Patent Publication Number: US-2012040451-A1

Title: Molecules able to modulate the expression of at least a gene involved in degradative pathways and uses thereof

Description:
FIELD OF THE INVENTION 
     The invention refers to molecules able to modulate the expression of at least a gene involved in degradative pathways so to enhance the cellular degradative pathways and prevent or antagonize the accumulation of toxic compounds in a cell. 
     BACKGROUND OF THE INVENTION 
     Lysosomes are specialized to degrade macromolecules received from the secretory, endocytic, autophagic and phagocytic pathways (1). Lysosomal storage disorders and neurodegenerative diseases such as Alzheimer&#39;s, Parkinson&#39;s, and Huntington&#39;s share as a common feature the progressive accumulation of undigested macromolecules within the cell, either proteins that tend to form pathogenic aggregates, or intermediates of the cellular catabolism. This ultimately results in cellular dysfunction and clinical manifestations with variable association of visceral (hepatosplenomegaly), skeletal (joint limitation, bone disease and deformities), hematologic (anemia, lymphocyte vacuolization and inclusions), and, most importantly, neurological involvement, with often irreversible damage and invalidating or fatal consequences. Since all of these disorders share a reduced digestive capability of the cell, it would be of great medical interest to identify molecules able to act as general enhancers of degradative pathways. 
     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 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 (GAGs) and the pathogenic protein causing Huntington 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. 
     Prior art reports the description of a system to increase the activity of some cathepsins following the inhibition of the lysosomal system; however, these results are rather partial, controversial, and the molecular mechanism has not been analyzed into details. In the published literature there are no papers that reveal the presence of a lysosomal gene network or that identify TFEB as a possible modulator of the lysosomal activity. 
     DESCRIPTION OF THE INVENTION 
     The authors of the invention identified a gene network that comprises the genes encoding lysosomal proteins of critical importance for the degradation of toxic compounds. These proteins are involved, directly or indirectly, in a high number of human diseases. The regulatory element responsible for the modulation of these genes has been identified in their promoter sequences. Such regulatory element, which authors called CLEAR, represents itself a target for the modulation—and therefore the enhancement—of the production of the lysosomal proteins responsible for the degradation of toxic compounds. Finally, a transcription factor, called TFEB, (NCBI GeneID=7942; nt=NM — 007162.1, protein=NP — 009093.1 (aa. 1-476 of Seq Id No. 228) and variants thereof) has been identified as a protein able to bind to the CLEAR element and to modulate the expression of target genes. Authors demonstrated that the lysosomal activity can be modulated by increasing or decreasing the amount of TFEB. In particular, the lysosomal enhancement resulting from the increase in TFEB levels is able to clear the cell from the toxic protein responsible for the neurodegenerative Huntington&#39;s disease. 
     The enhancement of the cellular degradative pathways by the activation of the lysosomal system may be advantageously used for the therapy of lysosomal storage disorders and of neurodegenerative diseases. 
     Such treatment may be performed by using: 
     1) TFEB or synthetic or biotechnological derivatives thereof, as peptide fragments, chimeric peptides etc., acting directly on the CLEAR element, responsible for the modulation of the expression of lysosomal genes and other genes involved in degradative pathways, in order to enhance the cellular degradative pathways and prevent or antagonize the accumulation of toxic compounds; and/or
 
2) molecules, as peptides, microRNAs, microRNA inhibitors, or any other chemicals, able to act directly or indirectly on the TFEB protein or on its amount; and/or
 
3) vectors for gene therapy containing TFEB, microRNAs, microRNA inhibitors, or other genes able to modulate the CLEAR regulatory network, in order to enhance the cellular degradative pathways.
 
     CA 2525255 A1 describes the use of TFEB for cancer treatment and for modulating cell proliferation or differentiation. 
     WO 2007/070856 claims the use of TFEB for treating immune dysfunction. The document discloses the suppression of CD40L expression by blocking TFEB via interfering RNA molecules; moreover the document discloses the suppression of TFEB by TFEB-dimers. None of the above relates to the enhancement of TFEB amount/activity to target genes. Esumi Noriko et al., The Journal of Biological Chemistry 1997, 282, 3, 1838-1850 discloses effects of siRNA on TFEB, which correlates with the expression of VMD2. The activation of degradative pathways via the TFEB/CLEAR network is not disclosed nor suggested in the document. 
     US2005/255450 discloses a method for screening candidate agents to identify lead compounds for the development of therapeutic agents for treatment of neurodegenerative diseases. The document discloses experiments with yeast cells, that identified several modificators of the clearance of neurotoxic peptides, suggesting that some putative human orthologs of yeast genes should act in the same way. A possible link between TFEB expression and clearance of neurotoxic peptides, in a diagnostic perspective, is suggested, with no data. As a matter of fact HMS1, the described yeast protein, is not the yeast ortholog of TFEB. 
     Finally, the CLEAR regulatory element—allowing the lysosomal system modulation—is not disclosed in any prior art documents. 
     Technologies able to enhance the lysosomal activity have not been described so far. Authors defined molecular events involved in the modulation of the lysosomal system through the regulatory element CLEAR or the TFEB protein. 
     In the instant invention, lysosomal storage disorders are intended as inherited diseases in which a defect in one of many proteins participating in lysosomal biogenesis or metabolism leads to the intralysosomal storage of undegraded molecules, as described in “Lysosomes”, author: Paul Saftig, Landes Bioscience, 2005. 
     It is an object of the invention a molecule being able to enhance the cellular degradative pathways to prevent or antagonize the accumulation of toxic compounds in a cell, characterized by: 
     a) acting either directly or indirectly on a CLEAR element to enhance the expression of at least a gene involved in cellular degradative pathways, said CLEAR element comprising at least one repeat of a nucleotide sequence having Seq Id No. 110 as consensus sequence; and
 
b) belonging to the group of: the TFEB protein, synthetic or biotechnological functional derivative thereof, peptide fragments thereof, chimeric molecules comprising the TFEB protein, synthetic or biotechnological functional derivative thereof; modulator of the TFEB protein activity and/or expression level.
 
     For the TFEB protein it is intended the NCBI GeneID=7942; nt=NM — 007162.1, protein=NP — 009093.1 (aa. 1-476 of Seq Id No. 228), and variants thereof. 
     In a particular aspect of the invention the CLEAR element comprises at least one repeat of a nucleotide sequence having Seq Id No. 111 as consensus sequence. 
     Preferred CLEAR elements are those comprising at least one repeat of a nucleotide sequence selected from the group from Seq Id No. 1 to Seq Id No. 109, most preferred CLEAR elements are those comprising at least one repeat of a nucleotide sequence selected from the group of: Seq Id No. 3, Seq Id No. 9, Seq Id No. 13, Seq Id No. 26, Seq Id No. 28, Seq Id No. 30, Seq Id No. 32, Seq Id No. 34, Seq Id No. 36, Seq Id No. 47, Seq Id No. 50, Seq Id No. 53, Seq Id No. 59, Seq Id No. 62, Seq Id No. 77, Seq Id No. 78, Seq Id No. 84, Seq Id No. 85, Seq Id No. 88, Seq Id No. 92, Seq Id No. 94, Seq Id No. 95, Seq Id No. 98, Seq Id No. 108. Such sequences belong to genes that are responsive either by microarray and/or realtime PCR experiments. 
     In a particular aspect of the invention the chimeric molecule comprises the TFEB protein and a nuclear localization signal (NLS), more preferably the chimeric molecule has the sequence of Seq Id No. 228. 
     In another particular aspect of the invention, the modulator of the TFEB protein is a microRNA or a microRNA inhibitor, preferably the modulator of the TFEB protein is the miR-128 or a miR-128 inhibitor. 
     In a preferred aspect, the molecule of the invention acts either directly or indirectly on a CLEAR element to enhance the expression of at least a gene expressing a lysosomal protein, involved in cellular degradative pathways. 
     In a preferred aspect, the molecule of the invention is for medical use. 
     In a preferred aspect, the molecule of the invention is for neurodegenerative disorders&#39; treatments. 
     Neurodegenerative diseases comprise but are not limited to the following: Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, Creutzfeldt-Jakob disease, Spinocerebellar Ataxia (SCA). 
     Preferably the neurodegenerative disorder belongs to the group of Alzheimer, Parkinson and Huntington diseases. 
     In an alternative preferred aspect, the molecule of the invention is for lysosomal storage disorders&#39; treatments. 
     Lysosomal storage disorders comprise but are not limited to the following: Activator Deficiency/GM2 Gangliosidosis; Alpha-mannosidosis; Aspartylglucosaminuria; Cholesteryl ester storage disease; Chronic Hexosaminidase A Deficiency; Cystinosis; Danon disease; Fabry disease; Farber disease; Fucosidosis; Galactosialidosis; Gaucher Disease (including Type I, Type II, and Type III); GM1 gangliosidosis (including Infantile, Late infantile/Juvenile, Adult/Chronic); I-Cell disease/Mucolipidosis II; Infantile Free Sialic Acid Storage Disease/ISSD; Juvenile Hexosaminidase A Deficiency; Krabbe disease (including Infantile Onset, Late Onset); Metachromatic Leukodystrophy; Pseudo-Hurler polydystrophy/Mucolipidosis IIIA; MPSI Hurler Syndrome; MPSI Scheie Syndrome; MPS I Hurler-Scheie Syndrome; MPS II Hunter syndrome; Sanfilippo syndrome Type A/MPS III A; Sanfilippo syndrome Type B/MPS III B; Sanfilippo syndrome Type C/MPS III C; Sanfilippo syndrome Type D/MPS III D; Morquio Type A/MPS IVA; Morquio Type B/MPS IVB; MPS IX Hyaluronidase Deficiency; MPS VI Maroteaux-Lamy; MPS VII Sly Syndrome; Mucolipidosis I/Sialidosis; Mucolipidosis IIIC; Mucolipidosis type IV; Multiple sulfatase deficiency; Niemann-Pick Disease (including Type A, Type B, and Type C); Neuronal Ceroid Lipofuscinoses, including CLN6 disease; Atypical Late Infantile, Late Onset variant; Early Juvenile Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease; Finnish Variant Late Infantile CLN5; Jansky-Bielschowsky disease/Late infantile CLN2/TPP1 Disease; Kufs/Adult-onset NCL/CLN4 disease; Northern Epilepsy/variant late infantile CLN8; Santavuori-Haltia/Infantile CLN1/PPT disease; Beta-mannosidosis; Pompe disease/Glycogen storage disease type II; Pycnodysostosis; Sandhoff disease/Adult Onset/GM2 Gangliosidosis; Sandhoff disease/GM2 gangliosidosis; Infantile Sandhoff disease/GM2 gangliosidosis; Juvenile Schindler disease; Salla disease/Sialic Acid Storage Disease; Tay-Sachs/GM2 gangliosidosis; Wolman disease. 
     Preferably the lysosomal storage disorder belongs to the group of Pompe disease and Multiple Sulfatase Deficiency (MSD). 
     It is another aspect of the invention a nucleic acid containing a sequence encoding for the molecule according as above disclosed, 
     It is another aspect of the invention a vector comprising under appropriate regulative sequence the above nucleic acid, preferably for gene therapy. 
     The invention shall be described with reference to experimental non limitating evidences. 
    
    
     
