Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. Gene transcription is regulated by the coordinated interaction of genetic, epigenetic and trans-acting factors. The chromatin environment at gene promoters, including positioned nucleosomes that may display functional histone modifications, is a key regulator of gene expression, contributing to transcriptional activation and repression. In addition to sense-strand transcription of gene sequences, antisense transcription is prevalent at gene promoters. Often resulting in a short-lived non-coding RNA transcript, the function of antisense transcription is poorly understood. Using next-generation sequencing techniques, we characterized transcription in human breast cancer cells and found extensive correlations between antisense transcription and the chromatin environment at promoters. We found that downstream antisense transcription initiates from between regularly positioned nucleosomes and that those nucleosomes between sense and downstream antisense transcription start sites display histone modifications associated with active gene promoters. Chromatin remodelers and other protein complexes responsible for creation and maintenance of the promoter chromatin environment associate with this same region, suggesting an important role of antisense transcription in the regulation of gene expression. The promoter region is intimately tied to the transcription of genes, providing the initial site of transcriptional machinery binding and assembly. Comprised of DNA elements in defined spatial arrangements , promoteDivergent transcription has emerged as a common feature of mammalian promoters –7. In diIn addition to divergent transcription, convergent transcription has been observed at genes in a variety of systems, ranging from fly and yeast to mammals –14. ConvTo characterize transcription initiation in human T47D/A1-2 cells, Start-seq was performed on nascent RNA transcripts . In a St−6). To ensure reproducibility, a biological replicate was prepared from the same cell-line, and similar read densities were found at the identified TSSs . This enrichment suggests cell-line specificity in downstream antisense transcription.daTSSs identified in T47D/A1-2 cells were investigated in other cell lines using other experimental data types associated with nascent transcription , part of the P-TEFb complex involved in the regulation of Pol II elongation, selectively associates to the area between gene TSSs and daTSSs and shows statistically significant enrichment compared to equivalent regions at genes without daTSSs . Like daTSSs, a variety of trans-regulatory factors associate to uaTSS positions , a similar proportion remain overlapped with introns when gene models are randomly shuffled (75%), indicating that daTSSs are not significantly enriched within introns . There iRather than describing a functionally distinct element, e.g. proximal enhancers, downstream antisense transcription seems to be a feature of promoters themselves. Along with antisense transcription upstream of gene TSSs, downstream antisense transcription may be an intrinsic feature at many mammalian promoters . There aThough both are proximal to promoters, daTSSs and uaTSSs exist in distinct epigenetic environments . daTSSs Despite differences in epigenetic features, tendency for association with transregulatory factors, and capacity to produce stable RNA transcripts, all three classes of TSS described in this work display similarities in sequence content, including enrichment for GC content and Pol II-associated sequence motifs . As suchWe characterized downstream antisense transcription initiating near gene promoters in human T47D/A1-2 cells. daTSSs fall between regularly positioned nucleosomes downstream of gene TSSs. Histones within this region are enriched for marks closely associated with active promoter regions, such as H3K4me3 and H3K27ac modifications. Chromatin remodeling complexes show enriched binding upstream of observed daTSS positions, suggesting that antisense transcription contributes to the establishment and maintenance of a promoter-specific chromatin environment. Downstream antisense transcription is common to many human promoters, and daTSSs correlate with the downstream edge of promoter-associated chromatin features. Coincidence of daTSSs with these features suggests interplay between antisense transcription and regulatory pathways.T47D/A1-2 cells were cultured in DMEM containing 10% FBS. Prior to RNA isolation, cells were cultured in medium supplemented with 5% charcoal dextran-treated serum for at least 24 hours. The A1-2 cell line is a previously described derivative of the T47D breast cancer cell line that overexpresses rat glucocorticoid receptor (GR) and contains a stably-integrated MMTV luciferase reporter gene . Short cStart-seq reads were first filtered by quality score; reads