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A Gene Network Regulating Lysosomal Biogenesis and Function

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Science  24 Jul 2009:
Vol. 325, Issue 5939, pp. 473-477
DOI: 10.1126/science.1174447

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Cellular organelles allow the localized regulation of specialized processes. Under certain conditions, such as increased growth, organelles may be required to alter their function. Coordinated regulation of the gene networks required for mitochondrial and endoplasmic reticulum function has been observed. Now, Sardiello et al. (p. 473; published online 25 June) have discovered a gene network regulating the lysosome, the major organelle involved in the degradation of internalized macromolecules. Many lysosomal genes were regulated by a single transcription factor, TFEB. TFEB itself can be activated when the lysosome malfunctions and can regulate both the abundance of lysosomes found in the cell, as well as the ability to degrade complex molecules, including a mutant protein that accumulates in patients with Huntington's disease. These results may have implications for the treatment of human lysosomal storage disorders, which are characterized by the aberrant accumulation of macromolecules causing cellular dysfunction.

Abstract

Lysosomes are organelles central to degradation and recycling processes in animal cells. Whether lysosomal activity is coordinated to respond to cellular needs remains unclear. We found that most lysosomal genes exhibit coordinated transcriptional behavior and are regulated by the transcription factor EB (TFEB). Under aberrant lysosomal storage conditions, TFEB translocated from the cytoplasm to the nucleus, resulting in the activation of its target genes. TFEB overexpression in cultured cells induced lysosomal biogenesis and increased the degradation of complex molecules, such as glycosaminoglycans and the pathogenic protein that causes Huntington’s disease. Thus, a genetic program controls lysosomal biogenesis and function, providing a potential therapeutic target to enhance cellular clearing in lysosomal storage disorders and neurodegenerative diseases.

Lysosomes are specialized to degrade macromolecules received from the secretory, endocytic, autophagic, and phagocytic pathways (1). Because degradation requirements of the cell may vary depending on tissue type, age, and environmental conditions, we postulated the presence of a cellular program coordinating lysosomal activity. By using the g:profiler (2) tool, we observed that genes encoding lysosomal proteins, hereafter referred to as lysosomal genes, tend to have coordinated expression (figs. S1 and S2). Pattern discovery analysis of the promoter regions of the 96 known lysosomal genes (3) resulted in the identification of a palindromic 10–base pair (bp) GTCACGTGAC motif highly enriched in this promoter set (68 genes out of 96; P < 0.0001) (fig. S3). This motif is preferentially located within 200 bp from the transcription start site (TSS), either as a single sequence or as tandem multiple copies (fig. S4 and table S1). The distribution of this motif was determined around all human gene TSSs (Fig. 1A), and gene ontology analysis of the genes with at least two motifs within 200 bp from the TSS—suggesting that they are probably in a promoter—showed a significant enrichment for functional categories related to lysosomal biogenesis and function (table S2). Thus, we named this motif Coordinated Lysosomal Expression and Regulation (CLEAR) element. A luciferase assay showed that the CLEAR element mediates transcriptional activation (Fig. 1B).

Fig. 1

A regulatory gene network controlling the expression of lysosomal genes. (A) Genomic distribution of CLEAR elements (red dots) at human gene promoters. Scores are assigned based on the CLEAR position weight matrix. Blue dots indicate CLEAR elements in the promoters of lysosomal genes. The dashed box contains all the elements corresponding to the genes that were used for Gene Ontology analysis. (B) Luciferase assay using constructs carrying four tandem copies of either intact (top) or mutated (middle; mutations in red) CLEAR elements. (C) Expression analysis of lysosomal genes after TFEB overexpression and silencing. Blue bars show the fold change of the mRNA levels of lysosomal genes in TFEB- versus pcDNA3-transfected cells. Red bars show the fold change of mRNA levels in mimic-miR-128–transfected cells versus cells transfected with a standard control miRNA (mimic-miR-cel-67). Randomly chosen nonlysosomal genes were used as controls. Gene expression was normalized relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) ChIP analysis. The histogram shows the amount of the immunoprecipitated DNA expressed as a percentage of the total input DNA. Controls include promoters of housekeeping genes (ACTB, APRT, and HPRT), random genes lacking CLEAR sites (TXNDC4 and WIF1), and intronic sequences (int) of lysosomal genes. Lysosomal genes and controls were significantly different: Mann-Whitney-Wilcoxon test (P < 10–4). All experiments in (B), (C), and (D) were performed in triplicates (data represent mean ± SD). (E) Confocal microscopy showing colocalization of C1orf85-Myc (green) with the lysosomal membrane marker LAMP1 (red) in HeLa cells.

