Overall, our data demonstrate oligoTRAFTACs as a generalizable platform towards difficult-to-drug TFs and their degradability via the proteasomal pathway.Dysregulated transcription factors (TFs) that rewire gene expression circuitry are frequently identified as key players in disease. Although several TFs have been drugged with small molecules, the majority of oncogenic TFs are not currently pharmaceutically tractable due to their paucity of ligandable pockets. The first generation of transcription factor targeting chimeras (TRAFTACs) was developed to target TFs for proteasomal degradation by exploiting their DNA binding ability. In the current study, we have developed the second generation TRAFTACs (“oligoTRAFTACs”) composed of a TF-binding oligonucleotide and an E3 ligase-recruiting ligand. Herein, we demonstrate the development of oligoTRAFTACs to induce the degradation of two oncogenic TFs, c-Myc and brachyury. In addition, we show that brachyury can be successfully degraded by oligoTRAFTACs in chordoma cell lines. Furthermore, zebrafish experiments demonstrate  Targeted transcription factor degradation using oligonucleotide-based transcription factor targeting chimeras (TRAFTACs). Although PROTACs have the potential to induce the degradation of numerous proteins, the identification of small molecule recruiting ligands for several classes of proteins is still challenging and time-consuming.5 Therefore, the development of alternative, proximity-inducing strategies would help to target disease-causing proteins, such as transcription factors.Protein degradation is an integral component of cellular homeostasis which supports the healthy environment within the cell. The ubiquitin-proteasomal pathway is one of the key mechanisms by which unwanted or defective proteins are degraded.6,7 Many diseases such as neurological disorders, autoimmunity, developmental syndromes, and many cancers result from abnormalities in the TF-controlled gene-regulatory circuitry within the diseased cell.8–10 For instance, the TF c-Myc, the most frequently amplified oncogene, has been extensively studied and established as a direct mediator of tumorigenesis in numerous cancers.11 Although indirect approaches such as bromodomain protein inhibitors and PROTACs have been explored to control c-Myc levels, direct inhibition or degradation of c-Myc has been an as yet unrealized goal.12,13 In contrast to c-Myc, T-box transcription factor (“brachyury”) is expressed only in a restricted set of cancer types and minimally expressed in normal cells.14 Brachyury is a master developmental TF that plays a pivotal role during early embryonic development in vertebrates.15 Brachyury expression is limited only to embryonic developmental stages and, in general, expression levels are highly downregulated in adult tissues.16 In addition to its regulatory functions during development, brachyury expression in remnant notochord cells in adults has been shown to be a key oncogenic driver in the rare bone cancer, chordoma.17 Chromosomal aberrations such as chromosome 6 gains and partial polysomy have been identified as potential molecular mechanisms in brachyury-dependent chordoma tumors.18–21 Other TFs such as NF-kB, STAT3/5, the androgen receptor (AR) and the estrogen receptor (ER) are other known oncogenic drivers that also rewire transcriptional circuitry in various cancer types.22,23Transcription factors (TFs) control gene expression by binding to specific DNA elements within a given gene promoter or distal enhancer regions.24–26 Recently, we developed a generalizable TF degradation platform by taking advantage of the DNA binding ability of TFs.27,28 In this approach, we generated a chimeric DNA:CRISPR RNA molecule – a TRAnscription Factor TArgeting Chimera (TRAFTAC) – that binds a dCas9-HaloTag7 fusion adaptor, which in turn recruits an E3 ligase into proximity with the transcription factor of interest (TOI). Using TRAFTACs, we demonstrated for the first time that TF response elements could be used in degrader design to recruit and induce the degradation of TOI, while highlighting their generalizability as well. While that strategy could be readily used as a chemical biology tool to study the biology of understudied TFs, therapeutic application of the technology is limited.Although several TFs, such as STAT3, AR and ER have been successfully degraded by PROTACs, a plethora of disease-relevant yet traditionally “undruggable” TFs remain unaddressed.in vivo settings without the requirement for genetic modification  in the system.In this current report, we describe the development of second-generation TRAFTACs – “oligoTRAFTACs” that are based solely on an oligonucleotide sequence and E3 ligase-recruiting small molecule ligand to recruit TOI and the cellular ubiquitination machinery, respectively . A short29 Even though c-Myc has been targeted by indirect approaches, development of direct-targeting methods is hindered by the paucity of ligandable pockets and their highly disordered structure.30 Since c-Myc binds to specific DNA sequences with a conserved E-box sequence (CACGTG), we sought to incorporate a c-Myc binding consensus DNA-sequence into the oligoTRAFTAC (OT) design.31 We adapted a Myc binding consensus sequence (5′ CACGTGGTTGCCACGTG 3′) taken from one of its target gene promoters.32 Additionally, we included a flanking sequence at both 3′ and 5′ end of the c-Myc-targeting oligonucleotide sequence to facilitate successful double strand formation of the recognition sequence while providing a flexibility for oligoTRAFTACs. First, we synthesized a c-Myc-targeting oligonucleotide sequence with an orthogonal alkyne handle on either side as a reactive moiety to append an azide-containing VHL ligand . As the data indicate, the induction of OT7-mediated c-Myc degradation inhibited cell proliferation compared to scrambled control-transfected cells  as the brachyury-recruiting element in our new oligoTRAFTACs design.36 We synthesized oligonucleotides with flanking bases and terminal alkyne moieties at either end (3′ and 5′) of the oligonucleotide  is a DNA-interacting TF that regulates gene expression during early embryonic development in vertebrates. Brachyury is crucial for early mesoderm formation and plays a key role in notochord development where, as a homodimer, it recognizes and binds to a consensus palindromic DNA sequence.To generate brachyury-targeting oligoTRAFTACs, we synthesized two azido-VHL ligands with a long (5 PEG units) and a short (2 PEG units) linker  did not induce significant degradation of brachyury-GFP  for brachyury degradation in cells. Consistently, OT3 induced a robust brachyury-GFP