with an average Sanger score less than 20 were removed from analysis. Following quality filtering, Cutadapt (version 1.2.1) was used to remove adapter sequences . AlignmeIdentification of TSSs was performed based on methods described previously ,16, TSS TSSs were called within each window in which the strand-specific 5’ end read count at any given nucleotide position met or exceeded a threshold of 5 reads. This threshold was determined in a previously described method . In shorIn those windows where a single nucleotide position met or exceeded the read threshold, a gene TSS was called. The calling method aims to select as the TSS the position with the highest read counts in window region with the highest read density. To accomplish this, two potential TSSs were first determined. The first was the position with the most aligned 5’ ends across the entire window. For the second, the search window was divided into 200-nt bins at every 10 nt across the search window. At the 200-nt bin with the most overlapped 5’ ends, the second potential TSS was called as the position with the most aligned 5’ ends. Of the two putative sites, the TSS closest to the associated annotated RefSeq TSS was selected.Following gene TSS identification, uaTSSs and daTSSs were found. For uaTSSs and daTSSs, search spaces were defined in antisense orientation as 1 to 1000 nt upstream and 1 to 2000 nt downstream, respectively. uaTSSs and daTSSs were called at the position with the most aligned 5’ ends within the search window if the count at any single nucleotide position met or exceeded a threshold of 5 reads. To ensure that identified daTSSs were not simply mis-called gene TSSs or uaTSSs, additional criteria were used to filter daTSS calls. A daTSS call was filtered if (1) within 1000 nt upstream of the daTSS there was a genomic position with Start-seq reads greater than or equal to 10% of reads at the associated gene TSS on the same strand as the gene TSS, implicating the daTSS as a potential uaTSS for an uncalled gene TSS or (2) it was within 1000 nt of an annotated TSS on the same strand, implicating the daTSS as a potential gene TSS.Prior to alignment, Pol II ChIP-seq reads from MCF-7 cells  see als were filConsidering each TSS group separately, the nucleotide composition of each position in a -1000 to +999 window was determined and reported as a percentage. Logo plots were generated using Web Logo 3 considering sequences in a -5 to +5 window about identified TSSs .5 Pol II-associated and 107 known vertebrate motifs were identified. Motifs and additional information are available at http://jaspar.genereg.net/.To identify occurrences of Pol II-associated and known vertebrate motifs, FIMO (version 4.10.0) was used considering a p < 0.0001 significance cutoff and a 0-order Hidden Markov Model from promoter regions as background . PublicaDe novo motif discovery was performed using MEME (version 4.10.0) with default parameters [rameters . Sequencrameters .CpG island heatmaps reflect the intersection of annotated CpG islands retrieved from UCSC Genome Browser  with TSSSequence conservation heatmaps were generated using phyloP scores from placental mammal alignments retrieved from UCSC Genome Browser . Each poGRO-cap data from K562 and GM12878 cells  considerNuclei were harvested from cultured T47D/A1-2 cells and digested for 5 minutes at 37°C with a range of MNase (Worthington) concentrations. Reactions were stopped by the addition of EDTA and then treated with RNase and proteinase K. Digested DNA was isolated by phenol/chloroform extraction and ethanol precipitation. Libraries were prepared using an Illumina TruSeq sample preparation kit and sequenced on an Illumina HiSeq for paired-end 50 base reads.Publically available MNase-seq data  and dataFAIRE-seq data were collected as described previously . PublicaOver biological triplicates, total RNA was harvested using an RNeasy Kit (Qiagen) with on-column DNase treatment. RNA quality was validated by Bioanalyzer (Agilent). Paired-end strand-specific poly-A enriched libraries were sequenced on an Illumina HiSeq 2500 for 125 base paired-end reads.Prior to alignment, RNA-seq reads were filtered based on quality; reads with an average Sanger quality score less than 20 were removed from analysis. Following quality filtering, Cutadapt was used to remove adapter sequences . Insert For publically available data sets , read coverage files were retrieved from public depositories . Given tHeatmaps consider 40-bp/40-nt bins over TSS-centered windows. Unless otherwise noted, each position in a heatmap gives the number of reads or other features overlapping with that bin. Heatmap images were generated using Partek (version 6.6) . Two-dimTo generate the gene TSS-centered panels in The data sets supporting the results of this article are available in the GEO repository, GSE74308.