The CLEAR consensus sequence overlaps that of the E-box (CANNTG), a known target site for basic helix-loop-helix (bHLH) transcription factors (4). In particular, members of the microphthalmia–transcription factor E (MiT/TFE) subfamily of bHLH factors were found to bind sequences similar to the CLEAR consensus (5). The MiT/TFE subfamily is composed of four members in humans: MITF, TFE3, TFEB, and TFEC (6). To determine whether any of these proteins are able to modulate the expression of lysosomal genes, we transfected HeLa cells with plasmids carrying MITF, TFE3, TFEB, or TFEC cDNAs. We observed an increase in the mRNA levels of lysosomal genes (22 out of 23 genes tested) only after TFEB overexpression (Fig. 1C). Accordingly, we detected a significant increase in the activities of lysosomal enzymes β-glucosidase, Cathepsin D, and β-glucuronidase (fig. S5). Induction of lysosomal genes after TFEB overexpression was also observed in human embryonic kidney (HEK) 293 cells (fig. S6). We predicted that TFEB could be a target of the microRNA miR-128 (7), which was confirmed by luciferase experiments (fig. S7). TFEB silencing mediated by miRNA was associated with the downregulation of 18 out of the 23 lysosomal genes tested (Fig. 1C and fig. S8). Thus, TFEB regulates the expression of lysosomal genes.

To test whether lysosomal genes are direct targets of TFEB, we performed chromatin immunoprecipitation (ChIP) analysis on HeLa cells that stably express a TFEB 3xFLAG construct using an antibody to FLAG. The results demonstrated that TFEB binds to CLEAR sites (Fig. 1D). To identify genes responsive to TFEB on a genomic scale, we performed microarray analysis of the HeLa transcriptome after TFEB overexpression. We observed that 291 genes were upregulated and seven were down-regulated, at a false discovery rate of <0.1 (table S3). Upregulated genes were greatly enriched with lysosomal genes and genes related to lysosomal biogenesis and function (figs. S9 and S10 and table S4). Accordingly, gene set enrichment analysis (GSEA) showed a significant enrichment (enrichment score = 0.84; P < 0.0001) of lysosomal genes that contain CLEAR elements in their promoters among induced genes (fig. S11). Nonlysosomal genes involved in degradation pathways appear to be modulated by TFEB. These include RRAGC and UVRAG, which are key factors regulating autophagy (8, 9); CSTB, which plays a role in protecting against the proteases leaking from lysosomes (10); and M6PR and IGF2R, which mediate the import of proteins into the lysosome (11). To illustrate the feasibility of using the CLEAR network as a tool to identify genes involved in lysosomal function and to provide candidate genes for orphan lysosomal diseases (3), we determined the subcellular distribution of two randomly chosen proteins of unknown function, C1orf85 and C12orf49. The uncharacterized TFEB target, C1orf85, was found localized to lysosomes (Fig. 1E).

An expansion of the lysosomal compartment was detected in HeLa transfectants that stably overexpress TFEB (Fig. 2, A and B, and fig. S12). Accordingly, ultrastructural analysis revealed a significant increase in the number of lysosomes per cell (Fig. 2, C and D), indicating the involvement of TFEB in lysosomal biogenesis. This is similar to MITF, another member of the MiT/TFE family, which is involved in a related cellular function, melanosomal biogenesis (6).

