Insulin Receptor (InR) expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO). We have finely characterized this transcription factor binding sites in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, we have tested the effect of mutations in the characterized dFOXO sites of D. melanogaster in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription.The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. Here we have focused on the  Drosophila, Caenorhabditis and mammals. In Drosophila melanogaster, INR constitutes the first step of the insulin/TOR signal transduction pathway. The expression of the InR gene is controlled by a set of three distinct promoters  spread over a rather long region [InR transcription was initially considered to be stable [InR expression from P1 is actually more complex as it is influenced by four intronic enhancers, with two of them activating transcription in response to dFOXO and the other two repressing it [The insulin receptor (INR) of the insulin/TOR signal transduction pathway is one of the key sensors of nutrient availability that plays an important role in the control of cellular proliferation, cell size determination, and the response to nutrient availability in metazoans. This pathway is critical for determining body and organ size ,2—via reg region  —a homologue of Caenorhabditis elegans DAF-16 and a mammalian orthology group that includes FOXO4 and other duplications  [DAF-16 family-member Binding Element (DBE) and the Insulin Responsive Element (IRE) that had been characterized in the mouse, where their consensus sequences are 5’-TTRTTTKK and 5’-TTRTTTAC, respectively [D. melanogaster, the bioinformatic analysis of dFOXO targeted regions by ChIP revealed a certain enrichment in FORKHEAD (FKH)-like motifs [in vivo [In d FOXO6) . This red FOXO6)  and the ectively –13. In De motifs  as well e motifs . The bin[in vivo ,16. Eache. g., body size and life span) exhibit, nevertheless, differences even between closely related species of the Drosophila genus [InR P2 promoter could influence its rate of transcription and consequently, the number of INR molecules that can bind dILPs. This differential binding could underlie the observed differences in the multiple traits affected by the insulin/TOR pathway in Drosophila.The insulin/TOR pathway is highly conserved across the animal kingdom. The traits affected by this pathway , stabilnhancer; . There inhancer; .D. melanogaster genes and summarized in the DNase I footprint database that does not contain TFBSs for the InR gene [D. pseudoobscura and in species of the melanogaster group of TFBS turnover in the well characterized eve stripe 2 enhancer of D.melanogaster [cis-regulatory evolution is the proper alignment of non-coding sequences. This problem has generally been minimized studying closely related sibling species such as D. melanogaster and D. simulans [Adh expression [The high turnover of transcription factor binding sites (TFBSs) through evolutionary time  and the InR gene  and ii) nogaster ,32. One simulans ,28. Evenpression .InR gene in D. melanogaster and tested their functionality in transgenic flies. Moreover, the extremely high conservation of the FORKHEAD (FKH) DNA Binding Domain (DBD) of FOXO proteins across the animal kingdom and particularly across the Drosophila genus, has allowed us to use the D. melanogaster dFOXO protein to experimentally characterize the dFOXO binding sites in the 1.3 kb fragment upstream of the InR promoter P2 in four species across the Drosophila phylogeny: three species of the Sophophora subgenus—D. simulans, D. yakuba and D. pseudoobscura—and one species of the Drosophila subgenus—D. virilis. Our work has revealed that binding sites at the downstream  half of the D. melanogaster 1.3 kb fragment have a higher effect on InR expression from P2 than those at its upstream  half. It has also revealed i) that the number of binding sites in footprinted sequences varies across the 1.3 kb region of any particular species, and ii) that their distribution differs among species. Our results would therefore suggest that the uneven distribution of dFOXO binding sites across the 1.3 kb fragment detected in D. melanogaster might underlie the different contribution to InR expression from the P2 promoter of sites at the proximal and distal halves of the 1.3 kb fragment in this species, and possibly also across the Drosophila genus.DNase I footprinting leads to the identification of regions of local DNA protection from DNase I cleavage and it is the method of choice to identify TFBSs at the base-pair resolution level  with itsInR expression in D. melanogaster, we finely characterized the InR P2 promoter region. DNA footprinting of the 1.3 kb fragment upstream of this promoter was performed using several overlapping fragments (300–500 bp long) covering this region. Each strand of these fragments was FAM labeled and subsequently incubated with either dFOXO or BSA. dFOXO binding sites are protected from DNase I cleavage, which results in clusters of protected residues in each FAM labeled strand that can be identified by a DNA analyzer machine. Automated footprinting of the 1.3 kb region upstream of P2 revealed that dFOXO produces 18 footprints in this region, which include a total of 444 bp. The heterogeneity detected in the number of protected residues within and outside footprints  would support the threshold used to consider a residue protected , Dwt_Pmut , Dmut_Pwt  and finally mut_ChIP . In thesulators . In the sulators  set the base.org ) neithereGFP, InR and eIF-1A have efficiencies ranging from 0.98 to 1.05 (eGFP) gene expression relative to a reference gene. In the four lines with mutations in the transgene, the relative expression level of the eGFP reporter gene . The significantly more reduced expression detected in the Dwt_Pmut than in the Dmut_Pwt line points to a lesser role of the dFOXO binding sites located in the distal half of the 1.3 kb InR P2 upstream region on levels of expression from this promoter. Moreover, the significantly more reduced expression detected in the Dwt_Pmut line than in either the Dmut_Pmut or mut_ChIP lines would suggest that in the latter lines the effect of mutations in the distal half of the 1.3 kb region might compensate for the effect of mutations in its proximal half.Amplicons for  to 1.05  as requiter gene  is greatD. melanogaster, D. simulans, D. yakuba, D. pseudoobscura and D. virilis–revealed their identity except for one amino-acid difference in D. virilis. The high conservation detected allowed us to use the D. melanogaster protein for the automated footprinting of the 1.3 kb region upstream of the InR P2 promoter in each of the other four Drosophila species that exhibit increasing times of divergence to D. melanogaster. The number of footprints produced by dFOXO in these four species is 19, 19, 14 and 17, respectively  of the 1.3 kb fragment confirmed these results except for the distal part of D. pseudoobscura sequence that is inverted in this species. Footprints and binding motifs in reliably aligned regions can be considered homologous but not those in other regions. Homologous footprints are numbered according to the D. melanogaster footprint number . In D. virilis, only two footprints (V1 and V15) can be confidently aligned with all other sequences, and V15 is also present twice (labeled V15 and V15’).The multiple alignment of the 110-aminoacid dFOXO DNA-binding domain sequences  of the fectively . These fer group . In D. pT search . The Mult number . In the melanogaster group, nine footprints  have DBE motifs in the three species but only in five footprints  are these motifs aligned in all three. Relative to D. melanogaster, four and eight of the footprinted regions exhibit an additional DBE motif in D. simulans  and D. yakuba  respectively, whereas three in D. simulans  and six in D. yakuba  exhibit one less. In D. pseudoobscura, DBE motifs are present in the six alignable footprinted regions  and in four of the eight non-alignable footprints . Also in D. virilis, they are present in the three alignable footprinted regions  and in ten of the fourteen non-alignable footprints . Furthermore, and as shown in In order to characterize the binding sites of the different footprints, we searched for the presence of DBE core motifs as well as of FKH consensus sequences . In the melanogaster group, the central part of this region exhibits the lowest density of motifs  and three proximal footprints (M1 to M3) flanking the TATA box. It should be, however, added that footprints M14 and M15 are in the region that had been previously reported to be bound by dFOXO in vivo [The identification of the TFBSs contained in  in vivo ,16 whereInR gene . Previou in vivo ,11 but f in vivo  of the d in vivo , such vaD. melanogaster, FKH core sequence TRTTK is present not only in some footprints with DBE but also in some footprints lacking this element. The distribution of DBE and FKH core motifs is not homogeneous across the 1.3 kb region. Indeed, its most proximal (~0.65 kb) and distal (~0.26 kb) parts exhibit higher densities of DBE motifs  its proximal part that encompasses the TATA box can be reliably aligned throughout the five species, ii) the distal part of the D. pseudoobscura sequence can be reliably aligned when reverse-complemented given that it is inverted in this species, and iii) the homology revealed by the MultiZ alignment (http://genome.ucsc.edu) across the five species at the beginning of the second exon of the nc-RNA gene that is present ~300-bp from the 1.3 kb distal end.The characterization of DBE motifs by DNase I footprinting upstream of the D. pseudoobscura to 36 in D. virilis. These motifs distribution is rather uneven both within any given species and among species. The core motifs detected in footprinted sequences are most similarly distributed in D. melanogaster and D. simulans. In these species as well as in D. yakuba and D. virilis, it is the central part of the 1.3 kb region that exhibits the lowest density of motifs  used in the initial Drosophila genome projects [InR promoter 2 upstream region as well as the orthologous sequences of the dFOXO DNA-binding domain were obtained from FlyBase (http://flybase.org) [The projects –43 were ase.org) .InR P2 promoter was amplified from purified DNA  using the AmpliTaq Gold polymerase , and primers designed to directionally clone the fragment in the pET101/D-TOPO plasmid vector . From each species clone, overlapping 300 to 500 bp long subclones were obtained through the amplification of the corresponding fragment with AmpliTaq Gold polymerase and suitable primers  according to manufacturer’s conditions. Sequencing products were separated on an ABI PRISM 3730 sequencer (Applied Biosystems). All sequences were obtained on both strands and assembled using the DNASTAR package . SequencThe four steps required for automated footprinting  are desc5’-AGGGTTAGGGATAGGCTTACCT) and reverse (5’-AGCGGATAACAATTCCCCTCTA) primers were designed to anneal at the pet101/D-TOPO vector regions flanking the subcloned insert. 