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.S1 FigReported data (red) and validation data (blue) are shown anchored at identified TSS positions in both heatmaps and two-dimensional plots. Validation data are taken from biological replicates or from similar cell lines. Read density of a Start-seq biological replicate (T-47D/A1-2 cells) (A), FAIRE-seq in T-47D/A1-2 cells  (B), and(TIF)Click here for additional data file.S2 Fig(A) Observed transcription at gene Rpe. Genomic context is given by RefSeq annotation (green), Pol II ChIP-seq coverage (red), and counts for the 5’ ends of Start-seq reads in both sense and anti-sense orientations (black). Observed transcription start sites (TSSs) are given for the gene TSS, upstream antisense TSS (uaTSS), and downstream antisense TSS (daTSS). (B) Heatmaps of counts for the 5’ ends of Start-seq reads over gene TSS, uaTSS, and daTSS positions. (Right panel) Heatmaps of counts for the 5’ ends of Start-seq reads and fragment centers of Pol II ChIP-seq reads. Heatmaps are centered on gene TSS positions and sorted by gene TSS-uaTSS (left) or gene TSS-daTSS distance (right). Only TSS positions with called uaTSS or daTSS positions, respectively, are included on the heatmaps.(TIF)Click here for additional data file.S3 Fig(A) Average RNA-seq coverage in T47D/A1-2 cells across identified TSS positions. (B) Heatmaps of read density from Pol II-associated sequencing approaches. Pol II ChIP-seq, NET-seq, GRO-seq, GRO-cap, and PRO-seq were performed over a variety of cell lines (indicated on figure). Each heatmap is centered on observed gene TSS position and sorted by increasing gene TSS-daTSS distance. (C) Categorization of T47D/A1-2-called daTSSs by presence of GRO-cap signal in heterologous cell lines. daTSSs were placed into a separate category if no GRO-cap signal was found within 10 nt of the observed daTSS. 987 (33%) and 971 (33%) daTSSs called in T47D/A1-2 cells were found to have no significant GRO-cap signal in GM12878 and K562 samples, respectively. (D) Plots of average occurrences of Pol II-associated sequence motifs. Motif occurrences were identified using FIMO . Motif p(TIF)Click here for additional data file.S4 Fig(A) Empirical cumulative distributions of gene RNA-seq FPKM values for genes displaying only daTSSs (blue) and only uaTSSs (cyan). Inset p-value was determined by Kolmogorov-Smirnov test. (B) Box plots of RNA-seq FPKM values for all genes, genes without uaTSSs, and genes without daTSSs. Reported p-values were determined by Kolmgorov-Smirnov tests. (C) Average MNase-seq read densities at TSSs of genes with (red) and without (blue) identified daTSSs.(TIF)Click here for additional data file.S5 FigFor each modification, observed gene TSS-centered heatmaps of ChIP-seq read counts are shown sorted by increasing distance to uaTSSs or daTSSs. Average densities are shown centered on uaTSS and daTSS positions (“uaTSS-centered” and “daTSS-centered”). To reflect the genomic context of transcription factor binding at promoters, plots of average density at antisense TSSs are transposed and shifted by median distance to gene TSSs (illustrated at bottom of figure). uaTSS- and daTSS-centered densities are plotted relative to observed gene TSS positions. In these plots (“Gene TSS-centered”), antisense plots were first transposed about the antisense TSS (left-most points became the right-most points and vice-versa) and then shifted by median distances observed between gene TSSs and antisense TSSs. Each plot considers 5,519 gene TSS-uaTSS or 2,956 gene TSS-daTSS pairs.(TIF)Click here for additional data file.S6 FigTBP ChIP-seq data were collected in GM12878 cells; GATA3 in MCF7 cells; SP1 in A549 cells; NFIC in GM12878 cells; c-Fos in K562 cells; c-Jun in K562 cells (data sources outlined in (TIF)Click here for additional data file.S7 FigCHD1-A (HAT SAGA complex) ChIP-seq data were collected in K562 cells; Sap30 (Sin3-HDAC) in K562 cells; BRG1 (SWI/SNF) in HeLa cells; INI1 (SWI/SNF) in HeLa cells; BAF155 (SWI/SNF) in HeLa cells; BAF170 (SWI/SNF) in HeLa cells (data sources outlined in (TIF)Click here for additional data file.S8 Figp300 ChIP-seq data were collected in MCF7 cells; CTCF in A549 cells (data sources outlines in (TIF)Click here for additional data file.S9 FigFor any given data type, as listed in the leftmost column, average read density or occurrences are plotted over a +/-1 kb window across TSS calls. Each plot is oriented relative to the gene with larger values corresponding to more downstream sequences relative to the gene TSS. Experimental cell lines are noted under the data type.(TIF)Click here for additional data file.


