Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes.We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses.Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4.We identified Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.The online version contains supplementary material available at 10.1186/s12931-021-01679-y. In the mammalian lung, trachea, bronchi and bronchioles form a tree-like system of tubes that conduct the air to thin-walled terminal sacs, the alveoli, where the exchange of carbon dioxide and oxygen occurs. This elaborate epithelial system arises from a simple outgrowth of the foregut endoderm by a complex program of specification, proliferative expansion, branching morphogenesis, proximal–distal patterning and differentiation during embryonic development . All of Orchestration and interpretation of these reciprocal signaling cascades require the activity of transcription factors that regulate the signals and their activities in time and space but also impinge onto the cell-cycle machinery to assure the pro-proliferative undifferentiated state in either tissue compartment. T-box proteins are members of a large, evolutionary conserved family of transcriptional regulators that share a highly conserved DNA-binding region, namely the T-box . TranscrTbx2 and the closely related Tbx3 gene are predominantly expressed in mesenchymal precursors that surround the distal endodermal tips. The expression largely depends on epithelial SHH signals with modulatory input from epithelial BMP4, mesenchymal TGFs, and WNTs possibly emerging from both compartments [Tbx2 and even more, the combined loss of Tbx2 and Tbx3 in mice, results in arrest of mesenchymal proliferation, premature mesenchymal differentiation and an arrest of epithelial branching morphogenesis leading to lung hypoplasia at birth. Prolongation of TBX2 expression into adulthood leads to hyperproliferation and maintenance of mesenchymal progenitor cells. These cellular changes were traced to a molecular function of TBX2 to directly repress expression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, which in turn increases pro-proliferative WNT signaling [Our previous work characterized the T-box transcription factor TBX2 as a mesenchymal regulatory hub during lung development. artments , 14. Losignaling , 15.Despite these important molecular insights, we still lack a survey of all direct target genes of TBX2 in the mesenchyme of the developing lung and of the nature and configuration of DNA-binding sites present in these genes. Moreover, we do not know with which other transcription factors, corepressors and chromatin remodeling complexes TBX2 interacts to achieve target gene specificity and repression in this developmental context.Ccn4 and Il33, and describe the consensus binding site of TBX2 in the developing lung. Additionally, we identified and characterized protein binding partners of TBX2 that may aid in specific repression of these target genes.Here, we set out to experimentally address these questions. Using a combination of transcriptional profiling by microarrays and ChIP-Seq technology, we identified additional targets of TBX2 activity including Tbx2tm1.1(cre)Vmc (synonym: Tbx2cre) [Tbx2tm2.2Vmc (synonym: Tbx2fl) [Gt(ROSA)26Sortm4Luo/J (synonym: R26mTmG) [Tbx2cre/+ males with R26mTmG/mTmG;Tbx2fl/fl or Tbx2cre/+ females. To time the pregnancy, vaginal plugs were checked on the morning after mating and noon was taken as embryonic day (E) 0.5. On the day of harvest, pregnant females were sacrificed by cervical dislocation. Embryos and lungs were dissected in PBS. For both in situ hybridization and immunofluorescence analyses, embryos were fixed in 4% PFA/PBS, transferred to methanol and stored at − 20 °C. PCR genotyping was performed on genomic DNA prepared from ear clips of adult mice or from embryonic tissues.All mouse strains used in this study: Tbx2cre) , Tbx2tm2 Tbx2fl) , Gt(ROSAR26mTmG)  were maiAll animal work conducted for this study was approved by the local authorities  and was performed at the central animal laboratory of the Medizinische Hochschule Hannover in accordance with the National Institute of Health guidelines for the care and use of laboratory animals.For ChIP-Seq analysis, a total of 100 E14.5 wildtype lungs were minced in PBS into pieces of 100–500 µm. The tissue was incubated in 1.6% formaldehyde/PBS for 20 min before glycine was added to a final concentration of 1% and incubation continued for 10 min at room temperature. After a washing step with PBS, the tissue was stored at − 80 °C until further use. ChIP reactions were performed with the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)  following manufacturer’s instructions. Nuclease treatment for fragmentation of chromatin was prolonged to 30 min and nuclease concentration was doubled to obtain fragments of 300 bp in average. The DNA-containing supernatants were incubated with a ChIP grade anti-TBX2 antibody , anti-Histone H3  or an IgG control  for 1 h at room temperature, and together with ChIP-Grade ProteinG Magnetic Beads  overnight at 4 °C.https://usegalaxy.org). Gene ontology (GO) term analysis was performed with Genomic Regions Enrichment of Annotations Tool . De novo motif analysis on the data was performed with the FIMO tool in Galaxy  [http://meme-suite.org/tools/tomtom) [http://jaspar.genereg.net) and footprintDB (http://floresta.eead.csic.es/footprintdb) databases.The DNA precipitates were passed to the Research Core Unit Genomics of Hannover Medical School. Library preparation was performed with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®  and next generation sequencing was performed on Illumina NextSeq High Output 500/550 flow cells with a reading depth of 15 million 75 bp paired-end reads  using NEBNext® Multiplex Oligos for Illumina®   following manufacturer’s instructions. ChIP peaks were mapped against the GRC38/mm10 genome (NCBI BioProject Accession: PRJNA20689) using MACS2 callpeak integrated in Galaxy version 2.1.1.20160309.1 . ChIP peaxy.org)  for pali/tomtom) , using ahttps://david.ncifcrf.gov) [Lists of gene symbols were imported into DAVID Bioinformatics Resources version 6.8 (crf.gov)  with anncrf.gov)  were anaTbx2-mutant lungs was isolated as described for ChIP-seq experiments and subjected to PCR amplification of gene-specific peak regions. Primers for a peak in Ccn4, chr15:66,883,385–66,883,657 were: 5′- CCAGAGAATGTCACACTCCAC-3′ and 5′- GCAGCTACTGGGTCTCTCA-3′. For peak #1 in Il33 : 5′-TGGTTCTCTGCCAAGTTCTG-3′ and 5′- TGCTCCACAGGTCCTAAGAT-3′; for peak #2 in Il33 : 5′- GGCTAAGGCAAGAAGATCATG-3′ and 5′-CCTGCCAATGTTACTGTTATC-3′.Chromatin of ~ 20 wildtype and Three independent proteomic analyses were performed using material of 100 E14.5 lungs each. The lung tissue was fixed and stored until further use as described for ChIP-seq assays. Tissue dissociation was achieved following the RIME protocol  utilizinApart from minor modifications, LC–MS/MS was performed as previously described . Peptidehttps://string-db.org) [Protein network analysis was performed using the STRING protein–protein interaction networks functional enrichment analysis tool v11 (-db.org)  with MCLNon-radioactive in situ hybridization analysis of gene expression was performed on 10-μm paraffin sections of embryos using digoxigenin-labeled antisense riboprobes as described previously . For eac2O2 for 20 min. Antigen retrieval was achieved by citrate-based heat unmasking . The following primary antibodies were used: anti-CBX3 , anti-CHD4 , anti-HDAC1 , anti-HDAC2 , anti-HMGB2 , anti-PBX1 , anti-TBX2 , anti-TBX2 . Primary antibodies were detected by directly labeled fluorescence- or biotin-conjugated secondary antibodies . The signal was amplified using a tyramide signal amplification (TSA) system  according to the manufacturer’s instruction. Nuclei were stained with 4′,6-diamidino-2-phenylindole .Detection of antigens was performed on 5-µm or 10-µm frontal sections through the lung region of paraffin-embedded embryos. Endogenous peroxidases were blocked by incubation in 6% HTbx2-mutant embryos was performed with the proximity ligation assay [Analysis of direct protein interactions on 10 µm frontal lung sections of E14.5 wildtype and on assay , 33 usin2. The transient transfections were performed with the calcium phosphate method as previously described [pcDNA3 vector.HEK293 cells  were cultured in DMEM medium with GlutaMax™  containing 10% FCS , 100 units/ml penicillin, 100 µg/ml streptomycin , 5% sodium pyruvate  and 5% non-essential amino acids  and kept in an incubator at 37 °C with 5% COescribed . For thipcDNA3.huTBX2.HA encoding N-terminally HA-tagged full-length human TBX2; pCS2.Pbx1b encoding full-length mouse PBX1B, ; pd2EGFP-N1 (EGFP-expression vector) [pCMV6-Entry.Cbx3-Myc-DDK encoding full-length mouse CBX3 ; pCMV-SPORT6.Hdac1 encoding full-length mouse HDAC1 ; pCMV6-Entry.Hdac2-Myc-DDK encoding full-length mouse HDAC2 ; pCMV-SPORT6.Chd4 encoding full-length mouse CHD4 ; pCMV-Entry.Hmgb2-GFP encoding full-length mouse HMGB2 .We used the following expression vectors for transfections in HEK293 cells:  vector) ; pCMV6-ECell lysates were obtained following the RIME protocol  as descrhttps://imagej.nih.gov). Western blots Blots were documented on a LAS-4000 luminescent Image Analyzer .Lung sections were documented with a DM5000 microscope  equipped with a Leica DFC300FX digital camera. Images were processed and analyzed with Adobe Photoshop CS5  and ImageJ software (http://great.stanford.edu/public/html) [To obtain an unbiased view of TBX2-bound genomic regions in the pseudoglandular stage of lung development, we performed in vivo ChIP-Seq analysis on E14.5 wildtype lungs using an anti-TBX2 antibody. Mapping of sequenced tags using MACS2 callpeak  identifiic/html) . With reGene ontology (GO) annotation of biological function and processes by GREAT revealed enrichment of peak-associated genes with various mouse phenotypes. ”Abnormal pulmonary trunk morphology” and “dilated respiratory conducting tubes” were the top enriched clusters in mouse phenotypes indicating significant affiliation of TBX2-bound regions to pulmonary development. Additional peak clusters were affiliated with the terms “abnormal digit development”, “failure of palatal shelf elevation”, “development of the urogenital system” and “limbs” reflecting known functions of TBX2 in mouse development –40. “Abnhttp://jaspar.genereg.net) and footprintDB (http://floresta.eead.csic.es/footprintdb) databases. We found five enriched binding motifs in our ChIP-Seq data set with three strongly resembling previously described binding sites for T-box proteins. Two of them, one palindromic, the other non-palindromic, demonstrated high similarity to a known binding motif for TBX2 (entry MA0688.1 in Jaspar)  and Il33  against the control (immunoprecipitates in absence of the anti-TBX2 primary antibody) was found for 219 proteins ) [To identify protein interaction partners that may explain target specificity and transcriptional repressive activity of TBX2 in the pulmonary mesenchyme, we used an in vivo co-immunoprecipitation (Co-IP) approach from E14.5 lungs with subsequent liquid chromatography–tandem mass spectrometry analysis (LC–MS/MS) Fig. a. For thdb.org))  uncoveredb.org)) , 47. Prodb.org)) , the homdb.org)) , the HMGdb.org)) , the DNAdb.org)) , and thedb.org)) .Fig. 3LCThe second cluster  as well as the proteins possibly associated with this complex  since they are likely to explain the target specificity and repressive activity of TBX2 in the pulmonary mesenchyme.Co-immunofluorescence analysis of the candidate proteins and TBX2 on transverse sections of E14.5 lungs revealed that all six candidates were widely coexpressed with TBX2 in the nuclei of pulmonary mesenchymal cells  subfamily of the connective tissue growth factor/CCN family of matricellular proteins. CCN proteins, which are secreted, interact with cell surface receptors  and extracellular matrix components to modulate cellular functions. CCN4 can stimulate proliferation, adhesion, invasion, metastasis and epithelial-to-mesenchymal transition of cells [Ccn4, and thus, of these cell programs in the lung mesenchyme cannot be answered at this point. Il33 codes for a cytokine which mediates inflammatory responses [The intersection of transcriptional profiling and ChIP-seq data sets provided a list of 39 genes that might be directly regulated by TBX2. In line with our previous phenotypic characterization, we found enrichment of genes annotated with proliferation and stress control, indicating that TBX2 predominantly represses anti-proliferative genes. To our surprise, we failed to detect increased expression of most candidate genes in the pulmonary mesenchyme of of cells . The sigesponses . Its repOur de novo motif analysis of the TBX2-ChIP-seq data set did not only reveal binding sites highly similar to the consensus binding site(s) of the T-box DNA-binding domain , 53 but Hmgb2 do not exhibit lung defects, while Pbx1-deficiency results in lung hypoplasia and alveolar defects [Hmgb1 and Pbx2-4) may conceal the requirement of these genes in these cellular programs.Most satisfyingly, we identified the homeobox transcription factor PBX1 and the HMG-box protein HMGB2 that have both been implicated in transcriptional repression , 60, amo defects , 62. In Although our de novo motif analysis found an enrichment of an ETS-domain binding motif in the TBX2-ChIP peaks, we did not identify a member of this protein family in our proteomic screen. This seems plausible since members of the ETS transcription factor family  act as transcriptional activators –65 and wIt is important to note that interaction of TBX2 and the closely related TBX3 with HMG-box and homeobox proteins has been documented before for other developmental contexts in which these closely related T-box proteins act –68 whileNDRG1. To do so, TBX2 recruits the DNA methyltransferase DNMT3B and histone methyltransferase complex components to set a repressive chromatin mark (H3K9me3) within the proximal promoter of NDRG1 [Cdkn1a, Cdkn2a, Adam10, Pten and muscle-specific genes in different cancer cell lines or myoblasts cells depends on recruitment of HDAC1, hence, deacetylation of lysine residues in N-terminal tails of histones [Cdkn1a and Cdkn2a [It is long known that TBX2 acts as repressor of target gene transcription both in vitro and in vivo –45 but eof NDRG1 . In conthistones –73. The d Cdkn2a , 75.Cdkn1a and Cdkn1b in the lung mesenchyme. The two proteins were enriched in our proteomic screen, they bound to TBX2 in HEK cells, and they were found in short distance, direct association with TBX2 in nuclei of lung mesenchymal cells. Our proteomic analysis further identified RBBP4, RBBP7 and CHD4 which are known to interact with HDAC1 and HDAC2 in the CHD/NuRD complex [Our proteomic analysis argues that HDACs namely, HDAC1 and HDAC2, are also involved in repression of TBX2 target genes including  complex , 47 implCdkn1a by TBX2 [NDRG1 in tumor cells [NDRG1 by TBX2, we found that a DNA methyltransferase, namely (maintenance) DNMT1 coprecipitated with TBX2 from lung tissue implicating DNA methylation in transcriptional repression by TBX2 [Frzb) and of Cdkn1a has been reported, substantiating the relevance of TBX2 interaction with these components [CHD proteins like CHD4 are known to bind to methylated histone tails (H3K9me3) most likely via their PHD2 finger . Similar by TBX2 , 77, 78.or cells . The ide by TBX2 . Intrigumponents –82.We also found MYBBP1A as an interaction partner in the proteomic analysis. MYBBP1A acts as a corepressor for different transcription factors and is possibly involved in chromatin compaction by recruiting negative epigenetic modifiers, such as HDAC1/2 and histone methyltransferase , 83. FinHDACs have an important role in regulation of gene expression. The removal of acetyl groups from lysine residues at histone tails leads to chromatin compaction, which inhibits the binding of transcription machinery at the promoter region, thus, represses gene transcription. Expression of many HDACs is increased in malignant cells, and HDACs have been linked with acquisition of malignant phenotypes in cancerogenesis by stimulation of proliferation, cell survival and motility, inhibition of differentiation and promotion of angiogenesis , 85. SimCdkn1a is significantly decreased by the upregulation of HDACs and the NuRD complex whereas transient overexpression of HDACs induce Cdkn1a repression [Increased proliferation in tumor cells frequently correlates with reduced expression levels of cyclin-dependent kinase inhibitors, including CDKN1A. Transcription of pression , 88. Forpression , 90.Cdkn1a in embryonic lung mesenchyme. Interestingly, TBX2 and the closely related TBX3 gene are reactivated and/or overexpressed in several cancers including small cell lung carcinoma melanoma, breast, pancreatic, liver, and bladder cancers [Cdkn2a and Cdkn1a to bypass senescence [HDACs are recruited to specific gene loci by tissue-specific transcription factors. Our work has characterized TBX2 as a factor that recruits HDAC1,2 and the NuRD complex to anti-proliferative target genes including  cancers . Moreovenescence , 70. It Il33 and Ccn4 as additional direct target genes of TBX2 in the lung mesenchyme. It revealed combinations of T-box binding elements with bindings sites for HMG-box and homeobox proteins in the TBX2 genomic binding peaks, and characterized the transcription factors PBX1 and HMGB2, and components and interaction partners of the NuRD complex as TBX2 protein binding partners. We suggest TBX2 cooperates with homeobox and HMG-box transcription factors in transcriptional repression of anti-proliferative genes in the lung mesenchyme, and that this repressive activity relies on histone deacetylation and chromatin remodeling mediated by the NuRD complex but also on DNA methylation, histone H3K9 trimethylation and subsequent heterochromatin formation by CBX3 at the nuclear lamina (Fig. Our work identified ina Fig. . Our finAdditional file 1: Figure S1. Expression analysis of candidate genes with increased expression in microarray analyses of Tbx2-deficient lungs. Figure S2. Derepression of TBX2 target genes occurs around E12.5 in Tbx2-deficient pulmonary mesenchyme.Additional file 2: Table S1. TBX2 ChIP-seq peaks. Table S2. Functional annotation of enriched TBX2 ChIP-seq peaks. Table S3. Functional annotation of enriched TBX2 ChIP-seq peaks. Table S4. Functional annotation of enriched TBX2 ChIP-seq peaks. Table S5. Genes with decreased expression in the microarrays of E14.5 control vs Tbx2-deficient lungs. Table S6. Genes with increased expression in the microarrays of E14.5 control vs Tbx2-deficient lungs. Table S7. GO-Term analysis of upregulated genes in microarray analysis. Table S8. Functional annotation analysis of upregulated genes in microarray analysis. Table S9. Mass Spectrometry. Table S10. GO-Term analysis of interaction candidates.


