malat1-knockout mouse model (Malat1 −/− ) described in our previous study, in which a transcriptional terminator was inserted downstream of the transcriptional start site of Malat1, causing the loss of Malat1 RNA expression without altering expression levels of Malat1's adjacent genes 22 .Also, we previously engineered mice with targeted transgenic Malat1 expression from the ROSA26 locus (Malat1 Tg/Tg ), which enabled us to conduct genetic rescue studies in Malat1 −/− mice by generating Malat1 −/− ;Malat1 Tg/Tg animals 22 .We collected various tissues, including bone marrow, stomach, colon, small intestine, liver, and pancreatic tissues, from Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice and measured Malat1 expression levels by qPCR.This analysis confirmed Malat1 depletion in Malat1 −/− mice and its re-expression in Malat1 −/− ;Malat1 Tg/ Tg mice, although the levels of Malat1 restoration varied among different tissues (Supplementary Fig. 2a).
Next, to determine the role of Malat1 in modulating pathological bone loss, we used a well-established mouse model of inflammatory bone resorption, which involves the injection of LPS into the subcutaneous space over the calvarial bones 36 .As gauged by μCT imaging, administration of LPS to 8-week-old Malat1 −/− mice resulted in significantly aggravated erosions on the surface of the calvarial bones, compared with Malat1 +/+ or Malat1 −/− ;Malat1 Tg/Tg mice (Fig. 2h, i).TRAP staining and quantification revealed that after LPS injection, Malat1 −/− mice had higher osteoclast numbers per bone perimeter (Oc.N/B.Pm, Fig. 2j, k) and more osteoclast surface per bone surface (Oc.S/BS, Fig. 2j, l) than either Malat1 +/+ or Malat1 −/− ;Malat1 Tg/Tg mice.Collectively, these findings indicate that Malat1 deficiency promotes osteoporosis under both physiological and inflammatory conditions.Untreated osteoporosis is associated with accelerated progression of bone metastasis in cancer patients [3][4][5] .Drugs for osteoporosis therapy, such as bisphosphonates that inhibit osteoclast-mediated bone resorption, have been used to treat bone diseases, including bone metastases 37 .To determine whether Malat1 in the host confers protection from bone metastases, we injected luciferase-labeled B16F1 melanoma cells into the tibiae of 6-month-old male Malat1 +/+ , Malat1 −/− , or Malat1 −/− ;Malat1 Tg/Tg mice, and we found that bone metastases were markedly exacerbated by Malat1 loss in the hosts, a phenotype that was rescued by Malat1 re-expression, as gauged by bioluminescent imaging of live animals (Fig. 3a and Supplementary Fig. 2i) and dissected bones (Fig. 3b, c), as well as gross examination of visible tumors in the bone (Fig. 3d).
Given that bone is a frequent metastasis site for breast cancer, we performed intratibial injection of 3-month-old female Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice with the EO771 cell line, a cell line derived from a mouse mammary tumor on a C57BL/6 background 38 .Before injecting tumor cells, we conducted μCT scanning and confirmed that at this age, only female Malat1 −/− mice, but not female Malat1 +/+ and Malat1 −/− ;Malat1 Tg/Tg mice, exhibited signs of osteoporosis (Supplementary Fig. 2j-n).After injection with 2 × 10 5 luciferase-labeled EO771 cells, bioluminescent signals showed no significant difference in baseline levels among the three animal groups on the injection day.At the endpoint, we observed significantly higher signals in Malat1 −/− mice compared with Malat1 +/+ and Malat1 −/− ;Malat1 Tg/Tg mice (Fig. 3e and Supplementary Fig. 2o).After euthanasia, we collected the tibiae for ex vivo imaging (Fig. 3f, g), which confirmed in vivo imaging results.We also performed X-ray imaging of the tibiae and found that Malat1 −/− mice had more osteolytic lesions (Fig. 3h).Moreover, H&E staining of bone sections demonstrated higher tumor burdens in the tibiae of Malat1 −/ − mice, as evidenced by more cancerous lesions in the cortical bone near the growth plate and deeper extension of tumor areas into the distal bone marrow cavity (Fig. 3i).Immunohistochemical staining of RFP (co-expressed with luciferase) supported the histologic analysis (Fig. 3i).In addition, TRAP staining revealed elevated osteoclast numbers in the tibiae of Malat1 −/− mice compared with Malat1 +/+ and Malat1 −/− ;Malat1 Tg/Tg mice (Fig. 3j-l).Taken together with the results from the B16F1 model, these findings collectively suggest that loss of Malat1 in host mice exacerbates metastatic bone colonization by melanoma and breast cancer cells.
Because bone homeostasis is maintained by osteoclastic bone resorption and osteoblastic bone formation, we next determined whether Malat1 modulates the number and differentiation potential of osteoblasts.To this end, we stained bone sections with toluidine blue 39 (Supplementary Fig. 3a), which revealed no significant difference in the numbers of osteoblasts per bone perimeter (N.Ob/B.Pm) among Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice (Supplementary Fig. 3a, b).Further, we isolated mouse mesenchymal stem cells (MSCs) from these three groups and cultured them in osteogenic differentiation medium 40 ; we observed comparable osteogenic differentiation among all groups, as gauged by calcium mineralization (via alizarin red staining, Supplementary Fig. 3c) and alkaline phosphatase (ALP, Supplementary Fig. 3d, e).Moreover, we found no significant difference in bone formation rates among Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice, as gauged by dynamic histomorphometry measurements through sequential labeling with calcein, a fluorescent chromophore that binds to calcified skeletal structures 41,42 (Supplementary Fig. 3f, g).Taken together, our results suggest that Malat1 inhibits osteoclast differentiation and protects against osteoporosis and bone metastasis without affecting osteoblastic bone formation.

Single-cell transcriptome analysis of bone tissues from patients with osteoporosis, osteosarcoma, or breast cancer bone metastasis
To assess the clinical relevance of MALAT1 in osteoporosis and bone metastasis, we analyzed single-cell RNA-seq data from human bone tissues.The datasets included GSE190772 with samples from two patients with breast cancer bone metastases 38,43 , GSE162454 with samples from six osteosarcoma patients 44,45 , and GSE169396 featuring bone tissues from a non-osteoporotic individual and three osteoporosis patients (femoral head collected during hip replacement surgery) 46 .Osteosarcomas and breast cancer bone metastases often exhibit osteolytic features.We used the "Harmony" method 47 to remove batch effects between samples, subsequently applying dimensionality reduction to annotate cell types based on marker genes (Supplementary Fig. 4a-c).These analyses defined the cell cluster-specific transcriptome of different patient groups.We then analyzed the expression of MALAT1 in pre-osteoclasts (including monocytes and macrophages) and mature osteoclasts of the non-osteoporotic individual (Fig. 4a, b), osteoporosis patients (Fig. 4c, d), osteosarcoma patients (Fig. 4e, f), and patients with breast cancer bone metastases (Fig. 4g, h).Within each group, MALAT1 expression levels were significantly lower in osteoclasts compared with pre-osteoclasts (Fig. 4b, d, f, h).Moreover, across the four patient groups, MALAT1 expression levels in pre-osteoclasts and osteoclasts were significantly lower in patients with osteoporosis, osteosarcoma, or breast cancer bone metastasis than in the nonosteoporotic individual (Fig. 4i-k).These findings indicate that reduced MALAT1 expression in the osteoclast lineage is associated with osteoporosis and bone lesions, including breast cancer metastases and osteosarcomas.