Fig. 2

TFEB overexpression induces lysosomal biogenesis. Comparison of HeLa stable transfectants of either TFEB or empty pcDNA3 vector (control). (A) Confocal microscopy after staining with an antibody against the lysosomal marker LAMP1. (B) Fluorescence-activated cell sorting (FACS) analysis after staining with lysosome-specific dye Lysotracker. The analysis was performed on four independent clones (TFEB#1 to -4) (fig. S12). Blue bars indicate the proportion of cells with fluorescence intensity greater than the indicated threshold (P4 gate). 30,000 cells per clone were analyzed. (C) Electron microscopy analysis. Thin sections exhibit more lysosome profiles (arrows) with typical ultrastructure (inset, details corresponding to dash boxed area) in TFEB–overexpressing transfectants over the control. Scale bar, 720 nm. (D) Number of lysosomes in thin sections (average ± SE, n = 20 cells).

An increase of the expression levels of lysosomal genes and of genes involved in cholesterol biosynthesis and intracellular trafficking was previously reported in a sucrose-induced vacuolation model (12, 13). We used this model to test whether the TFEB-CLEAR network responds to lysosomal storage of undegraded molecules. An increase of the binding events of TFEB to lysosomal promoters (Fig. 3A) and of the mRNA levels of lysosomal genes, and to a lesser extent of TFEB, was detected upon sucrose supplementation to the culture medium (Fig. 3B). The addition of sucrose also determined the progressive translocation of TFEB from a diffuse localization in the cytoplasm, where it predominantly resides in untreated cells, to the nucleus (Fig. 3C), suggesting that nuclear translocation is an important mechanism for TFEB activation.

Fig. 3

The CLEAR network is activated by lysosomal storage. (A) ChIP analysis following lysosomal storage of sucrose. The histogram shows the ratio (expressed as fold change) between the amounts of FLAG-immunoprecipitated chromatin in sucrose-treated versus nontreated cells. Lysosomal genes show an average two- to three-fold increase of immunoprecipitated chromatin, whereas no significant changes are observed for control genes. (B) Expression analysis of lysosomal genes after sucrose supplementation. The diagram shows a time-course analysis of the mRNA levels of lysosomal genes and of TFEB. Gene expression was monitored by means of real-time quantitative polymerase chain reaction (PCR) and normalized relative to GAPDH. All experiments in (A) and (B) were performed at least in duplicates (data represent mean ± SD). (C) Immunofluorescence microscopy analysis of TFEB subcellular localization after sucrose supplementation. HeLa clones stably expressing TFEB-3xFLAG were stained with an antibody to FLAG at various time points after the addition of sucrose in culture medium. (D) Immunofluorescence microscopy analysis of TFEB localization in mouse embryonic fibroblasts (MEFs) from mouse models of three different types of LSDs. MEFs from LSD or wild-type (WT) mice were transiently transfected with a TFEB-3xFLAG construct and stained with an antibody to FLAG. The percentages of nuclei positive for FLAG staining were estimated by examining 100 cells per cell type in two different transfection experiments (data represent mean ± SD).

Over 40 lysosomal storage disorders (LSDs) are characterized by the progressive accumulation of undigested macromolecules within the cell, resulting in cellular dysfunction that leads to diverse clinical manifestations (1, 14, 15). We investigated TFEB subcellular localization in embryonic fibroblasts obtained from mouse models of three different LSDs, Mucopolysaccharidoses types II and IIIA (MPSII and MPSIIIA) and Multiple Sulfatase Deficiency (MSD) (1618). A predominant nuclear localization of TFEB was detected in cells from all three LSD mouse models (Fig. 3D), suggesting that the TFEB signaling pathway is activated after the intralysosomal storage of undegraded molecules. Such activation could be part of the cellular physiological response to lysosomal stress and could serve degradation needs by enhancing the lysosomal system.