BMP15 gene, the regulatory sequence of porcine BMP15 was investigated in this study. The cloned BMP15 promoter retains the cell-type specificity, and is activated in cells derived from ovarian tissue. The luciferase assays in combination with a series of deletion of BMP15 promoter sequence show that the −427 to −376 bp region of BMP15 promoter is the primary regulatory element, in which there are a number of transcription factor binding sites, including LIM homeobox 8 (LHX8), newborn ovary homeobox gene (NOBOX), and paired-like homeodomain transcription factor 1 (PITX1). Determination of tissue-specific expression reveals that LHX8, but not PITX1 and NOBOX, is exclusively expressed in pig ovary tissue and is translocated into the cell nuclei. Overexpression of LHX8 in Chinese hamster ovary (CHO) cells could significantly promote BMP15 promoter activation. This study confirms a key regulatory element that is located in the proximal region of BMP15 promoter and is regulated by the LHX8 factor.Bone morphogenetic protein 15 (BMP15) is secreted by the mammalian oocytes and is indispensable for ovarian follicular development, ovulation, and fertility. To determine the regulation mechanism of  Bmp15 homozygous mutant causes subfertile, showing the ovulation defects and reduction of oocytes [Bmp15 null mice, in sheep, the naturally-occurring homozygous mutants (IFecX) that has point mutations in the coding region of BMP15 gene causes the infertile due to an arrest at the primary stage of folliculogenesis. On the other hand, the heterozygous females show increased ovulation rate and multiple pregnancies. Thus, BMP15 is related to infertility and super-fertility in a dosage-sensitive manner in sheep [BMP15 mRNA level in individual denuded oocytes from single-to-triple ovulation-rate species  was much lower than that from high ovulation-rate species . But the ratios of BMP15:GDF9 expression levels were significantly species-specific indicating a unique mechanism by which the species-specific ovulation-rate may be regulated [BMP15 gene.Reproductive capability is a vital economic factor for sows, for instance the Chinese Taihu breed farrows at least five more piglets per litter than the Large White breed . Many st oocytes . In contin sheep ,10. Receegulated . A tissuegulated . Thus, iBMP15 gene expression occurs during early follicular development in either primordial or primary follicles depending on animal species [BMP15 expression [BMP15 gene has not been fully investigated yet.As a member of the BMP superfamily, BMP15, also called growth and differentiation factor 9B (GDF9B), plays a vital role in ovarian follicular development, ovulation and fertility ,14. The  species . Functio species . Followipression ,18,19,20BMP15 gene expression, we analyzed porcine BMP15 gene 5ʹ untranslated and promoter regions, screened the primary regulatory sequence, and verified the potential transcription factors that could upregulate the expression of porcine BMP15.To understand the regulatory mechanism of pig BMP15 gene, indicating that BMP15 may be a downstream target of those transcription factors  was subcloned into pEGFP-1 and pGL3-basic plasmid to make the reporter vector pE2.2 and pL2.2 (BMP15 promoter was only activated in Chinese hamster ovary (CHO) cells, but not in C2C12 and NIH3T3 cells , LIM homeobox 8 (LHX8), newborn ovary homeobox gene (NOBOX), and paired-like homeodomain transcription factor 1 (PITX1), were found in the promoter sequence of  factors . We alsor region . To furtnd pL2.2 A,B. The T3 cells C, indicapromoter D.BMP15 promoter  were amplified from pL2.2, and were then inserted into pGL3-basic plasmid to construct reporter vectors pL1.8, pL1.3, pL0.8, pL0.5, pL0.4, pL0.3 and pL0.2  and pL0.4–0.3 that has no CAP and FOXA2 sites (−460 to −332 bp), were constructed  also showed that BMP15 was only expressed in ovary [BMP15 gene, we constructed the porcine BMP15 reporter vectors pE2.2 and pL2.2. Since a porcine ovarian cell line is not available and the primary-cultured ovarian cells are difficult to be obtained and handled, we, alternatively, used a CHO cell line to perform BMP15 assays using pE2.2 and pL2.2. The CHO cell line was derived from the ovary of the Chinese hamster, though it is an immortalized cell line that may not physiologically represent the ovarian epithelia, CHO cells were used to study Bmp15 promoter activation and signaling pathways [BMP15 promoter was exclusively activated in CHO cells, but not in myoblasts and fibroblasts, indicating that the cloned BMP15 promoter retains the cell-type specificity.The previous studies of reatment . In huma outcome . Our prein ovary . To invepathways ,34. The BMP15 promoter there exists sites for transcription factors LHX8, NOBOX, and PITX1, which are the potential positive regulators to activate pig BMP15. Although LHX8, NOBOX, BMP15, and GDF9 are highly expressed during ovarian follicle development [BMP15 c.