       FIGURE LEGENDS 
         FIG. 1 . A regulatory gene network controlling the expression of lysosomal genes. (A) Genomic distribution of CLEAR elements (red spots) at human gene promoters. Scores are assigned based on the CLEAR position weight matrix. Blue spots indicate CLEAR elements in the promoters of lysosomal genes. Dashed box contains all the elements corresponding to the genes that were used for Gene Ontology analysis (see text). (B) Luciferase assay using constructs carrying four tandem copies of either intact (upper) or mutated (middle, mutations in red) CLEAR elements. (C) Expression analysis of lysosomal genes following TFEB overexpression and silencing. Blue bars show the fold change of the mRNA levels of lysosomal genes in TFEB- vs. pcDNA3-transfected cells. Red bars show the fold change of mRNA levels in mimic-miR-128-transfected cells vs. cells transfected with a standard control microRNA (mimic-miR-cel-67). Randomly chosen non-lysosomal genes were used as controls. Gene expression was normalized relative to GAPDH. (D) Chromatin immunoprecipitation (ChIP) analysis. The histogram shows the amount of the immunoprecipitated DNA expressed as percentage of total input DNA. Controls include promoters of housekeeping genes (ACTB, APRT, HPRT), random genes lacking CLEAR sites (TXNDC4, WIF1) and intronic sequences (int) of lysosomal genes. Lysosomal genes and controls were significantly different: Mann-Whitney-Wilcoxon test (P&lt;10-4). All experiments in (B), (C) and (D) were performed in triplicates (data represent mean±s.d.). (E) Confocal microscopy showing colocalization of C1orf85-Myc (green) with the lysosomal membrane marker LAMP1 (red) in HeLa cells. 
         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) FACS analysis after staining with lysosome-specific dye Lysotracker. The analysis was performed on four independent clones (TFEB#1-4) (see  FIG. 18 ). 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 (see details in inset corresponding to dash boxed area) in TFEB overexpressing transfectants over the control. Scale bar, 720 nm. (D) Number of lysosomes in thin sections (average±s.e., N=20 cells). 
         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 non-treated 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 following sucrose supplementation. The diagram shows a time-course analysis of the mRNA levels of lysosomal genes and of TFEB. Gene expression was monitored by real-time qPCR and normalized relative to GAPDH. All experiments in (A) and (B) were performed at least in duplicates (data represent mean±s.d.). (C) Immunofluorescence microscopy analysis of TFEB subcellular localization following sucrose supplementation. HeLa clones stably expressing TFEB-3×FLAG were stained with an anti-FLAG antibody 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-3×FLAG construct and stained with an anti-FLAG antibody. 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±s.d.). 
         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. Asterisk, P&lt;0.05. Experiments were performed in triplicates (data represent mean±s.d.). (B and C) Clearance of polyQ expanded huntingtin (HTT) following TFEB overexpression. (B) Immuno blot analysis of TFEB-EGFP-positive (+) and TFEB-EGFP-negative (−) HD43 cells separated by FACS 24 h after electroporation. The graph of densitometric analysis shows a strong decrease of polyQ expanded huntingtin in TFEB-EGFP-positive cells compared to controls. (C) Immunocytochemical analysis of TFEB and HTT in HD43(Q105) cells transfected with 3×FLAG-TFEB construct showing little huntingtin staining in cells positive for 3×FLAGTFEB staining. 
         FIG. 5  Lysosomal genes display coordinated expression behaviour. The diagram reports a visual representation of the expression correlation of 40 lysosomal disease genes with all known lysosomal genes. Each column represents the ˜22,500 gene probes of the Affymetrix HG-U133A platform ranked by their correlation of expression with the gene indicated at the top. Blue bars represent the position of lysosomal genes within the ranked lists. The analysis shows that there is an enrichment of lysosomal genes within the first 5th percentile of ranked lists of expression correlation. 
         FIG. 6  Detailed view of the expression correlation among lysosomal genes. The columns include the first 100 gene probes of the expression correlation lists for selected lysosomal genes. Lysosomal genes are highlighted in orange. Other genes associated to the lysosomal function are highlighted in yellow. It should be noted that in a randomly ranked list the probability of finding a lysosomal gene probe is ˜1:100. 
         FIG. 7  Logo representation of the CLEAR element. The conservation of each residue within columns is visualized as the relative height of symbols. 
         FIG. 8  Distribution of CLEAR elements at the promoter regions of a subset of lysosomal genes. The CLEAR elements are clustered, often in multiple copies, around the transcription start site. The legend to colour code is reported as a schematic diagram in the figure. 
         FIG. 9  Enzymatic activities. Quantification of the activities of lysosomal enzymes β-glucosidase, cathepsin D and β-glucuronidase in HeLa cells stably overexpressing TFEB and controls. Asterisk, P&lt;0.05. All measures were performed in triplicates (data represent mean±s.d.). 
         FIG. 10  Expression analysis of lysosomal genes following TFEB overexpression in HEK293 cells. Blue bars show the fold change of the mRNA levels of monitored genes in TFEB- vs. pcDNA3-transfected cells. Gene expression was normalized relative to GAPDH. 
         FIG. 11  Validation of TFEB as a target gene of miR-128 by dual luciferase assay. The 3′UTR region of TFEB was cloned into a firefly luciferase sensor construct and transfected into HeLa cells along with a Renilla Luciferase control. Luciferase activities were measured in the presence or absence of a plasmid construct containing the precursor sequence of hsa-miR-128. EZH2 and LRIG1 genes, which were not predicted targets of miR-128, were used as negative controls. All experiments were performed in triplicates (data represent mean±s.d.). 
         FIG. 12  Expression analysis of lysosomal genes following mimic-miR-128 transfection into HeLa cells stably expressing a TFEB transgene lacking the 3′UTR region. To verify that the downregulation of lysosomal genes following mimic-miR-128 transfection was due to TFEB silencing, mimic-miR-128 was transfected into HeLa clones stably expressing a TFEB transgene lacking the TFEB 3′UTR region, which contains the miR128 binding site. Blue bars show the fold change of monitored genes in mimic-miR-128-transfected cells vs. cells transfected with a standard control microRNA (mimic-miR-cel-67). No significant changes were observed for any of the genes tested. Gene expression was normalized relative to GAPDH. 
         FIG. 13  Analysis of transcriptome changes following TFEB transient transfection in HeLa cells. The graph shows a Gene Ontology analysis by ‘Cellular Compartment’ category of up regulated genes with false discovery rate&lt;0.1. 
         FIG. 14  Venn diagram showing the overlap between lysosomal genes and genes induced by TFEB overexpression in HeLa cells at an FDR&lt;0.10. The diagram shows that 20 genes, all containing CLEAR sites in their promoters, are represented in both categories. This is likely to be an underestimate as it is based on highly stringent statistical criteria and on a single cell type. A more comprehensive view of the response of lysosomal genes to TFEB induction is shown in  FIG. 15  (Gene Set Enrichment Analysis). 
         FIG. 15  Gene Set Enrichment Score Analysis (GSEA) of transcriptome changes following TFEB overexpression. The graph shows the enrichment plots generated by GSEA analysis of ranked gene expression data (left: upregulated, red; right: down-regulated, blue). The enrichment score is shown as a blue line, and the vertical blue bars below the plot indicate the position of lysosomal genes carrying CLEAR sites in their promoters. The analysis shows that lysosomal genes with CLEAR sites are mostly grouped in the fraction of up-regulated genes (Enrichment Score=0.84; P&lt;0.0001). 
         FIG. 16  FACS analysis after staining with lysosome-specific dye lysotracker of HeLa stable transfectants of TFEB (TFEB#1-4). Blue bars indicate the proportion of cells with fluorescence intensity greater than the indicated threshold (P4 gate). 30,000 cells per clone were analyzed. 
         FIG. 17  Microscopy analysis of MSD cells at 48 hours following the transfection of an empty vector (left) or a TFEB vector (right). The arrows indicate the storage of glycosaminoglycans in untreated MSD cells. The experiment shows that cells treated with TFEB no longer display accumulation of undigested glycosaminoglycans. 
         FIG. 18  Electron microscopy analysis of MSD cells at 48 hours following the transfection of an empty vector (left) or a TFEB vector (right). Untreated cells show an extensive vacuolization due to the storage of undigested glycosaminoglycans. Cells treated with TFEB show that the cellular vacuolization is largely reversed. 
         FIG. 19  Immunofluorescence analysis of Pompe disease cells treated with a TFEB-3×FLAG vector. Transfected cells (arrows) show a strong reversal of the extensive vacuolization found in non-transfected cells (on the right) due to the accumulation of glycogen. 
         FIG. 20  Inhibition of miR-128 results in the transcriptional activation of the CLEAR network. Cultured HeLa cells were transfected with a specific inhibitor of miR-128 (Dharmacon) or with a standard control (inhibitor of miR-cel-167) that has no target in human cells. Real-time qPCR was performed to monitor the expression of TFEB, its lysosomal target PSAP, two housekeeping genes (HPRT and GAPDH) and two random genes (ARPP-19 and HOXA9) 48 hours after transfection. The graph shows the ratio between the expression levels of monitored genes in cells transfected with the inhibitor of miR-128 versus control. The results show an increase in the expression of both TFEB and its target PSAP, and no changes in control genes. Gene expression was normalized relative to HPRT. 
         FIG. 21  Amino acid sequence of the engineered analog of TFEB, TFEB-NLS (Seq Id No. 228). TFEB-NLS was obtained by the addition of a nuclear localization signal (NLS) at the C-terminus of the protein. The nuclear localization signal has sequence PKKKRK (underlined in the figure). 
         FIG. 22  TFEB-NLS localizes in the nucleus. Immunofluorescence analysis of the TFEB analog TFEB-NLS showing a complete nuclear localization of the TFEB-NLS construct. Two series of images are reported as representative of the subcellular localization of TFEB-NLS. In each series, on the left cell nuclei are stained with the DAPI dye (specific for the DNA); on the right, cells are stained for TFEB. 
     
    
    
     MATERIAL AND METHODS 
     Genome Analysis 
     Human genomic sequences were retrieved from the Ensemble database (http://www.ensembl.org) and analyzed by using the Regulatory Sequence Analysis Tool (28). Iterative analyses led to the identification of a consensus sequence of the CLEAR element. A position weight matrix (PWM) was built by assembling all CLEAR elements found within 200 bp from the transcription start site of lysosomal genes. Human gene promoters were searched with the CLEAR PWM using the PatSer tool (28) with default parameters. Gene Ontology (GO) analyses were performed with the web tool DAVID (http://david.abcc.ncifcrf.gov) using default parameters. Only non-redundant terms with a value≦0.01 and Fold Enrichment≧2 were retained. 
     Expression Correlation Analysis 
     Expression correlation analysis was performed as previously described (29), with minor modifications. Briefly, lysosomal genes were analyzed by using the g:Sorter tool, which is part of the g:Profiler package (30). For a selected gene probe, g:Sorter can retrieve a number of most similar coexpressed profiles in a specified GEO data set. The analysis was carried out on a total of 160 heterogeneous microarray experiments, based on the HG-U133A GeneChip array. g:Sorter was queried with the gene probes for a representative set of lysosomal genes. For each analyzed probe, the first 3% of most correlated gene probes was retrieved for each microarray data set. Subsequently, all HG-U133A gene probes were ranked based on their occurrence in the 160 different lists of most correlated genes. Genes with an equal number of occurrences were sub-ranked according to their average ranking within the various experiments. The procedure resulted in lists of gene probes ranked by their expression correlation to the investigated genes. 
     Cell Culture and Transfection 
     HeLa cells and mouse embryonic fibroblasts from mouse models of MPSII (31), MPSIIIA (32), and MSD (33), were grown in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM, Euroclone), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, Hyclone). Where indicated, the medium was supplied with sucrose to a final concentration of 100 mM. Cells were seeded in six-well plates at 10% confluence before transfection. Transfection was performed by using PolyFect Transfection Reagent (Qiagen) or Interferin (PolyPlus transfection) according to the manufacturer&#39;s protocols. Transfectants for full-length TFEB and TFEB-3×FLAG were selected with 1 mg/ml G418 (Sigma). For microRNA experiments, cells were transfected with 200 nM miRIDIAN Dharmacon miRNA Mimics (miR-128, or negative control cel-miR-67) and harvested after 48 h for total RNA extraction. 
     Luciferase Assays 
     To test the ability of the CLEAR site to promote transcription, HeLa cells were transfected with pGL3-basic luciferase reporter plasmids containing four tandem copies of either the sequence (4×CLEAR consensus sequences as in Seq Id No. 111 in bold characters) Seq Id No. 112: 
                    CCGG GTCACGTGAC CCCAGG GTCACGTGAC CCTGCGG GTCACGTGAC CCT               GCGG GTCACGTGAC CCCC            
or the sequence (4×control sequences in bold characters) Seq Id No. 113:
 
     
       
         
           
               
            
               
                 CCGG GAATCGTGAC CCCAGG GAATCGTGAC CCTGCGG GAATCGTGAC CCT 
               
               
                   
               
               
                 GCGG GAATCGTGAC CCCC. 
               
            
           
         
       