5’-6 FAM labeled and unlabeled primers were synthesized by SIGMA . In order to label each fragment at either end, fragments were amplified alternating which primer was 5’-6-FAM labeled. PCR reactions were performed at the same conditions for all fragments . The amplified fragments were purified with QIAquick PCR columns (QIAGEN) and its concentration quantified with Qubit (Invitrogen).For FAM labeling (step 1), forward  and 20nM DNA. Four reactions were performed: two with BSA at either a 600 nM or 1200 nM concentration, and two with dFOXO purified by GenScript  at either of those concentrations. Reactions were incubated at 20°C for 30 min.Binding reactions (step 2) were performed in a 20 μl total volume using a modified version of the Brent (2008) buffer  containing 0.03 Katz units of DNase I  to each binding reaction and posterior incubation at 20°C for 1 min. Reactions were stopped with 40 μl of 0.5 M EDTA and placed on ice. DNA was extracted with phenol: chloroform, chloroform and purified with a QIAquick PCR column (QIAGEN). Digested DNA concentration was quantified with Qubit and diluted to 0.6 ng/μl.DNase I reactions (step 3) were performed by adding 20 μl of DNase I dilution buffer  of each peak across the five replicates of each sample both for standards and experimental DNA, and it then calculates the mean height for each peak. The mean height of all standard peaks in a particular sample was used to normalize the values of mean height of each experimental peak in that sample. The BSA/dFOXO ratio was calculated for each peak. In order to define a footprint, it should be first noted that DNase I does not recognize sequence per se and its cutting rates vary along a given DNA sequence depending on global variation in helix groove width and radial asymmetry, and local variation in phosphate accessibility [Automated footprinting (step 4) was performed adding 3.75 μl autoclaved water, 5.85 μl HiDiFormamide and 0.15 μl 600 LIZ (Applied Biosystems) to 1.25 μl of digested DNA. Five replicates of each DNA sample were run in an ABIPRISM 3730 DNA analyzer (injection time 30 sec. and voltage 3.0 kV) with the Genemapper50_POP7 and DFACE software. Results were analyzed with the Peak Scanner (Applied Biosystems) version 1.0 software package (S1-S25 Datasets and sibility . It shougypsy) elements—effective in preventing position effects—flanking the mini-white gene, and the reporter eGFP gene [D. melanogaster wild type 1.3 kb fragment upstream of the InR P2 transcription start site (TSS), and iii) with different mutated versions of the 1.3 kb fragment. Sequences with changes relative to the wild type were synthesized by Gene Script .The pGreen Rabbit reporter vector that contains insulator  by injection into the attP 51D platform line. For each transgenic line, insert location was verified through PCR amplification—using primers flanking the integration site (at section 51D) and the corresponding insert—, and subsequently sequencing the amplification product. Insert identity was similarly verified through its PCR amplification and subsequent sequencing (Accession numbers LT838819 –LT838822).InR and dFoxo genes (modENCODE Development RNA-Seq in FlyBase) [Real Time RT-PCR was used for gene expression quantification taking into account MIQUE guidelines . Total RFlyBase) . An addieGFP reporter gene in each transgenic line, the expression of two endogenous genes (InR and elF-1A) was also measured. The expression of the InR gene from promoter P2 was determined because the 1.3 kb region upstream of this promoter should bind the same transcription factors (other than dFOXO) than the InR insert in the tested constructions, which would therefore allow to correct for any minor differences in developmental stage that might occur among embryo samples. Endogenous gene elF-1A, was used because of its previously reported high expression stability value [Three technical replicates of transgenic line RNA samples were analyzed in a 7900HT Sequence Detection System (Applied Biosystems). The RNA samples were first diluted to 200 ng / 4.43 μl. Subsequently, either four or three 1:4 dilutions were obtained depending on the construct. In each experiment, the standard curve for each gene was obtained from the pGR line, using four total RNA dilutions  . Amplifity value .D. melanogaster footprinted sequences and those of the other species. Indeed, results from BLAST, allowed us to notice that D. pseudoobscura has a microinversion in this sequence.The MUSCLE software was initially performed to obtain the multiple alignment of the 1.3 kb fragment in the five species. This alignment was refined manually with the help of the outputs obtained from the Align Sequences Nucleotide BLAST utility (NCBI webpage) that was used to search for similarities between the The Peak Scanner (Applied Biosystems) version 1.0 software package was used to display the electropherogram images  and to eOne- and two-tailed t tests as well as ANOVA were performed using the R package .S1 Data(ZIP)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


Adk gene as well as regulatory mechanisms that direct the cell-type and isoform specific expression of ADK have never been investigated. Here we analyzed potential gene regulatory mechanisms that may influence Adk expression including DNA promoter methylation, histone modifications and transcription factor binding. Our data suggest binding of transcription factor SP1 to the Adk promoter influences the regulation of Adk expression.The ubiquitous metabolic intermediary and nucleoside adenosine is a “master regulator” in all living systems. Under baseline conditions adenosine kinase (ADK) is the primary enzyme for the metabolic clearance of adenosine. By regulating the availability of adenosine, ADK is a critical upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. ADK protein exists in the two isoforms nuclear ADK-L, and cytoplasmic ADK-S, which are subject to dynamic expression changes during brain development and in response to brain injury; however, gene expression changes of the  Adenosine kinase  is an evolutionarily conserved phosphotransferase converting the purine ribonucleoside adenosine into 5′-adenosine-monophosphate (AMP) .Wild-type 4 and 14 day old male and female Wistar rats  were used. All rats in the present study were bred and maintained at the local animal center in accordance with the guidelines of the European Federation for Laboratory Animal Science Associations (Directive 2010/63/EU) and with the German Animal Welfare Act. The study was approved by the local animal care and use committee (TS 1/13). Animals were housed in breeding cages under controlled environmental conditions , decapitated, and skulls opened. Whole brain was removed and placed into a cold petri dish containing hippocampus dissection buffer  until preparation. Preparation of cell suspensions was performed as described elsewhere with modifications  according to the manufacturer's instructions with the modification to completely dry the pellet at 56°C for 1 h, and followed by DNase treatment (Life Technologies) to avoid contamination with genomic DNA. First-strand cDNA synthesis was performed using the SuperScript II Reverse Transcriptase Kit (Life Technologies) according to the manufacturer's instructions. Quantitative real-time PCR was performed using the 7500 Fast Real-Time PCR System (Life Technologies) with Power SYBR Green PCR Master Mix (Life Technologies) as fluorescent dye according to the manufacturer's protocol. The following primers were used to selectively amplify each ADK isoform and both isoforms at the same time: AdkRT_1 fw- CCAGAAGCGCTGAGTGAAAAT, rev- GTCTTCGGCCAAGATCTGGT; AdkRT_2 fw- ATGACGTCCACCAGTGAAAAT, rev- GTCTTCGGCCAAG ATCTGGT; AdkRT_3 fw- GTGGCAACCGGTCTCTTGTT, rev- AAACTCTGGCTTTCTCTACCAA; GAPDH fw- GGCTGGCATTGCTCTCAATG, rev- CATGTAGGCCATGAGGTCCA. GAPDH quantification was used as internal reference gene for normalization. Fold differences of mRNA levels were calculated using the ΔΔCt method. No-template controls for each primer were included on every plate and melt curve analysis was performed to exclude unspecific amplification. All PCR reactions were repeated three independent times  from three different cell preparations .For ChIP experiments hippocampi of rat pups derived from one litter were used for preparation and cell suspensions were pooled for fixation. Chromatin immunoprecipitation was performed according to the X-ChIP protocol from Abcam with some modifications. In detail, neuronal cell pellets were