Coexpression network and trans-activation analyses of maize reproductive phasiRNA loci.

The anther-enriched phased, small interfering RNAs (phasiRNAs) play vital roles in sustaining male fertility in grass species. Their long non-coding precursors are synthesized by RNA polymerase II and are likely regulated by transcription factors (TFs). A few putative transcriptional regulators of the 21- or 24-nucleotide phasiRNA loci (referred to as 21- or 24-PHAS loci) have been identified in maize (Zea mays), but whether any of the individual TFs or TF combinations suffice to activate any PHAS locus is unclear. Here, we identified the temporal gene coexpression networks (modules) associated with maize anther development, including two modules highly enriched for the 21- or 24-PHAS loci. Comparisons of these coexpression modules and gene sets dysregulated in several reported male sterile TF mutants provided insights into TF timing with regard to phasiRNA biogenesis, including antagonistic roles for OUTER CELL LAYER4 and MALE STERILE23. Trans-activation assays in maize protoplasts of individual TFs using bulk-protoplast RNA-sequencing showed that two of the TFs coexpressed with 21-PHAS loci could activate several 21-nucleotide phasiRNA pathway genes but not transcription of 21-PHAS loci. Screens for combinatorial activities of these TFs and, separately, the recently reported putative transcriptional regulators of 24-PHAS loci using single-cell (protoplast) RNA-sequencing, did not detect reproducible activation of either 21-PHAS or 24-PHAS loci. Collectively, our results suggest that the endogenous transcriptional machineries and/or chromatin states in the anthers are necessary to activate reproductive PHAS loci.

The CaPti1-CaERF3 module positively regulates resistance of Capsicum annuum to bacterial wilt disease by coupling enhanced immunity and dehydration tolerance.

Bacterial wilt, a severe disease involving vascular system blockade, is caused by Ralstonia solanacearum. Although both plant immunity and dehydration tolerance might contribute to disease resistance, whether and how they are related remains unclear. Herein, we showed that immunity against R. solanacearum and dehydration tolerance are coupled and regulated by the CaPti1-CaERF3 module. CaPti1 and CaERF3 are members of the serine/threonine protein kinase and ethylene-responsive factor families, respectively. Expression profiling revealed that CaPti1 and CaERF3 were upregulated by R. solanacearum inoculation, dehydration stress, and exogenously applied abscisic acid (ABA). They in turn phenocopied each other in promoting resistance of pepper (Capsicum annuum) to bacterial wilt not only by activating salicylic acid-dependent CaPR1, but also by activating dehydration tolerance-related CaOSM1 and CaOSR1 and inducing stomatal closure to reduce water loss in an ABA signaling-dependent manner. Our yeast two hybrid assay showed that CaERF3 interacted with CaPti1, which was confirmed using co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that upon R. solanacearum inoculation, CaPR1, CaOSM1, and CaOSR1 were directly targeted and positively regulated by CaERF3 and potentiated by CaPti1. Additionally, our data indicated that the CaPti1-CaERF3 complex might act downstream of ABA signaling, as exogenously applied ABA did not alter regulation of stomatal aperture by the CaPti1-CaERF3 module. Importantly, the CaPti1-CaERF3 module positively affected pepper growth and the response to dehydration stress. Collectively, the results suggested that immunity and dehydration tolerance are coupled and positively regulated by CaPti1-CaERF3 in pepper plants to enhance resistance against R. solanacearum.

A high-throughput method to identify trans-activation domains within transcription factor sequences.

Even though transcription factors (TFs) are central players of gene regulation and have been extensively studied, their regulatory trans-activation domains (tADs) often remain unknown and a systematic functional characterization of tADs is lacking. Here, we present a novel high-throughput approach tAD-seq to functionally test thousands of candidate tADs from different TFs in parallel. The tADs we identify by pooled screening validate in individual luciferase assays, whereas neutral regions do not. Interestingly, the tADs are found at arbitrary positions within the TF sequences and can contain amino acid (e.g., glutamine) repeat regions or overlap structured domains, including helix-loop-helix domains that are typically annotated as DNA-binding. We also identified tADs in the non-native reading frames, confirming that random sequences can function as tADs, albeit weakly. The identification of tADs as short protein sequences sufficient for transcription activation will enable the systematic study of TF function, which-particularly for TFs of different transcription activating functionalities-is still poorly understood.

Amyloid formation by intrinsically disordered trans-activation domain of cMyb.