Malat1 deficiency promotes osteoclastogenesis through the activation of Nfatc1
Because the Malat1 −/− and Malat1 Tg/Tg animals used in our study are whole-body knockout and transgenic mice, the osteoporotic phenotype observed above may or may not be a direct effect of Malat1 loss in osteoclast precursors.To address this issue, we isolated primary bone human placental macrophages were differentiated into multinucleated giant cells (MGCs) in culture.Both CD14 + macrophages and MGCs were subjected to highthroughput RNA sequencing (RNA-seq).n = 6 biological replicates per group.Data source: GSE38747.a Heatmap of differentially expressed genes between CD14 + macrophages and MGCs.b Volcano plot of genes upregulated (red) or downregulated (blue) in MGCs relative to CD14 + macrophages.Cutoff values: |log 2 (fold change)| >1 and adjusted P value < 0.001.Statistical significance was determined by a linear model with Benjamini-Hochberg correction.c Relative expression levels of MALAT1 and osteoclast markers were quantitated from the RNA-seq results.d Gene set enrichment analysis (GSEA) of the RNA-seq data, showing the top 13 Gene Ontology (GO) pathways.Statistical significance of the pathway enrichment score was determined by an empirical phenotype-based permutation test.Normalized enrichment scores (NES) and enriched pathways with an adjusted P value < 0.05 are listed in Supplementary Data 1. e-h qPCR of Nfatc1 (e), Ctsk (f), Trap5 (g), and Malat1 (h) in RAW264.7 cells treated with soluble RANKL (50 ng/mL) for the indicated times.n = 3 biological replicates per group.i Schematic representation of the treatments used to evaluate the osteoclastogenic activity of RANKL, LPS, and TNFα, with or without pretreatment (priming) with RANKL.j TRAP staining images (left panel) and quantification (right panel) of RAW264.7 cells treated with RANKL or LPS, with or without pretreatment with RANKL.n = 3 wells per group.k qPCR of Nfatc1, Ctsk, and Malat1 in the cells described in j. n = 3 biological replicates per group.l TRAP staining images (left panel) and quantification (right panel) of RAW264.7 cells treated with RANKL or TNF-α, with or without pretreatment with RANKL.n = 3 wells per group.m qPCR of Nfatc1, Ctsk, and Malat1 in the cells described in l. n = 3 biological replicates per group.Statistical significance in c, e-h, and j-m was determined by a two-tailed unpaired t test.Error bars are s.e.m.Source data are provided as a Source Data file.marrow-derived macrophages (BMMs) from Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice, and then treated these osteoclast precursors with M-CSF and RANKL for 4-6 days to induce their differentiation into osteoclasts.Genetic ablation and restoration of Malat1 expression in BMMs were confirmed by qPCR (Fig. 5a).After M-CSFand RANKL-induced differentiation, we detected osteoclasts by TRAP staining, finding that knockout of Malat1 led to a prominent increase in the number of TRAP-positive multinucleated osteoclasts, and that reexpression of Malat1 reversed the observed induction of osteoclastogenesis (Fig. 5b).The mRNA levels of osteoclast markers Ctsk and Trap5 were much higher in Malat1 −/− cells than in Malat1 +/+ and Malat1 −/− ;Malat1 Tg/Tg cells after RANKL treatment (Fig. 5c, d).
We also used CRISPR interference (CRISPRi) to knockdown Malat1 in cell lines.Eleven single guide RNAs (sgRNAs) targeting mouse    Malat1 were tested by using the mouse B16F1 cell line (Supplementary Fig. 5a).sgRNA-2 and sgRNA-3 were chosen to knockdown Malat1 in RAW264.7 cells, which was validated by qPCR (Fig. 5e), and the two resulting Malat1-knockdown stable cell lines were named Malat1 KD1 and Malat1 KD2 .After RANKL-induced differentiation, both Malat1 KD1 and Malat1 KD2 cells gave rise to more TRAP-positive multinucleated osteoclasts than the control RAW264.7 cells (Fig. 5f).Fluorescent staining of F-actin rings (microfilament structures that are characteristic of Osteoclast progenitor Osteoclast MALAT1 expression levels mature osteoclasts 48,49 ) and nuclei, by phalloidin and DAPI, respectively, revealed that Malat1 KD1 and Malat1 KD2 cells had higher numbers of nuclei per osteoclast than the control cells (Fig. 5g).Moreover, Ctsk and Trap5 mRNA levels were upregulated by knockdown of Malat1 in RANKL-treated RAW264.7 cells (Fig. 5h, i).Collectively, the results from primary BMMs and RAW264.7 cells suggest that Malat1 deficiency in osteoclast precursors promotes RANKL-induced osteoclastogenesis.
Upon binding to the RANK receptor, RANKL stimulates multiple signaling cascades, including nuclear factor-κB (NF-κB) signaling, mitogen-activated protein kinase (MAPK) signaling, and activator protein-1 (AP-1, whose major components are c-Jun and c-Fos proteins) signaling, leading to activation of downstream transcription factors, such as Nfatc1, Mitf, and Creb1 14 .To understand how Malat1 inhibits osteoclastogenesis, we first stimulated BMMs with RANKL for short periods (5-60 min) and examined the phosphorylation events in the signaling pathways mentioned above, finding no substantial difference in the phosphorylation levels of p65 (also known as RelA), Erk1/2, Jnk, or c-Jun among the BMMs isolated from Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice (Supplementary Fig. 5b).Thus, Malat1 loss does not affect the early kinase signaling events during RANKLinduced osteoclast differentiation.
We also sought to determine the effect of Malat1 overexpression.Consistent with Malat1 being a highly abundant lncRNA, we tested various methods and could only overexpress Malat1 in RAW264.7 cells by using the piggyBac transposon system and electroporation.The resulting overexpression level was approximately a 1.7-fold increase over the endogenous expression level (Supplementary Fig. 6a), which did not lead to significant differences in RANKL-induced osteoclastogenesis or the expression of Nfatc1, Trap5, and Ctsk (Supplementary Fig. 6b-g).Moreover, BMMs from Malat1 Tg/Tg mice exhibited approximately a 1.5-fold increase in Malat1 expression relative to BMMs from Malat1 +/+ mice (Supplementary Fig. 6h).Compared with Malat1 +/+ mice, Malat1 Tg/Tg mice did not display any significant difference in bone density or other bone parameters based on μCT analysis (Supplementary Fig. 6i-m).The challenge of achieving substantial Malat1 overexpression in wild-type cells and mice limited a comprehensive examination of its overexpression effects.However, considering that reduced MALAT1 expression in pre-osteoclasts and osteoclasts is associated with osteoporosis and bone metastasis (Fig. 4i-k), our lossof-function approach, coupled with re-expression of Malat1 in Malat1deficient mice and cell lines, is suitable for this investigation.
To further extend our study to human osteoclastogenesis, we treated the U937 human pre-osteoclast/monocyte cell line with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 2 days, followed by 12-14 days of human M-CSF (50 ng/mL) and RANKL (100 ng/mL) treatment, as described previously 53,54 .NFATC1 expression showed an initial upregulation in the first 5 days, followed by a decrease, while the osteoclast marker TRAP exhibited a progressive elevation (Supplementary Fig. 7a).To determine the role of MALAT1 in human osteoclast differentiation, we used CRISPRi to knockdown MALAT1.Five sgRNAs (sg1-5) that target the human MALAT1 promoter were tested in HEK293T cells, and four out of five sgRNAs showed ~50% knockdown efficiency (Supplementary Fig. 7b).We used sg2 and sg4 to deplete MALAT1 in U937 cells, achieving over 95% knockdown efficiency in this cell line (Supplementary Fig. 7c).Subsequently, osteoclastogenesis assays revealed a higher number of TRAP-positive osteoclasts in the MALAT1 knockdown group compared with the control group (Supplementary Fig. 7d, e).Moreover, NFATC1 and TRAP expression levels were elevated in MALAT1-knockdown U937 cells during osteoclast differentiation (Supplementary Fig. 7f-h).Hence, MALAT1 functions as a suppressor of both mouse and human osteoclastogenesis.