cross-linked in 0.8% formaldehyde for 10 min at room temperature. The crosslinking reaction was stopped by adding glycine to a final concentration of 0.125 M and centrifuged at 1000 rpm for 8 min at 4°C. Pellet was washed twice in cold PBS and snap frozen in liquid nitrogen. Cells were lysed in SDS-lysis buffer  for 1 h and lysis was facilitated by passing the lysate through a 0.4 mm needle several times. The lysate was sheared using a Diagenode Bioruptor on high power for 4 × 5 cycles (30 s on-off). Equal amounts of chromatin lysate (2 μg) were diluted 10 times with RIPA buffer  and pre-cleared with Dynabeads Protein G magnetic beads (Life Technologies) for 1 h. Immunoprecipitation was performed overnight with the corresponding primary IgG antibody along with the Dynabeads Protein G magnetic beads. Immune complexes were washed three times with ChIP wash buffer , once with ChIP final wash buffer  and were then eluted from the beads with 120 μl elution buffer . Protein-DNA cross-links were reverted by overnight incubation at 65°C and DNA was extracted using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's protocol. DNA quantification was done by quantitative real-time PCR using the same protocol as mentioned above. The following antibodies were used: 4 μg H3K9ac , 4 μg H3K4me3 , 10 μg H4ac , 10 μl H3S10phK14ac , 4 μg H3K27me3 , 8 μg SP1  and IgG (Millipore). Primers used for amplification of immunoprecipitated DNA are summarized in Table Genomic DNA was extracted from neuronal cell pellets using the QIAamp DNA Micro Kit , followed by bisulfite conversion of 1 μg of genomic DNA using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's protocol. Regions of interest were amplified using the TaKaRa EpiTaq HS Kit  and cloned using the TOPO TA Cloning Kit (Life Technologies) doing blue/white screening. White colonies were selected and grown in LB medium overnight. The plasmid was purified using the GeneJET Plasmid Miniprep Kit  and clones were sequenced by the Sanger sequencing method . Sequences were quality controlled and aligned using the CLC sequence viewer (CLC bio). The following primers were used to amplify bisulfite converted DNA at the promoter region of the first ADK isoform: AdkBB_1.1 fw AGGGATATTTTTGGAGTTTTAGAGAG, rev-TCAAAAAATACCAATCTCCCTATTC; AdkBB_1.2 fw-AATAGGGAGATTGGTATTTTTTGAG, rev-TTCCACCTTAAACTTCTTAAACTTC; AdkBB_1.3 fw-AGTTTAAGAAGTTTAAGGTGGAAG, rev- AAAATCCAAAACCAAAAAACACTAA, and at the promoter region of the second isoform: AdkBB_2.1 fw-AGTTTGAGGTTTTTTAGGTGGTTG, rev ATCCTAATAACCACCTCCCCTTAC; AdkBB_2.2 fw GTTAGTTTTTTTGTTGGTTAGTTAGTT, rev CAATAATTCTCCCTAAATCTACAAACTC.Firefly Luciferase vector  to produce recombinant constructs (pGL4.Adk-500 and pGL4.Adk-500mut). A construct derived from the pGL4.10 vector containing the Herpes Simplex Virus Thymidine kinase (HSV-TK) minimal promoter in front of the Firefly Luciferase gene served as positive control (pGL4.TK). The pRL-TK Vector (Promega) encoding the Renilla Luciferase under control of the HSV-TK promoter was used as transfection control. pGL4.Adk-500 as well as pGL4.Adk-500mut vectors were each co-transfected with the pGL4.TK reporter vector into HEK293 cells, with control plasmids encoding Renilla Luciferase, using Lipofectamine Transfection Reagent . 24 h following transfection HEK cells were incubated for 3 h with different concentrations of recombinant SP1 protein  in the growth medium. Luciferase activities were assayed using the Dual-Luciferase Reporter Assay (Promega) according to the manufacturer's protocol. Thereby Firefly Luciferase measurements were normalized to Renilla Luciferase activity. All Luciferase assays were performed in triplicate.A ~500 bp fragment of the rat Adk-L minimal promoter including transcription start and ATG triplet (start codon) and an analog fragment with mutated SP1 binding sites, both flanked by restriction sites for Nhe1 and EcoRV , were digested with respective restriction endonucleases  and cloned into the pGL4.10 t-test comparisons, one per row . Data from reporter gene assays was analyzed using Friedman statistic. A p value < 0.05 was considered significant. All data is presented as mean ± standard deviation.Statistical analysis was performed using SPSS for Windows version 17.0 and GraphPad Prism6. Differences in gene expression and DNA methylation were compared using Mann-Whitney test, whereas differences in transcription factor binding and chromatin modifications were analyzed using Wilcoxon matched-pairs test. Statistical significance of individual data pairs was further determined using multiple p < 0.05), whereas the short isoform was not affected , phosphoacetylated H3 (H3S10phK14Ac), and trimethylation of lysine 4 or 27 of histone H3  were applied. Following the enrichment step quantitative real-time PCR with Adk-specific primers was performed to prove binding of histones with respective modifications to the Adk promoters. The amplified regions cover large parts of each promoter region  as depicted in Figures We identified histone acetylation of histone H3 and H4 as well as phosphoacetylation at histone H3 at the promoter regions of both Adk isoforms in P4 and in P14 rat hippocampal neurons, especially upstream to the TSS Figures , 3B. AddWe next focused on promoter DNA methylation. The EMBOSS Cpgplot tool was used for CpG island prediction. Two consecutive CpG islands were identified around the TSS of the long Adk isoform enclosing a region of 755 bp. Furthermore, a single CpG island of 588 bp was predicted to be located within the presumed promoter of the short Adk isoform Figures , 3A.Harvested DNA from P4 and P14 rat hippocampal neurons was used for amplification (subsequent to bisulfite treatment) and sequencing of the rat Adk promoters including 5′ untranslated region (UTR) and first exon of long (Pos. −588 to +298) and short isoforms (Pos. −391 to +293), respectively. However, both promoters were unmethylated in P4 as well as in P14 rat hippocampal neurons  along the promoter identified significantly increased SP1 binding at position −102 to +50 relative to the TSS in P14 neurons . This region covers the three putative SP1 binding sites  containing a CpG island and transcription factor binding sites . Its coding sequence is highly conserved in vertebrates. The rat Adk gene, as all known mammalian Adk genes, consists of 11 short exons and exceptionally large introns. There are two ADK isoforms, long and short (ADK-L and ADK-S), expressed in mammalian cells  and stable astrocytic expression (P14) of Adk. Epigenetic gene regulation is very complex, which ensures nuclear processes  to be directed to the required region of the genome at appropriate time points mediating unique cellular responses and biological outcomes. This same complexity is what makes analysis and interpretation of epigenetic modifications difficult. However, some signals are more understood than others, e.g., promoters of active genes are commonly associated with acetylated H3K9 (H3K9ac) and H4. Likewise, phosphoacetylation of histone H3  concomitant with gene activation is well established and has been observed at several inducible genes  in close proximity to the transcriptional start site (TSS) of the long Adk isoform. SP1 binds with high affinity to GC-rich motifs and regulates the expression of a large number of genes involved in a variety of processes including cell growth, apoptosis, and differentiation  of adenosine, to control Adk gene expression. Such archaic mechanisms might be meaningful, because the long Adk primary transcripts  sequester significant amounts of adenosine, suggesting that Adk gene transcription might be regulated by the availability of adenosine, an interesting possibility that warrants further exploration.KKo conceived experiments, performed statistical analysis, and prepared figures. KKi and JJ performed experiments and prepared figures. All authors  wrote the manuscript.Our work was supported by the German Research Council (DFG Bl 421/3-1) as part of the European Science Foundation EUROCORES Programme EuroEPINOMICS , by the European Union's Seventh Framework Program  as well as through NIH grant NS084920 (DB).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Furthermore, RNA pull-down assay indicated the decreased level of RB1 induced by Linc00441 was associated with the incidental methylation by DNMT3A recruited by Linc00441. On the contrary, the transcription factor (TCF-4) enhanced H3K27 acetylation and direct transcription factor for Linc00441 was responsible for the upregulation of Linc00441 in HCC. In conclusion, the epigenetic interaction between Linc00441 and bidirectional transcripted neighbor RB1 may be a de novo theory cutting-point for the inactivation of RB1 in HCC and may serve as targeting site for tumor therapy in the future.The retinoblastoma gene (RB1), a known tumor-suppressor gene (TSG), was decreased in multiple cancers including hepatocellular carcinoma (HCC). Here we focused on the bidirectional transcripted long noncoding RNA (Linc00441) with neighbor gene RB1 to investigate whether Linc00441 is involved in the suppression of RB1 in HCC. We found that aberrant upregulated intranuclear Linc00441 was reversely correlated with RB1 expression in human HCC samples. The gain- and loss-of-function investigation revealed that Linc00441 could promote the proliferation of HCC cells  Annually, 748 300 new liver cancer cases were diagnosed and 695 900 cancer deaths occurred worldwide. Unfortunately, nearly half of these cases and deaths were estimated to take place in China.5 These are considered as non-protein-coding transcripts longer than 200 nucleotides and widely expressed in various organs as well as tissues of the body.7 Depending on their proximity to the nearest protein-coding transcripts, lncRNAs can be classified