The cMyb trans-activation domain is one of the model systems to understand the folding and binding mechanisms in intrinsically disordered proteins. cMyb (291-315) TAD (cMyb TAD) upon interaction with KIX plays a crucial role in transcriptional regulation. However, nothing is known regarding its aggregation behaviour on change of buffer conditions or stressed environment. Notably, most of the disease-associated amyloid-forming proteins such as Aß, Tau, α-synuclein, and amylin are natively unstructured. Nevertheless, to date, very fewer evidence on aggregation behaviours on TAD domains are available. Therefore, this is necessary to investigate the aggregation propensity of intrinsically disordered cMyb TAD domain in isolation. As an essential step in that direction, we have extensively studied the aggregation behaviour of cMyb TAD using the standard approaches for aggregation studies and systematically probed the amyloid conformations. These aggregates are ThT and ANS-positive whose amyloid nature was also confirmed by Far-UV CD spectroscopic studies suggesting that cMyb TAD fibrils are rich in ß-sheet secondary structure, transmission electron microscopy revealed the formation of characteristic long branched amyloid fibrils of 6-16 nm diameter, and MTT assay in SH-SY5Y neuroblastoma cells suggest that these aggregates are cytotoxic. This amyloid nature of cMyb TAD may affect its binding with KIX and alter cMyb function (transcriptional regulation) under acidic/stressed conditions.

Cloning and Functional Analysis of BcMYB101 Gene Involved in Leaf Development in Pak Choi (Brassica rapa ssp. Chinensis).

As one of the largest transcription factor families, MYB transcription factors are widely present, and they are involved in a diverse range of physiological activities in plants, such as leaf development. GAMYB genes belong to the R2R3-MYB subfamily, which includes the MYB33/65/101 gene, and these genes are studied well in seed germination and flowering, but their roles in leaf development are poorly understood. In the current study, we isolated a GAMYB transcription factor from pak choi, BcMYB101, and analyzed its characteristics and function. The sequence structure analysis indicated that BcMYB101 has a highly conserved R2R3 DNA-binding domain in the N-terminal region and three GAMYB-specific motifs (Box1, Box2, and Box3). The expression pattern of diverse tissues revealed that BcMYB101 has a higher transcript level in the petiole, leaf, root, and floral organs. Furthermore, the expression level was significantly elevated after GA (gibberellin) treatment, suggesting that the BcMYB101 response was positively regulated by GA. Subcellular localization exhibited that BcMYB101 was only present in the nuclear region, consistent with the characterization of the transcription factor. The overexpression of BcMYB101 elucidated that BcMYB101 increased leaf number and resulted in downward-curling cauline leaves. Moreover, the virus-induced BcMYB101 silencing displayed that BcMYB101 is involved in the regulation of curly leaves. Furthermore, we discovered that BcMYB101 has two trans-activation activities and one interaction protein, BcTCH4, using a trans-activation activity assay and a yeast two-hybrid assay, respectively. In this study, we firstly isolated the BcMYB101 gene and explored its function in leaf development, thereby providing a solid foundation for further research on the regulatory mechanism of leaf shape in Brassica or other species.

PML/RARa blocks the differentiation and promotes the proliferation of acute promyelocytic leukemia through activating MYB expression by transcriptional and epigenetic regulation mechanisms.

The promyelocytic leukemia (PML)/retinoic acid receptor-alpha (RARα) onco-fusion protein that is generated from t(15;17) chromosome translocation is crucial for the leukemogenesis of acute promyelocytic leukemia (APL) and is well documented as a transcriptional repressor. To understand the relationship between PML/RARα and the oncogene in the development of APL, we investigate the regulation mechanism of PML/RARα to MYB proto-oncogene and the role of this regulation on the proliferation and differentiation of APL cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays show that MYB expression was significantly higher in PML/RARα positive cell lines. Microarray data verify that the MYB expression was significantly higher in APL patient samples than in normal promyelocyte samples. Further expression analysis from RT-qPCR and microarray data verifies that the expression of MYB is upregulated by PML/RARα. Transcriptional factor binding analysis shows that MYB is directly bound by PML/RARα and its cofactors. Luciferase assays show that PML/RARα transactivated MYB promoter activity through the RARα binding site and the coexistence of CCAAT enhancer binding protein ε. We also find that PML/RARα increases the acetylation level of the promoter region of MYB. Further evidence demonstrates that PML/RARα regulates MYB expression through long-range interaction. Functionally, PML/RARα increases the cell proliferation and blocks the differentiation through activating MYB expression. Collectively, this study uncovers a novel mechanism of PML/RARα-mediated transcriptional activation and enriches our knowledge of the onco-fusion protein-mediated transcription activation.