degradation, whereas neither OT5 nor OT6 induced significant brachyury degradation . We next37 The addition of extra non-bridging sulfur atoms in the inter-nucleotide phosphate group has been shown to have both increased stability against nucleases and improved cell permeability.37 A brachyury-targeting oligoTRAFTAC, OT17, was synthesized by incorporating PS bonds throughout the oligo sequence. Increasing OT17 concentrations were transfected into chordoma cells (UM-Chor1) for 24 h following which the cells were lysed and probed for brachyury levels – western blotting showed that OT17 induced significant degradation of endogenous brachyury even at 15 nM  based on an oligonucleotide and E3 ligase recruiting intermediate protein, dCas9-Halotag7, by exploiting its ability to recruit both TOI and E3 ligase in the presence of TRAFTAC and a concurrently administered HaloPROTAC.27 Following the first generation TRAFTAC study, a couple of other studies were published describing the use of oligonucleotides for TF degradation.40,41 In addition to TF degradation, a separate study was reported showing the applicability of the oligonucleotide-based PROTAC platform for RNA-binding proteins as well.42The majority of transcription factors, key mediators of gene expression, are considered undruggable. In this study, we developed a method for oligonucleotide-dependent TF recruitment and degradation by the proteasomal pathway. We have coupled TF-binding short DNA sequences from target gene promoters with VHL ligands to create bifunctional molecules for the targeted degradation of those same TFs. Since the binding sequences already have been identified for many TFs, and since synthetic routes for oligonucleotide synthesis are well established and economical, these oligonucleotide sequences can be rapidly employed in oligoTRAFTAC design to use as a versatile tool for both basic discovery biology and therapy development.via the proteasomal pathway. Although washout experiments indicated that oligoTRAFTACs are not as kinetically efficient as conventional PROTACs, the transient binding nature of oligoTRAFTACs to both TF and VHL proteins suggests the probability of a similar catalytic degradation mechanism. Moreover, to our knowledge the current study provides the first evidence of the degradability of untagged, endogenous brachyury in multiple chordoma cell lines. In the first attempt to test oligoTRAFTAC in vivo activity, OT3 (containing a phosphodiester backbone) could not induce the intended defective tail phenotype in zebrafish. It is noteworthy that first generation TRAFTACs, where oligonucleotides are partly protected by the ribonuclearcomplex, induced defective tails in zebrafish. Therefore, a potential reason for the failure of OT3 to induce the intended phenotype within the embryos may be their high sensitivity and exposure of oligoTRAFTACs to embryonic nucleases present at the early stages of development. Therefore, phosphodiester oligoTRAFTACs might not endure in vivo at the intracellular concentrations needed to achieve a significant brachyury degradation and the intended phenotype. Previous studies suggested that synthesis of oligonucleotide-based entities with a phosphorothioate backbone largely benefitted from nuclease resistance. Therefore, the microinjection of phosphorothioate backbone-containing OT17 resulted in a significantly high occurrence rate of the defective tail phenotype compared to the mock or scrambled oligoTRAFTAC (OT20), confirming the increased stability and resistance of OT17 to nucleases.Although first generation TRAFTACs are widely applicable as chemical biology tools for discovery biology, therapeutic applicability needed further improvements. In this current study, we discuss the development of second generation TRAFTACs by replacing the intermediatory E3 ligase recruiting dCas9-Halotag7 in the first generation of TRAFTACs with a VHL-recruiting small molecule ligand. OligoTRAFTACs targeting c-Myc TF displayed a robust degradation. Addition of VHL ligand to either side of the oligonucleotide did not significantly alter its ability to induce c-Myc degradation. This can be partially attributed to the flexibility of oligoTRAFTACs provided by the extra flanking nucleotides between the VHL ligand and TF-recruiting oligonucleotide. Other oligoTRAFTACs used in this study induced the degradation of ectopically expressed brachyury-GFP and endogenous brachyury 43 Since TFs, such as TCF3, also recognizes a similar E-box consensus sequence as c-Myc, the evaluation of TCF3 degradation indicated a sequence-dependent off-target activity of OT7  protein levels in response to brachyury-targeting OT17. We observed a consistent brachyury degradation in OT17-treated cells. However, since p65 does not recognize brachyury-binding DNA sequence, OT17 did not induce p65 degradation. Therefore, the data suggest a sequence-dependent brachyury degradation by OT17 (Fig. S2G, ESIin vivo stability and demonstrated oligoTRAFTAC activity in zebrafish. However, similar to other oligonucleotide-based drugs, the therapeutic use of oligoTRAFTACs will be significantly challenged by their size and negatively charged nucleotides.44 Therefore, optimization of nanocarriers should be implemented to increase the cellular uptake and endosomal escape of oligoTRAFTACs. However, due to the faster degradation profile and catalytic nature, oligoTRAFTACs may exhibit improved therapeutic benefits than other oligonucleotide-based strategies such as RNAi. Overall, oligoTRAFTACs are programmable heterobifunctional molecules comprised of a TF-binding oligonucleotide sequence and a VHL binding ligand, which induce TF degradation in cells and displayed robust in vivo activity. Due to the simple and modular nature of their structure, oligoTRAFTACs can be rapidly designed for many non-ligandable DNA-binding TFs both for use as a chemical biology tool as well as a potential therapeutic strategy.It has been shown that multiple TFs recognize and bind to similar DNA sequences.C. M. C is founder, shareholder, and consultant to Arvinas, Inc. and Halda, LLC, which support research in his laboratory.CB-003-D2CB00138A-s001