into the following categories: sense, antisense, bidirectional, intronic and intergenic lncRNAs.9 In addition, it has been reported that various carcinogenic processes, such as proliferation, apoptosis, invasion and metastasis, were closely associated with the aberrant expression of the lncRNAs.11 LncRNA dysregulation may promote these process through modulation of chromatin remodeling, epigenetic modification, regulation of gene transcription, post-transcriptional mRNA processing, protein function and localization, and intercellular signal transduction.13 However, the functions of most annotated lncRNAs in liver diseases remain unexplored.In recent years, more attention has been paid to investigating on long noncoding RNAs (lncRNAs).14 When cells are stimulated by growth stimulation signals, the induced cyclin D/cdk4 can contribute to phosphorylation of RB1, which leads to the release of E2Fs from RB1-E2Fs complex and S phase entry.17 The inactivation of an upstream RB regulator (CDNKN2a/INK4a/p16) selectively occurred in these RB+/+ tumors resulting in almost 100% inactivation of the RB/p16 pathway in these tumors.18 Whether there are some other mechanisms participating in RB1 pathway inactivation is unclear.The retinoblastoma gene (RB1) is the first identified tumor-suppressor gene (TSG) that is frequently inactivated in several major cancers. Researchers have revealed that the pRb protein has a key role in cell cycle regulation, particularly during the G1–S transition.19 Among human genome, nearly 11% genes are arranged as bidirectional gene pair in the pattern of head to head, divergent manner and controlled by bidirectional promoters.21 Majority of bidirectional promoters regulate the co-expression of a bidirectional gene pair, whereas a minority of bidirectional promoters induce the transcription initiation of one gene while inhibiting transcription in other direction.20 In addition, a larger part of the two genes sharing the bidirectional promoter include lncRNAs and protein-coding genes,19 which indicated that lncRNAs may modulate the expression of neighbor protein-coding genes in a bidirectional transcription fashion. Owing to Linc00441 and RB1 having a bidirectional promoter, we hypothesis that Linc00441 could regulate the level of RB1 protein in HCC carcinogenesis process via bidirectional pattern.Advancement of sequencing and annotation of whole genomes, it has been noted that bidirectional organization of coding genes is common in the whole human genomes.In this study, we aimed to explore the role of Linc00441 in hepatocarcinogenesis process such as cell proliferation and apoptosis. In addition, we also delineate the definite mechanisms by which the Linc00441 regulate the expression of RB1 and how epigenetic mechanisms influence the overexpression of Linc00441 in the progress of HCC.P<0.001 and R2=0.45). We next analyzed the subcellular location of Linc00441 by probe hybridization in HepG2 and MHCC-97H based on the RNA expression in various cell lines listed in The bidirectional transcriptional location of Linc00441 and RB1 as predicted was presented in in vitro based on the aberrant expression of Linc00441 in different HCC cell lines. After the ectopic expression of Linc00441 in Hep3B cells was confirmed  to investigate whether the RB pathway was concerned. As presented in Supplementary Figure 3, both the p73 and cyclin E could be activated by overexpression of Linc00441 while suppressing by knocking down Linc00441.The remarkable oncogeneic function of Linc00441 from  7 cells , whereas 7 cells . BesidesFrozen sections obtained from the xenotransplantation tumor mentioned above were also used with RB1 staining. We found a aberrant lower fluorescence of RB1 in the slice resected from the Linc00441 overexpressed Hep3B-induced tumor . On the As Linc00441 could induce an inactivation of TSG RB1, we would like to unveil the detailed mechanism how Linc00441 can regulate the bidirectional transcripted neighbor gene RB1. The RNA pull-down assay was further conducted. At first, the biotin-labeled lncRNA and the antisense as control was used followed by mass spectrum validation with protein band collected at the ~120 kDa location. Through the mass spectrum screening, we found that peptide DNMT3A was most significant . Based oRNA immunoprecipitation (RIP) by using antibody targeting DNMT3A was also conducted. We designed two independent primer of Linc00441 during the PCR assay after hybridization (primer 1 targeting the +20 to 300 nt region while primer 2 as control). By comparing with the control groups, we found that the Linc00441 could be bound by DNMT3A .Both RNA pull-down assay and RIP assay revealed that Linc00441 could recruit the DNMT3A protein. We next checked whether the location of DNMT3A was close to Linc00441. The green fluorescently labeled DNMT3A was stained in the same monoplast in both MHCC-97H and HepG2. As presented in In vivo expression of DNMT3A led to hypermethylation in the genome.24 Coincidentally, the anergy of tumor-suppressor function of RB1 was also highly associated with the methylation in the promoter region in multiple human malignant tumors, including HCC.26 After analyzing the correlation between the expression of Linc00441 and the methylation rate of RB1 in HCC samples from matched patients, we found a positive correlation between two parameters  assay. We found that Linc00441high patients displayed a lower stain of RB1, whereas Linc00441low patients exhibited a higher expression of RB1 , which mainly bind with the promoter region of Linc00441 instead of RB1 to check if there may be a special TF involved in the aberrant expression of Linc00441. As predicted, we found that TCF-4, acting as the potential TF for special transcription of Linc00441 with three potential binding sites. The dual luciferase reporter gene assay was conducted, as presented in 30 In cancer initiation and advancement, lncRNA's one of the function is epigenetic regulation.9 For instance, lncRNA may promote methylation of CpG island in the promoter region of a gene via recruiting DNMTs11 or mediates the trimethylation of H3K27 at the promoter region.31 Furthermore, a part of lncRNAs have been demonstrated to perform their function in the methylation process by binding to EZH2, which is the core component of polycomb repressive complex.33Over the past years, many researchers have demonstrated the critical role of aberrant expression of lncRNA in occurrence and progress of several kinds of cancers including HCC.et al.34 have shown that LncRNA HOTTIP could regulate HOXA13 expression in HCC through bidirectional transcriptional pattern. Besides, Imamura et al.35 have demonstrated that LncRNA Khps1 controlled by bidirectional transcriptional manner could influence neighbor gene (Sphk1) expression through manipulating methylation pattern of the Sphk1 CpG island. Similarly, Linc00441 share a bidirectional promoter with RB1. So we explored the specific mechanisms involving Linc00441 regulation with RB1 expression. We discovered that aberrant upregulated intranuclear Linc00441 was inversely correlated with RB1 expression in human HCC samples. In in vitro experiments, we found that overexpression of Linc00441 could decrease the expression level of TSG RB1. In addition, analysis of the subcellular localization of Linc00441 and RB1 further confirms the expression relationship.As described in the introduction, bidirectional transcription may involve in physiological function and progression of diseases. For example, Quagliata 1 phase arrest and inhibit proliferation of cells.37 We have known that p16/p14 is responsible for the control of cyclin D/cdk4 kinase activity. Thus, in the absence of p16/p14, cyclin D/cdk4 activity is elevated, leading to Rb1 phosphorylation and E2Fs accumulation.38 Besides, there are some studies reported that inactivation of the p16INK4A gene, owing to several reasons including allelic deletions of this locus and/or hypermethylation of p16/14 promoter, was found in HCCs.42 In this study, we found that overexpression and decreased expression of Linc00441 may inversely regulate the G1 phase arrest and HCC cell lines proliferation in vitro and in vivo. Besides, we also discovered that Linc00441 can also enhance the ability of HCC cells to resist apoptosis. Taken together, Linc00441 could regulate RB1 expression, cell cycle proliferation and antiapoptotic activity.The retinoblastoma gene (RB1) could regulate cell proliferation in a cell cycle-dependent manner by forming RB1/E2Fs complex, which can repress the function of TF E2Fs, so high level of RB1 will increase the proportion of G>70%), encompassing a wide range of tumor types.44 Various kinds of mechanisms have been identified for loss or inactivation of RB1 gene expression in different types of cancers. For instance, bialleic loss of RB1 and mutations has been recognized in retinoblastoma lesions and lung cancers.46 However, in prostate carcinoma, RB1 gene is inactivated by a deletion within its promoter region.47 Furthermore, CpG island methylation in the promoter of RB1 may result in the inactivation of this gene, leading to oncogenesis.48 As there are more CpG islands in bidirectional promoters than in non-bidirectional promoters. Thus, we hypothesis that lower expression of RB1 in HCC may be due to promoter hypermethylation in RB1 gene through the recruitment of DNMTs by Linc00441. In accordance, we found that increased expression of Linc00441 could enhance the methylation rate in the promoter region of RB1, which could be reversed by DNMTs antagonist 5-aza-2'-deoxycytidine and ASO-DNMT3A. Altogether, Linc00441 may promote epigenetic repression of RB1 gene via promoter hypermethylation. It is well known that DNMT3A is essential for de novo methylation and inactivated DNMT3A may