Botrytis cinerea, we performed a chromatin immunoprecipitation (ChIP) analysis showing the increase in H3K9ac mark along the early induced genes SlyDES, SlyDOX1, and SlyLoxD encoding oxylipin-pathway enzymes, and SlyWRKY75 coding for a transcriptional regulator of hormonal signaling. This histone mark showed a more distinct distribution than the previously studied H3K4me3. The RNAPol-ChIP analysis reflected the actual gene transcription associated with increased histone modifications. A different pattern of marks in the oxylipin-related genes against P. syringae supported a pathogen-specific profile, while no significant differences occurred in SlyWRKY75. The epigenetic regulation of SlyWRKY75 by the intron-binding miR1127-3p was supported by the presence of SlyWRKY75 pre-mRNA in control plants. Interestingly, mRNA was found to be accumulated in response to B. cinerea and P. syringae, while reduction in miRNA only occurred against B. cinerea. The intronic region presented a similar pattern of marks than the rest of the gene in both pathosystems, except for H3K4me3 in the miRNA binding site upon B. cinerea. We located the gene encoding Sly-miR1127-3p, which presented reduced H3K4me3 on its promoter against B. cinerea. Research into the relationship between epigenetic regulation and resistance to biotic stresses provides alternatives for plant protection and crop improvement. To unravel the mechanisms underlying tomato responses to  Arabidopsis thaliana plants infected with biotrophic pathogens. Infection of Arabidopsis by Pseudomonas syringae pv. maculicola primed defensive genes by NPR1-dependent post-translational modifications of histone H3 at their promoter regions . Th. Th28]. promoter . The autpromoter . There aed genes . HoweverP. syringae showed a completely different pattern of both histone marks in SlyDES, SlyDOX1, and SlyLoxD genes. We observed a slight increase in H3K9ac and H3K4me3 along these oxylipin-related genes, which supports a pathogen-specific profile of epigenetic marks. The presence of RNAPII did not significantly increase in the highly induced SlyDES and SlyDOX1 upon P. syringae. However, the induction of SlyLoxD in this pathosystem was associated with increased RNAPII presence in the gene body. Therefore, the obtained results might be explained by different transcriptional and epigenetic regulation in response to B. cinerea or P. syringae leading to changes in transcript stability that deserve further research. Regarding SlyWRKY75, the increase in both epigenetic modifications along the gene was even higher than in response to B. cinerea. Interestingly, upon P. syringae infection, SlyWRKY75 showed a significant increase in RNAPII binding along the gene, supporting its transcriptional induction in this pathosystem too. In this case, the polymerase presence was higher than that observed in response to B. cinerea, correlating with the higher accumulation of SlyWRKY75 transcript and H3K9ac mark in response to the bacterial infection. The similar increase of H3K9ac and H3K4me3 along this gene in these two pathosystems suggests that this epigenetic profile is associated with a general response to biotic stresses. This is particularly interesting because this transcription factor might be a regulator of the JA pathway in tomato, which is a key signaling in plant defense against pathogens . Relative expression was determined as described in Pfaffl (2001) [Ct). Three replicates were performed with at least eight plants per genotype/condition with similar results. The normalized data are provided in Total RNA extraction and cDNA generation were performed as described by Crespo-Salvador et al., (2018) . Quantitl (2001) . ExpressExtraction, quantification, and determination of the SlymiR11273p expression levels were performed as described by López-Galiano et al. (2018) . The norS. lycopersicum leaves (2 g) were harvested and cross-linked with formaldehyde by the vacuum infiltration method [2 and ground into fine powder. The chromatin was extracted following several centrifugation and cleaning steps [Both protocols are described by Crespo-Salvador et al. (2018) . Brieflyn method . Tissue ng steps  before bProtein G-Dynabeads  were used along with chromatin to perform immunoprecipitation. Three main incubations were performed: first the pre-cleaning of both dynabeads and chromatin; second, the binding of antibodies with chromatin; third, the binding of the complexes formed in the previous incubation with dynabeads. Several cleaning steps with growing stringency buffers were performed to clean the sample of nonspecific binding particles. Finally, specifically bound chromatin was released from the dynabeads and its DNA was purified with the GeneJET PCR Purification Kit  for further qPCR analyses.The employed antibodies were anti-H3K4me3 , anti-H3K9ac , anti-H3 , and anti-RNAPII . The negative control was performed with no antibody.B. cinerea and P. syringae infection, respectively.The novel primer sequences are detailed in F test (p < 0.05) was performed to determine the equality of variances of each infected and control condition combination. Consequently, a two-tailed Student’s t test (p < 0.05) was evaluated to determine the statistically significant difference between the mean values . For the ratio comparisons, we normalized the data by transforming them into 10-base logarithms and then performing a one-tailed Student’s t test (p < 0.05), as shown in From the ChIP-qPCR signals, which resulted from each antibody and the INPUT samples, backgrounds were subtracted with their corresponding negative controls (no antibody), as described by Haring et al. (2007) . The res


The RNA-binding protein SRSF6 recognizes a purine-rich consensus motif consisting of GAA triplets, and its downregulation in human pancreatic β-cells affects alternative splicing in a position-dependent manner. SRSF6 down-regulation promotes β-cell demise through splicing dysregulation of central genes for β-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic β-cell line EndoC-βH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in β-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic β-cells.In pancreatic β-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene.  Alternative splicing (AS) is a key co- and post-transcriptional mechanism regulating eukaryotic gene expression that determines which transcript and protein isoforms are formed under specific physiological and cellular contexts . More thDiabetes is a chronic metabolic dysfunction characterized by deterioration and loss of the insulin-producing pancreatic β-cells, resulting in hyperglycemia and long-term complications . The twoGLIS3, a susceptibility gene for T1D and T2D development that is also causal for monogenic forms of the disease  in EndoC-βH1 cells, we have previously shown that SRSF6 modulates the splicing of genes involved in different biological processes, such as β-cell survival, insulin secretion, and c-Jun N-terminal kinase (JNK) signaling  with SRSF6-associated AS profiles to identify the mechanisms of SRSF6-mediated splicing regulation in the human pancreatic β-cell line EndoC-βH1. This combined approach unveiled how SRSF6 specifically recognizes its binding sites and promotes exon inclusion, thereby regulating the splicing of diabetes susceptibility genes. These findings disclose a novel layer of regulation in the genetic predisposition to diabetes.SRSF6 gene expression in EndoC-βH1 cells was comparable with HeLa and HEK293 cells (SRSF6 knockdown (KD) using a previously validated siRNA which leads to ≥50% SRSF6 depletion at mRNA and protein level (SRSF6 KD (2) induced the appearance of cross-linked SRSF6–RNA complexes , we first adapted the conditions for EndoC-βH1 cells. The EndoC-βH1 cell line is an established in vitro model that resembles human islet β-cells in terms of gene expression, functionality and metabolic properties . SRSF6 g93 cells . We confin level . WesternSRSF6 KD . Next, womplexes  However,omplexes , lane 5,omplexes , lane 4 omplexes , lane 6.0 mJ/cm2 .CCDC50 gene   data from EndoC-βH1 cells upon SRSF6 KD  which weSRSF6 KD , we obse > 0.05) . The spl > 0.05) . Most AS > 0.05) .Significantly changed cassette exons after SRSF6 KD in EndoC-βH1.Table S1 SRSF6 KD displayed more SRSF6 binding on the regulated exons . To avoid biases in the iCLIP signal that arise from differences in wild-type inclusion levels, we sampled two PSI-matched background sets of randomly selected exons with a comparable PSI distribution in wild-type EndoC-βH1 cells . In lineed exons  and S4A.ed exons  and S4B.ed exons .In essence, our findings suggest that SRSF6 regulates AS in a position-dependent manner. As described for other SR proteins , bindingSRSF6 KD impacted on gene expression , a genome-wide association studies (GWAS) catalog  and two recent publications  selected CEs , two in T2D susceptibility genes (RBM6 and STARD10), and one gene associated with both T1D and T2D (BCAR1)  . The splus study .SRSF6 KD , we found a strong positive correlation between GLIS3 and SRSF6 gene expression  showed a trend in the expected direction, probably reflecting the milder SRSF6 mRNA level reduction upon GLIS3 depletion as compared with the direct SRSF6 KD GLIS3 in d siRNAs , GLIS3 dpression . Importanscripts . Three oSRSF6 KD . For STASRSF6 KD . Finallyfication . As a whsurvival , which mAltogether, we found that SRSF6 acts downstream of GLIS3 and affects the expression of multiple T1D and T2D susceptibility genes via AS changes and/or differential gene expression. The functional impact of these susceptibility genes deserves further molecular studies.LMO7 was particularly interesting. LMO7 is located on 13q22 and harbors an intronic SNP (rs539514) that is significantly associated with T1D . The aim was to shift the splicing of radation . The ASOradation . Importaskipping . This unSequence of the antisense oligonucleotides (AONs).Table S3 GLIS3, a susceptibility gene for T1D and T2D and monogenic forms of the disease  and RNA immunoprecipitation experiments . It doesSRSF6 KD, supporting that the binding of SRSF6 mainly contributes to exon inclusion. This is in line with early in vitro studies reporting that SR proteins bind pre-mRNAs mainly at intronic splicing enhancers and exonic splicing enhancers and recruit spliceosomal components to the neighboring splice sites .We previously reported that SRSF6 regulates a network of AS events in pancreatic β-cells which impact on key processes like β-cell survival and insulin secretion . Importace sites . To datece sites . Moreovece sites , SRSF6 bce sites . Alternace sites . Althougce sites . Surprisproteins . A possiproteins . A primeSRSF6 KD influences the expression of many other splicing regulators, including other SR and hnRNP proteins. Similar to, for example, SRSF1   . AlthougLMO7 because it harbors an intronic SNP (rs539514) that is highly associated with T1D  . EndoC-βSRSF6 as described previously  and interferon-γ  for 48 h, as previously described .The percentage of viable EndoC-βH1 cells was assessed using fluorescence microscopy after 20 min incubation with the DNA-binding dyes Hoechst 33342  and propidium iodide , as described . ViabiliTotal protein extracts were obtained from EndoC-βH1 using Laemmli buffer, and separated on 8% SDS–PAGE. The nitrocellulose membranes were probed using specific primary antibodies, anti-SRSF6  diluted 1:1,000, anti-α-Tubulin  diluted 1:5,000, in 1× TBST  with 5% nonfat dry milk. After overnight incubation with the primary antibodies at 4°C, the membranes were probed for 1 h at room temperature with peroxidase-conjugated secondary antibodies (listed in Table S5). Detection of immunoreactive bands was performed using chemiluminescent substrate  using a Bio-Rad chemi DocTM XRS+ system (Bio-Rad Laboratories). The densitometric values were obtained by ImageLab software (Bio-Rad Laboratories) quantification and corrected against α-Tubulin as loading control, after background subtraction.List of primary and secondary antibodies used.Table S5 6 EndoC-βH1  (dedup function of the bamUtils tool suit (http://github.com/statgen/bamUtil). Marked duplicates with identical random barcodes were removed as they were considered technical duplicates, whereas biological duplicates showing distinct random barcodes were kept for further analysis.Initial quality checks were applied to all reads before and after quality filtering using FastQC  . Reads wfastqc/) . Sample fastqc/) , requirifastqc/)  using STfastqc/)  with twoProcessed read counts from all four replicates were merged into a single file for each strand and subjected to a peak calling step using PureCLIP (version 1.0.0)  with defIn brief, this was performed by clustering all significant cross-link sites closer than 8-nt into regions. Resulting regions that were shorter than 3-nt were removed. Equal-sized binding sites were achieved by either extending regions shorter than 9-nt, or by iteratively splitting up regions larger than 9-nt. In both cases, the binding site center was defined by the position with the highest number of cross-link events (peak summit). We also required each binding site to contain at least two positions covered with significant cross-link sites from the initial PureCLIP analysis. Next, reproducibility was established by requiring support of at least three of four replicates for each binding site, meaning that the binding site harbored more cross-link events from the replicate-specific threshold. This threshold was set to the 20% quantile of the distribution of cross-link events in all binding sites for the given replicate. This procedure yielded a total of 185,266 binding sites.STARD10, including those in the region of the regulated alternative exon. For all following analysis, only binding sites overlapping a single protein-coding gene were retained. On transcript level, a binding site was assigned to one of the four regions: intron, CDS, 3′ UTR, or 5′ UTR. Overlaps with multiple different transcript regions were resolved by applying a majority vote followed by a hierarchical rule, selecting intron > CDS > 3′ UTR > 5′ UTR. For Gene and transcript annotations were obtained from GENCODE (release 29), and filtered for gene support (≤2) and transcript support level (≤3). Binding sites were overlaid with the filtered annotations, and overlapping annotations were removed. Of note, the latter filter excluded most SRSF6 binding sites in the T2D susceptibility gene k-means clustering with three centroids of frequency profiles for top 300 pentamers with highest frequency in SRSF6 binding sites in the CDS. To dissect the binding sequence further, the number of GAA versus UUC triplets was counted in the 49-nt-wide windows and compared with the respective PureCLIP score    on 201-ns/dreme) .SRSF6 KD (siSRSF6) used in the present study were previously published by our group (The five independent RNA sequencing (RNA-seq) experiments of EndoC-βH1 cells exposed to control (siCTL) or ur group  , yielding a total of 4,126 differentially expressed genes. An additional filter on the absolute log2-transformed fold-change (|LFC| > 1) resulted in 106 differentially expressed genes. Genes encoding for splicing regulators were identified based on association with the Gene Ontology term GO:0008380 “RNA splicing”  . Mapped plicing” .SRSF6 silencing. Thus, in the present study only CE events, identified based on junction-spanning reads, were considered for further analyses. An alternative splice event was considered significant if passing below a false discovery rate < 0.05. Potential hits were further filtered by the absolute change in their “percent spliced-in” (PSI) value (|ΔPSI| > 0.05) as well as a log2-transformed sum of junction-spanning reads supporting the event >5 (mean between replicates). This process yielded 1,212 significantly alternatively spliced exons.AS analysis was performed using rMATS-turbo (version 4.0.1)  with defhttp://fasterdb.ens-lyon.fr/ExonOntology/) (Exon types were annotated using the software Exon Ontology  (http://tology/) . The strtology/)  were caltology/) . Non-regFor the