     
     To validate TFEB as a target of miR-128, HeLa cells were transfected with firefly luciferase reporter plasmids containing the 3′UTR regions of either TFEB or control genes (EZH2 and LRIG1) and with a psiUx plasmid (34), construct containing the precursor sequence of hsa-miR-128. Luciferase assays were performed 48 h after transfection using Dual Luciferase Reporter Assay System (Promega), normalized for transfection efficiency by cotransfected Renilla luciferase. 
     Molecular Biology 
     Full-length human MITF, TFE3, TFEB and TFEC were cloned into the pcDNA3.1 vector (Invitrogen). Full-length TFEB was also cloned into the p3×FLAG-CMV-10 vector. Full-length C1orf85 was cloned into the pcDNA3.1/c-Myc vector (Invitrogen). RNA samples were obtained using either the RNeasy or the miRNeasy kit (Qiagen) according to the manufacturer&#39;s instructions. RNA was quantified using the NanoDrop 8000 (Thermo Fischer). cDNA was synthesized using QuantiTect Reverse Transcription kit (Qiagen). 
     Chromatin Immunoprecipitation Assay (ChIP) 
     ChIP assays were carried out using formaldehyde-fixed nuclei isolated from HeLa transfectants carrying a TFEB-3×FLAG transgene or a control HeLa cell line without any tagged transgene (mock). Each ChIP experiment required 10 7  cells. ChIP was performed using the ANTI-FLAG M2 Affinity Gel (Sigma) according to the manufacturer&#39;s protocol. 
     Quantitative Real-Time PCR 
     Real-time quantitative RT-PCR on cDNAs or sonicated chromatin was carried out with the LightCycler 480 SYBR Green I mix (Roche) using the Light Cycler 480 II detection system (Roche) with the following conditions: 95° C., 5 min; (95° C., 10 s; 60° C., 10 s; 72° C., 15 s)×40. For expression studies the qRT-PCR results were normalized against an internal control (GAPDH). Oligonucleotide sequences are reported in Table 5. 
     Microarray Experiments 
     Total RNA from TFEB-transfected HeLa cells was used to prepare cDNA for hybridization to the Affymetrix Human Gene 1.0 ST array platform. Hybridizations were performed in triplicates at the Coriell Genotyping and Microarray Center, Coriell Institute for Medical Research, Camden, N.J., USA. A false discovery rate&lt;0.1 was used to assess significant gene differential expressions. Gene Set Enrichment Analysis was performed as previously described (35). The cumulative distribution function was constructed by performing 1,000 random gene set member-ship assignments. A nominal P value&lt;0.01 and an FDR&lt;10% were used to assess the significance of the Enrichment Score (ES). 
     Confocal Imaging 
     Transfected HeLa cells were grown on glass coverslips for 24 h, washed with PBS containing 100 mM MgCl 2  and 100 mM CaCl 2  (PBS/Ca/Mg), and fixed with 4% paraformaldehyde (PFA; Sigma) for 10 min. After washing and quenching PFA with 50 mM NH 4 Cl for 15 min, cells were washed with PBS and permeabilized in blocking buffer (0.05% saponin/0.2% BSA in PBS/Ca/Mg) for 20 min. Coverslips were then incubated O/N with appropriate primary antibodies and for 1 h with Alexa-594 and Alexa-488 conjugated secondary antibodies (Molecular Probes). Coverslips were mounted on glass slides with Vectashield (Vector Laboratories). Images were taken using a confocal microscope (LSM510; Carl Zeiss, Inc.) using a Plan-Neofluar 63× immersion objective (Carl Zeiss, Inc.). 
     Electron Microscopy 
     Control and TFEB-overexpressing HeLa cells were washed with PBS, and fixed in 1% glutaraldehyde dissolved in 0.2 M Hepes buffer (pH 7.4) for 30 min at room temperature. The cells were then postfixed for 2 h in OsO 4 . After dehydration in graded series of ethanol, the cells were embedded in Epon 812 (Fluka) and polymerized at 60° C. for 72 h. Thin sections were cut at the Leica EM UC6, counterstained with uranyl acetate and lead citrate. EM images were acquired from thin sections using a Philips Tecnai-12 electron microscope equipped with an ULTRA VIEW CCD digital camera (Philips, Eindhoven, The Netherlands). Quantification of lysosomes was performed using the AnalySIS software (Soft Imaging Systems GmbH, Munster, Germany). Selection of cells for quantification was based on their suitability for stereologic analysis, i.e. only cells sectioned through their central region (detected on the basis of the presence of Golgi membranes) were analyzed. Lysosomal profiles were detected on the basis of typical ultrastructural characteristics such as high electron density, presence of multiple internal luminal vesicles, concentric and myelinoid bodies. 
     Huntingtin Clearance 
     Huntingtin inducible striatal cells [HD43(Q105)] were cultured at 33° C. in DMEM high glucose, supplemented as described previously (36). HD43(Q105) cells were electroporated with a pCIG2-TFEB vector containing an IRES2-EGFP cassette, or with an empty pCIG2 vector as a control, using a Gene Pulser II electoporator (BioRad). Immediately after the electoporation, cells were plated in presence of 0.2 μg/ml doxycycline (Sigma) in order to induce the transgene for expanded huntingtin. Twenty-four hours post-induction, GFP-positive cells were sorted by flow cytometry using the BD FACSAria cytometer (BD Biosciences) and used for immuno blot analysis. 
     FACS Analysis 
     Cells were kept in 50 nM acidotropic dye LysoTracker Red DND-99 (Molecular Probes) for 40 min. Red lysosomal fluorescence of 30,000 cells per sample was determined by flow cytometry using the BD FACSAria cytometer (BD Biosciences). 
     GAG Clearance 
     HeLa cells were grown in RPMI medium (Gibco, Invitrogen, Grand Island) supplemented with 10% FCS in the presence of 7 μCi/ml  3 H-glucosamine hydrochloride (Perkin Elmer, 37.75 Ci/mmol, Boston) for 3 days, washed extensively with PBS and chased for variable times. At each time point cells were harvested, homogenized and subject to chromatography on Sephadex G-25 columns (GE Healthcare, Sweden) to eliminate unincorporated  3 H-glucosamine hydrochloride. The amounts of incorporated radioactivity was measured by liquid scintillation in a Beckman L56500 counter (Beckman Instruments, Fullerton, Calif., USA). 
     Immuno-Blot 
     Cells were lysed in cold lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% TritonX-100) in the presence of protease inhibitors (SIGMA) for 30 min on ice. 20 mg of protein samples were separated on SDS-PAGE acrylamide gel and transferred onto nitrocellulose membrane (Amersham Pharmacia Biotech). Primary and (HRP)-conjugated antibodies were diluted in 1% BSA TBS-T. Bands were visualized using the ECL detection reagent (Pierce) and normalized against actin. Proteins were quantified by the Bradford method. Antibodies: Huntingtin, MAb2166 (Chemicon, Temecula, Calif.); Actin (Sigma). 
     Enzymatic Activities 
     Cathepsin D activity was determined with the Cathepsin D Assay Kit (Sigma) following manufacturer&#39;s instructions. β-glucosidase activity was determined by incubating cell homogenates (10 7  cells, ˜10 μg proteins) with 5 mM 4-MU-beta-D-glucopyranoside in 0.1 M acetate buffer, pH 4.2, for 3 hrs at 37° C. β-glucuronidase activity was determined by incubating cell homogenates (2.5×10 7  cells, ˜25 μg proteins) with 10 mM 4-MU-glucuronide in 0.2 acetate buffer, pH 4.8, for 1 hr at 37° C. Both reactions were stopped with 1 ml glycine-carbonate buffer, pH 10.7. Fluorescence was read at 365 nm (excitation) and 450 nm (emission) on a Turner Modulus fluorometer. 
     Data Analysis 
     Most data are presented as the mean±s.d. Statistical comparisons were made using analysis of variance (ANOVA). A P value&lt;0.05 was considered statistically significant. 
     Results 
     As stated above, lysosomes are specialized to degrade macromolecules received from the secretory, endocytic, autophagic and phagocytic pathways (1). As degradation requirements of the cell may vary depending on tissue type, age, and environmental conditions, authors postulated the presence of a cellular program coordinating lysosomal activity. By using the g:profiler (2) tool authors observed that genes encoding lysosomal proteins, hereafter referred to as lysosomal genes, tend to have coordinated expression ( FIGS. 5 and 6 ). Pattern discovery analysis of the promoter regions of the 96 known lysosomal genes (3) resulted in the identification of a palindromic 10-bp GTCACGTGAC motif highly enriched in this promoter set (68 genes out of 96; P&lt;0.0001) ( FIG. 7 ). 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. 8  and Table 1). 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 they are likely in a promoter—showed a significant enrichment for functional categories related to lysosomal biogenesis and function (Table 2). Thus, authors named this motif Coordinated Lysosomal Expression And Regulation (CLEAR) element. A luciferase assay showed that the CLEAR element mediates transcriptional activation ( FIG. 1B ). 
     The CLEAR consensus sequence shown as Seq Id No. 110 overlaps that of the E-box (CANNTG), a known target site for bHLH transcription factors (4). In particular, members of the 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, authors transfected HeLa cells with plasmids carrying MITF, TFE3, TFEB, or TFEC cDNAs. Authors observed an increase in the mRNA levels of lysosomal genes (22 out of 23 genes tested) only following TFEB overexpression ( FIG. 1C ). Accordingly, authors detected a significant increase in the activities of lysosomal enzymes β-glucosidase, Cathepsin D and β-glucuronidase ( FIG. 9 ). Induction of lysosomal genes following TFEB overexpression was also observed in HEK293 cells ( FIG. 10 ). Authors predicted that TFEB could be a target of the micro-RNA miR-128 (7), which was confirmed by luciferase experiments ( FIG. 11 ). MicroRNA-mediated TFEB silencing was associated with the downregulation of 18 out of the 23 lysosomal genes tested ( FIGS. 10 and 12 ). Thus, TFEB regulates the expression of lysosomal genes. 
     The inhibition of miR-128, performed with a specific miRNA inhibitor (Dharmacon), resulted in the increase of the expression of TFEB and of its target lysosomal gene PSAP ( FIG. 20 ), demonstrating that the modulation of the expression of miR-128 can directly influence the activation of the CLEAR network. 
     To test whether lysosomal genes are direct targets of TFEB authors performed chromatin immunoprecipitation (ChIP) analysis on HeLa cells stably expressing a TFEB 3×FLAG construct using an anti-FLAG antibody. The results demonstrated that TFEB binds to CLEAR sites ( FIG. 1D ). To identify genes responsive to TFEB on a genomic scale authors performed microarray analysis of the HeLa transcriptome following TFEB overexpression. Authors observed that 291 genes were up-regulated, and 7 down-regulated, at a false discovery rate&lt;0.1 (Table 3). Up-regulated genes were greatly enriched with lysosomal genes and genes related to lysosomal biogenesis and function ( FIGS. 13 and 14 , Table 4). Accordingly, Gene Set Enrichment Analysis (GSEA) showed a significant enrichment (Enrichment Score=0.84; P&lt;0.0001) of lysosomal genes that contain CLEAR elements in their promoters among induced genes ( FIG. 15 ). Interestingly, non-lysosomal 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); 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), authors 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 stably overexpressing TFEB ( FIGS. 2 , A and B and  FIG. 16 ). Accordingly, ultrastructural analysis revealed a significant increase in the number of lysosomes per cell ( FIGS. 2 , C and D), indicating the involvement of TFEB in lysosomal biogenesis. 
     Authors used a sucrose-induced vacuolization model (12, 13) 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. 
     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). Authors 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) (16-18). 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 following the intra-lysosomal 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. In order to obtain a TFEB molecule able to completely and directly localize into the nucleus, authors designed a TFEB analog (chimeric molecule) by adding a nuclear localization signal (NLS) at the C-terminus of the TFEB protein (Seq Id No. 228,  FIG. 21 ). Immunofuorescence analysis of HeLa cells transfected with the TFEB-NLS construct demonstrated that it indeed localize into the nucleus ( FIG. 22 ), with no needs for storage conditions. 
     Lysosomal storage disorders are caused by the intracellular accumulation of undigested material due to mutations in genes participating to lysosomal function. In Multiple Sulfatase Deficiency (MSD), a severe human disorder, a defect in sulfatases impairs the ability of the cell to degrade sulfated compounds, with the subsequent accumulation of glycosaminoglycans that induce extensive cellular vacuolization and finally prove to be toxic for the cells. Authors used cells derived from a mouse model of MSD to test the clearance capability of TFEB in this disease. They transfected MSD cells with a TFEB vector or an empty vector and monitored the accumulation of glycosaminoglycans 48 hours post-transfection. They found that TFEB was able to promote the clearance of stored glycosaminoglycans ( FIG. 17 ) and to reverse the subsequent cellular vacuolization, as demonstrated by electron microscopy analysis ( FIG. 18 ). Authors tested the clearance capability of TFEB on an additional model of lysosomal storage disorder, the Pompe disease, in which a defect in the acid alpha-glucosidase gene leads to the intralysosomal accumulation of glycogen and subsequent extensive vacuolization of the cell. Authors transfected human fibroblasts derived from a Pompe patient with a TFEB-3×FLAG vector and monitored the shape and the number of lysosomes in the cells. Cells transfected with TFEB-3×FLAG were found to diminish the amount of undigested glycogen, as demonstrated by the decreased number of lysosomal vesicles compared to non-transfected cells ( FIG. 19 ). Together, these data indicate that the enhancement of the lysosomal activity by acting on the CLEAR network can provide in principle a polyvalent therapy against different lysosomal storage disorders. 
     To test the ability of TFEB to enhance lysosome-dependent degradation pathways authors analyzed the degradation of glycosaminoglycans (GAGs) in a pulse-chase experiment. TFEB stable transfectants displayed a faster rate of GAG clearance compared to controls ( FIG. 4A ). Authors also investigated the ability of TFEB to induce the degradation of the polyglutamine (polyQ) expanded huntingtin protein responsible for Huntington 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 compared to controls ( FIG. 4B ). In a parallel experiment, induced HD43 cells were electroporated with a 3×FLAG-TFEB construct. Immunofluorescence analyses showed that the cells that are positive for 3×FLAG-TFEB show little, if any, huntingtin accumulation ( FIG. 4C ). 
     Authors 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 (20-22). Interestingly, 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. Of particular interest is also the observation that miR-128, which authors used for TFEB downregulation, is significantly up-regulated in the brain of patients with Alzheimer&#39;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. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Distribution of CLEAR elements in the promoters of human lysosomal genes. 
               
            
           
           
               
               
               
               
               
            
               
                 Gene 
                   
                   
                   
                 Seq Id 
               
               
                 symbol 
                 Gene name 
                 CLEAR element 
                 Position* 
                 No. 
               
               
                   
               
            
           
           
               
            
               
                 Membrane transporters 
               
            
           
           
               
               
               
               
               
            
               
                 ABCA2 
                 ATP-binding cassette, sub-family A (ABC1), 
                 GTCGCGTGAC 
                 −187 
                 1 
               
               
                   
                 member 2 
                   
                   
                   
               
               
                 ABCB9 
                 ATP-binding cassette, sub-family B (MDR/TAP), 
                 CTCACCTGGT 
                 94 
                 2 
               
               
                   
                 member 9 
                   
                   
                   
               
               
                 CLCN7 
                 chloride channel 7 
                 
                   ATCACGTGGC 
                 
                 −103 
                 3 
               
               
                   
                   
                 GTCACGTGGC 
                 −83 
                 4 
               
               
                 CLN3 
                 ceroid-lipofuscinosis, neuronal 3, juvenile 
                 AGCACGTGAT 
                 −24 
                 5 
               
               
                   
                   
                 GTCACGTGAT 
                 6 
                 6 
               
               
                 CLN5 
                 ceroid-lipofuscinosis, neuronal 5 
                 CTCAAGTGTG 
                 50 
                 7 
               
               
                   
                   
                 TTCAGGTGCC 
                 74 
                 8 
               
               
                 CTNS 
                 cystinosis, nephropathic 
                 
                   GTCAGGTGGC 
                 
                 −32 
                 9 
               
               
                   
                   
                 GTCAGGTGAC 
                 −18 
                 10 
               
               
                 LAPTM4A 
                 lysosomal-associated protein transmembrane 4 
                 GTCACGTTAT 
                 −372 
                 11 
               
               
                   
                 alpha 
                   
                   
                   
               
               
                   
                   
                 GTGACGCTTC 
                 −356 
                 12 
               
               
                 LMBRD1 
                 LMBR1 domain containing 1 
                 — 
                 — 
                   
               
               
                 MCOLN1 
                 mucolipin 1 
                 
                   GTCACGTGAG 
                 
                 −47 
                 13 
               
               
                   
                   
                 GTCACGTGAC 
                 −20 
                 14 
               
               
                   
                   
                 ATCAGCTGAT 
                 0 
                 15 
               
               
                 MFSD8 
                 major facilitator superfamily domain containing 8 
                 GTCAGGTGCG 
                 −15 
                 16 
               
               
                 NPC1 
                 Niemann-Pick disease, type C1 
                 TTCAGGTGAC 
                 −383 
                 17 
               
               
                 SCARB2 
                 scavenger receptor class B, member 2 
                 CTCAGGCGCC 
                 −134 
                 18 
               
               
                   
                   
                 GGCACATGAC 
                 −57 
                 19 
               
               
                 SLC17A5 
                 solute carrier family 17 (anion/sugar 
                 GCCAGGTGGC 
                 47 
                 20 
               
               
                   
                 transporter), member 5 
                   
                   
                   
               
               
                   
                   
                 CTCACGTAGG 
                 68 
                 21 
               
               
                 SLC36A1 
                 solute carrier family 36 (proton/amino acid 
                 AGCACGTGAC 
                 −44 
                 22 
               
               
                   
                 symporter), member 1 
                   
                   
                   
               
               
                   
                   
                 ATCACGTGAT 
                 −9 
                 23 
               
               
                   
               
            
           
           
               
            
               
                 Hydrolases 
               
            
           
           
               
               
               
               
               
            
               
                 ACP2 
                 acid phosphatase 2 
                 — 
                 — 
                   
               
               
                 ACP5 
                 acid phosphatase 5, tartrate resistant 
                 CTCACCTGGG 
                 8 
                 24 
               
               
                 AGA 
                 aspartylglucosaminidase 
                 — 
                 — 
                   