Malat1 binds Tead3 to inhibit Nfatc1 activity and osteoclastogenesis
How does Malat1 regulate Nfatc1?The binding of Nfatc1 to other proteins can lead to either activation or inhibition of the transcriptional activity of Nfatc1 55 , while lncRNAs often exert their functions by interacting with proteins, and this mode of action has been demonstrated for Malat1 15,17,22,56 .We speculated that Malat1 might regulate the Nfatc1 auto-amplification loop by interacting with Nfatc1 and/or its binding proteins, and thus we searched a database of protein-protein interactions, Mentha (http://mentha.uniroma2.it/index.php).Of all potential NFATC1-interacting proteins (Supplementary Fig. 8a), TEAD was of particular interest, because our previous chromatin isolation by RNA purification coupled to mass spectrometry (ChIRP-MS) analysis captured an endogenous Malat1-Tead interaction in mouse tissues, which was validated by ChIRP-Western, RNA pulldown, and RNA immunoprecipitation (RIP) assays 22 .Therefore, we hypothesized that Malat1 might regulate Nfatc1 through Tead.
We examined the protein levels of the four Tead family members (Tead1-4) in BMMs and RAW264.7 cells, along with several other mouse cell lines.Interestingly, Tead1 and the Tead co-activator Yap were undetectable in BMMs and RAW264.7 cells but were abundantly expressed in the mouse melanoma cell line B16F1, mouse embryonic fibroblasts (MEF), MSC, and the mouse fibroblast cell line L929 (Fig. 6a).In contrast, Tead3 showed a relatively specific expression pattern in primary BMMs (Fig. 6a).To determine whether Malat1 interacts with Nfatc1 in pre-osteoclasts, we performed RIP assays, finding that Malat1 was enriched in both pan-Tead and Tead3 immunoprecipitates from RAW264.7 cells (Fig. 6b, c), which validated the interaction between Malat1 and Tead3 in these osteoclast precursors.To determine whether Malat1 directly binds to Tead3, we performed RNA pulldown assays with six non-overlapping biotinylated fragments of Malat1 (P1-P6; 1.1-1.2kb each) generated by in vitro transcription 22 , and we found that all six Malat1 fragments, but not an unrelated nuclear RNA U1, bound to Tead3 protein (Fig. 6d), suggesting that the q, r Control and Malat1-knockdown RAW264.7 cells were transfected with negative control (NC) or Nfatc1 siRNA.After 24 h, the cells were treated with RANKL for 5 days, followed by TRAP staining and quantification (q).Scale bars, 100 μm.Cell lysates were subjected to immunoblotting of Nfatc1, Ctsk, and β-actin (r).n = 3 wells per group.Statistical significance in a-i, l, m, and o-q was determined by a two-tailed unpaired t test.Error bars are s.e.m. n = 3 biological replicates in a, c-e, h, i, l, m, o, and p.The experiments in j, k, and r were repeated independently three times, yielding similar results.Source data are provided as a Source Data file.
Tead3-binding sites may be distributed diffusely on Malat1.Consistent with this, ectopic expression of each of the six Malat1 fragments in MALAT1-depleted U937 cells partially reversed, while re-expression of full-length Malat1 completely reversed RANKL-induced osteoclastogenesis (Supplementary Fig. 8b-d).
Interestingly, RANKL treatment of RAW264.7 and U937 cells upregulated Tead3, but not other Tead family members (Fig. 6e, f).Coimmunoprecipitation (co-IP) assays revealed that Tead3, but not Yap, interacted with Nfatc1 (Fig. 6g and Supplementary Fig. 8e).After validating the interaction of Tead3 with Malat1 and Nfatc1, we sought to determine whether Malat1 modulates the binding of Tead3 to Nfatc1.To this end, we generated MALAT1-knockout HEK293T cells (Supplementary Fig. 9a, b) and transfected these cells with Tead3 and Nfatc1.Co-IP assays showed that Malat1 loss significantly increased the interaction between Tead3 and Nfatc1 (Fig. 6h, i).To further corroborate this result, we re-expressed Malat1 in MALAT1-knockout HEK293T cells (Supplementary Fig. 9c), finding that restoring Malat1 expression reduced the Tead3-Nfatc1 interaction (Fig. 6j, k).Besides TEAD, our Mentha database search also revealed other candidate NFATC1-interacting proteins, among which FOS, JUN, and CREB1 have been reported to regulate osteoclast differentiation 57,58 (Supplementary Fig. 8a).We pulled down NFATC1 from the control, MALAT1-knockout, and Malat1-restored HEK293T cells, followed by immunoblotting with antibodies against FOS, JUN, and CREB1.While we did not detect an interaction of FOS with NFATC1, we observed interactions of JUN and CREB1 with NFATC1; however, unlike the TEAD3-NFATC1 interaction, these interactions were not affected by MALAT1 (Supplementary Fig. 9d, e).
Nfatc1 protein contains four domains: two transcription activation domains (TAD) in N-terminal and C-terminal regions, a central DNAbinding domain (DBD), and an N-terminal regulatory domain (NHR) 55 (Fig. 6l).Tead3 protein mainly consists of two domains: an N-terminal DBD (also known as the TEA domain) and a C-terminal YAP-binding domain 59 .Accordingly, we generated truncation mutants of Nfatc1 and Tead3 (Fig. 6l) and performed co-IP assays, finding that both the N-terminal region (containing a TAD and the NHR domain) and the central DBD, but not the C-terminal TAD of Nfact1, could bind Tead3 (Fig. 6m).In addition, co-IP assays using truncated Tead3 mutants and full-length Nfatc1 demonstrated that the TEA domain of Tead3, but not the YAP-binding domain, was responsible for interaction with Nfatc1 (Fig. 6n).
Similar to RAW264.7 cells (Fig. 6e), during osteoclast differentiation of the human pre-osteoclast cell line U937, TEAD3 was also the most upregulated TEAD family member (Fig. 6f).Thus, we examined function in U937 cells, finding that shRNA-mediated knockdown of TEAD3 impaired human osteoclastogenesis (Supplementary Fig. 10a-c) and downregulated the expression of NFATC1 and TRAP at both mRNA and protein levels (Supplementary Fig. 10d-f).It should be noted that MALAT1 depletion did not affect the upregulation of TEAD3 during osteoclast differentiation (Supplementary Fig. 10g), suggesting that MALAT1 does not regulate TEAD3's expression levels (but instead inhibits the TEAD3-NFATC1 interaction).Taken together with the results from RAW264.7 cells, these findings collectively suggest that TEAD3 promotes both mouse and human osteoclastogenesis.