overlap of SRSF6-regulated CEs and SRSF6 binding , all binSignificantly alternatively spliced exons were grouped by their decreased or increased inclusion based on their ΔPSI values. The 5′ and 3′ splice sites of the regulated and the flanking up- and downstream exons were used as reference point to span a symmetric window of 200-nt. On each nucleotide position in each window, all exons with at least one cross-link event at this position were counted and divided by the total number of exons. The resulting relative SRSF6 cross-link frequency is shown in SRSF6 KD must show less inclusion in the control situation and thus will show fewer iCLIP cross-link counts , GWAS catalog , and (T1D and T2D susceptibility genes were identified from ImmunoBase (18), and . This yihttps://string-db.org/) was used to identify PPI networks  and the respective information about the exon-exon junction read counts  were downloaded from the GTEx Portal .The gene read counts of the RNA-Seq GTEx version 8 data set  buffer solution (Promega) and stored at −80°C. EndoC-βH1 cells were transfected for 6 h with 50 μM of each AOS using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions. An ASO with a scrambled sequence that has no homology in the human genome was used as a negative control (The sequence of the antisense oligonucleotide (ASO) (Eurogentec) molecules used in this study are shown in Table S3. All ASOs contained 2′- control . The celPoly(A)+ mRNA was isolated using the Dynabeads mRNA DIRECT Kit (Invitrogen), according to the manufacturer’s instructions. The mRNA molecules were recovered in Tris–HCl elution solution and reverse-transcribed using the Reverse Transcriptase Core kit (Eurogentec), according to the manufacturer’s instructions. The quantitative PCR amplification was conducted using IQ SYBR Green Supermix (Bio-Rad) using Rotor-Gene Q (QIAGEN). The PCR product concentration was calculated as copies per μl using the standard curve method , and theSequences of primers used for splicing analyses and quantitative RT-PCR.Table S7 t test or by ANOVA followed by Bonferroni correction as indicated. P-values < 0.05 were considered statistically significant. Statistical tools used for the analysis of iCLIP and RNA-seq are described above.Data are shown as means ± SEM. Significant differences between experimental conditions were determined by paired Supplemental Data 1).RNA sequencing (RNA-seq) data of EndoC-βH1 cells were previously published  and are Supplemental Data 1.The script files contain the custom code to: (1) define reproducible binding sites from the SRSF6 iCLIP data (BindingSiteDefinition.R), (2) analyze the motif content around SRSF6 binding sites (MotifDefinition.R), (3) filter the rMATS output for significantly alternatively spliced exons (AsEventDefinition.R), (4) count crosslink events around regulated exons and draw the RNA splicing map (RnaMap.R). Each script has an associated function script with the required function definitions.


Komagataella phaffii , Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression. Despite highly similar open reading frames in the MUT pathways of the four yeasts, the regulation of the respective promoters varies strongly between species. While most endogenous Pp MUT promoters remain tightly repressed after depletion of a repressing carbon, Hp, Cb and Pm MUT promoters are derepressed to up to 70% of methanol induced levels, enabling methanol free production processes in their respective host background. Here, we have tested a series of orthologous promoters from Hp, Cb and Pm in Pp. Unexpectedly, when induced with methanol, the promoter of the HpMOX gene reached very similar expression levels as the strong methanol, inducible, and most frequently used promoter of the Pp alcohol oxidase 1 gene (PPpAOX1). The HpFMD promoter even surpassed PPpAOX1 up to three-fold, when induced with methanol, and reached under methanol-free/derepressed conditions similar expression as the methanol induced PPpAOX1. These results demonstrate that orthologous promoters from related yeast species can give access to otherwise unobtainable regulatory profiles and may even considerably surpass endogenous promoters in P. pastoris.Methylotrophic yeasts such as  Escherichia coli, Saccharomyces cerevisiae, filamentous fungi, and cells of higher eukaryotes have been widely used as expression hosts since the advent of recombinant protein production. Over the past three decades, the methylotrophic yeasts Pichia pastoris (Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) have emerged as powerful alternatives, enabling high cell density fermentation and simple, pure secretion of heterologous proteins  is only activated at 2–4% compared to methanol induced levels  in H. polymorpha (PHpMOX) shows derepressed expression up to 70% of methanol induced levels, even in presence of glycerol whereas PPpAOX1 is fully repressed by glycerol. Also the promoters of the orthologous genes in C. boidinii  and P. methanolica  were reported to be activated by derepression, reaching up to 70% of methanol induced levels  pathway  and carefully transferring the liquid without touching the pelleted cells. For MeHNL activity measurements, cell free extracts were generated in fourfold replicates from independently grown cultivations of the same strain , resuspending the pellet in 200 µL Y-PER (Thermo Scientific), shaking for 30 min followed by 30 min (3000g) centrifugation. The resulting supernatant was typically diluted at least tenfold for the MeHNL activity measurement [as described in , as previously noticed for extended lengths of the P. pastoris CAT1 promoter  strain, as higher yields have been reported  and subsequently induced with methanol . Inoculation was performed with ~ 10 µL of frozen glycerol stocks . The BMD and BMM minimal media contain 0.2 M/L potassium phosphate buffer (pH 6), 13.4 g/L yeast nitrogen base and 0.4 mg/L biotin and only differ in the carbon source (as indicated above).To avoid clonal variation due to different copy numbers of integrated expression cassettes as well as different integration sites and genomic alterations that can bias expression strength comparisons in Orthologous promoters (GenBank): MA887959, MA887960, MA887981, MA887982, MA887983, MA887984; Codon-optimized MeHNL gene: MA887980.HpFMD, HpMOX, CbFLD1, CbAOD1, PmMOD1 and PmMOD2 genes for functional evaluation in P. pastoris  considerably and upon methanol induction PHpFMD even outperformed PPpAOX1 ca. 2.1-fold.PHpFMD seemed to give also a very weak reporter fluorescence signal under glucose repressed conditions, hinting slight constitutive activity. Expression from the PHpMOX and P. pastoris PAOX1 and PCAT1 was undetectable. In experiments in shake flasks measuring also glucose levels  and Candida antarctica lipase B  and an intracellularly expressed hydroxynitrile lyase from Manihot esculenta (MeHNL)  Fig. . Yields HNL Fig. c than thHNL Fig. a, b. We HNL Fig. . SimilarHNL Fig. , where mPHpFMD was consistently reproducible using four reporter genes , demonstrating that orthologous promoters from related organisms can be valuable tools for protein production even exceeding endogenous promoters.The strong expression from PHpFMD compared to the strongest endogenous P. pastoris MUT promoters. Interestingly, although regulating genes of proteins with high sequence similarity and similar enzymatic function, the respective orthologous promoters show highly divergent sequences from P. pastoris. None of the orthologous promoters tested show clear identity to the P. pastoris genome when performing a BLAST search (using standard parameters) and also alignments to their P. pastoris orthologs did not exhibit clear identities (data not shown). A similar lack of sequence identity between similarly regulated promoters was reported for P. pastoris MUT promoters  did not maintain its tight repression if transferred to H. polymorpha. PPpAOX1 showed in Hp derepression similar to endogenous H. polymorpha promoters , S2 , S3 (screening data), and S4 (list of plasmids and strains).