-9C>G results in modification of its binding sites for PITX1 that trans-activates BMP15 promoter and increases the recruitment of an elevated number of follicles/oocytes early in a woman’s life [GDF9 promoter during human fetal ovary development [Nobox is a downstream target gene of SOHLH1 because in Sohlh1 null oocytes Nobox expression is defective resulting in a loss of genes downstream of the NOBOX pathway, such as Gdf9 and Oct4 [Lhx8 is also a direct transcriptional target of SOHLH1. In Lhx8−/− mouse, ovaries fail to maintain the primordial follicles, and the transition from primordial to growing follicles does not occur, in which the expression of oocyte-specific genes, such as Gdf9, Bmp15 and Oct4, are significantly reduced [Lhx8 conditional knockout (cKO) oocytes, Lhx8 represses Lin28a expression indirectly interacting with the PI3K-AKT pathway and blocking the primary to secondary follicle transition [Yy1, a multifaceted polycomb group gene, cKO oocytes exhibit reduced expressions of Lhx8, Gdf9 and Bmp15, indicating that Yy1 function is required at each of these genes to act as an activator [BMP15, indicating that LHX8 is an upstream positive factor of BMP15. Further investigation is needed to verify whether LHX8 can directly bind to BMP15 promoter and play a role in the regulation of folliculogenesis and granulosa cell growth in pig.Based on bioinformatics analysis and promoter deletion analysis of this study, we found that within the −427 to −376 bp region of the pig elopment ,35, the n’s life ,30,36. Rand Oct4 . Lhx8 is reduced . In Lhx8ansition . On the ctivator . HoweverBMP15 promoter did not have the typical TATA box sequence. The results of promoter deletion analysis indicated that the sequence within −332 to −175 bp region had a CAP site (−303) and a transcription start site for pig BMP15 gene, which was consistent with the transcription start site of rat BMP15 [In addition, we found that the porcine at BMP15 ,40.BMP15 promoter. The transcription factor LHX8 can specifically enhance BMP15 promoter activation. Understanding of LHX8 to BMP15 transactivation signaling can facilitate in vitro maturation of porcine oocytes, which are widely used for porcine cloning, because BMP15 treatment in vitro could promote porcine oocyte maturation and cumulus expansion [In conclusion, a key consensus sequence is identified within −427 to −376 bp region in porcine 2 in a humidified atmosphere and the media were changed every 2 days.Chinese hamster ovary (CHO) cells were grown in DMEM/F-12 medium  with 10% fetal bovine serum . NIH3T3 fibroblasts and C2C12 myoblasts were cultured in DMEM (Gibco) with 10% FBS. All these cells were growing at 37 °C, 5% COI and HindIII and formed the reporter vector pL2.2. A series of truncated DNA fragments from 2.2 kb sequence were listed in To construct luciferase reporter vector, a 2166 bp (ENSSSCG00000012310) DNA fragment from pE2.2 vector that was constructed by this laboratory as described previously [LHX8) was cloned from porcine ovary, inserted into the pGEM-T Easy Vector (Promega), and confirmed by DNA sequencing. LHX8 CDS was then subcloned into XhoI/BamHI sites of pEGFP-C1  to generate pEC1-LHX8 that could express GFP-LHX8 fusion protein.The coding DNA sequence (CDS) of LIM homeobox 8 gene  according to the manufacturer’s instructions. The total RNAs were examined by detecting the OD260/280 ratio and the RNAs with a ratio of 2.0 were then used for reverse transcription. One microgram RNAs were reverse transcribed using oligo-dT primers, reverse inhibitor, reverse transcriptase and RNA-free water . PCR reactions using PhantaTM Super-Fidelity DNA Polymerase kit  in a 50 µL reaction volume were performed for 35 cycles at 94 °C 30 s, 60 °C 30 s, and 72 °C 40 s. The products of RCR were separated by 1.5% agarose gel electrophoresis. GAPDH was used as internal reference. Primers used in this study were listed in BMP15 promoter, 2 × 104 cells were seeded in a 48-well plate 24 h before transfection. When these cells reached 80% confluence, the plasmids of pL2.2 and the control pRL-TK (10 ng) were cotransfected into CHO, NIH3T3, and C2C12 cells, respectively, by Lipofectamine 2000 Reagent (Invitrogen). For GFP fluorescence assay, the pE2.2 vector was transfected into cells for 48 h, and then the cells were fixed by 4% paraformaldehyde in PBS for 10 min. After washing 3 times with PBS, nuclei were stained with DAPI , and the fluorescence images were documented with the fluorescence microscope . To screen the primary regulatory sequence of BMP15 promoter, the truncated constructs, including pL1.8, pL1.3, pL0.8, pL0.5, pL0.4, pL0.3 and pL0.2, were transfected into CHO, C2C12 and NIH3T3 cells, respectively. In order to determine whether the GFP-LHX8 fusion protein located in cellular nuclei, pEC1-LHX8 and pEGFP-C1 was transfected into CHO and 3T3 cells for 48 h, and then the cells were fixed by 4% paraformaldehyde in PBS for 10 min. After washing 3 times with PBS, nuclei were stained with DAPI and the fluorescence images were documented with the fluorescence microscope. To verify the interaction of LHX8 