Trans-activation-based risk assessment of BRCA1 BRCT variants with unknown clinical significance.

BACKGROUND: Deleterious variants in the tumour suppressor BRCA1 are known to cause hereditary breast and ovarian cancer syndrome (HBOC). Missense variants in BRCA1 pose a challenge in clinical care, as their effect on protein functionality often remains unknown. Many of the pathogenic missense variants found in BRCA1 are located in the BRCA1 C-terminal (BRCT) domains, domains that are known to be vital for key functions such as homologous recombination repair, protein-protein interactions and trans-activation (TA). We investigated the TA activity of 12 BRCA1 variants of unknown clinical significance (VUSs) located in the BRCT domains to aid in the classification of these variants. RESULTS: Twelve BRCA1 VUSs were investigated using a modified version of the dual luciferase TA activity assay (TA assay) that yielded increased sensitivity and sample throughput. Variants were classified according to American College of Medical Genetics and Genomics (ACMG) criteria using TA assay results and available data. In combining our TA-assay results and available data, in accordance with the ACMG guidelines for variant classification, we proposed the following variant classifications: c.5100A>G, c.5326C>T, c.5348T>C and c.5477A>T as likely benign (class 2) variants. c.5075A>C, c.5116G>A and c.5513T>G were likely pathogenic (class 4), whereas c.5096G>A likely represents a likely pathogenic variant with moderate penetrance. Variants c.5123C>T, c.5125G>A, c.5131A>C and c.5504G>A remained classified as VUSs (class 3). CONCLUSIONS: The modified TA assay provides efficient risk assessment of rare missense variants found in the BRCA1 BRCT-domains. We also report that increased post-transfection incubation time yielded a significant increase in TA assay sensitivity.

AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation.

RNA polymerase III control elements are required for trans-activation by the murine retroviral long terminal repeat sequences.

RNA leukemia viruses induce T-cell lymphoblastic lymphomas or myeloid leukemias. Infection of cells with Moloney murine leukemia virus (M-MuLV) up-regulates the expression of a number of cellular genes, including those involved in T-lymphocyte activation. Previously, we demonstrated that this up-regulation occurs via the trans-activation activity of the M-MuLV long terminal repeat (LTR) sequences which produce an LTR-encoded transcript. Sequence analysis of the LTR revealed a potential transcription unit for RNA polymerase III (Pol III) within the U3 region that is actively occupied by Pol II factors. Here, we provide the direct evidence of involvement of Pol III in the trans-activation process and demonstrate the precise localization of the intragenic control elements for accurate and active Pol III transcription. Deletions of a copy of the directed repeats and further immediate upstream sequences significantly abrogated the generation of LTR-encoded transcript and abolished the trans-activational activity, whereas the deletion of a copy of directed repeats alone proportionally reduced the transcript size, but still retained moderately high trans-activational activity. In electrophoretic mobility shift assay, the fraction containing a multiple transcription factor TFIIIC complex strongly bound to the LTR-U3 probe containing the essential control elements. The specificity of the DNA-TFIIIC interaction was confirmed by conducting competition assays with DNA fragments containing a genuine Pol III-transcribed gene, VA1, and by vaccinia virus infection which stimulates the expression of Pol III factors. However, a deletion mutant lacking an essential control element bound to the TFIIIC complex poorly, consequently resulting in weak Pol III transcription as assessed by an IRES-GFP reporter system. This correlation strongly supports the possibility that the generation of LTR-encoded transcript is directed by Pol III. Therefore, this finding suggests the involvement of Pol III transcription in the retrovirus-induced activation of cellular genes, potentially contributing to leukemogenesis.

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.

Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

Large-scale screening of transcription factor-promoter interactions in spruce reveals a transcriptional network involved in vascular development.

This research aimed to investigate the role of diverse transcription factors (TFs) and to delineate gene regulatory networks directly in conifers at a relatively high-throughput level. The approach integrated sequence analyses, transcript profiling, and development of a conifer-specific activation assay. Transcript accumulation profiles of 102 TFs and potential target genes were clustered to identify groups of coordinately expressed genes. Several different patterns of transcript accumulation were observed by profiling in nine different organs and tissues: 27 genes were preferential to secondary xylem both in stems and roots, and other genes were preferential to phelloderm and periderm or were more ubiquitous. A robust system has been established as a screening approach to define which TFs have the ability to regulate a given promoter in planta. Trans-activation or repression effects were observed in 30% of TF-candidate gene promoter combinations. As a proof of concept, phylogenetic analysis and expression and trans-activation data were used to demonstrate that two spruce NAC-domain proteins most likely play key roles in secondary vascular growth as observed in other plant species. This study tested many TFs from diverse families in a conifer tree species, which broadens the knowledge of promoter-TF interactions in wood development and enables comparisons of gene regulatory networks found in angiosperms and gymnosperms.