               
               
                 ARSA 
                 arylsulfatase A 
                 GCCAAGTGAC 
                 80 
                 25 
               
               
                 ARSB 
                 arylsulfatase B 
                 
                   
                 
                 288 
                 26 
               
               
                 ARSG 
                 arylsulfatase G 
                 GCCACGTGTG 
                 183 
                 27 
               
               
                 ASAH1 
                 N-acylsphingosine amidohydrolase 1 
                 
                   GTCACGCGGC 
                 
                 −41 
                 28 
               
               
                 CPVL 
                 carboxypeptidase, vitellogenic-like 
                 GTCATGTGAG 
                 −123 
                 29 
               
               
                 CTBS 
                 di-N-acetyl-chitobiase 
                 — 
                 — 
                   
               
               
                 CTSA 
                 cathepsin A 
                 
                   GTCACGTGGC 
                 
                 −50 
                 30 
               
               
                   
                   
                 TTCACGTGAC 
                 −33 
                 31 
               
               
                 CTSB 
                 cathepsin B 
                 
                   GTCACGTGGG 
                 
                 −7 
                 32 
               
               
                 CTSC 
                 cathepsin C 
                 TTCACCTGAC 
                 −343 
                 33 
               
               
                 CTSD 
                 cathepsin D 
                 
                   CCCACGTGAC 
                 
                 16 
                 34 
               
               
                   
                   
                 GTCAGCTGAT 
                 48 
                 35 
               
               
                 CTSF 
                 cathepsin F 
                 
                   CCCACGTGCC 
                 
                 −83 
                 36 
               
               
                 CTSH 
                 cathepsin H 
                 CCCAGTTGAC 
                 30 
                 37 
               
               
                 CTSK 
                 cathepsin K 
                 GTCACATGTG 
                 −650 
                 38 
               
               
                   
                   
                 TTCAAGTGCT 
                 −615 
                 39 
               
               
                 CTSL1 
                 cathepsin L1 
                 GTCAGGCGAA 
                 43 
                 40 
               
               
                 CTSS 
                 cathepsin S 
                 CTCAAGTGAT 
                 −66 
                 41 
               
               
                 CTSZ 
                 cathepsin Z 
                 TTCAGGTGCC 
                 −166 
                 42 
               
               
                 DNASE2 
                 deoxyribonuclease II, lysosomal 
                 GCCAGGTGCC 
                 63 
                 43 
               
               
                 ENTPD4 
                 ectonucleoside triphosphate 
                 — 
                 — 
                   
               
               
                   
                 diphosphohydrolase 4 
                   
                   
                   
               
               
                 FUCA1 
                 alpha-L fucosidase 
                 — 
                 — 
                   
               
               
                 GAA 
                 alpha-glucosidase 
                 GTCACGTGAC 
                 20 
                 44 
               
               
                   
                   
                 GTCACGTGAC 
                 65 
                 45 
               
               
                 GALC 
                 galactosylceramidase 
                 GTCATGTGAC 
                 1 
                 46 
               
               
                 GALNS 
                 galactosamine (N-acetyl)-6-sulfate sulfatase 
                 
                   
                 
                 −147 
                 47 
               
               
                   
                   
                 GTCACGCGGC 
                 −128 
                 48 
               
               
                   
                   
                 GTCACGTGGC 
                 −5 
                 49 
               
               
                 GBA 
                 beta-glucosidase 
                 
                   GTCATGTGAC 
                 
                 −64 
                 50 
               
               
                   
                   
                 ATCACATGAC 
                 −44 
                 51 
               
               
                 GGH 
                 gamma-glutamyl hydrolase 
                 CTCACGCGAG 
                 −31 
                 52 
               
               
                 GLA 
                 alpha-galactosidase 
                 
                   CTCACGTAAG 
                 
                 −223 
                 53 
               
               
                   
                   
                 ATCACGTGAG 
                 −207 
                 54 
               
               
                   
                   
                 GTCATGTGAG 
                 −190 
                 55 
               
               
                   
                   
                 GTCACGTGAG 
                 −174 
                 56 
               
               
                 GLB1 
                 beta-galactosidase 
                 GTCACGCGGC 
                 −139 
                 57 
               
               
                   
                   
                 GTCAAGTGAC 
                 −3 
                 58 
               
               
                 GNS 
                 glucosamine (N-acetyl)-6-sulfatase 
                 
                   GTCACGTGAC 
                 
                 −42 
                 59 
               
               
                   
                   
                 CTCACGTGAT 
                 −2 
                 60 
               
               
                 GUSB 
                 beta-glucuronidase 
                 GTCACGCGAC 
                 −49 
                 61 
               
               
                 HEXA 
                 beta-hexosaminidase subunit alpha 
                 
                   GTCACGTGAT 
                 
                 −3 
                 62 
               
               
                   
                   
                 CTCACCTGAC 
                 33 
                 63 
               
               
                   
                   
                 CTCACGTGGC 
                 49 
                 64 
               
               
                 HEXB 
                 beta-hexosaminidase subunit beta 
                 GTCATCTGAC 
                 3 
                 65 
               
               
                 HGSNAT 
                 heparan-alpha-glucosaminide N- 
                 — 
                 — 
                   
               
               
                   
                 acetyltransferase 
                   
                   
                   
               
               
                 HPSE 
                 Heparanase 
                 GCCAGGTGAG 
                 84 
                 66 
               
               
                 HYAL1 
                 hyaluronoglucosaminidase 1 
                 — 
                 — 
                   
               
               
                 HYAL2 
                 hyaluronoglucosaminidase 2 
                 GTCACCTGGC 
                 −194 
                 67 
               
               
                 IDS 
                 Iduronate-2-sulfatase 
                 — 
                 — 
                   
               
               
                 IDUA 
                 alpha-L-iduronidase 
                 GTCACATGGG 
                 1 
                 68 
               
               
                 LGMN 
                 legumain 
                 — 
                 — 
                   
               
               
                 LIPA 
                 acid lipase 
                 ATCAGATGCC 
                 34 
                 69 
               
               
                 LYPLA3 
                 lysophospholipase 3 
                 GTCACCTGAG 
                 −431 
                 70 
               
               
                 MAN2B1 
                 alpha-mannosidase, class 2B, member 1 
                 CTCCCGTGAG 
                 −87 
                 71 
               
               
                 MAN2B2 
                 alpha-mannosidase, class 2B, member 2 
                 — 
                 — 
                   
               
               
                 MANBA 
                 beta-mannosidase 
                 CTCAGCTGAC 
                 −47 
                 72 
               
               
                 NAAA 
                 N-acylethanolamine acid amidase 
                 — 
                 — 
                   
               
               
                 NAGA 
                 alpha-N-acetylgalactosaminidase 
                 CCTTCGTGAG 
                 −23 
                 73 
               
               
                   
                   
                 CTCACTGGAA 
                 −5 
                 74 
               
               
                   
                   
                 ATCAGGTTAC 
                 18 
                 75 
               
               
                   
                   
                 GTCAGAAGCG 
                 37 
                 76 
               
               
                 NAGLU 
                 alpha-N-acetylglucosaminidase 
                 
                   
                 
                 178 
                 77 
               
               
                 NEU1 
                 sialidase 1 
                 
                   GTCACGCGCT 
                 
                 −116 
                 78 
               
               
                   
                   
                 GTCAGCTGAC 
                 69 
                 79 
               
               
                 NEU4 
                 sialidase 4 
                 GTCATTTGAG 
                 −336 
                 80 
               
               
                 P76 
                 mannose-6-phosphate protein p76 
                 GTCACGTGAC 
                 −12 
                 81 
               
               
                 PPT1 
                 palmitoyl-protein thioesterase 1 
                 GTCATGTGAC 
                 39 
                 82 
               
               
                 PPT2 
                 palmitoyl-protein thioesterase 2 
                 — 
                 — 
                   
               
               
                 RNASET2 
                 ribonuclease 6 
                 GGCAGGTGAG 
                 −41 
                 83 
               
               
                 SCPEP1 
                 serine carboxypeptidase 1 
                 
                   GTCACGTGAT 
                 
                 −26 
                 84 
               
               
                 SGSH 
                 N-sulfoglucosamine sulfohydrolase 
                 
                   
                 
                 −85 
                 85 
               
               
                 SIAE 
                 sialic acid acetylesterase 
                 — 
                 — 
                   
               
               
                 SMPD1 
                 sphingomyelin phosphodiesterase 
                 ATCAGCTGTC 
                 −14 
                 86 
               
               
                   
                   
                 GTCAGCCGAC 
                 51 
                 87 
               
               
                 TMEM55B 
                 transmembrane protein 55B 
                 
                   AACACGTGAC 
                 
                 −288 
                 88 
               
               
                   
                   
                 GTCACGTGCA 
                 −193 
                 89 
               
               
                   
                   
                 GTCATGTGAC 
                 −154 
                 90 
               
               
                   
                   
                 ATCACGTGCT 
                 −36 
                 91 
               
               
                 TPP1 
                 tripeptidyl peptidase I 
                 CTCATGTGAT 
                 −15 
                 92 
               
               
                   
                   
                 GTCACATGAC 
                 −3 
                 93 
               
               
                   
               
            
           
           
               
            
               
                 Signaling 
               
            
           
           
               
               
               
               
               
            
               
                 CREG1 
                 cellular repressor of E1A-stimulated genes 1 
                 — 
                 — 
                   
               
               
                 LITAF 
                 lipopolysaccharide-induced TNF factor 
                 — 
                 — 
                   
               
               
                 TMEM9 
                 transmembrane protein 9 
                 — 
                 — 
                   
               
               
                   
               
            
           
           
               
            
               
                 Other functions 
               
            
           
           
               
               
               
               
               
            
               
                 CD63 
                 CD63 molecule 
                 
                   GTCACATGAG 
                 
                 14 
                 94 
               
               
                 CD68 
                 CD68 molecule 
                 
                   TCAACTGCCC 
                 
                 −82 
                 95 
               
               
                   
                   
                 CCCATGTGAC 
                 −55 
                 96 
               
               
                 GM2A 
                 GM2 ganglioside activator 
                 — 
                 — 
                   
               
               
                 IFI30 
                 interferon, gamma-inducible protein 30 
                 CTCACGTGCC 
                 −174 
                 97 
               
               
                 LAMP1 
                 lysosomal-associated membrane protein 1 
                 
                   GTCACGTGGG 
                 
                 −196 
                 98 
               
               
                   
                   
                 GTCACGTGCC 
                 −180 
                 99 
               
               
                   
                   
                 GTCACGTGCC 
                 −163 
                 100 
               
               
                   
                   
                 GTCACGTGTC 
                 −146 
                 101 
               
               
                   
                   
                 ATCACGTGAC 
                 −32 
                 102 
               
               
                   
                   
                 CTCACGTGAC 
                 −5 
                 103 
               
               
                 LAMP2 
                 lysosomal-associated membrane protein 2 
                 — 
                 — 
                   
               
               
                 LAMP3 
                 lysosomal-associated membrane protein 3 
                 — 
                 — 
                   
               
               
                 MPO 
                 myeloperoxidase 
                 ATCAGGTGAG 
                 7 
                 104 
               
               
                 NCSTN 
                 nicastrin 
                 — 
                 — 
                   
               
               
                 NPC2 
                 Niemann-Pick disease, type C2 
                 CTCAGCTGTG 
                 −19 
                 105 
               
               
                   
                   
                 GTCGCCTGAC 
                 5 
                 106 
               
               
                   
                   
                 GTCTTGTGAC 
                 49 
                 107 
               
               
                 OSTM1 
                 osteopetrosis associated transmembrane 
                 — 
                 — 
                   
               
               
                   
                 protein 1 
                   
                   
                   
               
               
                 PCYOX1 
                 prenylcysteine oxidase 1 
                 — 
                 — 
                   
               
               
                 PSAP 
                 prosaposin 
                 
                   ATCAGCTGAC 
                 
                 5 
                 108 
               
               
                 TMEM74 
                 transmembrane protein 74 
                 — 
                 — 
                   
               
               
                   
               
            
           
           
               
            
               
                 Unknown function 
               
            
           
           
               
               
               
               
               
            
               
                 C2orf18 
                 chromosome 2 open reading frame 18 
                 GTCACGTGAC 
                 −33 
                 109 
               
               
                 C7orf28A 
                 chromosome 7 open reading frame 28A 
                 — 
                 — 
                   
               
               
                 EPDR1 
                 ependymin related protein 1 
                 — 
                 — 
                   
               
               
                 LAPTM5 
                 lysosomal-associated multispanning membrane 
                 — 
                 — 
                   
               
               
                   
                 protein 5 
                   
                   
                   
               
               
                 TMEM92 
                 transmembrane protein 92 
                 — 
                 — 
                   
               
               
                   
               
               
                 *Position refers to the transcription start site 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Gene Ontology (GO) analysis of CLEAR genes. 
               