Aedes aegypti mosquito (AaFEcR), the vector of the world’s most devastating human diseases such as dengue and Zika. The His-Pro-rich segment of AaFEcR was recently shown to form the unique poly-proline helix II (PPII) in the presence of Cu2+. Here, using widefield microscopy of fluorescently labeled AaFEcR, Zn2+- and Cu2+-induced liquid-liquid phase separation (LLPS) was observed for the first time for the members of NRs. The perspectives of this finding on future research on the F domain are discussed, especially in relation to other NR members.The superfamily of nuclear receptors (NRs), composed of ligand-activated transcription factors, is responsible for gene expression as a reaction to physiological and environmental changes. Transcriptional machinery may require phase separation to fulfil its role. Although NRs have a similar canonical structure, their C-terminal domains (F domains) are considered the least conserved and known regions. This article focuses on the peculiar molecular properties of the intrinsically disordered F domain of the ecdysteroid receptor from the  Nuclear receptors (NRs) are a specific superfamily of the most abundant class of ligand-activated transcription factors. These transcription factors play a critical role in the modulation of the expression of target genes by selective binding to appropriate genomic DNA response elements. They are responsible for the regulation of fundamental biological processes such as development, tissue and metabolic homeostasis, cell proliferation, metamorphosis, and reproduction in animals ,2. The aThe N-terminal domain (NTD) is responsible for ligand-independent transactivation AF-1). The most characterized NTDs to date exhibit features of intrinsically disordered proteins (IDPs) ,17,18,19. The mosTropical and subtropical areas stand out in terms of their lush vegetation, various animal species, and changing weather conditions. Most of the world’s population live in these areas and struggle with the health problems that frequently occur there. Environmental conditions are favorable for insect reproduction, and therefore for insect-dependent disease transmission.Flaviviridae family, is Zika virus—first reported in 1947 in Uganda [Plasmodium falciparum sporozoites (formed through fertilization) are transferred to human hepatocytes. Later, merozoites invade human erythrocytes, in turn causing erythocytic schizogony [Caused by one of the 4 serotypes of the dengue virus, dengue is one of the greatest threats in the world. Annually, about 390 million people get infected, with 96 million of them developing symptoms ,25. Mostn Uganda , sequencn Uganda . Until in Uganda . Some pan Uganda . This isn Uganda . Yet anon Uganda . After thizogony . Shivershizogony .The regulation of gene expression requires the balanced orchestration of adjustable complexes of many specific transcription factors, modulators and signal transduction. Modulation of gene expression profiles by NRs is an allosteric process where binding ligands and specific DNA sequences initiate the recruitment of diverse transcription factors, co-activators, co-repressors and elements of the chromatin remodeling machinery to transactivate or transrepress the expression of target genes. Symptoms of dengue, Zika or malaria diseases are extensive and caused by a series of metabolic pathways. Some of them involve transcription factors to maintain the balanced orchestration of gene expression.CREB3 (cyclic AMP-responsive element-binding protein 3), which enhances the transcription of genes involved in secretory pathways, takes part in the cellular response to Zika and dengue virus infections ,36. AfteAnopheles, the expression of antimicrobial peptides (AMPs), i.e., gambicin or attacin, is controlled by the Rel1 and Rel2 transcription factors. Their translocation to the nucleus depends on the activation of the Toll and Imd pathways, respectively [Plasmodium to human, A. gambiae shows an immune response to the presence of the parasite. Disrupted parasite development is achieved by an increased level of reactive nitric oxide (NO). A high level of NO is caused by NO synthase (NOS), the transcription of which is mediated by STAT-A in A. gambiae. Interestingly, the mRNA level of STAT-A is controlled by its ortholog—STAT-B [A. aegypti to dengue infection [P. falciparum parasites in A. gambiae females is linked to physiological processes controlled by 20-hydroxyecdysone (20E), which is a physiological ligand of the insect ecdysteroid receptor (EcR) during egg development [What is interesting is that the parasite itself is able to trigger a mechanism that will enable it to survive in the host’s cells ,39,40. Tectively . In mosqg—STAT-B . The so-nfection . Most ofelopment . The anaelopment . HoweverRecently, liquid-liquid phase separation (LLPS) emerged as a mechanism in which cells organize their interior, separate, and then segregate biochemical processes ,45,46,47LLPS is important for the regulation of various aspects of gene expression, including transcription ,67,68,69A. aegypti EcR (AaEcR) (ca. 107 aa) or the EcR from D. melanogaster (ca. 190 aa), the F domains of mammalian NRs are relatively short  [When compared to the F domain of esidues) . As was esidues) . Althougesidues) .Concerning the F domain role in the functioning of NRs, two papers during the last five years are worth paying attention to. Bianchetti and co-workers showed that the F domain of the glucocorticoid receptor-α (GRα) forms a steric hindrance to the well-known canonical LBD dimer assembly . AccordiA. aegypti mosquitoes have been the main vectors of the world’s most devastating human diseases in recent years, such as dengue, Zika, yellow fever and chikungunya [Ultraspiracle protein (Usp) [A. aegypti (AaEcR) contains one of the longest F domains in known members of NRs  . The EcRs of NRs , and it s of NRs .2+ and Cu2+ ions [2+ and AaFEcR-Cu2+ complexes and their stoichiometry. Coordination of Cu2+ and Zn2+ ions to this domain did not induce any statistically relevant changes in its overall secondary structure [2 and Ac-QQLTPNQQQHQQQHSQLQQVHANG-NH2) contained in the sequence of AaFEcR. The most fascinating result was observed for the interaction between the Ac-HGPHPHPHG-NH2 peptide and Cu2+ ions, which resulted in the formation of the rare and unique polyproline type II helical structure (PPII) [2+ binding-induced formation of PPII—a structure, which had not been reported for any NR superfamily member before. PPII helixes responsible for protein-protein and protein-DNA interactions [It was previously shown that recombinant AaFEcR is mainly disordered with residual ordered secondary structures and takes the conformation of a premolten globule (PMG) . It can u2+ ions . Mass sptructure . Furthere (PPII) . Our finractions ,90,91,92A. aegypti and D. melanogaster, which possess one of the longest F domains, indicates that both N-terminal domains can probably lead to the formation of liquid condensates , the solution remains in a one-phase regime  in a Tris buffer containing 150 mM sodium chloride; next we tested the F domain (70 µM) in a buffer containing different sodium chloride concentrations ranging from 150–700 mM. Finally, the protein at 70 µM was analyzed in the presence of various buffer additives such as glycerol, polyethylene glycol 300 and 8000, ficol, hexanediol , which was approved by the FDA in 2019, is not recommended for people who have not been infected with the virus in the past [A. aegypti may use its intrinsically disordered F domain with the Cu2+-induced PPII helical structure as the platform for multiple interactions with modulatory proteins controlling gene expression under the control of specific ligands. By modulating the availability of metal ions, the LLPS formation could be disrupted in the appropriate signaling pathway. The 20-hydroxyecdysone signaling is strongly believed to be a promising target for the chemical control of malaria vectors [P. falciparum inflammation. Various pathways are under their control, and therefore transcription factors, together with NRs, can be treated as an attractive target to strengthen an organism’s immune response. Deepening the knowledge about each element of the NRs’ structure will reveal the detailed mechanism of their action. A significant part of previous studies of the NRs’ structure was conducted using in vitro methods and recombinant proteins. Single-molecule in vivo studies of full-length NRs, supported by the recent technological advances in live-cell microscopy, should be applied to this new area of science. We believe that detailed studies of the mechanisms of LLPS in the activities of NRs during transcriptional events will significantly contribute to the development of new strategies to combat not only mosquito-transmitted diseases, but also cancer and other pathological disorders.Mosquito transmitted diseases stand out for their universality and scale, but they have still not yet been overcome. There is no successful vaccine against Zika virus. The vaccine against all 4 serotypes of the dengue virus (the past . The RTSthe past ,101. Conthe past . One can vectors . It also vectors . Thus, i