To test the ability of TFEB to enhance lysosome-dependent degradation pathways, we analyzed the degradation of glycosaminoglycans (GAGs) in a pulse-chase experiment. TFEB stable transfectants displayed a faster rate of GAG clearance as compared with that in controls (Fig. 4A). We also investigated the ability of TFEB to induce the degradation of the polyglutamine (polyQ)–expanded huntingtin protein that is responsible for Huntington’s disease using the rat striatal cell model HD43 that carries an inducible transgene for mutant huntingtin (19). Immunoblot analyses showed a strong decrease of mutant huntingtin in TFEB-overexpressing cells as compared with those in controls (Fig. 4B). In a parallel experiment, induced HD43 cells were electroporated with a 3xFLAG-TFEB construct. Immunofluorescence analyses showed that the cells that are positive for 3xFLAG-TFEB show little if any huntingtin accumulation (Fig. 4C).

Fig. 4

TFEB enhances cellular clearance. (A) Comparison of the kinetics of GAG clearance in HeLa-stable clones of either TFEB or empty pcDNA3 vector (control). The graph shows relative amounts of 3H-glucosamine incorporated into GAGs over time. 1 = 3H-glucosamine levels at time zero. *P < 0.05. Experiments were performed in triplicates (data represent mean ± SD). (B and C) Clearance of polyQ-expanded huntingtin (HTT) after TFEB overexpression. (B) Immunoblot analysis of TFEB-EGFP–positive (+) and TFEB-EGFP–negative (–) HD43 cells separated by FACS 24 hours after electroporation. The graph of densitometric analysis shows a strong decrease of polyQ-expanded huntingtin in TFEB-EGFP–positive cells as compared with that in controls. (C) Immunocytochemical analysis of TFEB and HTT in HD43(Q105) cells transfected with 3xFLAG-TFEB construct showing little huntingtin staining in cells positive for 3xFLAG-TFEB staining.

We have discovered a cellular program that regulates lysosomal biogenesis and participates in macromolecule clearance. Lysosomal enhancement as a cellular response to pathogenic accumulation has been observed in neurodegenerative diseases (2022). Cathepsin D (23, 24), one of the key enzymes involved in the degradation of neurotoxic proteins, belongs to the CLEAR network and is induced by TFEB overexpression. Also, miR-128 (which we used for TFEB downregulation) is significantly upregulated in the brain of patients with Alzheimer’s disease (25) and in both prion- and chemical-induced neurodegeneration (26, 27). An appealing perspective would be the use of the CLEAR network as a therapeutic target to enhance cellular response to intracellular pathogenic accumulation in neurodegenerative diseases.

Note added in proof: While this study was in proof, a report was published by Schieweck et al. (28) in which was shown a lysosomal localization for NCU-G1, the mouse ortholog of C1orf85.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1174447/DC1

Materials and Methods

Figs. S1 to S12

Tables S1 to S5

References

References and Notes

  1. This paper is dedicated to the memory of Ms. Susanna Agnelli, President of the Italian Telethon Foundation. We thank S. Anand, M. V. Barone, L. Cutillo, M. Morleo, A. Pignata, R. M. Tuzzi, and S. Pepe for technical assistance. We also thank B. Amati, E. Guccione, and P. De Camilli for helpful suggestions and P. Di Fiore, G. Diez-Roux, A. Luini, and C. Missero for comments on the manuscript. This work was supported by the European Union, 7th Framework Program “Euclyd—a European Consortium for Lysosomal Storage Diseases” (health F2/2008 grant agreement 201678) and by the Italian Ministry of Research (PRIN 2006064337). The contributions of the Italian Telethon Foundation and of the National MPS Society USA are gratefully acknowledged. The authors have no conflicts of interest. Expression microarray data are available at the Gene Expression Omnibus repository under accession number GSE16267. A patent application on the discovery of a gene network regulating lysosomal biogenesis and function has been filed to the European Patent Office (patent application EP 091527788). A.B. and M.S. are inventors on this patent.
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