Discussion
This study identified Malat1 as an osteoporosis-suppressing and bone metastasis-inhibiting lncRNA that is downregulated during RANKLtriggered osteoclastogenesis.RANKL stimulates multiple signaling pathways, most of which (such as MAPK and NF-κB pathways) can also be activated by other factors, and yet RANKL is indispensable and irreplaceable in osteoclastogenesis 14 , which could be explained by Nfatc1's role as a specific master regulator of osteoclast differentiation.As a transcriptional factor of its own coding gene and other osteoclastspecific genes, the binding of Nfatc1 to other nuclear proteins can lead to synergistic activation of gene transcription 55 , as exemplified by the AP-1 transcription factor complex, which interacts with Nfatc1 to boost the transcriptional activity of Nfatc1 60 .Here, we identified Tead3 as a macrophage-osteoclast-specific Tead family member and a binding partner of Nfatc1, and our data suggest a model (Fig. 8) in which Malat1 binds and sequesters Tead3, blocking Tead3 from associating with Nfatc1 and inducing the transcription of Nfatc1 target genes, including Nfatc1 itself and Ctsk.In response to RANKL stimulation, downregulation of Malat1 releases Tead3, thereby enhancing both the Tead3-Nfatc1 interaction as well as the transcription factor occupancy of Nfatc1 target genes, which leads to activation of Nfatc1-mediated gene transcription and osteoclast differentiation.
In addition to hyperactivation of osteoclastic bone resorption, suppression of osteoblastic bone formation can also contribute to low BMD and osteoporosis.Several previous publications reported that MALAT1 promotes osteoblast differentiation by acting as a competing endogenous RNA (ceRNA) to microRNAs (miRNAs), i.e., a "miRNA sponge", based on MALAT1 shRNA or siRNA knockdown in cell culture [61][62][63][64][65] .How a nuclear lncRNA could bind miRNAs is unclear.Considering the pitfalls in using RNAi, large genomic deletion (MALAT1, a single-exon gene, is ~7 kb in mice and ~8 kb in humans), promoter deletion, and RNase H-dependent antisense oligonucleotide approaches to deplete nuclear lncRNAs 15,16,[66][67][68] , we used different cell lysates, and pulled down with streptavidin beads.The bound proteins were immunoblotted with a V5-specific antibody (lower).e, f Immunoblotting of NFATC1, Ctsk or TRAP, and TEAD1-4 in RANKL-treated RAW264.7 (e) or U937 (f) cells.SE short exposure, LE long exposure.g HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with S-protein beads or a MYC-specific antibody and immunoblotting with antibodies against MYC and FLAG.h, i Control and MALAT1-knockout HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with a MYC-specific antibody (h) or S-protein beads (i) and immunoblotting with antibodies against MYC and FLAG.j, k MALAT1-knockout and Malat1-restored HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with a MYC-specific antibody (j) or S-protein beads (k) and immunoblotting with antibodies against MYC and FLAG.
l Mouse Nfatc1, Tead3, and truncation mutants.m HEK293T cells were cotransfected with MYC-Tead3 and SFB-Nfatc1 (full-length or truncated), followed by pulldown with S-protein beads and immunoblotting with antibodies against MYC and FLAG.n HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3 (full-length or truncated), followed by pulldown with a MYC-specific antibody and immunoblotting with antibodies against MYC and FLAG.o, p Luciferase activity in HEK293T cells co-transfected with Tead3, constitutively active Nfatc1 (Nfatc1-CA), Renilla luciferase, and a firefly luciferase reporter containing tandem Nfatc1binding sites (o) or the Ctsk promoter (p).q, r Luciferase activity in wild-type, MALAT1-knockout, and Malat1-restored HEK293T cells co-transfected with Tead3, Nfatc1-CA, Renilla luciferase, and a firefly luciferase reporter containing tandem Nfatc1-binding sites (q) or the Ctsk promoter (r).Statistical significance in b, c, and o-r was determined by a two-tailed unpaired t test.Error bars are s.e.m. n = 3 biological replicates.The experiments in a, d-k, m, and n were repeated independently three times, yielding similar results.Source data are provided as a Source Data file.methods for loss-of-function analyses of Malat1 in vitro and in vivo, including CRISPRi, double gRNA-mediated focal deletion in the 5′ region (without affecting the promoter), and insertional inactivation, along with genetic rescue experiments.In the present study, loss of Malat1 in pre-osteoclasts (including RAW264.7 cells and primary BMMs from Malat1 −/− mice) promoted osteoclastogenesis, a phenotype that could be reversed by restoration of Malat1 expression.On the other hand, the results from Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice, as well as osteoblast differentiation assays of MSCs isolated from these animals, showed no evidence for the regulation of osteoblastogenesis by Malat1 (Supplementary Fig. 3a-g).
Via its C-terminal YAP-binding domain, the TEAD family of transcription factors binds to the transcriptional co-activator YAP to turn on the expression of TEAD-YAP target genes 69 .In doing so, TEAD proteins and YAP are involved in several processes, including organ growth, regeneration, tumor progression, and metastasis 69 .Potentially druggable sites in the protein-protein interaction between YAP and TEAD, as well as a highly conserved palmitoylation pocket in TEADs, have been identified and exploited for drug development 70 .In an ongoing clinical trial (NCT04665206), the first-in-class TEAD inhibitor was well tolerated with durable antitumor responses in patients with advanced mesothelioma or other cancers harboring NF2 mutations.However, whether the TEAD family can function in a YAP-independent manner is elusive.In this study, Tead3, but not other Tead family members, exhibited a specific expression pattern in primary bone marrow macrophages (pre-osteoclasts), whereas Tead1 and Yap were barely detectable in these cells (Fig. 6a).We further found that Tead3 binds and activates Nfatc1 via its N-terminal TEA domain (but not its C-terminal YAP-binding domain), which is required for RANKL-induced osteoclastogenesis, thus revealing a non-canonical function of Tead that is mediated by Nfatc1 and is controlled by Malat1 lncRNA.Our findings suggest the therapeutic potential of developing agents that disrupt the TEAD3-NFATC1 interaction for treating osteoporosis and bone metastasis.For example, the TEAD inhibitor could emerge as a drug that only elicits antitumor responses through the tumor cell-intrinsic mechanism, but also inhibits bone metastasis through the tumor cell-extrinsic mechanism.
Future studies should address the following issues: first, we found that Malat1 expression is downregulated during osteoclast differentiation; yet, how this lncRNA is regulated by pro-osteoclastogenic factors under physiological and pathological conditions is unknown.Second, our study identified a Malat1-Tead3-Nfatc1 axis that regulates osteoclastogenesis, but it is possible that additional binding partners of Malat1 and Tead3 could also be involved in osteoclast differentiation.Third, our previous study 22 and the present study collectively demonstrate that Malat1 binds to Tead to inactivate Yap-Tead's prometastatic function in cancer cells and to inhibit Nfatc1-Tead3's proosteoclastogenic function in pre-osteoclasts, revealing both tumorintrinsic and tumor-extrinsic mechanisms of Malat1 in metastasis suppression.Whether Malat1 suppresses metastasis at other anatomic sites (in addition to the lung and bone) and in cancer types in addition to breast cancer and melanoma warrants further investigation.Finally, whether Malat1 regulates other types of niche cells to control metastasis remains an open question.

Genetically engineered mouse models
All animal studies were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of MD Anderson Cancer Center.Animals were housed at 70 °F-74 °F (set point: 72 °F) with 40-55% humidity (set point: 45%).The light cycle of animal rooms is 12 h of light and 12 h of dark.Malat1-knockout mice with targeted inactivation of Malat1 (Malat1 −/− ) and mice with targeted transgenic expression of Malat1 from the ROSA26 locus (Malat1 Tg/Tg ) were described in our previous paper 22 .To restore Malat1 expression in Malat1 −/− mice, we bred Malat1 −/− mice to Malat1 Tg/Tg mice and further mated their heterozygous offspring to produce Malat1 −/− ;Malat1 Tg/Tg mice.All mice described here were on a C57BL/6 background.Primers for PCR genotyping were listed in our previous paper 22 .