Eukaryotic and archaeal RNA polymerase II (POL II) machinery is highly conserved, regardless of the extreme changes in promoter sequences in different organisms. The goal of our work is to find the cause of this conservatism. The representative sets of aligned promoter sequences of fifteen organisms belonging to different evolutional stages were studied. Their textual profiles, as well as profiles of the indexes that characterize the secondary structure and the mechanical and physicochemical properties, were analyzed. The evolutionarily stable, extremely heterogeneous special secondary structure of POL II core promoters was revealed, which includes two singular regions—hexanucleotide “INR” around TSS and octanucleotide “TATA element” of about −28 bp upstream. Such structures may have developed at some stage of evolution. It turned out to be so well matched for the pre-initiation complex formation and the subsequent initiation of transcription for POL II machinery that in the course of evolution there were selected only those nucleotide sequences that were able to reproduce these structural properties. The individual features of specific sequences representing the singular region of the promoter of each gene can affect the kinetics of DNA-protein complex formation and facilitate strand separation in double-stranded DNA at the TSS position. The heterogeneity of the three-dimensional structure of the double-stranded DNA plays an important role in the regulation of genetic processes. This heterogeneity is modulated by the nucleotide sequence. Proteins may recognize the shape of DNA (“indirect readout”) or the unique chemical signatures of the DNA bases (“direct readout”) . As a ruRNA polymerase II (Pol II) in eukaryotes is responsible for the transcription of messenger RNA and some non-protein-coding small nuclear RNAs. Pol II core promoters are fragments of genomic DNA, about 100 bp long, surrounding the transcription start site (TSS). Transcription initiation occurs when TATA-binding protein (TBP) binds to the eight base-pair TATA elements of Pol II core promoter, coordinating accretion of class II initiation factors and Pol II into a functional preinitiation complex (PIC). This process is a slow stage of transcription; it leads to the formation of a long-lived protein-DNA complex .The mechanical, thermodynamic, and structural properties of Pol II promoter regions have long attracted the attention of researchers ,6,7,8,9.The nucleotide sequences of the core promoters are usually represented by the DNA coding strand . The TSS position is taken as coordinates −1, +1 (there is no nucleotide with zero coordinates). In all organisms, positions −2, +4 are occupied by the initiator element (INR). At this region, the complementary strands of the double helix diverge, and Pol II recognizes the template strand. TATA element in the promoters of most organisms is located at a distance of about −28 bp from the TSS.H. sapiens, M. musculus, D. melanogaster, D. rerio, C. elegans, A. thaliana, S. cerevisiae, S. pombe), were constructed. The analysis of these profiles allowed us to reveal the common scheme of the animal and plant core promoter architecture. The promoters of the unicellular fungus S. pombe were found to correspond to the same structural scheme, but the structure of the core promoter of another unicellular fungus, S. cerevisiae, turned out to be different , taken from DiProDB  or from the work   around TSS and the octanucleotide (“TATA element”) upstream. The strength of each promoter, to some extent, depends on the nucleotide sequences forming its singular regions. In our opinion, all of the data presented here correspond to the bottom-up approach conception of evolution , startin


Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe3O4) that under microoxic to anoxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of > 30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear.One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth’s magnetic field. In the alphaproteobacterium mam and mms gene clusters, specifically controlling magnetosome biosynthesis, were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ70 dependent promoters in other Alphaproteobacteria.Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the > 4300 genes was significantly enhanced during magnetosome formation. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource (GEO Series accession number GSE197098).Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism The online version contains supplementary material available at 10.1186/s12864-022-08913-x. Magnetosomes, which are formed by magnetotactic bacteria (MTB) as sensors for geomagnetic navigation in their aquatic habitat, represent an example for one of the most complex organelles found in prokaryotic cells –3. TheirMagnetospirillum gryphiswaldense and related MTB, magnetosomes consist of a monocrystalline core of magnetite (Fe3O4) bounded by a dedicated proteo-lipid membrane /23% [0% dO2]), suggesting that most regulators remain unidentified. Taken together, it seems that expression regulation under the tested conditions in M. gryphiswaldense is based to a significant degree on cis-regulatory elements like riboswitches as sensors for environmental cues, as suggested by the 5′-UTR length.Within the 5′-UTR we found a conserved aaGGAG motif serving as an RBS with in average 8 nt as spacer to the corresponding start codon in both oxic and anoxic datasets. This architecture resembles the optimized RBS (AGGAG followed by an 8 nt spacer) for expression in entified , 73. Note genome . New eleThe transcriptome under conditions of highest magnetosome biosynthesis revealed an interplay between generic metabolic processes, such as anaerobic respiration, as well as increased biosynthesis and maturation of cytochrome c proteins and hemerythrins; some of these pathways have already been implicated in magnetosome biosynthesis. In addition, in highly magnetic cells, the transcriptional complexity is reduced compared to oxic, nonmagnetic cells. Furthermore, magnetosome genes mostly exhibit a constitutively high expression, which is only weakly affected by growth conditions.Our study sheds light on the genome-wide complex transcriptional organization during magnetosome biosynthesis as a model for the formation of an intricate prokaryotic organelle with relevance for the research at system level. Furthermore, the insights can be used for engineering promoters as well as entire cellular pathways, thereby enabling rational design of synthetic magnetosome operons for targeted magnetosome production.Magnetospirillum gryphiswaldense strain MSR-1 (DSM 6361) .Additional file 4: Figure S2. Distribution of classified transcription start sites (TSS) under anoxic (0% dO2) and oxic (95% dO2) conditions in the whole genome .Additional file 5: Table S1. Mapping statistics of the different RNA-seq datasets including the three library preparation techniques, namely Cappable-seq, whole transcriptomic shotgun sequencing and term-seq sequencing.Additional file 6: Table S2. Pearson correlation coefficients R2 of the biological replicates.