            
           
           
               
               
               
               
            
               
                   
                 Gene 
                 Fold 
                   
               
               
                 GO Term 
                 Count 
                 enr. 
                 P value 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Cellular Compartment 
                   
                   
                   
               
               
                 GO: 0005764~lysosome 
                 23 
                 7.2 
                 1.03E−12 
               
               
                 GO: 0016471~vacuolar proton-transporting 
                 3 
                 37.3 
                 2.48E−03 
               
               
                 V-type ATPase complex 
               
               
                 GO: 0005768~endosome 
                 10 
                 3.2 
                 4.34E−03 
               
               
                 Biological Process 
               
               
                 GO: 0007040~lysosome organization and 
                 7 
                 24.3 
                 2.56E−07 
               
               
                 biogenesis 
               
               
                 GO: 0016192~vesicle-mediated transport 
                 20 
                 2.6 
                 2.73E−04 
               
               
                 GO: 0032940~secretion by cell 
                 13 
                 3 
                 1.43E−03 
               
               
                 GO: 0006643~membrane lipid metabolic 
                 11 
                 3.4 
                 1.56E−03 
               
               
                 process 
               
               
                 GO: 0046034~ATP metabolic process 
                 6 
                 6.7 
                 1.94E−03 
               
               
                 GO: 0006644~phospholipid metabolic 
                 9 
                 3.7 
                 3.12E−03 
               
               
                 process 
               
               
                 GO: 0045045~secretory pathway 
                 11 
                 3 
                 3.49E−03 
               
               
                 Molecular Function 
               
               
                 GO: 0016787~hydrolase activity 
                 58 
                 1.6 
                 2.39E−04 
               
               
                 GO: 0016298~lipase activity 
                 8 
                 5.3 
                 7.98E−04 
               
               
                 GO: 0016798~hydrolase activity, acting 
                 9 
                 4.2 
                 1.37E−03 
               
               
                 on glycosyl bonds 
               
               
                 GO: 0016805~dipeptidase activity 
                 3 
                 20.2 
                 9.07E−03 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Genes differentially expressed following TFEB transient overexpression. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Fold 
               
               
                 Gene Symbol 
                 Protein 
                 Process 
                 change 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 ATP6V0D2 
                 ATPase, H+ transporting, lysosomal 38kDa, V0 
                 Lysosomal 
                 2908 
               
               
                   
                 subunit d2 
                 acidification 
               
               
                 RASGRP3 
                 RAS guanyl releasing protein 3 (calcium and DAG- 
                 Signal transduction 
                 92.8 
               
               
                   
                 regulated) 
               
               
                 ZNF57 
                 zinc finger protein 57 
                 unknown 
                 60.7 
               
               
                 TRIM63 
                 tripartite motif-containing 63 
                 Protein degradation 
                 40.6 
               
               
                 SLC16A6 
                 solute carrier family 16, member 6 (monocarboxylic 
                 Drug disposition 
                 38.5 
               
               
                   
                 acid transporter 7) 
               
               
                 PER3 
                 period homolog 3 ( Drosophila ) 
                 Circadian rhythms 
                 37.7 
               
               
                 TM4SF19 
                 transmembrane 4 L six family member 19 
                 unknown 
                 23.6 
               
               
                 CPA2 
                 carboxypeptidase A2 (pancreatic) 
                 Protein degradation 
                 19.4 
               
               
                 C1orf54 
                 chromosome 1 open reading frame 54 
                 unknown 
                 17.2 
               
               
                 SULT1C2 
                 sulfotransferase family, cytosolic, 1C, member 2 
                 Sulfate conjugation 
                 13.9 
               
               
                 CTNS 
                 cystinosis, nephropathic 
                 Lysosomal carrier 
                 13.6 
               
               
                 NR1D1 
                 nuclear receptor subfamily 1, group D, member 1 
                 Circadian rhythms 
                 12.5 
               
               
                 UCA1 
                 urothelial cancer associated 1 
                 unknown 
                 12.3 
               
               
                 UPP1 
                 uridine phosphorylase 1 
                 Catabolism of 
                 11.1 
               
               
                   
                   
                 nucleotides 
               
               
                 SLC19A2 
                 solute carrier family 19 (thiamine transporter), 
                 Thiamin transport 
                 10.3 
               
               
                   
                 member 2 
               
               
                 GPR56 
                 G protein-coupled receptor 56 
                 Signal transduction 
                 9.8 
               
               
                 SLAMF7 
                 SLAM family member 7 
                 Immune response 
                 9.6 
               
               
                 PRKAG2 
                 protein kinase, AMP-activated, gamma 2 non- 
                 Energy metabolism 
                 8.6 
               
               
                   
                 catalytic subunit 
               
               
                 STS 
                 steroid sulfatase (microsomal), isozyme S 
                 Microsomal hydrolase 
                 8.4 
               
               
                 CCRL2 
                 similar to chemokine (C-C motif) receptor-like 2 
                 Immune response 
                 8.3 
               
               
                 MAP3K13 
                 mitogen-activated protein kinase kinase kinase 13 
                 Signal transduction 
                 7.8 
               
               
                 GIPR 
                 gastric inhibitory polypeptide receptor 
                 Insulin metabolism 
                 7.6 
               
               
                 SEMA3D 
                 sema domain, immunoglobulin domain (Ig), short 
                 Signal transduction 
                 7.4 
               
               
                   
                 basic domain, secreted, (semaphorin) 3D 
               
               
                 ANKRD1 
                 ankyrin repeat domain 1 (cardiac muscle) 
                 Signal transduction 
                 7.2 
               
               
                 BHLHB3 
                 basic helix-loop-helix domain containing, class B, 3 
                 Circadian rhythms 
                 6.8 
               
               
                 VASN 
                 vasorin 
                 Signal transduction 
                 6.5 
               
               
                 PTP4A3 
                 protein tyrosine phosphatase type IVA, member 3 
                 Cell growth 
                 6.4 
               
               
                 FNIP2 
                 folliculin interacting protein 2 
                 unknown 
                 6.3 
               
               
                 PLK3 
                 polo-like kinase 3 ( Drosophila ) 
                 Protein 
                 6.2 
               
               
                   
                   
                 phosphorylation 
               
               
                 CPA4 
                 carboxypeptidase A4 
                 Protein degradation 
                 6.1 
               
               
                 ST3GAL1 
                 ST3 beta-galactoside alpha-2,3-sialyltransferase 1 
                 Protein glycosylation 
                 6.1 
               
               
                 CSF1R 
                 colony stimulating factor 1 receptor, formerly 
                 Immune response 
                 5.8 
               
               
                   
                 McDonough feline sarcoma viral (v-fms) oncogene 
               
               
                   
                 homolog 
               
               
                 SUV39H1 
                 suppressor of variegation 3-9 homolog 1 
                 Chromatin 
                 5.7 
               
               
                   
                 ( Drosophila ) 
                 modification 
               
               
                 ZDHHC3 
                 zinc finger, DHHC-type containing 3 
                 unknown 
                 5.5 
               
               
                 IL6R 
                 interleukin 6 receptor 
                 Immune response 
                 5.5 
               
               
                 FAM27E3 
                 family with sequence similarity 27, member E3 
                 unknown 
                 5.5 
               
               
                 C1R 
                 complement component 1, r subcomponent 
                 Immune response 
                 5.5 
               
               
                 FAM102A 
                 family with sequence similarity 102, member A 
                 unknown 
                 5.4 
               
               
                 SECTM1 
                 secreted and transmembrane 1 
                 Immune response 
                 5.4 
               
               
                 FAM124A 
                 family with sequence similarity 124A 
                 unknown 
                 5.3 
               
               
                 RGS16 
                 regulator of G-protein signaling 16 
                 Signal transduction 
                 5.3 
               
               
                 RASD2 
                 RASD family, member 2 
                 Signal transduction 
                 5.3 
               
               
                 PLCXD1 
                 phosphatidylinositol-specific phospholipase C, X 
                 unknown 
                 5.2 
               
               
                   
                 domain containing 1 
               
               
                 AHNAK2 
                 AHNAK nucleoprotein 2 
                 unknown 
                 5.1 
               
               
                 ASAH1 
                 N-acylsphingosine amidohydrolase (acid 
                 Lysosomal hydrolase 
                 5.1 
               
               
                   
                 ceramidase) 1 
               
               
                 SLC26A11 
                 solute carrier family 26, member 11 
                 Sulfate transport 
                 5.1 
               
               
                 TMEM80 
                 transmembrane protein 80 
                 unknown 
                 5.1 
               
               
                 HEXA 
                 hexosaminidase A (alpha polypeptide) 
                 Lysosomal hydrolase 
                 5.1 
               
               
                 SLC26A9 
                 solute carrier family 26, member 9 
                 Sulfate transport 
                 5.0 
               
               
                 TGM5 
                 transglutaminase 5 
                 Epidermis 
                 5.0 
               
               
                   
                   
                 development 
               
               
                 MCOLN1 
                 mucolipin 1 
                 Lysosomal carrier 
                 5.0 
               
               
                 FLJ41484 
                 hypothetical LOC650669 
                 unknown 
                 5.0 
               
               
                 ALOXE3 
                 arachidonate lipoxygenase 3 
                 Inflammatory 
                 4.9 
               
               
                   
                   
                 response 
               
               
                 CHKA 
                 choline kinase alpha 
                 Lipid metabolism 
                 4.9 
               
               
                 C17orf80 
                 chromosome 17 open reading frame 80 
                 unknown 
                 4.7 
               
               
                 LIF 
                 leukemia inhibitory factor (cholinergic differentiation 
                 Immune response 
                 4.6 
               
               
                   
                 factor) 
               
               
                 ADFP 
                 adipose differentiation-related protein 
                 Adipocyte 
                 4.6 
               
               
                   
                   
                 differentiation 
               
               
                 SLC20A1 
                 solute carrier family 20 (phosphate transporter), 
                 Sulfate transport 
                 4.6 
               
               
                   
                 member 1 
               
               
                 DKFZp451A211 
                 DKFZp451A211 protein 
                 unknown 
                 4.6 
               
               
                 ATP6V0D1 
                 ATPase, H+ transporting, lysosomal 38kDa, V0 
                 Lysosomal 
                 4.5 
               
               
                   
                 subunit d1 
                 acidification 
               
               
                 DEXI 
                 dexamethasone-induced transcript 
                 unknown 
                 4.4 
               
               
                 FAM21B 
                 family with sequence similarity 21, member B 
                 unknown 
                 4.4 
               
               
                 PLEKHM1 
                 pleckstrin homology domain containing, family M 
                 Lysosomal 
                 4.4 
               
               
                   
                 (with RUN domain) member 1 
                 metabolism 
               
               
                 CEP72 
                 centrosomal protein 72kDa 
                 Centrosome 
                 4.3 
               
               
                   
                   
                 component 
               
               
                 DVL2 
                 dishevelled, dsh homolog 2 ( Drosophila ) 
                 Signal transduction 
                 4.3 
               
               
                 SNAI2 
                 snail homolog 2 ( Drosophila ) 
                 Development 
                 4.3 
               
               
                 LSS 
                 lanosterol synthase (2,3-oxidosqualene-lanosterol 
                 Cholesterol 
                 4.2 
               
               
                   
                 cyclase) 
                 metabolism 
               
               
                 HSPC159 
                 galectin-related protein 
                 unknown 
                 4.2 
               
               
                 RAET1E 
                 retinoic acid early transcript 1E 
                 Immune response 
                 4.2 
               
               
                 TCTEX1D2 
                 Tctex1 domain containing 2 
                 unknown 
                 4.2 
               
               
                 SERTAD2 
                 SERTA domain containing 2 
                 Cell proliferation 
                 4.2 
               
               
                 LOC201164 
                 similar to CG12314 gene product 
                 unknown 
                 4.1 
               
               
                 TMEFF1 
                 transmembrane protein with EGF-like and two 
                 Signal transduction 
                 4.1 
               
               
                   
                 follistatin-like domains 1 
               
               
                 VPS18 
                 vacuolar protein sorting 18 homolog ( S. cerevisiae ) 
                 Lysosomal trafficking 
                 4.1 
               
               
                 SYNJ2 
                 synaptojanin 2 
                 Metabolism 
                 4.1 
               
               
                 LOC100132929 
                 similar to hCG24378 
                 unknown 
                 4.1 
               
               
                 HLA-B 
                 major histocompatibility complex, class I, B 
                 Proteasome 
                 4.1 
               
               
                   
                   
                 degradation 
               
               
                 CRYAB 
                 crystallin, alpha B 
                 Apoptosis 
                 4.1 
               
               
                 CABLES1 
                 Cdk5 and Abl enzyme substrate 1 
                 Cell proliferation and 
                 4.0 
               
               
                   
                   
                 differentiation 
               
               
                 GRN 
                 granulin 
                 Inflammatory 
                 4.0 
               
               
                   
                   
                 response 
               
               
                 UVRAG 
                 UV radiation resistance associated gene 
                 Autophagy 
                 4.0 
               
               
                 CAMKK1 
                 calcium/calmodulin-dependent protein kinase kinase 
                 Immune response 
                 4.0 
               
               
                   
                 1, alpha 
               
               
                 SPINK1 
                 serine peptidase inhibitor, Kazal type 1 
                 Protease inhibitor 
                 4.0 
               
               
                 CLEC17A 
                 C-type lectin and transmembrane domain- 
                 unknown 
                 4.0 
               
               
                   
                 containing protein FLJ45910 
               
               
                 PPARGC1A 
                 peroxisome proliferator-activated receptor gamma, 
                 Energy metabolism 
                 3.9 
               
               
                   
                 coactivator 1 alpha 
               
               
                 TPP1 
                 tripeptidyl peptidase I 
                 Lysosomal hydrolase 
                 3.9 
               
               
                 SFXN3 
                 sideroflexin 3 
                 Mitochondrial carrier 
                 3.9 
               
               
                 HES1 
                 hairy and enhancer of split 1, ( Drosophila ) 
                 Development 
                 3.9 
               
               
                 EIF2C4 
                 eukaryotic translation initiation factor 2C, 4 
                 Gene silencing 
                 3.9 
               
               
                 VPS11 
                 vacuolar protein sorting 11 homolog ( S. cerevisiae ) 
                 Lysosomal trafficking 
                 3.9 
               
               
                 CTSF 
                 cathepsin F 
                 Lysosomal hydrolase 
                 3.9 
               
               
                 KCNAB2 
                 potassium voltage-gated channel, shaker-related 
                 unknown 
                 3.8 
               
               
                   
                 subfamily, beta member 2 
               
               
                 SETDB2 
                 SET domain, bifurcated 2 
                 Chromatin 
                 3.8 
               
               
                   
                   
                 modification 
               
               
                 PSG4 
                 pregnancy specific beta-1-glycoprotein 4 
                 Defense response 
                 3.8 
               
               
                 C12orf49 
                 chromosome 12 open reading frame 49 
                 unknown 
                 3.8 
               
               
                 BLVRB 
                 biliverdin reductase B (flavin reductase (NADPH)) 
                 Metabolism 
                 3.8 
               
               
                 APBB3 
                 amyloid beta (A4) precursor protein-binding, family 
                 APP metabolism 
                 3.8 
               
               
                   
                 B, member 3 
               
               
                 UCK1 
                 uridine-cytidine kinase 1 
                 Metabolism 
                 3.7 
               
               
                 HSPB8 
                 heat shock 22kDa protein 8 
                 Cell proliferation 
                 3.7 
               
               
                 LRRC8B 
                 leucine rich repeat containing 8 family, member B 
                 unknown 
                 3.7 
               