LPS-induced inflammatory osteoporosis model
The procedure for inflammation-induced bone destruction was performed as previously described 36 .Briefly, 8-week-old female mice were injected above the calvarium with 12.5 mg/kg of LPS (Sigma, L4391) or vehicle (phosphate buffered saline, PBS).After 6 days, calvariae were collected and analyzed by μCT, followed by embedding, sectioning, and TRAP staining.

μCT-based bone scanning and analysis
Mouse femurs were scanned on a Bruker microCT SkyScan 1276 (Bruker, Kontich, Belgium) with a source voltage of 55 kV, a source current of 200 μA, a filter setting of Al 0.2 mm, and a pixel size of 13 μm at 2016 × 1344.We used 435 ms exposure time and the step and shoot mode with rotation step 0.400 degrees.Backward projection datasets of all femurs were reconstructed by using Insta-Recon software (Bruker microCT, Kontich, Belgium).Parameters for reconstruction were windowing 0-0.08 intensity, ring artifact reduction 5, beam hardening 23%, and automatic post-alignment correction.The proximal end of the femur corresponding to a 0-1.3 mm region (consisting of 100 sections over the region of interest) below the growth plate was selected and analyzed by using CTAn software (Version 1.18 8.0+, Bruker microCT, Belgium) to determine the trabecular BMD, trabecular bone volume per tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.th).The threshold value for μCT was set at 150-250.All calculations were performed based on 3D standard microstructural analysis.For visualizing the femurs, a 3D model was created with CTVox software (Version 3.3.0,Bruker microCT, Belgium) based on the same region of the microstructural analysis.
Mouse calvariae were scanned on a Bruker microCT SkyScan 1276 (Bruker, Kontich, Belgium) with the same parameters as those used for scanning femurs, except the exposure time (400 ms), windowing (0-0.06 intensity), ring artifact reduction (3), and beam hardening (20).For visualizing the osteolytic area of the calvaria, a 3D model was created with CTVox software (Version 3.3.0,Bruker microCT, Belgium).A region of 8 mm × 8 mm centered at the midline suture was used for further quantitative analysis with ImageJ (Version 1.53 m).

Bone metastasis assay
B16F1 melanoma cells with stable expression of firefly luciferase (Addgene, 39196) were cultured to 70% confluence and harvested during the log phase of growth.Then, 5000 cells were resuspended in sterile PBS, and the tumor cell suspensions were injected into the left tibiae of 6-month-old male Malat1 +/+ , Malat1 −/− , or Malat1 −/− ;Malat1 Tg/Tg mice by using a 27-gauge needle under isoflurane anesthesia.Bioluminescence imaging was performed at days 0, 14, and 25 after intratibial injection under isoflurane anesthesia by using an IVIS 200 imaging platform (Perkin Elmer), following the intraperitoneal injection of 100 μl D-luciferin substrate (25 mg/mL in PBS, Perkin Elmer).The mammary tumor line EO771 was labeled with firefly luciferase and RFP and injected into the tibiae of 3-month-old female Malat1 +/+ , Malat1 −/− , or Malat1 −/− ;Malat1 Tg/Tg mice at 2 × 10 5 cells per mouse.Bioluminescence imaging was performed at day 0 and day 16 after intratibial injection.At the endpoint, the mice were euthanized, and the tibiae were collected for ex vivo bioluminescence imaging and photography.The imaging data were processed and quantitated with Living Image Software version 4.7 (Perkin Elmer).For detecting osteolytic lesions, the tibiae were scanned and processed with the Faxitron MX-20 Digital X-Ray System (Wheeling, IL).None of the following IACUC-approved euthanasia criteria was exceeded: (1) the maximum cumulative tumor burden of 2.0 cm in diameter; (2) the tumor impedes eating, urination, defecation, or ambulation; (3) very poor body condition.

Histology, TRAP staining, and toluidine blue staining of bone tissues
Following fixation in formalin for 2 days, the femurs, tibiae (with tumors), and calvariae of mice were decalcified in 12.5% EDTA solution for 5 days before being transferred to 70% ethanol.After paraffin embedding, the tissues were sectioned at 4 μm thickness.The slides were deparaffinized in xylene, rehydrated in gradients of ethanol, and immersed in PBS for 5 min.For TRAP staining, we used a staining kit (Sigma, 387A-1KT) according to the manufacturer's instructions.Briefly, the slices were incubated with the staining buffer containing Fast Garnet GBC solution, Naphthol AS-BI phosphoric acid solution, acetate solution, and tartrate solution in a 37 °C water bath protected from light for 1 h.After being rinsed with water for 5 min, the slices were counterstained with methyl green (Vector Laboratories, H-3402-500) for 1 min and rinsed with water for 5 min.The slides were mounted with VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000).For toluidine blue staining, the slides were stained with toluidine blue solution (Sigma, 89640), dehydrated and cleared with xylene, and coverslipped with DMX hydrophobic adhesive.All slides were scanned with an Aperio CS2 Digital Pathology Slide Scanner (Leica Biosystems).Bone histomorphometry was analyzed by using Bioquant OSTEO II software (Bioquant Nashville) on the subepiphyseal region 150 μm away from the distal growth plate and extending 1.3 mm into the bone compartment, at a distance of 150 µm from the cortical walls.

Immunohistochemical staining
Sections of tibiae (with tumors) were deparaffinized in xylene and degraded alcohols.Heat-induced epitope retrieval was performed by using a 2100-Retriever.Slides were rinsed with PBS, and a hydrophobic barrier was created around the tissue using a hydrophobic barrier pen (Vector Laboratories, H-4000-2).Then, slides were placed in an incubating chamber with blocking solution (Vector Laboratories, SP-6000) for 10 min and rinsed with PBS, followed by incubation with 20% horse serum (Vector Laboratories, PK-7200) for 20 min.Next, slides were incubated with the primary antibody against RFP (1:400, Abcam, ab62341, RRID: AB_945213) at 4 °C overnight and rinsed with PBS, followed by incubation with a Horse Anti-Rabbit IgG Antibody (H + L), Biotinylated, R.T.U.(Vector Laboratories, BP-1100-50) or goat IgG HRPconjugated antibody (R&D systems, HAF017) for 30 min.After being washed again with PBS, slides were incubated with the avidin-biotin detection complex (ABC; Vector Laboratories, SK-4100) for 30 min and were then developed with 3,3′-diaminobenzidine (DAB) solution (Vector Laboratories, SK-4100).Counterstaining was performed by using Hematoxylin QS (Vector Laboratories, H-3404).Slides were scanned with an Aperio CS2 Digital Pathology Slide Scanner (Leica Biosystems).

Calcein staining of bone tissues
For dynamic histomorphometric measures of bone formation, calcein (Sigma, C0875) was intraperitoneally injected twice into mice (at 5 days and 1 day before euthanasia) at a dose of 25 mg/kg to obtain double labeling of newly formed bones.The non-decalcified femur bones were embedded in methyl methacrylate.The tissues were sectioned at 5 μm thickness, and the images were acquired by using an inverted microscope.Bone histomorphometric analysis of mineral apposition rate (MAR) was done with Bioquant OSTEO II software (Bioquant Nashville).
was cultured with RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin.Cells were maintained in a humidified, 5% CO 2 atmosphere at 37 °C, and low-passage stocks were maintained in a centralized lab cell bank.Short tandem repeat profiling and mycoplasma tests were done by ATCC and MD Anderson's Cytogenetics and Cell Authentication Core.