with the BMP15 promoter, pL0.4A construct was cotransfected with pEC1-LHX8 into CHO and 3T3 cells, respectively.To verify the tissue specificity of porcine p < 0.05 and determined using one-way ANOVA followed by post-hoc tests (Newman–Keuls method).For dual-luciferase assays, cells were harvested at 36 h after liposome-mediated transfection and lysed for 15 min at room temperature. Luciferase activity was stimulated by adding luciferase assay reagents (Promega) and then detected by a BHP9504 Luminometer . The average ratio of firefly luciferase light units to Renilla luciferase light units was calculated and we conducted three independent experiments and then collected the data for analysis. Statistical significance was accepted at www.genomatix.de), JASPAR (www.jaspar.genereg.net), and Gene Regulation [BMP15 promoter (−2166 to −1 bp). ClustalW2 software (http://www.ebi.ac.uk) was used for multiple alignments of DNA sequences from different animal species .The online programs of MatInspector function of Genomatix Suite (gulation  were usep < 0.05) existed in a group of data. Values were represented as the mean ± SD, and statistical significance was indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001. Data were representative of at least three independent experiments.One-way ANOVA was used and Newman–Keuls multiple range tests were conducted as post-hoc tests if a significant difference (


Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.We reported pearl oyster  Pinctada fucata, widely distributed in tropic and subtropic marine coasts, is an important marine bivalve species that is cultured for pearl production and hot research topics in biomineralization, for its highly organized internal structure, chemical complexity, mechanical properties and optical effects of nacre P. fucata can generate the shell in vivo consisting of two different forms of calcium carbonate, i.e. aragonite in the inner nacreous layer and calcite in the outer prismatic layer. The organic matrix, secreted by the mantle epithelium, comprises 1–5% of the shell weight. At molecular level, the matrix proteins play a key role in the mineralization process, which is thought to direct the growth of calcium carbonate crystal and be responsible for the extraordinary properties of nacre and prism Pearl oyster, The MSX gene family is one of the oldest animal-specific homeodomain transcription factors which are confined to the Metazoa MSX genes have been isolated from major metazoan phyla MSX genes in each animal taxa is very limited except in mammals. Molecular phylogenetic analysis of wide-ranging groups of metazoan animals indicated that functionally important residues in MSX proteins are strongly conserved in all metazoan MSX homeodomains which is known to physically interact with DNAs or proteins that are essential for the molecular function Although Pinctada fucataPinctada fucata, designated as PfMSX, which is highly conserved in its homeodomain comparing with other known mammalian MSX proteins. These findings led us to speculate that PfMSX could be a transcription factor of the Pif gene, which may bind to the consensus MSX-binding site of the Pif promoter, further governs the expression of Pif gene and control the biomineralization of the shell formation.Homeodomain genes are among the most slowly evolving of all protein classes PfMSX which recognized a conserved MSX-binding site and played an important role in the Pif expression in Pinctada fucata.To address these hypotheses, we identified an ancestral conserved homeobox-containing gene Adult pearl oysters (shell length 4.5–5.5 cm) were sampled from Daya Bay Station  in Shenzhen, China. The field studies did not involve endangered or protected species. The specific location of our study: .P. fucata have been performed by our lab MSX gene homologous to Corbicula fluminea  was obtained. To obtain full-length cDNA of P. fucata MSX homolog, 5′and 3′ RACE were performed using SMART RACE cDNA Amplification Kit  following the manufacturer’s instructions. 5′-RACE was performed using gene-specific primers . 3′-RACE was performed using gene-specific primers . The PCR products were purified with Gel Extraction Kits  following the manufacturer’s instructions and sequenced . PfMSX sequences were analyzed using the BLAST algorithm at NCBI web site. SMART was used to analyze the deduced amino acid sequences of PfMSXP. fucata adduct muscle using Mollusc DNA Kit  following the instruction manual. Based on the PfMSX cDNA sequence, four primers  for PfMSX genomic sequence were designed, and four fragments were obtained by PCR and sequenced. The extrons and introns were determined by Splign program Gigabase-scale transcriptomes sequencing, assembly and functional annotation of pearl oyster P. fucata samples were isolated using Trizol . Total RNA (1 µg) was treated with DNase I  and subsequently