It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P.epi-CCDC163P–MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2.We unraveled the methylome architecture of the PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters.The PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.The antisense readthrough transcription of the mutated The online version contains supplementary material available at 10.1186/s13148-022-01271-1. For the control group, we used in silico data generated using the Infinium Human Methylation 450 k BeadChip array from the MARTHA cohort, which included 350 unrelated European-ancestry ambulatory subjects recruited in Marseille (France) between January 1994 and October 2005 [We performed a case–control EWAS to look for significant epigenomic signatures associated with an epi-escribed , 3. All t test with Bonferroni correction. Output data included the mean β values in each subgroup, the difference of β values, the nominal P-value, and the Bonferroni corrected P-value. We used the smoothed P-value transformation by converting nominal P-values obtained from the t test to smoothed P-values using a window radius of 3, as previously reported [We carried out bisulfite conversion of 600 ng of DNA extracted from whole blood using the EZ DNA Methylation kit . The genome-wide profiling of DNA methylome was determined using the Infinium Human Methylation 450 k BeadChip array  of the Infinium MethylationEPIC BeadChip array , according to the manufacturer’s instructions. The arrays were scanned on an Illumina iScan® system, and raw methylation data were extracted using Illumina’s Genome Studio methylation module. For each CpG probe, the methylation level was described as a β value, ranging between 0 (fully unmethylated CpG probe) and 1 (fully methylated CpG probe). Background correction and normalization were implemented using the SWAN method (R Package Minfi) [reported , 3, 6. AChIP-Seq was performed on patient’s fibroblasts and control fibroblast lines without inherited metabolic defects in the cobalamin pathway, as previously reported . AccordicblC cases, 3 cblC cases with the c.270_271insA mutation, 3 cblG cases (another inherited defect of vitamin B12 intracellular metabolism due to mutations in methionine synthase (MTR) gene) and 4 control fibroblasts using RiboMinus™  [cblC samples compiled 150-nucleotide-long paired-end reads. The control group originated from a previous study [TESK2 to PRDX1, we could not rely on reference-based transcriptome reconstruction methods, which would not efficiently report on the real isoform population and abundances in this region. We extracted the high-quality reads aligning inside the genomic interval chr1:45,443,800–45,543,000 (TESK2 to PRDX1) and performed de novo transcriptome assembly with Trinity v2.6.5 [cblC samples, 4 controls were suitable for the analysis and were used as controls against the composite epi-cblC cases. All samples presented with enough coverage in high-quality reads in the region of interest  to achieve transcript reconstruction, providing enough read depth for unbiased estimation of their expression levels. Reconstructed transcripts were then aligned with blastn  against a cDNA database obtained with BioMart (https://www.ensembl.org/biomart/) and comprising all known isoforms of the four genes TESK2, CCDC163, MMACHC, and PRDX1. Criteria for a successful transcript assignment to a known gene product of interest were as follows: i) biotype = protein-coding; ii) select the highest alignment length among candidates; iii) subject length = alignment length and is > 300 nt; iv) a number of mismatches allowed < 4; and v) a maximum number of gaps = 1. Finally, all transcripts were quantified using Kallisto v0.43 [As previously described, RNA depleted of rRNA transcripts was extracted from fibroblasts obtained by skin biopsies of CHU-12122 and WG-3838 epi-us study . All samus study . The alius study , that acy v2.6.5 . The TriMMACHC and TESK2 transcripts and PRDX1 aberrant transcript using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Fibroblasts from an epi-cblC case (CHU-12122) and control fibroblasts were cultured in DMEM medium supplemented with 10% v/v heat-inactivated fetal bovine serum, 1% v/v pyruvate, and 1% v/v penicillin–streptomycin. Cells were maintained at 37 °C and in 5% CO2. All the reagents were obtained from Sigma-Aldrich. RNA was extracted using NucleoSpin RNA Plus . After the extraction, a DNase digestion step was carried out to remove DNA contamination using the DNA-free™ DNA Removal kit . RNA was reverse-transcribed using PrimeScript™ RT Master Mix ; 2 µL of cDNA was used for qPCR with TB Green Premix Ex Taq (Tli RNase H Plus)  in a 20 µL reaction volume with the forward and the reverse primers specific for each of the studied genes  cblC phenype Fig. A. The viMMACHC and TESK2 loci. We observed a significant accumulation of trimethylated lysine 36 on histone H3 (H3K36me3) and no mark of H3K4me3 in the CCDC163–MMACHC bidirectional promoter region (CpG:33) and in the TESK2 promoter region (CpG:51) among patients with an epi-cblC phenotype in comparison with controls  but also through the TESK2 promoter region (CpG:51)  and TESK2 (fourfold decrease) mRNA, in RT-qPCR analyses normalized to GAPDH and TBP  concerns pathologies with multigenic etiology implicating more than one gene . Exampleequences . TherefoCCDC163P/MMACHC promoter was the most frequent among the 384 hypermethylated in the studied individuals [TESK2 and CCDC163P/MMACHC promoters [CCDC163P/MMACHC promoter [http://ewascatalog.org/?cpg=cg0036120) showed the association of hypermethylated CpG in CCDC163P/MMACHC and TESK2 promoters with sex, age, child abuse, rheumatoid arthritis, and five-year Increase in epigenomic biological age of subjects with chronic HIV Infection [Recently, a comprehensive genome-wide analysis of human epivariations analyzed in silico data generated from 23,116 individuals with the Illumina 450 k methylation array and reported 4,452 unique autosomal epivariations, including 384 in disease-causing OMIM genes . The epiividuals . Of noteromoters . This supromoter . In addinfection –37.cblC should be distinguished from cblC, with potential consequences on spermatogenesis and cancer risk produced by TESK2 silencing. The coverage of readthrough transcripts through a large region that encompasses MMACHC, CCDC163P and TESK2 involves the activation of numerous splice cryptic sites. Epi-cblC is an example of altered expression of two neighboring genes through a shared epigenetic pathomechanism. We propose introducing the term 'epi-digenism' to characterize this type of disorder.In conclusion, our results show that epi-Additional file 1. Supplemental Figure S1. Genome-wide density distribution of CpG probes in the 17 DNA methylome profiles of the 17 patients with MMACHC epimutation .Additional file 2. Supplemental Figure S2. (A) 2-D plot using the two top eigenvectors  derived from the primary component analysis on the genome-wide methylome landscape of the studied patients and controls. (B) 2-D plot using the PC1 and PC2 eigenvectors derived from the primary component analysis on the methylome landscape of chromosome 1 of the studied patients and controls.Additional file 3. Supplemental Table S1. Forward and reverse primers for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of MMACHC, TESK2 transcripts and PRDX1 aberrant transcript.