Osteoclast differentiation
Osteoclast differentiation from bone marrow-derived macrophages (BMMs) was induced as described previously 71 .Briefly, femurs, tibiae, and iliac bones were removed from mice after euthanasia.Small incisions were made at both the proximal and distal ends of the bones, and the bones were placed in a sterile tube and centrifuged at 10,000 × g at room temperature for 15 s.After purification with a 70 μm cell strainer, bone marrow cells were cultured in Minimum Essential Medium α (MEMα, Gibco, 41061029) containing 10% FBS for 1 day.Non-adherent cells were collected and seeded in 24-or 6-well plates and treated with 50 ng/mL of mouse M-CSF (Peprotech, 315-02) for 2 days, after which mouse soluble RANKL (Peprotech, 315-11) was added at a concentration of 100 ng/mL for additional culture for 4-6 days.Osteoclast differentiation from the RAW264.7 mouse cell line was induced as described previously 72 .Cells were seeded in 24-well or 6-well plates at a density of 2 × 10 4 or 5 × 10 5 cells per well.50 ng/mL of mouse soluble RANKL (Peprotech, 315-11) was used to induce differentiation, and the culture medium was changed every 2 days.Osteoclasts derived from BMMs or RAW264.7 cells were identified as multinucleated (more than three nuclei) cells by TRAP staining (Sigma, 387A-1KT).Osteoclast differentiation from the U937 human cell line was induced as described previously 53,54 .Cells were seeded in 24-well or 6-well plates at a density of 5 × 10 5 or 3 × 10 6 cells per well.Cells were treated with 100 ng/mL of PMA for 2 days, followed by 50 ng/mL of human M-CSF (Peprotech, AF-300-25) and 100 ng/mL human soluble RANKL (Peprotech, AF-310-01) for 12-14 days.The culture medium was changed every 2 days.Osteoclasts derived from U937 cells were identified as TRAP+ cells.Osteoclast markers (NFATC1, CTSK, and TRAP5) were examined by qPCR and immunoblotting.

Osteoblast differentiation
Osteoblast differentiation from bone marrow MSCs was induced as previously described 71 .Briefly, bone marrow cells were collected from the femurs, tibiae, and iliac bones of mice and plated for culture.After 48 h, non-adherent cells were removed, and attached cells were trypsinized and seeded in 12-well plates.When the cells reached 90% confluence, osteogenic differentiation medium (MEM containing 10% FBS, 5 mM β-glycerol phosphate, Selleck Chemicals, S3620, and 50 μg/ ml of ascorbic acid, Selleck Chemicals, S3114) was added, and cells were cultured for 10-21 days.Then, ALP staining was done by using a staining kit (Sigma, 86R-1KT) according to the manufacturer's protocol.For ALP activity detection in the medium, equal volumes of conditioned medium were analyzed with an Alkaline Phosphatase Activity Fluorometric Assay Kit (BioVision, K422-500).For alizarin red S (ARS) staining, cells were fixed with 4% polyoxymethylene for 15 min and stained with 1% ARS (pH 4.2, Sigma-Aldrich, A5533) for 10 min.The dye was then removed and the cells were washed three times with water and photographed with an inverted microscope.The calcium mineralization stained with ARS was dissolved with 10% acetic acid and heated at 85 °C for 10 min, followed by neutralization with 10% ammonium hydroxide.The samples were transferred to 96-well plates, and the absorbance at 405 nm was measured on a microplate reader (Biotek Synergy 2).
F-actin ring staining RAW264.7 cells were treated with RANKL to induce differentiation into osteoclasts, fixed with 4% paraformaldehyde for 20 min, rinsed with PBS, and permeabilized with 0.5% Triton X-100 at room temperature for 10 min.The cells were washed with PBS and blocked with 5% FBS at room temperature for 30 min.The fixed cells were stained with diluted phalloidin green 488 (1:100, BioLegend, 424201) in the dark for 20 min and mounted with the antifade mounting medium with DAPI (Vector Laboratories, H-1200-10).The slides were imaged with a Zeiss LSM880 confocal microscope and processed with Zen 2.6 (Zeiss) software.

Lentiviral transduction
Lentiviruses were produced in HEK293T cells by co-transfection with the viral vector and packaging plasmids (pMD2.G: Addgene, 12259; psPAX2: Addgene, 12260).Two days after transfection, viral supernatant was harvested, filtered through a 0.45 μm filter, and added to target cells in the presence of polybrene reagent (Sigma, R-1003-G) at 4 μg/mL.The infected cells were selected with puromycin, hygromycin B, or blasticidin, as indicated below.

Cytoplasmic-nuclear fractionation
Control and Malat1-knockdown RAW264.7 cells were plated in 6-cm dishes.At 12 h after seeding, the cells were treated with soluble RANKL (50 ng/mL) for 3 days.Nuclear and cytoplasmic proteins were fractionated by using the NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher Scientific, 78833) according to the manufacturer's protocol.After protein extraction, Western blot analysis was performed to detect Nfatc1 protein in the cytoplasmic and nuclear fractions.Gapdh and Lamin B1 were used as markers of the cytoplasm and the nucleus, respectively.

Protein pulldown and immunoprecipitation
HEK293FT cells were transfected with SFB (a triple-epitope tag containing S-protein, FLAG, and streptavidin-binding peptide)-tagged Nfatc1 (full-length or truncation mutants) and MYC-tagged Tead3 (fulllength or truncation mutants) and harvested 2 days after transfection.Cells were lysed in RIPA lysis buffer (Sigma, 20-188) at 4 °C for 15 min and sonicated.The lysates were centrifuged at 14,000 rpm at 4 °C for 15 min, and the supernatant was incubated with specific beads or antibodies.For the pulldown of SFB-tagged proteins, cell extracts were incubated with S-protein beads (EMD Millipore, 69704-3).For immunoprecipitation of MYC-tagged proteins, cell extracts were incubated with anti-MYC beads (Sigma, A7470, RRID: AB_10109522).After incubation at 4 °C overnight, the immune complexes were centrifuged and washed with PBS three times, and the bound proteins were eluted by boiling in Laemmli buffer at 95 °C for 10 min, followed by Western blot analysis with the indicated antibodies.

Chromatin immunoprecipitation (ChIP) assay
ChIP assays of control and Malat1-knockdown RAW264.7 cells were done with a ChIP assay kit (Millipore, 17-371) as described previously 22 .Briefly, 1 × 10 7 RAW264.7 cells were cross-linked by using 1% formaldehyde.Excess formaldehyde was quenched by glycine, and cell pellets were collected and lysed with SDS lysis buffer.The lysates were sonicated so that the chromosomal DNA fragments were 200-800 bp in length.Chromatin extracts were precleared with Protein G agarose, followed by immunoprecipitation with 8 μg of an Nfatc1-specific antibody (Invitrogen, MA3024, RRID: AB_2236037) or normal mouse IgG at 4 °C overnight.Immune complexes were collected on Protein G agarose beads and washed.The protein-DNA complexes were eluted with 1% SDS in 50 mM NaHCO3.After the reversal of protein-DNA crosslinks and removal of proteins, purified DNA was used for PCR amplification of the Ctsk, Nfatc1, and Acp5 promoter regions bound to Nfatc1.The Ctsk, Nfatc1, and Acp5 gene-specific primers were described previously 52,76,77 and are listed in Supplementary Data 5.