have close relationships with cancers.49 Owing to lncRNA biological role as a scaffold to recruit proteins, which can modulate the gene nearby, afterward, we perform RNA pull-down assay, protein mass spectrometry RIP assay to interact between Linc00441 and DNMT3A. Bioinformatics analysis and subcellular colocalization analysis of Linc00441 and RB1 also confirmed the interactive relation between them.Loss or inactivation of the RB1 genes have been identified in the majority of human tumors (51 Transcription factor 4 (TCF-4), also known as TCF7L2, is a member of the basic helix–loop–helix (bHLH) family of TFs, which could be involved in DNA binding via recognizing an E-box DNA-binding site and enhancing the transcription of genes.53 Chromatin immunoprecipitation (ChIP) sequence experiments in various cells revealed that most of cells have shown the majority of TCF-4 sites colocalized with H3K27Ac.54 Luciferase reporter gene assay confirmed that TCF-4 can enhance the expression of Linc00441 and elevated TCF-4 is associated with H3K27 acetylation detected by Chip-PCR in the promoter region of Linc00441. Besides, researchers have shown that TCF-4 can also promote the expression of cyclin D1 and S phase entry and TCF-4 depletion can render cancer cells sensitive to apoptotic stimulation.56 This may be a synergistic effect with Linc00441 on liver cancer initiation.Since Linc00441 and RB1 transcript in a bidirectional manner, why expression of them have shown a reverse tendency? To answer this query, we have found a 'gap' in the attachment area especially the enrichment of H3K27Ac and H3K4m3 according to the gene map. In addition, active promoter regions contain the histone modifications such as H3K4me3 and acetylation of H3K27 is considered to facilitate DNA unwinding and increase accessibility to TF binding.in vitro and in vivo. TCF-4 and H3K27 acetylation also contributed to Linc00441 upregulation. Collectively, all findings in this study are in favor of understanding the mechanism of lncRNA function in HCC carcinogenesis and advancement, which can enable us to explore novel prognosis markers and potential targets for cancer therapy in near future.In conclusion, our study found that elevated expression of Linc00441 that has an inverse relationship with neighbor tumor suppressor RB1 in human HCC. Linc00441 may decrease RB1 expression through enhanced CpG islands methylation in the promoter of RB1 gene by DNMT3A recruitment, and afterward, causing proliferation of HCC in both Eighty paired fresh HCC tissues and corresponding adjacent noncancerous tissue samples and related clinical information were obtained from the patients undergoing hepatectomy at The First Affiliate Hospital of Nanjing Medical University. The study was approved by our Institutional Ethics Committee. All patients provided their written informed consent to participate in this study. The fresh tissue samples, which were confirmed by the histopathological examination, were collected in the operating room and processed immediately. Each sample was frozen and stored at liquid nitrogen. The clinical characteristics of all patients are summarized in 7 cells/ml stably knocked down for Linc00441 expression in MHCC-97H, HepG2 and Hep3B-overexpressing Linc00441 and respective control cells (Linc00441-NC) were subcutaneously implanted into the left hind limb axillary of 10 BALB/C nude mice. Five weeks later, all mice were killed, the weight and size of the xenografts in each group was processed for further analysis. Animal work was permitted by the Nanjing Medical University animal studies committee.Forty BALB/c nude male mice whose age were about 2 months were randomly divided into six groups including the negative control group, Linc00441 knockdown groups and Linc00441 overexpression group. After acclimating to local environment for 2 weeks, a concentration of 1 × 10According to the manufacturer's instructions of Magna RIP RNA-Binding Protein Immunoprecipitation Kit , antibody for RIP assays of DNMT3A (ab13888) was used. The co-precipitated RNAs were detected by RT-PCR. Simultaneously, input controls and negative controls were performed to ensure that the detected signals were from RNAs specifically binding to DNMT3A.Genomic DNA was extracted from HCC cells, respectively, treated with the Linc00441 expression lentivirus, 5-aza-2'-deoxycytidine, DNMT3A knockdown and their control group cells. After adding sodium bisulfate from the EZ DNA Methylation-Gold Kit  into the DNA samples, we tested the methylation in the promoter of RB1 using the method of bisulfite sequencing PCR.For the stain of Linc00441 by ISH, 97H and HepG2 cells were seeded into small culture dish with glass bottom and fixed in 4% formaldehyde for 15 min next day. Cells were permeabilized in PBS containing 0.5% Triton X-100 and 5 mM vanadyl ribonucleoside complex (10 mM) , washed in PBS and rinsed once in SSC buffer. Hybridization was carried out using a digoxin-labeled  Linc00441 cDNA probes (0.5 μg/ml) in a moist chamber at 37 °C overnight. Linc00441 probe was ordered from the Abnova . After hybridization, cells were incubated with anti-digoxin–rhodamine mAb (Roche) and subjected to confocal microscopic imaging. For colocalization studies, after RNA-FISH, cells were again fixed for 5 min in 2% formaldehyde and subjected to IF staining using anti-RB1 or DNMT3A mAb (Abcam) and then stained with second antibody combined with green fluorescein and DAPI . Cells were then observed with a Zeiss (LSM 780) confocal laser scanning microscope . For ISH in tissues, we choose chromogenic ISH method to show the expression level of Linc00441.In order to determine the location of the RB1, DNMT3A as well as the relationship of one another, we added 0.5% Triton-100 to the frozen sections for 10 min and incubated the sections with the primary antibody respectively(1:200 dilution) overnight at 4 °C. The secondary antibody (red) was Alexa Fluor 546 goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (1/500 dilution) for 1 h at 37 °C. The sections were added with the mounting medium and detected by confocal microscopy (Zeiss).5 cells were plated into 24-well plates. A Renilla luciferase vector pRL-SV40 (5 ng) was also co-transfected to normalize the differences in transfection efficiency. For luciferase reporter gene assay detection, the promoter of RB1 was cloned into pGL4 vector in HCC cells and cells were treated with TCF7L2.The promoter sequence of Linc00441 predicted to interact with TCF7L2, which was inserted into pGL4 promoter vector. A mutated sequence with the predicted target sites was also conducted. In all, 1 × 107–5 × 107 cells were collected and cross-link the proteins to the DNA for 20–30 min with formaldehyde followed by sonication of lysate to shear DNA to an fragment size of 200–1000 bp. Fragment and pre-analyzed chromatin lysate was incubated overnight with the antibody anti-H3K27ac, anti-H3k27m3 and IgG, and protein A/G beads into the samples and incubated overnight at 4 °C. The crosslinking was reversed by incubation at 65 °C for 4 h. The DNA was recovered by phenol–chloroform extraction. The primers were used to detect the human RB1 promoter region by PCR.Chromatin immunoprecipitation was performed by using the ChIP assay kit according to the manufacturer's protocol . Nearly, 1 × 10t-test was used to assess the statistical differences between groups. All statistical data were carried out using Statistical Program for Social Sciences 18.0 software  and presented with Graphpad prism 5.0 . P-value <0.05 was considered as significant.All experimental assays were performed independently in triplicate. Two-tailed Student's Detailed Materials and methods are described in


Recent sequence-based profiling technologies such as high-throughput sequencing to detect fragment nucleotide sequence (Hi-C) and chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) have revolutionized the field of three-dimensional (3D) chromatin architecture. It is now recognized that human genome functions as folded 3D chromatin units and looping paradigm is the basic principle of gene regulation. To better interpret the 3D data dramatically accumulating in past five years and to gain deep biological insights, huge efforts have been made in developing novel quantitative analysis methods. However, the full understanding of genome regulation requires thorough knowledge in both genomic technologies and their related data analyses. We summarize the recent advances in genomic technologies in identifying the 3D chromatin structure and interaction, and illustrate the quantitative analysis methods to infer functional domains and chromatin interactions, and further elucidate the emerging single-cell Hi-C technique and its computational analysis, and finally discuss the future directions such as advances of 3D chromatin techniques in diseases. Although the diverse cell types of an organism share the same DNA information, their genomes undergo quite different structural and organizational changes during differentiation and replication; and such changes affect gene expression and cellular functions via different information routes.In vivo, human genome functions as a folded three-dimensional 3D) chromatin polymer. Nearly half a century ago, gene position on a single chromosome was considered a main determinant of gene activity due to the lack of an advanced profiling technique. The regulatory activities were simplified into a genetic one-dimensional sequence underlying the expression level . The 3D D chromatThrough genome-wide assessing of chromatin interaction and characterizing the global contacts between the regulatory elements and targets, the spatial architecture of the genome is found to be indispensable functional units in the nearly all of the transcription and translation