The microphthalmia/transcription factor E (MiTF/TFE) transcription factors are responsible for the regulation of various key processes for the maintenance of brain function, including autophagy-lysosomal pathway, lipid catabolism, and mitochondrial homeostasis. Among them, autophagy is one of the most relevant pathways in this frame; it is evolutionary conserved and crucial for cellular homeostasis. The dysregulation of MiTF/TFE proteins was shown to be involved in the development and progression of neurodegenerative diseases. Thus, the characterization of their function is key in the understanding of the etiology of these diseases, with the potential to develop novel therapeutics targeted to MiTF/TFE proteins and to the autophagic process. The fact that these proteins are evolutionary conserved suggests that their function and dysfunction can be investigated in model organisms with a simpler nervous system than the mammalian one. Building not only on studies in mammalian models but also in complementary model organisms, in this review we discuss (1) the mechanistic regulation of MiTF/TFE transcription factors; (2) their roles in different regions of the central nervous system, in different cell types, and their involvement in the development of neurodegenerative diseases, including lysosomal storage disorders; (3) the overlap and the compensation that occur among the different members of the family; (4) the importance of the evolutionary conservation of these protein and the process they regulate, which allows their study in different model organisms; and (5) their possible role as therapeutic targets in neurodegeneration. The transcription factors of the microphthalmia/transcription factor E (MiTF/TFE) family are crucial for the regulation of different cellular functions . Among tMultiple other roles have been associated with the members of the MiTF/TFE family, including the regulation of mitophagy , lipid cAutophagy is a crucial process in cellular physiology and is responsible for the degradation of unnecessary and defective cellular components . The autAutophagy is a multistep process that determines the engulfment of cytoplasmic components, such as protein aggregates, damaged organelles, and cell debris in double membrane vesicles called autophagosomes Fig. . These vAutophagy is ubiquitously performed at the basal level; what differs among cells and tissues is the regulation of the process and the speed of the autophagic flux, which measures the rate of autophagic degradation activity .Autophagic flux is finely regulated by multiple signaling pathways, which are activated by different stimuli, including nutrients, reactive oxygen species and calcium, and by energy imbalance , 16. AutAs the MiTF/TFE family is conserved in many different organisms, ranging from mice to fruit flies, and from zebrafishes to worms, their study in these model organisms can provide a better perspective for the interpretation of different findings also in the field of neurodegeneration. The possibility of exploiting different model organisms to characterize the function of the MiTF/TFE family, the processes that they can regulate and their role in neurodegeneration, is also crucial for the identification of new targets for the development of novel therapeutic strategies against neurodegeneration. Finally, the activation of MiTF/TFE transcription factors, particularly of TFEB, can promote the clearance of intracellular waste in both LSDs and more common neurodegenerative diseases –20. ThisCaenorhabditis elegans orthologue of the MiTF/TFE proteins HLH-30 has been directly associated to autophagic induction and increased lifespan .Fi.Fi106].FFor instance, analysis of postmortem PD midbrains revealed a selective loss of nuclear TFEB. In this frame, TFEB colocalizes with α-syn in Lewy bodies contained in surviving nigral neurons in PD human brains . α-Syn sTFEB and its target genes was found to be reduced also in the striatum of a mouse model of HD [The expression of el of HD . A reducel of HD , 109. Moel of HD .TFEB nuclear translocation appeared to be negatively affected in a dose-dependent manner in primary microglial cells treated with different concentrations of Aβ . All theA decline in TFEB levels with age in human immune cells has been recently reported , suggestThe opposite findings of TFEB being up- or downregulated during neurodegeneration suggest that the extent and length of the neurodegenerative process may impact differently TFEB expression, with an increase in the early phases and a reduction in the late stages of the disease. The same could be hypothesized for TFEB nuclear or cytoplasmic localization. A further degree of complexity is represented by cell type and tissue specificity, which are still understudied, and by the role of the different MiTF/TFE family members in neurodegeneration. The investigation of these aspects in both familial and sporadic forms of these diseases in suitable models may provide interesting clues about the role of these transcription factors in their etiology and further help in the identification of novel therapeutic targets for these disorders.Another group of diseases in which TFEB and the other transcription factors of the MiTF/TFE family seem to play an important role are lysosomal storage disorders (LSDs). They comprise more than sixty diseases caused by mutations in genes involved in lysosomal function, such as lysosomal hydrolases or lysosomal membrane proteins. About two-thirds of LSDs determine neurological symptoms and are counted among the neurodegenerative disorders , 117.Although these pathologies are characterized by lysosomal defects, in the majority of the cases, they can affect various stages of the autophagic process causing impairments in autophagosome maturation and in autophagosome-lysosome fusion and, eventually, accumulation of undigested material in cells . BecauseAs expected, both the activity of TFEB and its nuclear localization together with the autophagic process are affected in several lysosomal disorders Fig. .GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), and is characterized by a decrease in the degradation of autophagosome content after its fusion with lysosomes [Gaucher disease (GD) is the most common LSD and in the most severe cases it determines neurological defects. The pathology is caused by mutation in the ysosomes . Lysosomysosomes .Decreased levels of TFEB have been observed in induced pluripotent stem cell (iPSC)-derived neurons from GD patients, probably due to an increased proteasomal degradation of the transcription factor. In the same iPSC-derived model, the instability of TFEB was linked to the hyperactivation of mTORC1 . mTORC1-In another study, Sardiello and colleagues investigated the subcellular localization of TFEB in embryonic fibroblast from mouse models of three LSDs: mucopolysaccharidoses types II and III  and multiple sulfatase deficiency (MSD). The first two diseases belong to a group of metabolic disorders caused by impairment of lysosomal enzymes required for the degradation of glycosaminoglycans, while the latter is caused by the deficiency in the formylglycine-generating enzyme . As in tGAA gene. This disease is characterized by deficiency of the lysosomal enzyme acid