RNA pulldown assay
Full-length mouse Malat1 (NR_002847) was divided into six nonoverlapping pieces (P1-P6, 1.1-1.2kb each) and cloned into the pGEM-T vector as previously described 22 .The vector was linearized by NotI-HF (New England Biolabs, R3189s) and used as the template for the synthesis of biotin-labeled RNA by using an in vitro T7 transcription kit (New England Biolabs, E2040S).Biotin-16-UTP (Roche, 11388908910) was used to biotinylate the RNAs.Non-biotinylated RNAs and biotinylated U1 were synthesized as negative controls.After in vitro transcription, the products were purified by using a PureLink RNA Mini Kit (Invitrogen, 12183018 A) and digested with RNase-free DNase I (Invitrogen, 12185010) to remove the template DNA according to the manufacturer's protocols.3 µg of purified biotin-labeled or biotin-free RNA was heated at 90 °C for 2 min and chilled on ice for 2 min in the RNA structure buffer (2×: 20 mM Tris-HCl at PH 7.4, 0.2 M KCl, 20 mM MgCl 2 , 2 mM DTT) containing RNase inhibitor (Takara, 2313B).RNA samples were placed at room temperature for 20 min for proper secondary structure formation.HEK293T cells overexpressing V5-tagged Tead3 were lysed in RIPA lysis buffer (Sigma, 20-188) containing protease inhibitors (GenDEPOT) and RNase inhibitor and sonicated.Cell lysates were precleared with streptavidin agarose (Pierce, 20349) at room temperature.3 mg of precleared cell lysates were added to each folded RNA sample and incubated at room temperature overnight.Streptavidin agarose was added and incubated at room temperature for 1 h.The bound proteins were washed and eluted by boiling in Laemmli buffer at 95 °C for 10 min and subjected to Western blot analysis.

RIP assay
The RIP assay was done with an EZ-Magna RIP Kit (Millipore, 17-10522) according to the manufacturer's instructions.Briefly, ~4 × 10 7 RAW264.7 cells were lysed in the Complete Nuclei Isolation Buffer (included in the kit) with protease inhibitor cocktail and RNase inhibitor and centrifuged at 800 × g at 4 °C for 5 min.The nuclear pellet was resuspended with Complete RIP Lysis Buffer (included in the kit).After being treated with DNase I, the samples were precleared with Protein A/G magnetic beads and incubated with Protein A/G magnetic beads coated with a pan-Tead-specific antibody (Cell Signaling Technology, 13295 S, RRID: AB_2687902), a Tead3-specific antibody (Proteintech, 13120-1-AP, RRID: AB_2203068), or normal Rabbit IgG at 4 °C overnight.The beads were placed on a magnetic separator and washed with Nuclear RIP Wash Buffer, and the bead-bound immunoprecipitates were subjected to RNA purification by using a PureLink RNA Mini Kit (Invitrogen, 12183018 A), followed by DNase I treatment.Purified RNA samples were used for cDNA synthesis, followed by qPCR analysis with the primers listed in Supplementary Data 4. U6 was used as a negative control.The results are presented as fold enrichment (normalized to IgG).

Luciferase reporter assay
The pGL-NFAT reporter construct containing 3× NFAT-binding sites was from Addgene (17870).The mouse Ctsk promoter region was PCR-amplified (PCR primers are listed in Supplementary Data 2) and ligated into the linearized pGL3-basic plasmid by using an In-Fusion HD Cloning Kit (Takara Bio, 638909).MALAT1 wild-type, MALAT1-knockout, and Malat1-restored HEK293T cells were plated in triplicates in 96well plates.The next day, 16.67 ng of the indicated firefly luciferase vector, 50 ng of Nfatc1-CA-SFB, 1 ng of a Renilla luciferase vector, and 20 or 100 ng of Tead3-MYC were transfected per well.At 36 h after transfection, firefly and Renilla luciferase activities were measured by using a Dual-Luciferase Reporter Assay (Promega, E1910) on a microplate reader according to the manufacturer's protocol.Firefly luciferase activity was normalized to Renilla luciferase activity.

ELISA
Mouse blood was collected through intracardiac puncture immediately after euthanasia.The blood was transferred to a 1.5 mL tube and left at room temperature for 30 min.The clot was removed by centrifuging at 7000 rpm in a refrigerated centrifuge for 15 min.The resulting supernatant was aliquoted and stored in a −80 °C freezer.For the detection of serum TRAP5b, a TRAP5b ELISA Kit (Immunodiagnostic Systems, SB-TR103) was used according to the manufacturer's instructions.
RenameCells function was used to ensure unique cell labels.A globalscaling normalization method ("LogNormalize") was used to ensure equal total gene expression in each cell, with the scale factor set to 10,000.The top 2000 variably expressed genes were used in downstream analysis by using the FindVariableFeatures function.The Sca-leData function, "vars.to.regress" option UMI, and percent mitochondrial content were used to regress out unwanted sources of variation.Principal component analysis incorporating highly variable features was used to reduce the dimensionality of the dataset, and the first 30 PCs were identified for analysis.The "Harmony" method 47 was used to remove batch effects between samples.The scrublet algorithm was used to remove potential doublets.Cell clustering was performed based on the edge weights between any two cells, and a shared nearestneighbor graph was produced by using the Louvain algorithm, which is implanted in the FindNeighbors and FindClusters functions.The FindClusters function's resolution parameter was used repeatedly between 0.05 and 1.The clustree function was used to observe cell clustering trees at different resolutions.
Cell annotation was performed by using multiple methods.First, the Immune_All_High atlas in CellTypist software was used for automatic annotation.Then, the FindAllMarkers function in the Seurat package was used to identify markers for each cell type, and these cell markers were checked in the Cell Taxonomy database (https://ngdc.cncb.ac.cn/celltaxonomy/).The necessary manual adjustment was made to ensure reliable annotation results.

Statistical analysis
Except for the animal studies, each experiment was repeated at least three times with similar results.Statistical analyses were done with GraphPad Prism 9.0.0.Unless otherwise noted, data are presented as mean ± s.e.m., and a two-tailed unpaired t-test was used to compare two groups of independent samples.Statistical methods used for single-cell and bulk RNA-seq analyses and Expression Atlas data analysis are described above.P < 0.05 was considered statistically significant.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig. 1 | MALAT1 is downregulated during osteoclast differentiation.a-d CD14 + human placental macrophages were differentiated into multinucleated giant cells (MGCs) in culture.Both CD14 + macrophages and MGCs were subjected to highthroughput RNA sequencing (RNA-seq).n = 6 biological replicates per group.Data source: GSE38747.a Heatmap of differentially expressed genes between CD14 + macrophages and MGCs.b Volcano plot of genes upregulated (red) or downregulated (blue) in MGCs relative to CD14 + macrophages.Cutoff values: |log 2 (fold change)| >1 and adjusted P value < 0.001.Statistical significance was determined by a linear model with Benjamini-Hochberg correction.c Relative expression levels of MALAT1 and osteoclast markers were quantitated from the RNA-seq results.d Gene set enrichment analysis (GSEA) of the RNA-seq data, showing the top 13 Gene Ontology (GO) pathways.Statistical significance of the pathway enrichment score was determined by an empirical phenotype-based permutation test.Normalized enrichment scores (NES) and enriched pathways with an adjusted P value < 0.05 are

Fig. 2 |
Fig. 2 | Malat1 protects against osteoporosis.a, b Representative μCT images of 3D bone structures of the femurs from 6-month-old male (a) and female (b) Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice.c, d μCT-based measurements of the bone mineral density of the femurs from 6-month-old male (c; n = 5, 5, and 7 mice per group) and female (d; n = 7, 10, and 9 mice per group) Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice, with left and right femurs for each mouse measured.e Representative TRAP staining images of the femurs from 6-monthold male Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice.Scale bars, 700 μm in upper panels and 100 μm in lower panels.f, g Quantification of osteoclast numbers per bone perimeter (Oc.N/B.Pm, f) and osteoclast surface per bone surface (Oc.S/BS, g) in femurs of the mice described in e. n = 5 mice per group.h-l μCT images of the surface of calvariae (h), quantification of the relative resorption area (i), TRAP staining images of calvarial sections (j), the number of osteoclasts per bone perimeter (Oc.N/B.Pm, k), and osteoclast surface per bone surface (Oc.S/BS, l) in the calvarial bones from 8-week-old female Malat1 +/+ , Malat1 −/− , and Malat1 −/− ;Malat1 Tg/Tg mice after the administration of PBS or LPS to the calvarial periosteum for 5 days.n = 3 mice per PBS group, and n = 4, 3, and 4 mice per LPS group in i. n = 3 mice per group in k and l.Scale bars in j, 200 μm.Statistical significance in c, d, f, g, i, k, and l was determined by a two-tailed unpaired t test.Error bars are s.e.m.Source data are provided as a Source Data file.