               
                 NHEDC2 
                 Na+/H+ exchanger domain containing 2 
                 Mitochondrial carrier 
                 3.7 
               
               
                 TIAF1 
                 TGFB1-induced anti-apoptotic factor 1 
                 Apoptosis 
                 3.7 
               
               
                 FAM21A 
                 family with sequence similarity 21, member A 
                 unknown 
                 3.7 
               
               
                 STOM 
                 stomatin 
                 unknown 
                 3.7 
               
               
                 HEY1 
                 hairy/enhancer-of-split related with YRPW motif 1 
                 Development 
                 3.6 
               
               
                 BHLHB2 
                 basic helix-loop-helix domain containing, class B, 2 
                 Development 
                 3.6 
               
               
                 NUP50 
                 nucleoporin 50kDa 
                 Nuclear pore 
                 3.6 
               
               
                   
                   
                 component 
               
               
                 WDR81 
                 WD repeat domain 81 
                 unknown 
                 3.6 
               
               
                 ACBD3 
                 acyl-Coenzyme A binding domain containing 3 
                 Golgi transport 
                 3.6 
               
               
                 FBXO32 
                 F-box protein 32 
                 Ubiquitylation 
                 3.6 
               
               
                 GEM 
                 GTP binding protein overexpressed in skeletal 
                 Signal transduction 
                 3.6 
               
               
                   
                 muscle 
               
               
                 UGDH 
                 UDP-glucose dehydrogenase 
                 Biosiynthesis of 
                 3.6 
               
               
                   
                   
                 GAGs 
               
               
                 HOXB9 
                 homeobox B9 
                 Cell proliferation and 
                 3.6 
               
               
                   
                   
                 differentiation 
               
               
                 LOC100128975 
                 similar to Zinc finger protein 626 
                 unknown 
                 3.6 
               
               
                 LYPD5 
                 LY6/PLAUR domain containing 5 
                 Signal transduction 
                 3.6 
               
               
                 CLC 
                 Charcot-Leyden crystal protein 
                 Lipid metabolism 
                 3.6 
               
               
                 CD22 
                 CD22 molecule 
                 Immune response 
                 3.5 
               
               
                 NIT1 
                 nitrilase 1 
                 Metabolism 
                 3.5 
               
               
                 SRRD 
                 SRR1 domain containing 
                 unknown 
                 3.5 
               
               
                 VEGFA 
                 vascular endothelial growth factor A 
                 Development 
                 3.5 
               
               
                 MMP12 
                 matrix metallopeptidase 12 (macrophage elastase) 
                 Protein degradation 
                 3.5 
               
               
                 LAMA1 
                 laminin, alpha 1 
                 Cell proliferation and 
                 3.5 
               
               
                   
                   
                 differentiation 
               
               
                 HMOX1 
                 heme oxygenase (decycling) 1 
                 Metabolism 
                 3.5 
               
               
                 SLC25A16 
                 solute carrier family 25 (mitochondrial carrier; 
                 Mitochondrial carrier 
                 3.5 
               
               
                   
                 Graves disease autoantigen), member 16 
               
               
                 KIAA1632 
                 KIAA1632 
                 unknown 
                 3.5 
               
               
                 HK2 
                 hexokinase 2 
                 Energy metabolism 
                 3.5 
               
               
                 KIFC3 
                 kinesin family member C3 
                 Golgi organization 
                 3.5 
               
               
                   
                   
                 and biogenesis 
               
               
                 CD68 
                 CD68 molecule 
                 Lysosomal 
                 3.5 
               
               
                   
                   
                 metabolism 
               
               
                 CHUK 
                 conserved helix-loop-helix ubiquitous kinase 
                 Immune response 
                 3.5 
               
               
                 RAB17 
                 Ras-related protein Rab-17 
                 Signal transduction 
                 3.5 
               
               
                 CXCL16 
                 chemokine (C-X-C motif) ligand 16 
                 Immune response 
                 3.5 
               
               
                 KIAA1737 
                 KIAA1737 
                 unknown 
                 3.4 
               
               
                 CRY1 
                 cryptochrome 1 (photolyase-like) 
                 Circadian rhythms 
                 3.4 
               
               
                 NDRG1 
                 N-myc downstream regulated gene 1 
                 Cell proliferation and 
                 3.4 
               
               
                   
                   
                 differentiation 
               
               
                 NEDD4L 
                 neural precursor cell expressed, developmentally 
                 Ubiquitylation 
                 3.4 
               
               
                   
                 down-regulated 4-like 
               
               
                 KCNN4 
                 potassium intermediate/small conductance calcium- 
                 Defense response 
                 3.4 
               
               
                   
                 activated channel, subfamily N, member 4 
               
               
                 NAGK 
                 N-acetylglucosamine kinase 
                 Metabolism 
                 3.4 
               
               
                 FAM54A 
                 family with sequence similarity 54, member A 
                 unknown 
                 3.4 
               
               
                 PSEN2 
                 presenilin 2 (Alzheimer disease 4) 
                 APP metabolism 
                 3.4 
               
               
                 PPIF 
                 peptidylprolyl isomerase F (cyclophilin F) 
                 Mitochondrial 
                 3.4 
               
               
                   
                   
                 metabolism 
               
               
                 LOC654433 
                 hypothetical LOC654433 
                 unknown 
                 3.4 
               
               
                 DCPS 
                 decapping enzyme, scavenger 
                 mRNA metabolism 
                 3.4 
               
               
                 PDXDC2 
                 pyridoxal-dependent decarboxylase domain 
                 Metabolism 
                 3.4 
               
               
                   
                 containing 2 
               
               
                 PLCD1 
                 phospholipase C, delta 1 
                 Phospholipid 
                 3.4 
               
               
                   
                   
                 metabolic process 
               
               
                 STK19 
                 serine/threonine kinase 19 
                 unknown 
                 3.4 
               
               
                 LCN8 
                 lipocalin 8 
                 Metabolism 
                 3.4 
               
               
                 DUSP10 
                 dual specificity phosphatase 10 
                 Signal transduction 
                 3.3 
               
               
                 SBNO2 
                 strawberry notch homolog 2 ( Drosophila ) 
                 Immune response 
                 3.3 
               
               
                 LY6K 
                 lymphocyte antigen 6 complex, locus K 
                 unknown 
                 3.3 
               
               
                 GSTO1 
                 glutathione S-transferase omega 1 
                 Metabolism 
                 3.3 
               
               
                 SLC29A1 
                 solute carrier family 29 (nucleoside transporters), 
                 Metabolism 
                 3.3 
               
               
                   
                 member 1 
               
               
                 CD300C 
                 CD300c molecule 
                 Immune response 
                 3.3 
               
               
                 AVPI1 
                 arginine vasopressin-induced 1 
                 unknown 
                 3.3 
               
               
                 DAB2 
                 disabled homolog 2, mitogen-responsive 
                 Lysosomal trafficking 
                 3.3 
               
               
                   
                 phosphoprotein ( Drosophila ) 
               
               
                 SLCO4A1 
                 solute carrier organic anion transporter family, 
                 unknown 
                 3.3 
               
               
                   
                 member 4A1 
               
               
                 GSR 
                 glutathione reductase 
                 Metabolism 
                 3.3 
               
               
                 UST 
                 uronyl-2-sulfotransferase 
                 Metabolism 
                 3.3 
               
               
                 PTTG1IP 
                 pituitary tumor-transforming 1 interacting protein 
                 Signal transduction 
                 3.3 
               
               
                 ICAM1 
                 intercellular adhesion molecule 1 (CD54), human 
                 Immune response 
                 3.3 
               
               
                   
                 rhinovirus receptor 
               
               
                 NUFIP1 
                 nuclear fragile X mental retardation protein 
                 Transcription 
                 3.3 
               
               
                   
                 interacting protein 1 
               
               
                 RAB3IL1 
                 RAB3A interacting protein (rabin3)-like 1 
                 Exocytosis 
                 3.3 
               
               
                 TEAD3 
                 TEA domain family member 3 
                 Pregnancy 
                 3.2 
               
               
                 GDF15 
                 growth differentiation factor 15 
                 Signal transduction 
                 3.2 
               
               
                 PIM1 
                 pim-1 oncogene 
                 Cell proliferation 
                 3.2 
               
               
                 TAF4B 
                 TAF4b RNA polymerase II, TATA box binding 
                 Transcription 
                 3.2 
               
               
                   
                 protein (TBP)-associated factor, 105kDa 
               
               
                 MFSD1 
                 major facilitator superfamily domain containing 1 
                 unknown 
                 3.2 
               
               
                 CTSB 
                 cathepsin B 
                 Lysosomal hydrolase 
                 3.2 
               
               
                 EPS15L1 
                 epidermal growth factor receptor pathway substrate 
                 Endocytosis 
                 3.2 
               
               
                   
                 15-like 1 
               
               
                 SPTBN1 
                 spectrin, beta, non-erythrocytic 1 
                 Cytoskeleton 
                 3.2 
               
               
                   
                   
                 component 
               
               
                 CSTB 
                 cystatin B (stefin B) 
                 Protease inhibitor 
                 3.2 
               
               
                 HKDC1 
                 hexokinase domain containing 1 
                 Energy metabolism 
                 3.2 
               
               
                 LPAR5 
                 lysophosphatidic acid receptor 5 
                 Signal transduction 
                 3.2 
               
               
                 CTSD 
                 cathepsin D 
                 Lysosomal hydrolase 
                 3.2 
               
               
                 LINS1 
                 lines homolog 1 ( Drosophila ) 
                 unknown 
                 3.2 
               
               
                 IGF2R 
                 insulin-like growth factor 2 receptor 
                 Lysosomal trafficking 
                 3.2 
               
               
                 RCSD1 
                 RCSD domain containing 1 
                 unknown 
                 3.2 
               
               
                 CSPG4 
                 chondroitin sulfate proteoglycan 4 
                 Signal transduction 
                 3.2 
               
               
                 VAC14 
                 Vac14 homolog ( S. cerevisiae ) 
                 Signal transduction 
                 3.2 
               
               
                 CHRM4 
                 cholinergic receptor, muscarinic 4 
                 Signal transduction 
                 3.2 
               
               
                 IL16 
                 interleukin 16 (lymphocyte chemoattractant factor) 
                 Immune response 
                 3.2 
               
               
                 SLC25A40 
                 solute carrier family 25, member 40 
                 Mitochondrial carrier 
                 3.2 
               
               
                 MTMR10 
                 myotubularin related protein 10 
                 Signal transduction 
                 3.2 
               
               
                 RLTPR 
                 RGD motif, leucine rich repeats, tropomodulin 
                 unknown 
                 3.2 
               
               
                   
                 domain and proline-rich containing 
               
               
                 SH3RF2 
                 SH3 domain containing ring finger 2 
                 Ubiquitylation 
                 3.1 
               
               
                 PFKFB3 
                 6-phosphofructo-2-kinase/fructose-2,6- 
                 Energy metabolism 
                 3.1 
               
               
                   
                 biphosphatase 3 
               
               
                 TMEM16B 
                 transmembrane protein 16B 
                 unknown 
                 3.1 
               
               
                 DENND2D 
                 DENN/MADD domain containing 2D 
                 unknown 
                 3.1 
               
               
                 ADM 
                 adrenomedullin 
                 Signal transduction 
                 3.1 
               
               
                 SLC25A25 
                 solute carrier family 25 (mitochondrial carrier; 
                 Mitochondrial carrier 
                 3.1 
               
               
                   
                 phosphate carrier), member 25 
               
               
                 SLC2A1 
                 solute carrier family 2 (facilitated glucose 
                 Glucose transporter 
                 3.1 
               
               
                   
                 transporter), member 1 
               
               
                 ATP6V0B 
                 ATPase, H+ transporting, lysosomal 21kDa, V0 
                 Lysosomal 
                 3.1 
               
               
                   
                 subunit b 
                 acidification 
               
               
                 TOM1 
                 target of myb1 (chicken) 
                 Endocytic trafficking 
                 3.1 
               
               
                 DDI2 
                 DDI1, DNA-damage inducible 1, homolog 2 ( S.   
                 Protein degradation 
                 3.1 
               
               
                   
                   cerevisiae ) 
               
               
                 SLC25A22 
                 solute carrier family 25 (mitochondrial carrier: 
                 Mitochondrial carrier 
                 3.1 
               
               
                   
                 glutamate), member 22 
               
               
                 NAPA 
                 N-ethylmaleimide-sensitive factor attachment 
                 ER-Golgi transport 
                 3.1 
               
               
                   
                 protein, alpha 
               
               
                 ESCO1 
                 Establishment of cohesion 1 homolog 1 ( S.   
                 DNA metabolism 
                 3.1 
               
               
                   
                   cerevisiae ) 
               
               
                 SETD4 
                 SET domain containing 4 
                 unknown 
                 3.1 
               
               
                 RRAGC 
                 Ras-related GTP binding C 
                 Autophagy 
                 3.1 
               
               
                 ATP6V1C1 
                 ATPase, H+ transporting, lysosomal 42kDa, V1 
                 Lysosomal 
                 3.1 
               
               
                   
                 subunit C1 
                 acidification 
               
               
                 PDP2 
                 pyruvate dehydrogenase phosphatase isoenzyme 2 
                 Mitochondrial 
                 3.1 
               
               
                   
                   
                 metabolism 
               
               
                 HSPBAP1 
                 HSPB (heat shock 27kDa) associated protein 1 
                 unknown 
                 3.1 
               
               
                 SUNC1 
                 Sad1 and UNC84 domain containing 1 
                 unknown 
                 3.1 
               
               
                 ITPKB 
                 inositol 1,4,5-trisphosphate 3-kinase B 
                 Signal transduction 
                 3.1 
               
               
                 RPP25 
                 ribonuclease P/MRP 25kDa subunit 
                 RNA metabolism 
                 3.0 
               
               
                 CEP250 
                 centrosomal protein 250kDa 
                 Centrosome 
                 3.0 
               
               
                   
                   
                 component 
               
               
                 TACC2 
                 transforming, acidic coiled-coil containing protein 2 
                 Centrosome 
                 3.0 
               
               
                   
                   
                 component 
               
               
                 FAM83G 
                 family with sequence similarity 83, member G 
                 unknown 
                 3.0 
               