Trans-Activation of the Coactivator-Associated Arginine Methyltransferase 1 (Carm1) Gene by the Oncogene Product Tax of Human T-Cell Leukemia Virus Type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.

Activation of the CDK7 Gene, Coding for the Catalytic Subunit of the Cyclin-Dependent Kinase (CDK)-Activating Kinase (CAK) and General Transcription Factor II H, by the Trans-Activator Protein Tax of Human T-Cell Leukemia Virus Type-1.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The trans-activator protein Tax of HTLV-1 plays crucial roles in leukemogenesis by promoting proliferation of virus-infected cells through activation of growth-promoting genes. However, critical target genes are yet to be elucidated. We show here that Tax activates the gene coding for cyclin-dependent kinase 7 (CDK7), the essential component of both CDK-activating kinase (CAK) and general transcription factor TFIIH. CAK and TFIIH play essential roles in cell cycle progression and transcription by activating CDKs and facilitating transcriptional initiation, respectively. Tax induced CDK7 gene expression not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs) along with increased protein expression. Tax stimulated phosphorylation of CDK2 and RNA polymerase II at sites reported to be mediated by CDK7. Tax activated the CDK7 promoter through the NF-κB pathway, which mainly mediates cell growth promotion by Tax. Knockdown of CDK7 expression reduced Tax-mediated induction of target gene expression and cell cycle progression. These results suggest that the CDK7 gene is a crucial target of Tax-mediated trans-activation to promote cell proliferation by activating CDKs and transcription.

Inducible, Tissue-Specific Gene Expression in Arabidopsis Using GR-LhG4-Mediated Trans-Activation.

Inducible, tissue-specific gene expression is a potent tool to study gene regulatory networks as it allows spatially and temporally controlled genetic perturbations. To this end, we generated a toolkit that covers many cell types in the three main meristems: the root apical meristem, the shoot apical meristem, and the vascular cambium. The system is based on an extensive set of driver lines expressing a synthetic transcription factor under cell type-specific promoters. Induction leads to nuclear translocation of the transcription factor and expression of response elements under control of a cognate synthetic promoter. In addition, a fluorescent reporter incorporated in driver lines allows to monitor induction. All previously generated driver lines are available from the Nottingham Arabidopsis Stock Center. This protocol describes how users can create their own constructs compatible with the existing set of lines and as well as induction and imaging procedures.

The USR domain of USF1 mediates NF-Y interactions and cooperative DNA binding.

The trimeric CCAAT-binding NF-Y is a "pioneer" Transcription Factor -TF- known to cooperate with neighboring TFs to regulate gene expression. Genome-wide analyses detected a precise stereo-alignment -10/12 bp- of CCAAT with E-box elements and corresponding colocalization of NF-Y with basic-Helix-Loop-Helix (bHLH) TFs. We dissected here NF-Y interactions with USF1 and MAX. USF1, but not MAX, cooperates in DNA binding with NF-Y. NF-Y and USF1 synergize to activate target promoters. Reconstruction of complexes by structural means shows independent DNA binding of MAX, whereas USF1 has extended contacts with NF-Y, involving the USR, a USF-specific amino acid sequence stretch required for trans-activation. The USR is an intrinsically disordered domain and adopts different conformations based on E-box-CCAAT distances. Deletion of the USR abolishes cooperative DNA binding with NF-Y. Our data indicate that the functionality of certain unstructured domains involves adapting to small variation in stereo-alignments of the multimeric TFs sites.

Intra-Clade C signature polymorphisms in HIV-1 LTR region: The Indian and African lookout.

Polymorphisms occurring in LTR (Long Terminal Repeat) region can profoundly impact pathogenicity, transmission and biology of Human Immunodeficiency Virus Type 1 (HIV-1). We investigated intra-clade polymorphisms, associated with HIV-1 clade-C infections that occur in India and Africa. Plasma samples were obtained from 24 HIV-infected ART-experienced individuals. Next Generation Sequencing was performed on Illumina Hi Seq X system. Sequence analysis was done using MEGA v7. Transcription factor binding sites (TFBS) were investigated to unveil signature sequences. Signature nucleotides in Indian sequences were observed at 19 positions, of which 7 nucleotide signatures occurred in transcription binding sites (TFBS), namely NF-AT-II, NF-AT-III, USF, TCF- 1alpha, Sp1-I and TAR. Intra-clade C variations in HIV-1 LTR that inscribe signature nucleotides in Indian sequences lead to formation monophyletic cluster of Indian sequences. Moreover, occurrence of intra-clade signature nucleotides was observed at the key positions in the transcription factor binding sites in Indian and African clade-C sequences.