α-glucosidase and leads to the accumulation of lysosomal glycogen [Despite the nuclear translocation of TFEB observed in several LSDs, this compensatory mechanism does not seem to be enough to counteract the progression of the disease and TFEB activity is not sufficient to properly remove the intracellularly accumulated debris. As observed in GD, it is possible that the stability and accumulation of TFEB are affected by increased proteasomal degradation, resulting in a decreased total amount of the protein. This hypothesis is supported by an experiment performed on myotubes in a mouse model of Pompe disease, another LSD caused by mutation in the glycogen , 122. Whglycogen . Similarglycogen .As previously discussed, also the other members of the MiTF/TFE family are involved in the regulation of the autophagy and lysosomal activity. However, as in the case of the neurodegenerative disorders previously discussed, little is known about the involvement of TFEC and MITF in the onset and progression of LSDs. Recently, it has been demonstrated that the overexpression of TFE3 can induce lysosomal exocytosis and cellular clearance in a model of Pompe disease, suggesting that also this homologue of TFEB can play an important function in the regulation of cell fate in these disorders .Although the contribution of TFEB to the pathogenesis of neurodegenerative disorders is still under debate, several studies have evaluated the effects induced by the exogenous TFEB overexpression Fig. . In a moAnother study reported decreased levels of tau aggregates in the hippocampus and cortex upon neuron-targeted TFEB overexpression , togethesubstantia nigra pars compacta of a PD rat model overexpressing the human disease-associated A53T α-syn mutant reduced the accumulation and the aggregation of α-syn as well as astrogliosis and prevented the behavioral deficits typical of this PD model [Q175/Q7 mice reduced the levels of mutant HTT (mHTT) while preserving the levels of wild-type HTT. However, in this model, TFEB overexpression was also accompanied by ER stress and reactive gliosis [Extracellular tau is considered to be responsible for the spreading of tau pathology and represents the primary target for tau immunotherapy . InteresPD model . Further gliosis .In contrast with the later work, another study reported that the co-injection of human TFEB and mHTT in the mouse striatum has no impact on the level of mHTT aggregates even though autophagy appears to be activated. In this case, the accumulation of late autophagic structures seems to impair the global process .Overall, these results seem to indicate that TFEB might represent a promising therapeutic target for the treatment of neurodegenerative disorders. However, it is important to notice that autophagy must be strictly regulated to guarantee the correct homeostasis in each type of cell. Therefore, while a regulated induction of autophagic flux may have positive effects in neurons, the overactivation of this process may be deleterious in other cells. Thus, deciphering the physiological and pathological role of TFEB in the different cell types that constitute the central nervous system will be necessary to develop efficient and safe therapeutic strategies for neurodegenerative disorders.The possibility of modulating the activity of the MiTF/TFE factors, and in particular of TFEB, has also generated a great interest as a possible therapeutic strategy for LSDs. The role of TFEB as a potential target for the treatment of several LSDs has been largely investigated, and the overexpression of TFEB or its activation through the inhibition of mTORC1 has been reported to be beneficial in the rescue of the lysosome-associated pathological phenotype.. Also, this approach could be combined to other therapeutic approaches such as enzyme replacement therapy (ERT) or gene therapy.Despite the promising observations in cellular models of LSDs and the first proof of concept in vivo, additional data on the effects of chronic activation of TFEB in animal models is required. Thus, the uncontrolled expression of MiTF/TFE factors is linked to various human rare genetic cancers . In thisTo evaluate the effect of TFEB modulation in vivo, it would be very useful to take advantage of the different transgenic animal models already used to study LSDs, which are quite reliable compared to the transgenic mouse models for some age-related neurodegenerative diseases. Animal models can provide the opportunity to assess the effects of the constitutive long-term activation of TFEB, for example by evaluating in which way TFEB activation affects the lifespan, the homeostasis, and the function of neurons and of other CNS cell types. They could also help to understand to what extent the modulation of TFEB may be beneficial in the context of LSDs, and how to prevent possible negative effects.In recent years, several compounds able to modulate TFEB activity have been found to enhance autophagy and lysosomal biogenesis and might have therapeutic potential for the treatment of neurodegenerative diseases and LSD Table . Very reThe idea of enhancing autophagy to counteract neurodegeneration has been explored by many  and in mMITF gene amplification was found in in 20% of melanomas. Translocations and rearrangements of TFE3 and TFEB are associated with a rare subtype of kidney cancer termed translocation-renal cell carcinoma (tRCC) and alveolar soft part sarcomas (ASPS), a rare lung cancer variant [Another interesting aspect to evaluate is the fact that research is mainly focused on TFEB among all MiTF/TFE family members, but even though MiTF/TFE transcription factors display some functional overlap, it remains to be established to which extent they have common functions, whether they are complementary or differ, and which factors orchestrate their interplay. Therefore, despite the promising perspective to fight neurodegenerative diseases and LSDs by enhancing autophagy/lysosomal biogenesis via TFEB modulation, a better understanding of the factors that regulate TFEB activity as well as the interplay between TFEB and the MiTF/TFE transcription factors is strongly required. This would ensure a safe development of targeted therapies for the treatment of these diseases. In fact, enhancing autophagy and lysosomal activity may be beneficial for the treatment of neurodegenerative diseases and LSDs but could have adverse effects. For example, it is quite well established that altered regulation of MiTF/TFE proteins can be linked to cancer development.  