reverse transcribed with TOYOBO RT-PCR kit . The QPCR primers for tissue and developmental stages distribution were as follows: PfMSX, 5′-ATGCACCCGGTAGCTCTA-3′ and 5′-TCACCGACTCCGAAACAGG-3′; β-actin, 5′-TGGTATGGGACAGAAGGAC-3′ and 5′-GACAATGCCGTGCTCAAT-3′. QPCR was carried out using a LightCycler 480 Real-Time PCR System , with SYBR Green as the fluorescent dye, according to the manufacturer’s protocol . QPCR conditions were as follows: denaturation at 94°C for 1 min, followed by 40 cycles at 94°C for 15 s, 55°C for 15 s and 72°C for 60 s. We analyzed the relative gene expression by the typical Ct method (2−ΔΔCt method).Pif-specific primer 1 (5′-TTGTGTCGGTGTCAAATCTG-3′) and nested primer 2 (5′-GCAAGTTCCATCTATTCGAGTTG-3′) were used to clone the promoter of the Pif gene by genome walking with the Genome Walker kit . The longest fragment from the four genomic libraries  was gel-purified, and subcloned for sequencing. At least two clones were sequenced, and all were found to be virtually identical in the region directly upstream of the 5′ untranslated region of the Pif gene. The Pif promoter sequence for the 1358-bp EcoRV fragment was deposited in GenBank [accession no. KJ028207].5′-CGGGGTACCATGCACCCGGTAGCTCTA-3′, containing a KpnI restriction site (underscored), and 5′-CCGCTCGAGATGGTGATACGTCATACCTAC-3′, containing an XhoI restriction site (underscored). After the double digestion with KpnI and XhoI, the cDNA was cloned in-frame into the KpnI/XhoI sites of pcDNA3.1/myc-His (A) vector . The construct was verified by sequencing.The cDNA encoding the full-length PfMSX was amplified with the sequence specific primers Pif promoter fragment (corresponding to bases -1358 to -1) was subcloned into the KpnI and NheI sites of the pGL3basic luciferase reporter vector  to generate P1358Luc. The fragments of the oyster Pif gene between P1125Luc, P907Luc, P473Luc and P106Luc were amplified by PCR using P1358Luc as a template.A 1358 bp GGTACCGCATGAAGAAAGATCAGGTGATTCTTTA-3′; primer B, 5′-GCTTTTTCATTAAAATTATTTCATA-3′; and primer C, 5′-ATATGACTGGTGGAAAAATATGCAAATA-3′, KpnI site is underscored. “ACTAATTGG” deletion mutation amplified a 126 bp fragment using primer A–B and “GTAATTG” deletion mutation amplified a 727 bp fragment using primer A–C. The second set was primer D, 5′-ATTTTAATGAAAAAGCTACATATATG-3′; primer E, 5′-TATTTGCATATTTTTCCACCAGTCATAT-3′ and primer F, GCTAGCGACCCTTGTCTTATCT-3′5′-GCCCGG, NheI site is underscored, “ACTAATTGG” deletion mutation amplified a 1027 bp fragment using primer D–F and “GTAATTG” deletion mutation amplified a 419 bp fragment using primer E–F. These PCR products respectively were annealed in equal molar ratios and used as template for PCR with primers A and D. The 1116 bp overlap product of the “ACTAATTGG” deletion mutation and the 1118 bp overlap product of the “GTAATTG” deletion mutation were digested with KpnI and NheI, and ligated to similarly cut pGL3basic separately, creating P1125Δ1Luc or P1125Δ2Luc. The P1125ΔΔLuc created by “ACTAATTGG” and “GTAATTG” deletion mutation was constructed using the same strategy of P1125Δ2Luc basing on the P1125Δ1Luc.To engineer “ACTAATTGG” or “GTAATTG” deletion mutations, use overlap PCR with the following two sets of primers. The first set was primer A, 5′ -CGGSubcellular localization of PfMSX was performed by EGFP fusion protein expression and immunofluorescence. The 293T cells were seeded onto cover slips (10 mm×10 mm) in a 12-well plate. After the cell adhering for 18 h, the 293T cells were transiently transfected with recombinant pEGFP-PfMSX or empty pEGFP-C1. After transfection for 48 h, cells were washed with PBS (pH 7.4) and fixed with 4% paraformaldehyde for 20 min, and then stained with 6-diamidino-2-pheny-lindole (DAPI) (1 mg/ml) for 10 min. Finally, the cells were rinsed with PBS, mounted with 50% glycerol, and observed using fluorescence microscopy . For immunofluorescence localization, the 293T cells were fixed with 4% paraformaldehyde and then the coverslips were blocked using 2% bovine serum albumin (BSA) at room temperature for 30 min. Cells were incubated either with anti-myc antibody (1∶60) or preimmune mouse serum (1∶60) for 1 h, rinsed with PBS three times for 10 min and then incubated with FITC-conjugated goat anti-mouse antibodies  for a further hour. Finally, cells were stained with DAPI (1 mg/ml) and observed under fluorescence microscopy.PfMSX expression analysis in different tissues, three pearl oyster’s ovary, testes, gills, adduct muscle, mantle, heart, digestive gland were collected. For analyzing the developmental expression patterns of PfMSX, nine developmental stages including fertilized eggs, 2–4 cell, blastocyst, the trocophore, D-shaped larvae, and umbo larvae, eye-spot larvae, spats and juveniles were collected and stored at −80°C. β-actin was used as reference gene which was expressed stably in all tested tissues and developmental