Long non-coding RNAs (lncRNAs) represent a major fraction of the transcriptome in multicellular organisms. Although a handful of well-studied lncRNAs are broadly recognized as biologically meaningful, the fraction of such transcripts out of the entire collection of lncRNAs remains a subject of vigorous debate. Here we review the evidence for and against biological functionalities of lncRNAs and attempt to arrive at potential modes of lncRNA functionality that would reconcile the contradictory conclusions. Finally, we discuss different strategies of phenotypic analyses that could be used to investigate such modes of lncRNA functionality. At the beginning of this century, the central dogma of biology that posits genetic information flow from DNA to RNA to protein was challenged by the discovery of pervasive transcription in the human genome , 2. LongAmple body of research based on a variety of techniques supports the notion that lncRNAs do play biological roles in a variety of biological processes. Below, we attempt to review and summarize the main outcomes of these studies in the context of the different reverse-genetics Fig.  and evolMethods that specifically target an RNA molecule without altering its DNA sequence or transcription represent the most direct means of answering the question of the functionality of an lncRNA transcript per se without the potential confounding effects caused by disruption of an important DNA element or hampering progression of the RNA polymerase complex (see below) Fig. . PhenotyNeat1 by RNAi led to ablation of paraspeckles, suggesting the essential role of this lncRNA in the formation of these subnuclear compartments [VAD belonging to a class of very long intergenic non-coding RNAs (vlincRNAs) using transfected siRNAs has demonstrated importance of this lncRNA for the maintenance of cellular senescence [SPRY4-IT1 has showed the role of this transcript in modulation of apoptosis [ARLNC1 localized in the nucleus and cytosol using either RNAi or ASO technologies has revealed its roles in androgen receptor signaling and growth of prostate cancer cells [DGCR5 lncRNA that also localized in both cellular compartments using a mixture of siRNAs and ASOs has demonstrated involvement of this lncRNA in regulating a number of schizophrenia-related genes [Knockdown of a nuclear unspliced polyA+ lncRNA artments . Knockdonescence . RNAi-mepoptosis . Inhibiter cells . Knockdoed genes . Overalled genes , LncRNADed genes , or LNCied genes . An impoed genes .SPRIGHTLY  resulted in a decrease in anchorage-independent proliferation rate of cancer cells and the rate of tumor growth in a xenograft model [vlinc273 or ASAR6-141, in control of replication timing of that chromosome [Evidence of lncRNA functionality has also come from other experimental strategies, most notably those that create a complete knockout by altering the DNA sequence of an lncRNA locus , 116  and reduced lifespan [Caenorhabditis elegans, knocking out 155 out of 170 annotated long intergenic RNAs (lincRNAs) could associate 23 of those with at least one of the 6 analyzed traits [Furthermore, genome editing has also demonstrated functionality of lncRNAs in whole-animal in vivo studies in different animal models Table . In micelifespan . In nemalifespan . Importalifespan . In Caend traits . Just lid traits . More red traits . Furtherd traits .LOC646329 can regulate proliferation in human glioblastoma cells [Recent strategies based on targeting of transcriptional silencers or activators to specific promoters using the CRISPR/dead (d)Cas9 strategy (CRISPR interference (CRISPRi) or activation (CRISPRa)) have also contributed to phenotypic analyses of lncRNAs –132 , the availability of genome sequences for a multitude of species allows to estimate the ability of a genomic element to withstand natural processes of sequence change or loss during evolution. In other words, the genome of any given species  contains information on a myriad of past survival experiments conducted over millions of years of evolution and comparison of multiple genomes among or within multiple species allows to extract it. Any genomic sequence that changes less than expected from random chance  across genomes of multiple species is widely assumed to represent a genomic feature that contributes to survival even if the function of the latter is unknown. And, indeed, such assumption has been exhaustively validated on exons of protein-coding genes where the general trend of primary sequences conservation across multiple species is very obvious.Neat1 and Malat1 [In contrast, with notable exceptions of some highly conserved lncRNAs such as d Malat1 , in gened Malat1 . Such std Malat1 . Of specd Malat1 , transcrd Malat1 , relativd Malat1 . Specifid Malat1 . Strikind Malat1 . Furtherd Malat1 .Despite the ample body of evidence supporting general biological relevance of the lncRNA class of transcripts reviewed above, derived primarily from studies on cultured cells, multiple in vivo reverse-genetics probes into their function done in vertebrate organisms challenge this conclusion. The first indication that the non-coding genome is dispensable for their survival came in 2004 when Nobrega et al. reported on strains of mice engineered to have deletions of two large non-coding gene desert regions, 1511 kb and 845 kb in lengths, harboring 1243 non-coding sequences conserved between humans and rodents . Mice hoRecently, Han et al. knocked out 12 lncRNAs from the mouse genome, including 9 lncRNAs conserved syntenically in the human genome, 8 located near developmentally important genes, and 4 previously reported to play important biological roles based on studies in cultured cells . DespiteHotair, whose biological significance recently became a subject of debate [Hotair in the mouse genome led to homeotic transformation, derepression of genes including HoxDs, and skeletal malformations [Hotair deletion strain of mice from the Chang’s team and crossed it with animals of a different background [HoxD gene expression and no significant morphological alterations in the progeny harboring the homozygous deletion of the lncRNA [Hotair knockout reported by the Chang’s group, 2 could not be found by the Duboule’s team at all and one was found in a very subtle form and attributed to DNA-dependent events [Hotair knockout on gene expression [Gomafu lncRNA was associated with an anxiety-like behavior in mice where this transcript was knocked down in the medial prefrontal cortex using ASOs [Gomafu in the entire brain showed no difference in the same behavioral tests [In addition to the failure of generating in vivo phenotypes, reproducing those that could be obtained appears less than certain Table . Perhapsf debate , 145. Inrmations . Howeverckground . They foe lncRNA . Overallt events . Furtherpression . In anoting ASOs . Howeveral tests .Linc-p21 lncRNA has been extensively studied due to its involvement in p53 signaling, human diseases and has been reported to regulate various biological processes [cis [trans [Linc-p21 expression, suggesting that DNA enhancer elements in the Linc-p21 locus rather than the transcript itself are responsible for this effect [Hand2as lncRNA showed right ventricular hypoplasia and embryonic lethality phenotypes, thus associating this transcript with heart development [Hand2as gene body and promoter regions with the CRISPR/Cas9 technology found only the former to have heart-related defects, thus arguing that the phenotypes were caused by the DNA locus rather than the lncRNA [To further compound the problem with in vivo phenotypes, those initially believed to associate with lncRNAs can actually be caused by different mechanisms. For example, the rocesses , 147–149ses [cis  or transs effect . In anotelopment . HoweverXist, H19, roX, Neat1, Malat1, and Hotair are perhaps the most well-studied lncRNAs accounting for at least 4500 records in PubMed. For example, in the case of Malat1, a number of studies using cultured cells associated this lncRNA with the regulation of gene expression [Malat1 knockout mice and found the homozygous knockouts to be viable and fertile, with no obvious phenotypes or histological abnormalities, including no obvious defects in nuclear speckles where this lncRNA localizes. Furthermore, despite significant evidence of involvement of Malat1 in hypoxia response and specifically in renal ischemia-reperfusion injury, no discernable in vivo effect of the lncRNA on that condition was observed in a mouse knockout [Malat1 in brain tissues after ischemic stroke could be observed; however, no RNA rescue experiments have been conducted in those studies [Malat1 in vivo studies). In summary, strikingly, even among those transcripts, clear and uncontroversial evidence of biological function in vivo exists only for very few  against the lncRNA  present . Strikin present . Further deleted . Injectinockdown . The proenotypes .Furthermore, it is not clear which knockdown method is more specific. Unfortunately, non-specific effects are not limited to siRNAs; shRNAs have also been shown to have them, partially through interfering with the immune response and miRNA levels in the cell . By compcyrano or megamind lncRNAs produced similar phenotypes [Theoretically, targeting multiple sites within the same transcript should increase the reliability of assigning the phenotypes to the transcript. Multiple studies using siRNAs or ASOs rely on this strategy to account for the off-target effects, with as many as 5 independent ASOs per transcript . Presumaenotypes , these Menotypes , 167. StAirn, located in a well-characterized imprinted locus, is antisense to Igf2r and was believed to function by silencing this protein-coding gene [Airn lncRNAs showed that the overlap of the lncRNA transcription with the Igf2r promoter was responsible for the silencing and excluded a role of the lncRNA in this phenomenon [Multiple studies are pointing to the fact that an lncRNA locus may not necessarily function only via the transcript itself. For example, using genome-editing techniques, Engreitz et al. found that of the 5 lncRNA loci that can influence expression of the neighboring genes, none in fact required the transcripts to mediate this effect . Insteading gene , 175. Hoenomenon . These fWhile it is hard to estimate the fraction of lncRNAs whose reported phenotypes are affected by the issues described above, the consistent emergence of reports pointing to the problems with functional studies of lncRNAs suggest that this fraction might be significant. Moreover, the recent studies point to the fact that a true in vivo phenotype  of any given lncRNA knockout at least in vertebrates would likely be subtle if at all observable. This brings a natural question of whether these transcripts can be