variant . A recen variant . Moreove variant . All the variant , 157. ThOverall, given that (1) autophagy and TFEB translocation may already by overactivated in certain neurodegenerative diseases or LSDs to compensate for the defective mechanisms already in place and to remove undigested cellular waste, (2) uncontrolled boosting of autophagy may impact the overall cellular homeostasis, and (3) overactivation of TFEB may trigger downstream pathways other than ALP, the final goal of novel therapeutic strategies would be to restore the homeostatic regulation of these processes and the homeostasis of the MiTF/TFE transcription factors, rather than promoting their uncontrolled activation.Non-mammalian model organisms can have a role in the implementation of our current knowledge on MiTF/TFE transcription factors. All MiTF/TFE family members are conserved in vertebrates, while invertebrates have only a single MiTF orthologue Fig. . ConsideThe following paragraphs will describe the current knowledge about MiTF/TFE transcription factor in model organisms other than rodents, by focusing, when possible, on phylogenetic and mechanistic aspects.Caenorhabditis elegans is one of the most used model organisms in biology [C. elegans make this animal an advantageous model for biology research. First, it is relatively easy to produce transgenic worm lines that overexpress or lack a gene of interest; it is possible to limit the expression of genes to specific cell types and to study protein activity by tagging them with fluorescent probes. Several studies characterized TFEB activity in worm lines lacking the C. elegans TFEB orthologue gene or studied its subcellular localization by expressing the protein tagged with a GFP [The nematode  biology . Severalth a GFP –161. It th a GFP . Second,th a GFP .TFEB has been found in C. elegans, called hlh-30, which shares high homology to the human protein in both the DNA-binding and activation domain. The protein HLH-30 is the only member of the MiTF/TFE family, which is present in C. elegans and, like the human orthologue, it modulates autophagy and lysosomal function [A functional orthologue gene of human function .Interestingly, also the regulation of HLH-30 protein seems to be conserved, with the worm transcription factor that is modulated via post-transcriptional modifications, in a similar manner to its mammalian orthologues. In fact, the silencing of mTOR has been demonstrated to enhance the HLH-30 nuclear localization and to increase the expression of several genes that are the Nematoda orthologues of the human TFEB targets, including autophagy-related genes .C. elegans HLH-30 downstream genes are characterized by the presence in their promoter region of an E-box sequence that overlaps with that of the CLEAR motif, further confirming the conservation between the HLH-30 and its mammalian orthologues. This region in C. elegans is crucial for the specificity of HLH-30 binding to DNA [Moreover, the g to DNA , 164.Drosophila melanogaster, commonly known as fruit fly, is one of the most used model organism in biology and in biomedical research [D. melanogaster brain is developed enough to promote elaborated behavioral features, but it is still small enough and relatively simple, thus allowing the detailed analysis of its structure and functions.research . In partresearch . Synapseresearch . In addiDrosophila melanogaster contains a single Mitf, which shares several features and functions with its mammalian orthologues, providing evidence of high conservation of this gene throughout the evolution. Interestingly, Mitf is not phylogenetically closer to the mammalian MITF than to TFEB, suggesting that the ancestral gene underwent multiple duplication events after the separation of the invertebrate to the vertebrate lineages [The genome of lineages . The preDrosophila melanogaster Mitf can regulate gene expression through the binding to the DNA. The conservation of the DNA binding region in the protein suggests that the fly transcription factor recognizes the same target domain of its mammalian orthologues. This DNA region is represented by the already described CLEAR motif, which is crucial for the binding specificity to the DNA. The conservation of the CLEAR motif in Drosophila has been confirmed in the promoter region of genes whose expression resulted significantly upregulated by overexpression of Mitf protein [Like the mammalian transcription factors of the MiTF/TFE family,  protein , 23, 168In fruit flies, Mitf has been shown to play a role in the transcriptional regulation of lysosomal biogenesis, in autophagy, and in the catabolism of lipids, further supporting a functional similarity with its mammalian orthologues TFEB and TFE3 . MoreoveD. melanogaster Mitf is involved in eye development, a function that is performed by MITF in mammals. More specifically, Mitf is expressed in the eye-antennal imaginal disc during the second and third larval stages of the fruit fly and the expression of a dominant negative Mitf mutant impairs the correct development of the eye [Besides its role in autophagy,  the eye .D. melanogaster Mitf suggest that the protein function is highly conserved and that the distinct roles described for the mammalian proteins might coexist in the unique fly orthologue. This makes D. melanogaster a perfect asset to test drugs able to modulate Mitf activity in a cost-effective but valuable model organism, and before moving towards more complex organisms.Overall, the data concerning Mitf orthologue both in C. elegans and D. melanogaster points at them as good organisms to study this protein in a simple model in which the presence of only one protein of the MiTF/TFE family removes the potential uncertainty derived from the redundancy. On the other side, this hinders the possibility of studying the interplay among the different MiTF/TFE family members, which may be crucial for the understanding of the molecular mechanisms that regulate the downstream pathways of these transcription factors. Under these circumstances, it may be worthwhile to exploit more complex model organisms.The presence of a unique Danio rerio, commonly known as zebrafish, is one of the most studied model organisms [tfeb and tfec, it also presents two orthologues of the mammalian MITF (Mitfa and Mitfb) and two orthologues of the mammalian TFE3 (Tfe3a and Tfe3b). Since often-duplicated genes are characterized by tissue-specific expression or by the activation in precise developmental stages, the research may take advantage of the duplication of the genome to study in detail the role of the protein of interest in different cell types or at different times. This feature seems to apply to the proteins Mitfa and Mitfb that share high homology in their sequence, with the differences mostly located in their amino and carboxy termini. Interestingly, Mitfa seems to correspond to the mammal melanocytic “M” isoform, whereas Mitfb shares the highest homology with the mammalian “A” isoform. Moreover, the zebrafish Mitf genes have restricted expression profiles that approximate the localized expression of their mammalian orthologue [Among the vertebrates, rganisms . As a verganisms . Furtherrganisms . Genomicthologue .Regarding the role of tfeb, the major functions of this transcription factor are conserved. In zebrafish, as in mammals, it controls the network of genes involved in lysosomal biogenesis and autophagy. Moreover, similarly to its mammalian orthologue, tfeb activation is regulated by mTORC1 and Rag-GTPases. Like in mouse, tfeb can repress in zebrafish the process of myelination during the development of the CNS. More specifically, tfeb has been shown to upregulate several of its target genes in the oligodentrocytes, leading the authors to speculate that the activity of tfeb may impair the trafficking of endo-lysosomal organelles to the membrane and the synthesis of lipids, two crucial processes for the membranous myelin sheath. As the activity of tfeb might disrupt the process of myelination, it