processes ,6,7,8. TFurthermore, huge efforts have been made in developing novel analysis methods to better interpret the data and gain biological insights. However, the full understanding of genome regulation requires a thorough knowledge in both genomic technologies and their related data analyses.In this work, we summarize the recent advances in genomic technologies in identifying 3D chromatin structure and interaction, and illustrate the quantitative analysis methods on processing data and inferring functional domains or chromatin interactions, and further elucidate the newly emerging single-cell Hi-C technique and its computational analysis, and finally discuss the future directions such as advances of 3D chromatin profiling technique in diseases.During cell differentiation and transcription, the human genome functions as a folded 3D chromatin structure. These processes cover the large-scale folding of whole chromosomes or smaller genomic regions, and the reorganization of local interactions between enhancers and promoters, mediated by the binding of transcription factors and chromatin looping. The higher-order organization of chromatin is also influenced by the specificity of the contacts that it makes with nuclear structures such as the lamina .Within condensed chromatin compartments, there exist functionally genetic elements called TAD, serving as a pervasive structural feature in the genome organization. Moreover, the occurring topological domains keep stable across the diverse cell types, and remains highly conserved by cross-species studies, indicating that TADs are inherent and important functional units in mammalian genomes ,11,12.Thus, to uncover how the human genome is spatially organized and condensed within the cell nucleus is a prerequisite to understanding genetic regulation mechanisms in normal differentiation, development, and even dysregulation in disease.Diverse contact structures and models have been raised so far, such as the early proposed active chromatin hub (ACH) related to hypersensitive genomic sites . ACH is TAD is a discretely folded structural entity with specific chromatin activities, normally it takes the size of kilo to mega base pairs. The central regions in TADs have highly frequent chromatin interactions, supporting the viewpoint that TADs dictate tissue-specific genes’ and enhancers’ activities ,19,20. IWithin nucleus, the inner envelope is covered by lamins and other types of proteins, both of which form the nucleus laminas. Nucleus laminas have the key leverage on the spatial organization of chromosomes through contacts with chromatins while sustaining spatial positions on the inner nucleus. Lamina-associated domain (LAD) is a recently identified structural unit associated with condensed chromatin, often bound by nucleus lamina. LADs have a median size of ≈0.5 Mb, with a relatively low gene density where most of these genes are transcriptionally inactive, suggesting that the nuclear lamina has a repressive role in gene regulation. LADs also participate in the two main roles during cell differentiation when constitutive LADs (cLAD) keep attached to laminas, and facultative LADs (fLAD) become disaffiliated from laminas due to the corresponding activated genes. After mitosis, some LADs can return to the nucleolus periphery, which was reported as a sequence specificity although the underlying mechanisms remain unclear ,22.Noticeably, within TADs, there exists two highly dynamic architectures of submegabase scale: (I) smaller-size domains, named sub-TADs ,24,25, aDeciphering the mechanisms controlling chromosome folding and interpreting their roles in gene regulation become the focus in the current epigenetic study. Recent high-resolution microscopy and conformation capture-based techniques have revealed that chromatin has a complicated structure, varying across different organisms and cell types.Until now, sophisticated profiling platforms in mapping chromatin contacts generate high-throughput sequencing data with deep insights into the 3D formation of chromatin interactions, and into their roles in the chromatin organization and cellular function ,27. The PCR amplification-based ligation capture techniques that are mainly composed of the typical 3C-based technique and its extensions, such as Chromosome Conformation Capture on Chip (4C) ,30, and Given the diverse experimental protocols, there requires specific consideration in designing the computational algorithms and pipelines for characterizing 3D chromatin interaction. In this section, we illustrate the basic principle and general pipeline for preprocessing high-throughput profiling 3D data. A typical sequence-based 3D data preprocess procedure includes raw reads mapping, fragment filtering, read-pair filtering, normalization, and downstream analysis . For theIn short, In addition to noticeable advancements in profiling techniques, statistical models and quantitative analyses are a prerequisite in discovering the chromosome folding mechanism, together with its origin and function in diverse cellular activities. In this section, we review recent progresses in chromosome modeling with a focus on computational analysis of identifying chromatin interactions.The Strings and Binders Switch (SBS) model was introduced based on the hypothesis that the chromatin conformation occurs during the interaction of specific binding sites with diffusing binding factors. SBS model has shown that it can recapitulate scaling and dynamic folding properties of chromosome organization in the spatial and temporal dimensions ,38.To reveal the causal underpinnings of complex diseases, there is still an open challenge to distinguish the gene targets of a distal regulatory element from other adjacent coding genes. Whalen et al. presented a computational method, TargetFinder, to reconstruct the regulatory landscapes from multiple genome-wide features . It was Because 3D genome structures are highly plastic and diverse among cells even in an isogenic sample, it is still a major task to inferring structure-function linkages. Recently, based on ensemble-averaged and single-cell Hi-C data, Dai and their colleagues reported an approach to comprehensively identify 3D chromatin clusters occurring frequently across a population of genome structures . At the To assess the chromatin domains and their positional association, Molitor et al. introduced multiscale correlation evaluation (MCORE), based on the fluctuation spectrum of mapped sequencing reads to quantify and compare chromatin patterns with diverse scales. Through integrating multiple sources from chromatin immunoprecipitation, RNA expression, DNA methylation, and 3C experiments, the approach was claimed capable of revealing the positional relationships on different genomic scales .Despite plenty of cis-regulatory sequences annotated, it is still challenging to identify their target genes in the human genome. Previous strategy is to profile the long-range looping interactions for those elements with 3C-based techniques, but they lack either resolution or coverage depth satisfying the whole-genome and precise capture of chromatin interactions.Ren and his colleagues reported a comprehensive chromatin interaction map at 5–10 kb resolution for human fibroblasts using a genome-wide Hi-C sequencing technique . ThroughGenetic transcription activities depend on tissue-specific chromatin architecture and flexible localization among the involved transcription and regulation elements. The inherent organization and coordination mechanisms are still under investigation. Bortle et al. explored recent findings, and focused on highly conserved multiprotein complexes composed of insulator and Polycomb group proteins, which were identified with functions in interceding long-range interactions and nuclear organization . FurtherBoth the well-established light microscopy-based cell-imaging and most recent molecular 3C-based techniques provide researchers with the unprecedentedly precise insight into human genomics. The human genome exists as a stereoscopic entity within the nucleus, and lineage-specific transcriptional activities related to cell identity and fate are performed under the 3D context. Molecular 3C-based techniques have many unique advantages in 3D chromosome conformation studies, which make it possible to identify the conformations between cells in the population. Hi-C contact maps are the foundation for Hi-C data analyses of 3D spatial structure, as depicted in To correct the copy number bias in the Hi-C interaction matrix, Wu et al. recently proposed a linear regression-based chromosome-level adjustment method called caICB , which iTAD is a discretely folded domain with self-interacting chromatin in its central region, and normally such a structural entity ranges from kilo- to mega-basepairs. Since the current 3C-based sequencing data is inevitably contaminated with systematic biases, the ICE (iterative correction and eigenvector decomposition) technique was proposed to identify the local chromatin states, global chromosomal interactions, and the conserved chromatin organization . To infeProper gene expression requires communication with the corresponding regulatory elements scattered across the chromosome. The physics of chromatin fibers imposes a range of constraints on such communication. The molecular and biophysical mechanisms for chromosomal communication are key issues in the spatial organization of chromosomes. Dekker et al. proposed a topological machine with the claimed function of setting up and exploiting a 3D genome organization to both promote and censor intra- and inter-chromosome communication . ThroughTo infer 3D spatial associations within TADs from histone modification, chromatin accessibility and RNA-seq profiling resources, EpiTensor