The AC2 Protein of a Bipartite Geminivirus Stimulates the Transcription of the BV1 Gene through Abscisic Acid Responsive Promoter Elements.

Geminiviruses possess single-stranded, circular DNA genomes and control the transcription of their late genes, including BV1 of many bipartite begomoviruses, through transcriptional activation by the early expressing AC2 protein. DNA binding by AC2 is not sequence-specific; hence, the specificity of AC2 activation is thought to be conferred by plant transcription factors (TFs) recruited by AC2 in infected cells. However, the exact TFs AC2 recruits are not known for most viruses. Here, we report a systematic examination of the BV1 promoter (PBV1) of the mungbean yellow mosaic virus (MYMV) for conserved promoter motifs. We found that MYMV PBV1 contains three abscisic acid (ABA)-responsive elements (ABREs) within its first 70 nucleotides. Deleting these ABREs, or mutating them all via site-directed mutagenesis, abolished the capacity of PBV1 to respond to AC2-mediated transcriptional activation. Furthermore, ABRE and other related ABA-responsive elements were prevalent in more than a dozen Old World begomoviruses we inspected. Together, these findings suggest that ABA-responsive TFs may be recruited by AC2 to BV1 promoters of these viruses to confer specificity to AC2 activation. These observations are expected to guide the search for the actual TF(s), furthering our understanding of the mechanisms of AC2 action.

Multiple domains in the 50 kDa form of E4F1 regulate promoter-specific repression and E1A trans-activation.

The 50 kDa N-terminal product of the cellular transcription factor E4F1 (p50E4F1) mediates E1A289R trans-activation of the adenovirus E4 gene, and suppresses E1A-mediated transformation by sensitizing cells to cell death. This report shows that while both E1A289R and E1A243R stimulate p50E4F1 DNA binding activity, E1A289R trans-activation, as measured using GAL-p50E4F1 fusion proteins, involves a p50E4F1 transcription regulatory (TR) region that must be promoter-bound and is dependent upon E1A CR3, CR1 and N-terminal domains. Trans-activation is promoter-specific, as GAL-p50E4F1 did not stimulate commonly used artificial promoters and was strongly repressive when competing against GAL-VP16. p50E4F1 and E1A289R stably associate in vivo using the p50E4F1 TR region and E1A CR3, although their association in vitro is indirect and paradoxically disrupted by MAP kinase phosphorylation of E1A289R, which stimulates E4 trans-activation in vivo. Multiple cellular proteins, including TBP, bind the p50E4F1 TR region in vitro. The mechanistic implications for p50E4F1 function are discussed.

Cytoplasmic TDP43 Binds microRNAs: New Disease Targets in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, and incurable neurodegenerative disease. Recent studies suggest that dysregulation of gene expression by microRNAs (miRNAs) may play an important role in ALS pathogenesis. The reversible nature of this dysregulation makes miRNAs attractive pharmacological targets and a potential therapeutic avenue. Under physiological conditions, miRNA biogenesis, which begins in the nucleus and includes further maturation in the cytoplasm, involves trans-activation response element DNA/RNA-binding protein of 43 kDa (TDP43). However, TDP43 mutations or stress trigger TDP43 mislocalization and inclusion formation, a hallmark of most ALS cases, that may lead to aberrant protein/miRNA interactions in the cytoplasm. Herein, we demonstrated that TDP43 exhibits differential binding affinity for select miRNAs, which prompted us to profile miRNAs that preferentially bind cytoplasmic TDP43. Using cellular models expressing TDP43 variants and miRNA profiling analyses, we identified differential levels of 65 cytoplasmic TDP43-associated miRNAs. Of these, approximately 30% exhibited levels that differed by more than 3-fold in the cytoplasmic TDP43 models relative to our control model. The hits included both novel miRNAs and miRNAs previously associated with ALS that potentially regulate several predicted genes and pathways that may be important for pathogenesis. Accordingly, these findings highlight specific miRNAs that may shed light on relevant disease pathways and could represent potential biomarkers and reversible treatment targets for ALS.