appears to be specifically repressed by mTORC1 and other inhibitory kinases during myelination . This fuMitf and Tfeb, which are the most studied and characterized genes among the members of the MiTF/TFE family in zebrafish, much less is known about the two tfe3 genes. Tfe3a has been described to encode a protein of 539 amino acids that shares about 50% of homology with the human TFE3. As observed for the other members of the family, the most conserved region of Tfe3a is the helix-loop-helix/leucine zipper region. Like the human orthologue that has been shown to regulate immunoglobulin expression, tfe3a in zebrafish is present in the ventral mesoderm, which gives origin to blood cells, suggesting a possible functional conservation of this gene between different organisms [In comparison to rganisms .Tfe3 genes seem to be co-expressed with mitf genes in several tissues. In fibroblast cell cultures, the two genes share comparable activities, suggesting a possible redundant role. Nevertheless, in mitf knockout zebrafish models, Tfe3 has been demonstrated to support very inefficiently the role of Mitf [ of Mitf , indicat of Mitf .Tfec is the less characterized member of the Mitf family. The protein has been proposed to be a key regulator of zebrafish embryonic hematopoiesis, the process responsible for the formation of all types of blood cells from hematopoietic stem cells (HSCs) [In zebrafish, s (HSCs) . How andBeside the most common and studied animal models, orthologues of the MiTF/TFE family of transcription factors have been found in other organisms. Even though these organisms are considered irrelevant in biomedical research, they may inspire the study of alternative pathways or regulatory mechanisms in more conventional models.Paracentrotus lividus is a sea urchin that has an important phylogenetic position because it belongs to the phylum of Deuterostome, like vertebrates. Given the conservation of many important molecular pathways, the study of Mitf in Paracentrotus lividus may provide interesting information about the role and the signalling pathways of this transcription factor.Among the organisms in which an orthologue of Mitf has been described, D. melanogaster. Among the phosphorylation sites of Pl-Mitf, some of them are conserved, further providing evidence of a possible common pattern of regulation of this protein. Mitf has been found in the pigment cells of the sea urchin, coherently to the role of the mammalian orthologue in melanocyte. Moreover, while MITF in mammals is an important transcription factor in osteoclasts, Pl-Mitf is expressed in the presumptive mesenchymal cells (PMC) that are progenitor cells of the sea urchin larval skeleton. Some studies have highlighted similar features between PMC and osteoclasts: both cell types are involved in the skeleton development, have migratory capability, and can form multinucleated syncytia. These data may suggest an unknown role of Pl-Mitf in the skeletogenesis of sea urchin [Pl-Mitf protein is characterized by all the functional domains of the MiTF/TFE protein family, including the DNA binding domain and the bHLHzip domain. However, in this domain, only four out of five canonical leucine residues are observed and two of them are conservative substitutions. This imperfect leucine zipper has also been found in a urchin .Patinopecten yessoensis, is a large group of molluscs that live in the bottom of the northwestern Pacific Ocean. The genome and the transcriptome of Yesso scallop have been widely studied and a unique orthologue of MITF (Py-Mitf) has been found in this organism. It shares less homology with vertebrate organisms, highlighting the fact that the evolution of this gene is consistent with the species taxonomy. This gene is formed by eight exons, in contrast with the mammalian MITF that is organized into nine exons. As observed in other organisms, the most conserved region of the Py-Mitf gene is the bHLH-LZ motif [Py-Mitf has been shown to correlate with the shell color, further confirming the high degree of conservation of these genes among different organisms. Moreover, the higher level of Py-Mitf mRNA was detected in the mantel, the organ involved in shell color formation. Notably, even though the two valves of the same organism usually are characterized by different colors, no difference in the level of Py-Mitf expression was detected between the right and the left mantels. This result may indicate that Py-Mitf is strictly regulated, and a different regulation process may modulate the shell color in the same animal. Reports about Mitf involvement in autophagic regulation in these organisms are lacking; however, given the high conservation of autophagy throughout evolution and the similarities between mammalian MITF and the invertebrate orthologue, it is highly probable that this transcription factor may control ALP also in these invertebrates.Given the well-established role of MITF in the pigment cells, another group of organisms may be interesting to study the function of this gene. In fact, molluscs are characterized by a vast pattern of colors, mainly in shells. The yesso scallop, LZ motif . Shell cThe high degree of conservation of the MiTF/TFE transcription factors across evolution should be exploited for the research of modulators of these proteins. The possibility of being inspired not only of many classical model organisms but also of several non-canonical animal models may be very relevant to implement the in vivo characterization of these factors and represent very good tools to analyze in detail different aspects of the MiTF/TFE transcription factors that are still unclear.Autophagy dysfunction has been described in different neurodegenerative disorders. All the members of the MiTF/TFE family have been shown to participate in the regulation of autophagy, and in other processes that are relevant for brain physiology. However, many aspects related to the basic biology of these transcription factors remain unknown. For instance, MiTF/TFE transcription factors can form both homodimers and heterodimers with any other family member, but little is known about the functional difference between homodimers and heterodimers. Furthermore, with the exception of TFE3, all family members have alternative transcripts which display different tissue distribution patterns, and it remains to be determined the functional importance of these transcripts and whether this may result in cell-type–specific regulatory networks. As MiTF/TFE transcription factors are conserved across species, comparing MiTF/TFE protein function and regulation in different and appropriate animal models may provide a better understanding of their physiological function in the CNS. Moreover, the use of different model organisms may provide a valuable tool for understanding the roles of these transcription factors common to all forms of eukaryotic life and how their impairment may be implicated in neurodegeneration. Thus, it appears fundamental to decipher the factors that are responsible for MiTF/TFE transcription factor regulation and their interplay. This would allow modulating autophagy and other relevant pathways for brain cells via MiTF/TFE family members in a tissue/cell-specific manner thus avoiding negative side effects.