was proposed to computationally identify sets of hotspots as key elements stabilizing the 3D interaction. Through further study on diverse cell types, the identified hotspots were claimed to complete with higher chromatin, transcriptional activity, and enriched TF and ncRNA binding .Besides considerable genome-wide study on chromosomal architecture by 3C and Hi-C techniques, the recent in situ DNase Hi-C was reported to demonstrate the inactive murine X chromosome adopts a bipartite structure. The in situ DNase Hi-C relies on the endonuclease DNase I, rather than on a restriction enzyme to digest chromatin as traditional Hi-C does. Furthermore, through comparison with traditional Hi-C libraries, it is claimed that in situ DNase Hi-C has a higher effective resolution. The advantage brings forward much more opportunities in higher sequencing depth or hybrid capture techniques .Owing to the great improvement in the Hi-C protocol , single-Consensus methods transform the frequency of read pairs into pairwise distances and store the distances in a matrix, in which each element can be visualized as a point in 3D space. Interaction frequencies, nuclear envelope, nuclear pore complexes, and nucleoli have been used as constraints to the reconstruction of the 3D structures. However, each distance is based on an average of multiple structures in that population and is not suitable for the triangle inequality principle. As a result, the structure inferred from the average of millions of cells will differ from structures derived from the subpopulations of cells and will typically not represent any of the structures in individual cells ,63. SemiConsensus methods are usually used to identify unique 3D structures and assess chromosomal structural heterogeneity, but cannot fully capture the 3D structural heterogeneity within a cell population . They prTo identify structurally plausible, unobserved substructures, deconvolution methods are applied to seek an ensemble of 3D structural solutions and perform well in capture the inherent heterogeneity of chromosome structures in a cell population. There are two main deconvolution methods, namely structural deconvolution and matrix deconvolution. Structural deconvolution is applied at the 3D structure reconstruction level, and the resulting structural ensemble can be clustered to study the underlying structural variability and sub-population constituents. Matrix deconvolution is applied directly on contact frequency matrices and are usually faster than structural deconvolution, but the substructures recovered might not be physically plausible. Implementing solid-phase ligation is applied to improve the signal-to-noise ratio and enable a detailed analysis of the inter-chromosomal interactions . TADs arDeconvolution methods enable the inference of the main substructures that exist but require extensive computational resources. It is also not quite sure how accurate the deconvolution methods could be and whether the current data allows for the well-determined estimation of structural subpopulations.A crucial issue with single-cell Hi-C chromosome contact information is the inherent sparsity of the contact frequency maps ,70, as dThe genome coverage from the richest data sets was as low as approximately 2.5%, and the sparsity of genome coverage even led to concerns over the success rate of the single cell Hi-C protocol . One appAnother major inherent limitation for single-cell Hi-C experiments is the technical noise. Quality control of sequencing data is crucial to avoid technical artifacts. For single-cell sequencing, technical noise is mainly due to low amounts of starting material, often resulting in variable capture efficiencies . SequencAdditionally, the two copies of human autosomal chromosomes are difficult to be distinguished in single-cell Hi-C experiments, which can complicate the 3D structure reconstruction. The genomic distribution of the digestion sites of the restriction enzyme used can also be an important limitation to the Hi-C map resolution.Despite that we have witnessed a booming field of 3D genome organization studies within a short period, we should be cautious that some important caveats have been noted about the nature of the contacts detected by ligation-based methods, especially when interpreting results obtained with these techniques . It is nIn addition, many issues depend on the improvement of genomic techniques and the development of sophisticated statistical and computational methods. We want to point out the devoid of substantial applications of 3D chromatin structure and interaction in disease biology.With the development of 3D genome technology, many labs have promptly applied 3C-based high-throughput profiling techniques for disease research. Here, we review a few representative studies to demonstrate aberrant 3D chromatin structures and interactions in human diseases.Hi-C contact frequencies have been found to be tissue-specific and to influence the translocation partner selection in human diseases . ChromosNoticeably, the recent 3C-based technique revealed that genomic duplications in human patient cells and mice can lead to the formation of new chromatin domains (neo-TADs), and such a process determines their corresponding pathology outcomes . EnhanceIDH mutant glioma have dysfunction of chromosomal topological domains and allows oncogene expression [Due to hypermethylation at cohesin and CTCF binding sites human pression . The dispression .For validation and complement analysis on genomic 3C-based profiling techniques, the recent molecular imaging for noninvasive detection, such as quantitative 3D telomere fluorescence in situ hybridization (FISH) analysis and 3D super-resolution imaging (3D-SIM), are also a promising direction ,83.


Alternative polyadenylation (APA), a phenomenon that RNA molecules with different 3′ ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing (NGS)-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single cells. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative polyA (pA) site, the biological processes involved, and the corresponding consequences. We especially categorized the APA changes discovered so far and discussed their potential functions under given conditions, along with the possible underlying molecular mechanisms. With more in-depth studies on extensive samples, more signatures and functions of APA will be revealed, and its diverse roles will gradually heave in sight. Typically, mRNA molecules are reversely transcribed with oligo(dT) primer to produce cDNA ArabidopsisMethods in this category differ from each other in several aspects, such as 5′ adapting, fragmentation, and second-strand synthesis. Wu et al. have developed a method to study APA in Other than priming at poly(A) tail of RNA molecules, oligo(dT) primers can also anneal to internal A-rich sequences, a phenomenon termed internal priming, leading to the generation of false pA peaks. Most of these fake pA peaks can be removed computationally based on the successive As in their downstream genomic sequences. However, such strategy would also lead to the systematical loss of real pA sites flanking A-rich sequences, which account for ∼ 8% of total pA sites in mouse transcriptome The oligo(dT)-based methods have several shortcomings. Typically, when oligo(dT) primers are used in reverse transcription for 3′ end enrichment, the T-stretch of primers is retained during library construction, which has the benefit to preserve strand information, but can be problematic during sequencing. In Illumina sequencing platform, DNA molecules with homopolymers can probably lead to color imbalance and failure of cluster identification. Notably, base-calling quality significantly drops after sequencing through the T-stretch  [40]. ThSeveral approaches have been adopted to avoid sequencing directly through the T-stretch. Some methods used a custom sequencing primer, which contained several Ts at the 3′ end, to address this problem A [33], [RNA manipulation-based methods have been developed to circumvent internal priming caused by oligo(dT) priming. Typically, RNA fragments harboring pA site are enriched and adaptors are added to the 3′ end of these fragments. Then, primers annealing to the 3′ adaptors, rather than the oligo(dT) primers, are used for the reverse transcription B. In a m3′ region extraction and deep sequencing (3′ READS) is another high-throughput method to discriminate poly(A) tails from internal A-rich regions using chimeric U5 and T45 (CU5T45) and a stringent primer washing condition i.e., library barcode is designed into primers or adaptors. Fourth, internal priming should be avoided or minimized, while 3′-most fragments are efficiently enriched. Fifth, sequencing should start from the end of pA site or from both ends. And last, the library construction protocol should be easy to implement and can be adapted for commonly-used sequencing platforms.There are currently no approaches apparently superior to the others. Each method has its own advantages and shortcomings. Although RNA manipulation-based methods circumvent the internal priming issue and identify true pA sites experimentally, Oligo(dT) priming-based methods can also eliminate most of internal fake pA sites computationally. There are always valuable findings regarding APA regulation by applying any of the methods described above. Detailed comparison of all aforementioned 3′-enriched RNA-seq methods is presented in The newly-published method WTTS-seq outperforms other oligo(dT) priming-based methods in terms of minimizing the effect of internal priming during library construction, and surpasses RNA manipulation-based methods by circumventing RNA ligation and complicated hands-on procedure. Moreover, this method has been optimized to increase