Fig. 4 |
Fig. 4 | Single-cell transcriptome analysis of bone tissues from patients with osteoporosis, osteosarcoma, or breast cancer bone metastasis.a, b Single-cell analysis of the non-osteoporotic patient in the GSE169396 dataset (n = 1 patient).a t-SNE dimensionality reduction landscape and MALAT1 expression heatmap of monocytes, macrophages, and osteoclasts.Data represent n = 849 monocytes, n = 14 macrophages, and n = 2217 osteoclasts.b Violin plot of MALAT1 expression in osteoclast progenitors (monocytes and macrophages) and osteoclasts.c, d Single-cell analysis of the osteoporosis patient group in the GSE169396 dataset (n = 3 patients).c t-SNE dimensionality reduction landscape and MALAT1 expression heatmap of monocytes, macrophages, and osteoclasts.Data represent n = 3704 monocytes, n = 462 macrophages, and n = 6551 osteoclasts.d Violin plot of MALAT1 expression in osteoclast progenitors (monocytes and macrophages) and osteoclasts.e, f Single-cell analysis of the osteosarcoma patient group in the GSE162454 dataset (n = 6 patients).e t-SNE dimensionality reduction landscape and MALAT1 expression heatmap of monocytes, macrophages, and osteoclasts.Data represent n = 894 monocytes, n = 15,283 macrophages, and n = 4129 osteoclasts.f Violin plot of MALAT1 expression in osteoclast progenitors (monocytes and macrophages) and osteoclasts.g, h Single-cell analysis of the breast cancer bone metastasis patient group in the GSE190772 dataset (n = 4 samples from 2 patients).g t-SNE dimensionality reduction landscape and MALAT1 expression heatmap of monocytes, macrophages, and osteoclasts.Data represent n = 327 monocytes, n = 296 macrophages, and n = 80 osteoclasts.h Violin plot of MALAT1 expression in osteoclast progenitors (monocytes and macrophages) and osteoclasts.i-k Single-cell RNA-seq meta-analysis of GSE169396, GSE162454, and GSE190772 (n = 1 non-osteoporosis patient, 3 osteoporosis patients, 6 osteosarcoma patients, and 4 samples from 2 breast cancer bone metastasis patients).i t-SNE dimensionality reduction landscape and MALAT1 expression heatmap of macrophages, monocytes, and osteoclasts from the above datasets.Data represent n = 5774 monocytes, n = 16,055 macrophages, and n = 12,977 osteoclasts.j, k Violin plots of MALAT1 expression in osteoclast progenitors (j) and osteoclasts (k) across patients with non-osteoporosis, osteoporosis, osteosarcoma, and breast cancer bone metastasis.Statistical significance in b, d, f, h, j, and k was determined by the Wilcoxon ranksum test.The center line of the boxplot depicts the median, bounded by the interquartile range (IQR), 25th to 75th percentile, and the whisker represents 1.5 × IQR.

Fig. 6 |
Fig. 6 | Malat1 binds to Tead3 to inhibit Nfatc1 activity.a Immunoblotting of Yap and Tead1-4 in B16F1, MEF, BMM, MSC, L929, and RAW264.7 cells.b, c Total Tead (b) or Tead3 (c) was immunoprecipitated from RAW264.7 cells.Tead-or Tead3bound Malat1 was quantitated by qPCR.d Biotinylated (Btn) Malat1 fragments were synthesized in vitro (upper), incubated with V5-Tead3-overexpressing HEK293Tcell lysates, and pulled down with streptavidin beads.The bound proteins were immunoblotted with a V5-specific antibody (lower).e, f Immunoblotting of NFATC1, Ctsk or TRAP, and TEAD1-4 in RANKL-treated RAW264.7 (e) or U937 (f) cells.SE short exposure, LE long exposure.g HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with S-protein beads or a MYC-specific antibody and immunoblotting with antibodies against MYC and FLAG.h, i Control and MALAT1-knockout HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with a MYC-specific antibody (h) or S-protein beads (i) and immunoblotting with antibodies against MYC and FLAG.j, k MALAT1-knockout and Malat1-restored HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with a MYC-specific antibody (j) or S-protein beads (k) and immunoblotting with antibodies against MYC and FLAG.

Fig. 7 |
Fig. 7 | Tead3 promotes osteoclastogenesis and mediates the effect of Malat1 deficiency.a Immunoblotting of Cas9 and MCP in RAW264.7 cells transduced with lenti-dCas9-VP64 and lenti-MS2-P65-HSF1.b qPCR (upper) and immunoblotting (lower) of Tead3 in RAW264.7 cells with CRISPRa-mediated overexpression of Tead3.n = 3 biological replicates per group.c, d TRAP staining images (c) and quantification (d) of control and Tead3-overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 5 days.Multinucleated TRAP-positive cells (outlined by dashed lines) were counted.Scale bars, 100 μm.n = 3 wells per group.e-g qPCR of Nfatc1 (e), Trap5 (f), and Ctsk (g) in control and Tead3-overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 3 days.n = 3 biological replicates per group.h Immunoblotting of Nfatc1, Ctsk, and Hsp90 in control and Tead3overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 2 days and 5 days.i Immunoblotting of Tead3 and Hsp90 in RAW264.7 cells transfected with two independent Tead siRNAs or scrambled negative control (NC).j, k TRAP staining images (j) and quantification (k) of control and Tead3-knockdown RAW264.7 cells treated with RANKL (50 ng/mL) for 5 days.Multinucleated TRAPpositive cells (outlined by dashed lines) were counted.Scale bars: 100 μm.n = 3 wells per group.l Immunoblotting of Nfatc1, Ctsk, and β-actin in control and Tead3knockdown RAW264.7 cells treated with RANKL for 3 days and 5 days.m Immunoblotting of Tead3 and Hsp90 in control and Malat1-knockdown RAW264.7 cells transfected with Tead3 siRNA or scrambled negative control (NC).n-p Control and Malat1-knockdown RAW264.7 cells were transfected with Tead3 siRNA or scrambled negative control (NC).24 h after siRNA transfection, the cells were treated with RANKL for 5 days, followed by TRAP staining (n) and quantification (o) of multinucleated TRAP-positive cells (outlined by dashed lines).Scale bars, 100 μm.Cell lysates were subjected to immunoblotting of Nfatc1, Ctsk, and β-actin (p).n = 3 wells per group in o.Statistical significance in b, d-g, k, and o was determined by a two-tailed unpaired t test.Error bars are s.e.m.The experiments in a, h, i, l, m, and p were repeated independently three times, yielding similar results.Source data are provided as a Source Data file.