functional at all and, if so, how these functions can be reconciled with the abovementioned phenotypic studies. Below, we will review studies that potentially point to possible modes of biological functionality of lncRNAs that could in turn explain the controversial results described above.ANRIL, believed to function by regulating multiple genes in trans [RP11-634B7.4 have been associated with severity of pre-treatment pain in head and neck cancer patients [A hint to a mode of lncRNA functionality could potentially come from the genome-wide association studies (GWAS). The meta-analysis of these studies shows that most of the phenotype-associated polymorphisms lie in the non-coding parts of the genome  and theiin trans , 181. Sipatients . Furtherpatients .In such scenario, the small effect sizes typically observed in GWAS studies would be consistent with the subtle phenotypes of lncRNA knockouts. In this model, each lncRNA would contribute a small effect, yet due to their vast numbers, resulting in a significant cumulative biological impact of these transcripts .lncRNAs are well known to have highly cell-type-specific patterns of expression, much more so than protein-coding mRNAs. As shown by the ENCODE consortium, only 10% of lncRNAs were constitutively expressed as compared to 53% of mRNAs based on expression analysis across multiple human cell lines –187. AnaThe restricted expression feature of lncRNAs fits well with the abovementioned results of the CRISPRi phenotypic screen where 89% of the positive lncRNAs displayed the phenotypes exclusively in one cell type . ObviousCdk2, Cdk4, and others were found to be non-essential for survival in vivo [Cdk2 and Cdk4 caused embryonic lethality, demonstrating that Cdk2 and Cdk4 function redundantly to couple the G1/S phase transition to mitosis [Snx1 and Snx2 are viable and fertile, while the double mutant is embryonic lethal, indicating that these genes have essential yet redundant functions [Functional redundancy of genes is a strategy formed during evolution to counter adverse effects of mutations in genes encoding critical molecular components , 189. An in vivo , 193. Ho mitosis . Similarunctions . The PINunctions , 197, whunctions .Drosophila roX1 or roX2 genes is essential for survival, while the double mutant is male lethal [Malat1  [Neat1 in paraspeckles of the mammary gland luminal epithelial cells and essential for mice mammary gland development and lactation [Neat1 was also found to have in vivo effects under some other specific conditions inducing expression of this lncRNA [Neat1 can be induced by activation of p53 and ablation of this lncRNA can lead to impaired tumorigenesis in mice [The phenotype of a given lncRNA can be outside of the scope of the tests performed on the knockout animals. For example, ed cells , 199. Kned cells , 199, wh numbers . On the resolved . Later, e below) . In the actation . Later os lncRNA –57. For  in mice .C. elegans lncRNA knockouts [Special consideration has to be given to a possibility that a mutant phenotype may become apparent only in natural conditions as revealed by behavioral analyses of BC1 knockout mice . The mutnockouts . Only exnockouts .Neat1 and BC1  and other lncRNAs either in specific cell types or under specific environmental conditions, it is quite possible that some viable and fertile lncRNA knockout animals may harbor yet undiscovered issues associated with the absence of these transcripts.In summary, even in the protein-coding gene realm, it is common for a knockout animal to have either no observable phenotype or a phenotype revealed only under certain environmental or genetic conditions . AlthougBased on the examples described above, it would perhaps not be an exaggeration to state that for many if not most lncRNAs the authenticity of the reported phenotype-transcript associations in any system is still ambiguous. It is fairly clear that the main reason for it lies in the issues with the currently used reverse-genetics methods and strategies described above  that can be programmed to target a specific transcript via agRNA specifically designed against the latter [MALAT1 and XIST [First, development of new RNA-targeting knockdown methods with significantly reduced non-specific and off-target effects. One such promising approach is represented by the newly developed CRISPR/Cas13 system from e latter  and XIST . Furtherand XIST . Finallyand XIST . This feand XIST  and thusOur group applied the CRISPR/Cas13 technology to investigate the functionality of vlinc class of nuclear lncRNAs in a large-scale setting . For eacAnother promising new approach is insertion of self-cleaving ribozymes into lncRNA sequences Fig. . In factHand2as case above, a phenotype truly associated with an lncRNA should be found in deletions of both the gene body and promoter, while the lack of concordance likely indicates transcript-independent functions [BGLT3 lncRNA and found a reduction in transcription of the γ-globin genes that could be rescued by overexpressing BGLT3 in the knockout cells [BGLT3 transcription and found a reduction of the γ-globin transcription that could not by rescued by overexpressing BGLT3 in these cells [BGLT3 transcription and transcript can upregulate transcription of the γ-globin genes [Haunt downregulated the HOXA gene cluster, while depletion of the Haunt transcript by RNAi, polyA insertion, or promoter deletion upregulated the HOXA genes [ Haunt transcript via knock-in into the original genomic location in the background of the homozygous Haunt deletion mutant could not rescue the downregulation of the HOXA expression [Haunt genomic locus contains DNA elements with potential enhancer functions for the HOXA genes while the lncRNA can potentially silence them [Second, the realization that a combination of multiple approaches and/or mutant alleles is needed to fully understand the root cause of a phenotype Fig. . For exaunctions , 101. Inunctions . They fiut cells . The autse cells . Taken tin genes . A DNA lXA genes . Howeverpression . The autnce them .linc-HOXA1, only the former method affected a specific subset of linc-HOXA1 RNA molecules—those associated with sites of transcription—and led to the cis phenotype of suppression of the nearby Hoxa1 gene expression [In summary, a single reverse-genetics method is unlikely to yield a conclusive answer as to lncRNA biological function especially if it does not explicitly target RNA, calling for a combination of multiple approaches and careful analysis to separate different possible causes of the observed effects. However, caution should also be exercised when interpreting inconsistent results from different experimental approaches because each reverse-genetics method might target unique pools of transcripts derived from the same locus. For example, while both ASO- and RNAi-mediated knockdowns resulted in similar levels of depletion of the lncRNA pression .Fendrr lncRNA expression in the corresponding knockout rescued the majority of the abnormal phenotypes in the heart and body wall development [Firre lncRNA in the Firre knockout mouse rescued the defects in hematopoiesis and alterations in gene expression [H19 knockout mice previously attributed to the cis effect of the H19 locus on the local chromatin environment [H19 transcript itself [Xist and Neat1 have multiple isoforms, which may possess different functions [cis-regulatory lncRNAs which could be numerous in the human genome [megamind and cyrano lncRNAs were proven to be functional via RNA-based rescue [Still, presence of several DNA elements with different functions overlapping an lncRNA transcript—not an unlikely scenario—could theoretically mislead interpretation of even carefully constructed genome-editing experiments Fig. . As suchelopment . Likewispression . More inironment , 33 weret itself  . In another example, expression of the Firre lncRNA is the highest in the hematopoietic stem cells and, as expected, knockout of that lncRNA in mice caused defects in the hematopoiesis [A phenotype un-ambiguously attributable to an lncRNA in an animal model would always hold the key to answering the question of whether the lncRNA has biological function. However, obtaining such a phenotype is extremely challenging, not in a small part due to a highly restricted expression pattern of a typical lncRNA that makes obvious, global defects caused by its knockout less likely. Indeed, the phenotype(s) would likely associate with the cells or tissues expressing the lncRNA Fig. , as exemopoiesis . Still, opoiesis , GTEX [2opoiesis , TCGA [2opoiesis ), mouse opoiesis ) or bothopoiesis , 215), aOn the other hand, functional studies on cultured cells are unavoidable in a number of scenarios and also offer a number of advantages compared to the whole-animal studies Fig. . First, LINC00948 was later realized to represent an mRNA encoding a 46-amino acid micropeptide myoregulin [Finally, it should also be realized that an lncRNA may in fact encode short peptide(s) and thus represent an mRNA. Moreover, such peptides can have biological functions as revealed by the phenotypes of the corresponding knockouts. For example, a spliced human transcript originally annotated as the lncRNA oregulin . In vivooregulin . The numoregulin , 219. SiPerhaps the major challenge in the lncRNA field is to prove beyond a reasonable doubt the biological significance of these transcripts not only in cultured cells but also at the organismal level. As of now, in vivo phenotypes in reverse-genetics studies appear rather subtle and/or highly redundant for most of these transcripts. However, the challenges posed by these negative outcomes may also represent opportunities as we might be getting hints as to the actual modes of functioning of lncRNAs in vivo. However, novel experimental methods and strategies have to be adopted to match these challenges and to resolve the debate about the functionality of this fascinating class of RNAs.Additional file 1. Review history.