transcriptome coverage and reduce sequencing bias, thus exhibiting high efficiency for global pA site analysis. However, the currently-used adaptors of WTTS-seq are matched to the Ion Torrent Personal Genome Machine, which is not so commonly-used as Illumina platform. Therefore, a modification enabling the WTTS-seq protocol to be compatible with Illumina HiSeq platform is highly wanted. On the whole, we recommend WTTS-seq for detecting authentic pA sites globally if it can be compatible with the Illumina platform.After getting the read sequences that originate from the libraries for 3′ end of mRNA, a series of bioinformatics steps are needed to acquire the potential polyadenylation sites. First, the reads need to be aligned to the corresponding genome with or without the guide of annotated genes. Several tools can be used in this step, such as Bowtie After mapping the right reads back to genome, the next step is to filter out reads that originate from internal priming as described above, which would finally lead to the false pA peaks. Two categories of methods are adopted to filter out internal-priming reads. The most widely-used one is based on the position of the aligned reads. A straightforward and idiomatic way is to filter out reads, alignment locations of which are just upstream of genomic A-rich sequences. For example, a consecutive 6As or 15As in 20 nucleotides are used in some studies The next step is to predict the polyadenylation sites based on the aligned reads passing the filtration steps above. Peak-calling methods are usually adopted at this step. There are two strategies for peak calling, including window-based methods and density-based methods. Windows-based clustering methods were developed first, which count the number of reads within a window of certain length After the acquisition of the possible pA sites, some methods employ statistical analyses, such as calculating the false-discovery rate (FDR) and polyA score, to evaluate the precision and sensitivity of peak-calling, to achieve a more precise identification of real pA sites Single-cell whole-transcriptome sequencing methods have been applied in many research fields, revealing extensive diversity in RNA expression between seemingly identical cells Up till now, SCRS methods mainly focus on gene expression level, and few studies query post-transcriptional regulation mechanisms like APA, alternative transcription start site, and intron retention. It should be mentioned that to date, SCRS methods with the designed UMI are either 3′-enriched methods, such as CEL-seq2 Recently, a new method termed BATSeq, which combines a conventional SCRS protocol and a bulk population 3′-enriched RNA-seq protocol, has been developed to survey the genome-wide polyadenylation in single cells e.g., degradation rate, translation efficiency, export, and localization) and protein localization  polo, which encodes polo-like kinase in Drosophila, is such an example. The longer 3′UTR isoform of polo generates more protein than the shorter one polo gene with longer 3′UTR can’t be explained by the interaction with miRNAs. Previous studies have reported that RBPs or RNA secondary structure may be responsible for upregulation of many genes polo gene expression deserve further studies.Notably, although genes with longer 3′UTR tend to show a decreased steady-state expression level under certain condition Pdgfra) in fibro/adipogenic progenitors causes the elevated expression of a shorter transcript variant with a truncated kinase domain, which reduces tissue fibrosis through regulating pro-fibrotic pathways in muscle POLR2A), cyclin-dependent kinase inhibitor 1A (CDKN1A), and ephrin B2 (EFNB2). Such increased usage of intronic pA sites is caused by a decrease in U1 small nuclear ribonucleoprotein (U1 snRNP) levels in UV-induced DDR An increasing number of studies have reported that some alternative pA sites occur in genomic regions other than annotated pA sites and 3′UTRs in many eukaryotic organisms trans-acting factors associated with APA. For example, in human cells, U1 snRNP interacts with cleavage factor I (CF Im) Rsrp1) One line of compelling evidence indicating crosstalk between APA and AS under certain circumstance is that some factors involved in AS like snRNP proteins are reported to interact with Other than endogenous proteins, some peptides from viruses can also be responsible for interaction between APA and AS. For example, herpes simplex virus (HSV) infected cell polypeptide 27 (ICP27) promotes the 3′ end formation of co-transcriptional pre-mRNA using cryptic polyadenylation signals in introns, thus generating hundreds of novel, intron-less, GC-rich cellular transcripts that resemble HSV genes ACHE), probably by competing with the 3′ end processing factor cleavage stimulation factor 64 kDa (CstF64) ACHE, similar phenomena have also been observed in other genes as well In general, splicing factors promote the usage of UTR-APA but suppress the cleavage of intronic pA sites Recently, a full-length cDNA sequencing method termed ISO-seq has been established by Pacific Bioscience, allowing the direct sequencing of full-length transcripts without the need for assembly Global APA changes have been discovered in numerous physiological processes including development, cell differentiation cyclinD1 (CCND1), Dicer1, and the gene encoding insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1/IMP-1), show increased stability and produce more proteins IGF2BP1/IMP-1 could lead to cellular phenotypes such as oncogenic transformation, demonstrating the importance of APA regulation in cancer Studies have shown that genome-wide preference of promoter-proximal pA sites leads to global shortening of mRNA 3′UTRs, which occurs in a series of biological processes, especially in cancer. For example, Mayr and Bartel have revealed that a considerable number of oncogenes in cancer cells exhibit 3′UTR shortening phenomenon. The isoform of certain oncogenes with shorter 3′UTR, such as CCND1 locus, a point mutation downstream of stop codon creates a novel polyadenylation site and therefore leads to an CCND1 isoform with a shorter 3′UTR TP53), which converts the canonical pA signal ‘AATAAA’ into ‘AATACA’, inducing the damaged 3′-end formation of TP53 mRNA. This SNP is further identified to be associated with other tumors, such as prostate cancer, malignant glioma, and colorectal adenoma, but not breast cancer DNA mutations are usually considered as the cause of tumors, and have been recently discovered to impact APA switch in cancer. For example, in the Drosophila embryogenesis. Moreover, some extended 3′UTRs contain potential RBP recognition motifs that act as translational repressors, such as Pumilio regulatory element Conversely, genes favoring promoter-distal pA sites would generate transcripts with 3′UTR lengthening globally. Progressive lengthening of 3′UTRs was first discovered during the embryonic development process trans-acting factors has thus been proposed. Dynamically-regulated APA is also found during mouse retinal development and maturation process Despite the unidirectional trend of APA change described above, dynamic changes of 3′UTR length can also occur, especially during a long-period process with multiple time points. Zebrafish development is one of such examples. During the zygotic genome activation in early embryogenesis of zebrafish, a global shortening of 3′UTRs is observed, while a quick lengthening of mRNA 3′UTRs appears during the following stage of gastrulation Altogether, these aforementioned findings clearly demonstrate that APA changes can accompany many biological processes and probably play important roles. With more extensive studies on various samples, more featured pA sites and functional pathways involved will be discovered, and the biological significance of APA in many developmental or pathological processes would gradually heave in sight as well.Alternative polyadenylation, alternative promoter and alternative splicing, are the three major types for gene expression regulation at RNA level, covering the starting to the ending of transcription, which contribute to the diversity and complexity of transcriptome and proteome. Recent years have witnessed the spring up of APA studies and related interesting findings. For example, more than 70% of the human genes have multiple pA sites, suggesting the prevalence of APA across the genome. APA has also been found to accompany many biological processes and can play important roles under given conditions. Currently quite a few NGS-based methods have been developed for detecting pA sites genome-widely, with each having advantages and shortcomings of its own. More accurate and practical methods are still desired, such as the methods that can circumvent internal priming and that have the ability to identify real pA site precisely, and that are practical for study in single cells. APA has demonstrated its universal and important roles both at molecular function level and physiological level. Global APA regulation has been found involved in the increasing number of biological and pathological processes. However, omics studies need to be combined with candidate gene strategy to reveal the underlying biological function and detailed regulation mechanisms for given genes and conditions. For example, isoforms with longer 3′UTR for a considerable number of genes generate more protein than those with shorter 3′UTR. Do RBPs play a role in explaining this phenomenon? And if so, which RBP and how it functions to regulate? These concerns remain to be elucidated. With more researchers diving into this field, more functions and biological roles, along with in-depth regulatory mechanisms of APA, will be discovered.The authors declared that there are no competing interests.