               
                 ATP6V1B2 
                 ATPase, H+ transporting, lysosomal 56/58kDa, V1 
                 Lysosomal 
                 3.0 
               
               
                   
                 subunit B2 
                 acidification 
               
               
                 PDE2A 
                 phosphodiesterase 2A, cGMP-stimulated 
                 Signal transduction 
                 3.0 
               
               
                 NSMCE2 
                 non-SMC element 2, MMS21 homolog (( S.   
                 DNA metabolism 
                 3.0 
               
               
                   
                   cerevisiae ) 
               
               
                 WBP2 
                 WW domain binding protein 2 
                 Signal transduction 
                 3.0 
               
               
                 ATP6V0A1 
                 ATPase, H+ transporting, lysosomal V0 subunit a1 
                 Lysosomal 
                 3.0 
               
               
                   
                   
                 acidification 
               
               
                 LYPD3 
                 LY6/PLAUR domain containing 3 
                 unknown 
                 3.0 
               
               
                 CTSA 
                 cathepsin A 
                 Lysosomal hydrolase 
                 3.0 
               
               
                 MCCC1 
                 methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) 
                 Metabolism 
                 3.0 
               
               
                 ATP6V1H 
                 ATPase, H+ transporting, lysosomal 50/57kDa, V1 
                 Lysosomal 
                 3.0 
               
               
                   
                 subunit H 
                 acidification 
               
               
                 NR1D2 
                 nuclear receptor subfamily 1, group D, member 2 
                 Circadian rhythms 
                 3.0 
               
               
                 CLCN7 
                 chloride channel 7 
                 Lysosomal 
                 3.0 
               
               
                   
                   
                 acidification 
               
               
                 RYBP 
                 RING1 and YY1 binding protein 
                 Transcription 
                 3.0 
               
               
                 LOC643338 
                 hypothetical LOC643338 
                 unknown 
                 3.0 
               
               
                 CLCN6 
                 chloride channel 6 
                 Endosomal 
                 3.0 
               
               
                   
                   
                 component 
               
               
                 ZSCAN5A 
                 zinc finger and SCAN domain containing 5 
                 Transcription 
                 3.0 
               
               
                 FOLR1 
                 folate receptor 1 (adult) 
                 Metabolism 
                 3.0 
               
               
                 TRAF5 
                 TNF receptor-associated factor 5 
                 Apoptosis 
                 3.0 
               
               
                 HIF1A 
                 hypoxia-inducible factor 1, alpha subunit (basic 
                 Transcription 
                 3.0 
               
               
                   
                 helix-loop-helix transcription factor) 
               
               
                 PPP1R13B 
                 protein phosphatase 1, regulatory (inhibitor) subunit 
                 Apoptosis 
                 3.0 
               
               
                   
                 13B 
               
               
                 GBA 
                 glucosidase, beta; acid (includes 
                 Lysosomal hydrolase 
                 3.0 
               
               
                   
                 glucosylceramidase) 
               
               
                 ELOVL7 
                 ELOVL family member 7, elongation of long chain 
                 Metabolism 
                 3.0 
               
               
                   
                 fatty acids (yeast) 
               
               
                 TRPM7 
                 transient receptor potential cation channel, 
                 Calcium ion transport 
                 3.0 
               
               
                   
                 subfamily M, member 7 
               
               
                 GLA 
                 galactosidase, alpha 
                 Lysosomal hydrolase 
                 2.9 
               
               
                 MAFF 
                 v-maf musculoaponeurotic fibrosarcoma oncogene 
                 Inflammatory 
                 2.9 
               
               
                   
                 homolog F (avian) 
                 response 
               
               
                 UAP1L1 
                 UDP-N-acteylglucosamine pyrophosphorylase 1-like 
                 Metabolism 
                 2.9 
               
               
                   
                 1 
               
               
                 ZNF330 
                 zinc finger protein 330 
                 unknown 
                 2.9 
               
               
                 PIP4K2C 
                 phosphatidylinositol-5-phosphate 4-kinase, type II, 
                 unknown 
                 2.9 
               
               
                   
                 gamma 
               
               
                 FNBP1L 
                 formin binding protein 1-like 
                 Endocytosis 
                 2.9 
               
               
                 TNFAIP3 
                 tumor necrosis factor, alpha-induced protein 3 
                 Signal transduction 
                 2.9 
               
               
                 EPS8 
                 epidermal growth factor receptor pathway substrate 
                 Signal transduction 
                 2.9 
               
               
                   
                 8 
               
               
                 PTGES 
                 prostaglandin E synthase 
                 Signal transduction 
                 2.9 
               
               
                 SCPEP1 
                 serine carboxypeptidase 1 
                 Lysosomal hydrolase 
                 2.9 
               
               
                 GTF2H1 
                 general transcription factor IIH, polypeptide 1, 
                 Transcription 
                 2.9 
               
               
                   
                 62kDa 
               
               
                 INSIG1 
                 insulin induced gene 1 
                 Cholesterol 
                 2.9 
               
               
                   
                   
                 metabolism 
               
               
                 ARAP3 
                 ArfGAP with RhoGAP domain, ankyrin repeat and 
                 Cytoskeleton 
                 2.9 
               
               
                   
                 PH domain 3 
                 component 
               
               
                 TBC1D14 
                 TBC1 domain family, member 14 
                 Signal transduction 
                 2.9 
               
               
                 KCNK9 
                 potassium channel, subfamily K, member 9 
                 Potassium ion 
                 2.9 
               
               
                   
                   
                 transport 
               
               
                 TMCC3 
                 transmembrane and coiled-coil domain family 3 
                 unknown 
                 2.9 
               
               
                 AMPD3 
                 adenosine monophosphate deaminase (isoform E) 
                 Metabolism 
                 2.9 
               
               
                 NAGPA 
                 N-acetylglucosamine-1-phosphodiester alpha-N- 
                 Lysosomal trafficking 
                 2.9 
               
               
                   
                 acetylglucosaminidase 
               
               
                 GNS 
                 glucosamine (N-acetyl)-6-sulfatase (Sanfilippo 
                 Lysosomal hydrolase 
                 2.9 
               
               
                   
                 disease IIID) 
               
               
                 TMEM38B 
                 transmembrane protein 38B 
                 Potassium ion 
                 2.9 
               
               
                   
                   
                 transport 
               
               
                 SH3BP2 
                 SH3-domain binding protein 2 
                 Signal transduction 
                 2.9 
               
               
                 PMP22 
                 peripheral myelin protein 22 
                 Myelin component 
                 2.9 
               
               
                 TOB1 
                 transducer of ERBB2, 1 
                 Cell proliferation 
                 2.9 
               
               
                 GRAMD1B 
                 GRAM domain containing 1B 
                 unknown 
                 2.8 
               
               
                 ST3GAL4 
                 ST3 beta-galactoside alpha-2,3-sialyltransferase 4 
                 Golgi metabolism 
                 2.8 
               
               
                 NEU1 
                 sialidase 1 (lysosomal sialidase) 
                 Lysosomal hydrolase 
                 2.8 
               
               
                 GNPDA1 
                 glucosamine-6-phosphate deaminase 1 
                 Golgi metabolism 
                 2.8 
               
               
                 TMEM55B 
                 transmembrane protein 55B 
                 Lysosomal 
                 2.8 
               
               
                   
                   
                 component 
               
               
                 BRI3 
                 brain protein I3 
                 Cell differentiation 
                 2.8 
               
               
                 C5orf24 
                 hypothetical LOC134553 
                 unknown 
                 2.8 
               
               
                 CYB5R1 
                 cytochrome b5 reductase 1 
                 Metabolism 
                 2.8 
               
               
                 TMEM159 
                 transmembrane protein 159 
                 unknown 
                 2.8 
               
               
                 GGA2 
                 golgi associated, gamma adaptin ear containing, 
                 Golgi metabolism 
                 2.8 
               
               
                   
                 ARF binding protein 2 
               
               
                 RREB1 
                 ras responsive element binding protein 1 
                 Transcription 
                 2.8 
               
               
                 TRAPPC2L 
                 trafficking protein particle complex 2-like 
                 ER-Golgi transport 
                 2.8 
               
               
                 PCGF1 
                 polycomb group ring finger 1 
                 Transcription 
                 2.8 
               
               
                 STK17B 
                 serine/threonine kinase 17b 
                 Apoptosis 
                 2.8 
               
               
                 MPHOSPH10 
                 M-phase phosphoprotein 10 (U3 small nucleolar 
                 Ribosome biogenesis 
                 2.8 
               
               
                   
                 ribonucleoprotein) 
               
               
                 LOC440957 
                 hypothetical LOC440957 
                 unknown 
                 2.8 
               
               
                 CFB 
                 complement factor B 
                 Immune response 
                 2.8 
               
               
                 HTRA2 
                 HtrA serine peptidase 2 
                 Apoptosis 
                 2.8 
               
               
                 JPH1 
                 junctophilin 1 
                 unknown 
                 2.8 
               
               
                 SPG21 
                 spastic paraplegia 21 (autosomal recessive, Mast 
                 Signal transduction 
                 2.8 
               
               
                   
                 syndrome) 
               
               
                 CCDC43 
                 coiled-coil domain containing 43 
                 unknown 
                 2.8 
               
               
                 ZCCHC8 
                 zinc finger, CCHC domain containing 8 
                 RNA metabolism 
                 2.7 
               
               
                 RAD9A 
                 RAD9 homolog A ( S. pombe ) 
                 DNA metabolism 
                 2.7 
               
               
                 GPR175 
                 G protein-coupled receptor 175 
                 Signal transduction 
                 2.7 
               
               
                 SNX8 
                 sorting nexin 8 
                 Transport 
                 2.7 
               
               
                 WDTC1 
                 WD and tetratricopeptide repeats 1 
                 unknown 
                 2.7 
               
               
                 AXUD1 
                 AXIN1 up-regulated 1 
                 unknown 
                 2.7 
               
               
                 PEA15 
                 phosphoprotein enriched in astrocytes 15 
                 Apoptosis 
                 2.7 
               
               
                 CD63 
                 CD63 molecule 
                 Lysosomal 
                 2.7 
               
               
                   
                   
                 metabolism 
               
               
                 SPNS1 
                 spinster homolog 1 ( Drosophila ) 
                 unknown 
                 2.7 
               
               
                 LAMP1 
                 lysosomal-associated membrane protein 1 
                 Lysosomal 
                 2.7 
               
               
                   
                   
                 metabolism 
               
               
                 C7orf20 
                 chromosome 7 open reading frame 20 
                 unknown 
                 2.7 
               
               
                 LAMB3 
                 laminin, beta 3 
                 Cell adhesion 
                 2.7 
               
               
                 PSAP 
                 prosaposin (variant Gaucher disease and variant 
                 Lysosomal hydrolase 
                 2.7 
               
               
                   
                 metachromatic leukodystrophy) 
               
               
                 SNX27 
                 sorting nexin family member 27 
                 Endocytic trafficking 
                 2.7 
               
               
                 WIPI1 
                 WD repeat domain, phosphoinositide interacting 1 
                 Autophagy 
                 2.7 
               
               
                 ATP6V1E1 
                 ATPase, H+ transporting, lysosomal 31kDa, V1 
                 Lysosomal 
                 2.6 
               
               
                   
                 subunit E1 
                 acidification 
               
               
                 CDKN1A 
                 cyclin-dependent kinase inhibitor 1A (p21, Cip1) 
                 Cell cycle 
                 2.6 
               
               
                 C1orf85 
                 chromosome 1 open reading frame 85 
                 unknown 
                 2.6 
               
               
                 XRCC2 
                 X-ray repair complementing defective repair in 
                 DNA metabolism 
                 0.4 
               
               
                   
                 Chinese hamster cells 2 
               
               
                 DDX58 
                 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 
                 Immune response 
                 0.4 
               
               
                 BIRC3 
                 baculoviral IAP repeat-containing 3 
                 Apoptosis 
                 0.4 
               
               
                 DNAJB4 
                 DnaJ (Hsp40) homolog, subfamily B, member 4 
                 Stress response 
                 0.3 
               
               
                 LOC644714 
                 hypothetical protein LOC644714 
                 unknown 
                 0.3 
               
               
                 LCE2C 
                 late cornified envelope 2C 
                 Keratinization 
                 0.3 
               
               
                 LOC646993 
                 similar to high-mobility group box 3 
                 unknown 
                 0.2 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Gene Ontology (GO) terms enriched within the set of genes 
               
               
                 upregulated following TFEB transient overexpression. 
               
            
           
           
               
               
               
               
            
               
                   
                 Gene 
                 Fold 
                   
               
               
                 GO Term 
                 Count 
                 enr. 
                 P-value 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Cellular Compartment 
                   
                   
                   
               
               
                 GO: 0005764~lysosome 
                 17 
                 8.2 
                 2.50E−10 
               
               
                 GO: 0005765~lysosomal membrane 
                 8 
                 15.4 
                 7.65E−07 
               
               
                 GO: 0005768~endosome 
                 7 
                 4.9 
                 2.04E−04 
               
               
                 GO: 0048770~pigment granule 
                 10 
                 8.0 
                 2.33E−04 
               
               
                 GO: 0042470~melanosome 
                 7 
                 8.0 
                 2.33E−04 
               
               
                 Biological Process 
               
               
                 GO: 0015992~proton transport 
                 7 
                 7.0 
                 4.87E−04 
               
               
                 Molecular Function 
               
               
                 GO: 0022857~transmembrane transporter 
                 27 
                 2.5 
                 1.78E−05 
               
               
                 activity 
               
               
                 GO: 0003824~catalytic activity 
                 86 
                 1.4 
                 1.80E−04 
               
               
                 GO: 0019829~cation-transporting ATPase 
                 6 
                 10.3 
                 2.75E−04 
               
               
                 activity 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Sequences of oligos used in real-time qPCR analysis 
               
            
           
           
               
               
               
            
               
                 Gene name 
                 Forward primer 
                 Reverse primer 
               
               
                   
               
            
           
           
               
            
               
                 Expression analysis 
               
            
           
           
               
               
               
            
               
                 TFEB 
                 CCAGAAGCGAGAGCTCACAGAT 
                 TGTGATTGTCTTTCTTCTGCCG 
               
               
                   
                 Seq Id No. 114 
                 Seq Id No. 115 
               
               
                   
               
               
                 ARSA 
                 AGAGCTTTGCAGAGCGTTCAG 
                 ATACGCATGGTCTCAGGTCCA 
               
               
                   
                 Seq Id No. 116 
                 Seq Id No. 117 
               
               
                   
               
               
                 ARSB 
                 ATCAGTGAAGGAAGCCCATCC 
                 ACACGGTGAAGAGTCCACGAA 
               
               
                   
                 Seq Id No. 118 
                 Seq Id No. 119 
               
               
                   
               
               
                 ATP6V0E1 
                 CATTGTGATGAGCGTGTTCTGG 
                 AACTCCCCGGTTAGGACCCTTA 
               
               
                   
                 Seq Id No. 120 
                 Seq Id No. 121 
               
               
                   
               
               
                 ATP6V1H 
                 GGAAGTGTCAGATGATCCCCA 
                 CCGTTTGCCTCGTGGATAAT 
               
               
                   
                 Seq Id No. 122 
                 Seq Id No. 123 
               
               
                   
               
               
                 CLCN7 
                 TGATCTCCACGTTCACCCTGA 
                 TCTCCGAGTCAAACCTTCCGA 
               
               
                   
                 Seq Id No. 124 
                 Seq Id No. 125 
               
               
                   
               
               
                 CTSA 
                 CAGGCTTTGGTCTTCTCTCCA 
                 TCACGCATTCCAGGTCTTTG 
               
               
                   
                 Seq Id No. 126 
                 Seq Id No. 127 
               
               
                   
               
               
                 CTSB 
                 AGTGGAGAATGGCACACCCTA 
                 AAGAAGCCATTGTCACCCCA 
               
               
                   
                 Seq Id No. 128 
                 Seq Id No. 129 
               
               
                   
               
               
                 CTSD 
                 AACTGCTGGACATCGCTTGCT 
                 CATTCTTCACGTAGGTGCTGGA 
               
               
                   
                 Seq Id No. 130 
                 Seq Id No. 131 
               
               
                   
               
               
                 CTSF 
                 ACAGAGGAGGAGTTCCGCACTA 
                 GCTTGCTTCATCTTGTTGCCA 
               
               
                   
                 Seq Id No. 132 
                 Seq Id No. 133 
               
               
                   
               
               
                 GALNS 
                 TTGTCGGCAAGTGGCATCT 
                 CCAAACCACTCATCAAATCCG 
               
               
                   
                 Seq Id No. 134 
                 Seq Id No. 135 
               
               
                   
               
               
                 GBA 
                 TGGGTACCCGGATGATGTTA 
                 AGATGCTGCTGCTCTCAACA 
               
               
                   
                 Seq Id No. 136 
                 Seq Id No. 137 
               
               
                   
               
               
                 GLA 
                 AGCCAGATTCCTGCATCAGTG 
                 ATAACCTGCATCCTTCCAGCC 
               
               
                   
                 Seq Id No. 138 
                 Seq Id No. 139 
               
               
                   
               
               
                 GNS 
                 CCCATTTTGAGAGGTGCCAGT 
                 TGACGTTACGGCCTTCTCCTT 
               
               
                   
                 Seq Id No. 140 
                 Seq Id No. 141 
               
               
                   
               
               
                 HEXA 
                 CAACCAACACATTCTTCTCCA 
                 CGCTATCGTGACCTGCTTTT 
               
               
                   
                 Seq Id No. 142 
                 Seq Id No. 143 
               
               
                   
               
               
                 LAMP1 
                 ACGTTACAGCGTCCAGCTCAT 
                 TCTTTGGAGCTCGCATTGG 
               
               
                   
                 Seq Id No. 144 
                 Seq Id No. 145 
               
               
                   
               
               
                 MCOLN1 
                 TTGCTCTCTGCCAGCGGTACTA 
                 GCAGTCAGTAACCACCATCGGA 
               
               
                   
                 Seq Id No. 146 
                 Seq Id No. 147 
               
               
                   
               
               
                 NAGLU 
                 CAGAAGGAAGGAGCAGGAGT 
                 ATGTTCCCGAGGCTGTCAC 
               
               
                   
                 Seq Id No. 148 
                 Seq Id No. 149 
               
               
                   
               
               
                 NEU1 
                 CAGCACATCCAGAGTTCCGAGT 
                 TGTCTCTTTCCGCCATGAGGT 
               
               
                   
                 Seq Id No. 150 
                 Seq Id No. 151 
               
               
                   
               
               
                 PSAP 
                 GCCAACAGTGAAATCCCTTCC 
                 TCAGTGGCATTGTCCTTCAGC 
               
               
                   
                 Seq Id No. 152 
                 Seq Id No. 153 
               
               
                   
               
               
                 SCPEP1 
                 GATCTCCCCTGTTGATTCGGT 
                 AGCCCCTTATTTACGGCATTC 
               
               
                   
                 Seq Id No. 154 
                 Seq Id No. 155 
               
               
                   
               
               
                 SGSH 
                 TGACCGGCCTTTCTTCCTCTA 
                 GCTCTCTCCGTTGCCAAACTT 
               
               
                   
                 Seq Id No. 156 
                 Seq Id No. 157 
               
               
                   
               
               
                 TMEM55B 
                 GTTCGATGCCCCTGTAACTGTC 
                 CCCAGGTTGATGATTCTTTTGC 
               
               
                   
                 Seq Id No. 158 
                 Seq Id No. 159 
               
               
                   
               
               
                 TPP1 
                 GATCCCAGCTCTCCTCAATACG 
                 GCCATTTTTGCACCGTGTG 
               
               
                   
                 Seq Id No. 160 
                 Seq Id No. 161 
               
               
                   
               
               
                 GAPDH 
                 TGCACCACCAACTGCTTAGC 
                 GGCATGGACTGTGGTCATGAG 
               
               
                   
                 Seq Id No. 162 
                 Seq Id No. 163 
               
               
                   
               
               
                 HPRT1 
                 TGACACTGGCAAAACAATGCA 
                 GGTCCTTTTCACCAGCAAGCT 
               
               
                   
                 Seq Id No. 164 
                 Seq Id No. 165 
               
               
                   
               
               
                 ARPP-19 
                 AGGAAACGGTTGCAGAAAGG 
                 GTCTTGCGGAGTGGGAATGT 
               
               
                   
                 Seq Id No. 166 
                 Seq Id No. 167 
               
               
                   
               
               
                 C6orf211 
                 ACTCACCGTGGTTGTTGGTAGA 
                 TCGATTGGTGGACTCTGGATAA 
               
               
                   
                 Seq Id No. 168 
                 Seq Id No. 169 
               
               
                   
               
               
                 FBXO11 
                 GTGATGGACGAGGCCTTATTG 
                 TGCACATAAATCCCACCATGC 
               
               
                   
                 Seq Id No. 170 
                 Seq Id No. 171 
               
               
                   
               
               
                 HOXA9 
                 CCCCCATCGATCCCAATAA 
                 CCCTGGTGAGGTACATGTTGAA 
               
               
                   
                 Seq Id No. 172 
                 Seq Id No. 173 
               
               
                   
               
               
                 KPNA2 
                 TCCAAGCTACTCAAGCTGCCAG 
                 CCAGCCCGGATTATGTTGTCT 
               
               
                   
                 Seq Id No. 174 
                 Seq Id No. 175 
               
               
                   
               
               
                 MTDH 
                 CCTCTAAAACCCGTCCAAAACA 
                 TCGGTAGAAGTAGCAGGTGGAA 
               
               
                   
                 Seq Id No. 176 
                 Seq Id No. 177 
               
               
                   
               
               
                 MTX2 
                 TGCTGTTGACTGCAGAGCTGT 
                 CCTAGCATGAGTGATCTCCCCT 
               
               
                   
                 Seq Id No. 178 
                 Seq Id No. 179 
               
               
                   
               
               
                 ONECUT2 
                 ATGTGGAAGTGGCTTCAGGAG 
                 GGGACTTCTTCTGGGAATTGT 
               
               
                   
                 Seq Id No. 180 
                 Seq Id No. 181 
               
               
                   
               
               
                 STAT3 
                 GTCAGGTTGCTGGTCAAATTCC 
                 CAACGTCCCCAGAGTCTTTGTC 
               
               
                   
                 Seq Id No. 182 
                 Seq Id No. 183 
               
               
                   
               
            
           
           
               
            
               
                 ChIP assay 
               
            
           
           
               
               
               
            
               
                 ATP6V1H 
                 TCGGGAATCCAGTTGTCCG 
                 GCCGCACAGGTAGAAGGAA 
               
               
                   
                 Seq Id No. 184 
                 Seq Id No. 185 
               
               
                   
               
               
                 CLCN7 
                 CGTTGCAGGTCACATGGTC 
                 GGCTGCCCCCGTGTTTGT 
               
               
                   
                 Seq Id No. 186 
                 Seq Id No. 187 
               
               
                   
               
               
                 CTSA 
                 CCGTAGGGACCAAAGAAGG 
                 TGGAAGTCATGTGTACGAGTCA 
               
               
                   
                 Seq Id No. 188 
                 Seq Id No. 189 
               
               
                   
               
               
                 CTSD 
                 GCGTCATCCCGGCTATAAG 
                 TGAGGCTTCACCTGACGAG 
               
               
                   
                 Seq Id No. 190 
                 Seq Id No. 191 
               
               
                   
               
               
                 CTSF 
                 AAGCACGTGATAGAGGTCAGTG 
                 CCTGCGCGTTCTCTTGTT 
               
               
                   
                 Seq Id No. 192 
                 Seq Id No. 193 
               
               
                   
               
               
                 GBA 
                 TGTAACAGATGAGAGGAAGC 
                 ACACAGGAAGTGAGGCAATC 
               
               
                   
                 Seq Id No. 194 
                 Seq Id No. 195 
               
               
                   
               
               
                 GLA 
                 TAGCGAGACGGTAGACGAC 
                 ACCCGCCCTATTTCCATAC 
               
               
                   
                 Seq Id No. 196 
                 Seq Id No. 197 
               
               
                   
               
               
                 GNS 
                 ATCGCGCCTAGGGAGAAA 
                 AATAAAAAGCCGTGCCTTGA 
               
               
                   
                 Seq Id No. 198 
                 Seq Id No. 199 
               
               
                   
               
               
                 HEXA 
                 GTGAAAGGGCAGGGTGTG 
                 CGAATCACGTGACCAGAGG 
               
               
                   
                 Seq Id No. 200 
                 Seq Id No. 201 
               
               
                   
               
               
                 NEU1 
                 CTTCGAGATGCTGCGTGAT 
                 TCCCGGACTCTAATTGGTCTT 
               
               
                   
                 Seq Id No. 202 
                 Seq Id No. 203 
               
               
                   
               
               
                 MCOLN1 
                 AGGGGCTCTGGGCTACC 
                 GCCCGCCGCTGTCACTG 
               
               
                   
                 Seq Id No. 204 
                 Seq Id No. 205 
               
               
                   
               
               
                 PSAP 
                 TTGGGGCAGGGCAGATTTAT 
                 CAGGAGGAAGAGGGCGTACA 
               
               
                   
                 Seq Id No. 206 
                 Seq Id No. 207 
               
               
                   
               
               
                 SCPEP1 
                 CCGTCCGCCTCCGTCAC 
                 GGCAGCAGCAGCAACCAC 
               
               
                   
                 Seq Id No. 208 
                 Seq Id No. 209 
               
               
                   
               
               
                 TMEM55B 
                 TCCCAATAGCTTGCAGAACC 
                 TGTCACATGACCTGCCAGA 
               
               
                   
                 Seq Id No. 210 
                 Seq Id No. 211 
               
               
                   
               
               
                 TPP1 
                 AGAGGGGTAGTGGTGGTGGAA 
                 CAGGCTTGGAGTCCCATTCT 
               
               
                   
                 Seq Id No. 212 
                 Seq Id No. 213 
               
               
                   
               
               
                 PSAP(int) 
                 CACAGGCACCCACACAAA 
                 ACGCAGCTGGTCAGCAAT 
               
               
                   
                 Seq Id No. 214 
                 Seq Id No. 215 
               
               
                   
               
               
                 GNS(int) 
                 ACAAGGAATGGAAACAAAGATACC 
                 GATGCGTCTTCCCTTTTTCC 
               
               
                   
                 Seq Id No. 216 
                 Seq Id No. 217 
               
               
                   
               
               
                 ACTB 
                 ATGCAGCGATCAGTGGCGT 
                 TCCAGCTTCTTGTCACCACCTC 
               
               
                   
                 Seq Id No. 218 
                 Seq Id No. 219 
               
               
                   
               
               
                 APRT 
                 GCCTTGACTCGCACTTTTGT 
                 TAGGCGCCATCGATTTTAAG 
               
               
                   
                 Seq Id No. 220 
                 Seq Id No. 221 
               
               
                   
               
               
                 HPRT 
                 GCCACAGGTAGTGCAAGGTCTT 
                 TTCATGGCGGCCGTAAAC 
               
               
                   
                 Seq Id No. 222 
                 Seq Id No. 223 
               
               
                   
               
               
                 TXNDC4 
                 CCTCTCACACCCTCACTTCC 
                 TCTAGATGACGGACGACGTG 
               
               
                   
                 Seq Id No. 224 
                 Seq Id No. 225 
               
               
                   
               
               
                 WIF1 
                 GCCAGCTTTGCCAGTCTTAC 
                 CGAGTCGCGCAAGAAGAT 
               
               
                   
                 Seq Id No. 226 
                 Seq Id No. 227 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Positional weight matrix (PWM) describing CLEAR sequences 
               
               
                 (CLEAR PWM). 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 A 
                 15 
                 1 
                 0 
                 92 
                 6 
                 2 
                 0 
                 0 
                 79 
                 5 
               
               
                 C 
                 19 
                 9 
                 94 
                 0 
                 74 
                 12 
                 2 
                 0 
                 5 
                 55 
               
               
                 G 
                 55 
                 5 
                 0 
                 2 
                 12 
                 74 
                 0 
                 94 
                 9 
                 19 
               
               
                 T 
                 5 
                 79 
                 0 
                 0 
                 2 
                 6 
                 92 
                 0 
                 1 
                 15 
               
               
                   
               
            
           
         
       
     
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