stages. Three repetitions of the reaction were performed.Adult pearl oysters (shell length 4.5–5.5 cm) were sampled from Daya Bay Station  in Shenzhen, China. They were acclimated in indoor cement ponds at ambient seawater temperature for one week before experiment. For 2 using DMEM  supplemented with 10% FBS , 100 IU/ml penicillin and 100 µg/ml streptomycin . The cultures were split every 2 to 3 d. Lipofectamine 2000  was used for the DNA transfections according to the manufacturer’s protocol.The 293T human kidney cell line (HEK293T), and the C2C12 mouse myoblast cell line were cultured at 37°C in a humidified atmosphere of 5% COPfMSX expression vectors. At 48 h after transfection, the cultures were harvested and lysed. Luciferase assays were performed using 20 µl of cell extract and 100 µl of luciferin substrate  using a luminometer.C2C12 cells grown to 80% confluence in a 48-well plate were transfected with 100ng/well of the reporter construct along with 100 ng/well of pRL-TK plasmid  in the absence or presence of PfMSX gene transfectants. The following complementary oligonucleotides were used: Oligo Pif wildtype (wt): ACTAATTGGTACATA-3′5′-AAAAGC, and Oligo Pif mutated (mut): CCATGGACTTACATA-3′5′-AAAAGC. The nucleotide changes were shown in boldface and the MSX element is underlined. Sense and antisense oligonucleotide were 5′ end labeled in Takara Biotechnology (DaLian) CO., LTD. Binding experiments were performed with an electrophoretic mobility shift assay kit  according to the manufacturer’s protocol using 6 µg of nuclear protein and 1 µl of 25 fmol of biotin-labeled wild type oligonucleotides. In the competitive reactions, a 100-fold, 200-fold and 400-fold excess of unlabeled wildtype oligonucleotides were added separately. In the mutant reactions, a 100-fold, 200-fold and 400-fold excess of unlabeled mutant oligonucleotides were added separately. In the supershift experiments, protein extracts were incubated for 15 min with 1–2 µg of the anti-myc antibody  at room temperature before the addition of the probe. Chemiluminescent detection of biotin DNA on membranes was realized with the Chemi-Doc apparatus .Nuclear protein extract were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents  from HEK293T cells with or without PfMSX dsRNA were GCGTAATACGACTCACTATAGGGAGATTCATGGATCTCCAAAGACAAT (forward); GCGTAATACGACTCACTATAGGGAGAATGGTGATACGTCATACCTACG (reverse). The T7 promoter sequence was underlined. A RiboMAX Large Scale RNA Production System (T7) kit  was used to synthesize and purify the dsRNA. RNase-free DNase I  was used to digest the template DNA. The dsRNA was diluted to 40 µg/100 µl using PBS. Total RNA from the mantle tissue of each oyster was extracted in day 7 after injection and used to synthesize the first strand cDNA as described above. QPCR was used to quantify the expression levels of PfMSX and Pif, where β-actin was used as an internal reference. QPCR primers were designed for PfMSX and β-actin which were the same sequences as that in the distribution experiment above. The Pif QPCR primers were from RNA interference was performed as described in Suzuki et al.Data were analyzed by one-way analysis of variance (ANOVA) with default parameters or t-Student test to identify differences between groups. Differences were considered statistically significant when p values were lower than 0.05.PfMSX cDNA, deposited in GenBank under accession no.KJ028206, consisted of a 5′-untranslated region (UTR) of 127 bp, a 3′-UTR of 106 bp, and an open reading frame (ORF) of 912 bp encoding a polypeptide of 304 amino acids with an estimated molecular mass of 33.3 kDa and a theoretical isoelectric point of 9.92. A comparison of the predicted homedomain amino acid sequence with those of a representative set of protostomes and deuterostomes revealed a striking conservation (similarity 86.8%–100%)  . The phy8%–100%)  indicateThe subcellular localization of PfMSX was determined by GFP fusion protein expression and immunofluorescence assay in HEK293T cells. As shown in PfMSX mRNA was constitutively expressed in all detected tissues. The expression level of the PfMSX in mantle was higher than in other tissues, and the expression in heart and digest was in a lower level , using WT and mutated oligonucleotide probes encompassing residues −1000 to −1008 of the pearl oyster Pif promoter sequence. line 1 shows that a faint retardation complex was obtained with HEK293T cell nuclear extract without any transfection, which would relate to the high-expression of MSX1/MSX2 in HEK293T cells (see ). line 2 shows that a strong retardation complex with HEK293T nuclear extracts transfected with pHis/Myc-PfMSX construct. The anti-myc antiserum supershifted the PfMSX-wt complex (lane 9). When the TAAT core sequence of MSX-binding site in Pif promoter was competed with 100-fold, 200-fold, 400-fold excess of the unlabeled DNA fragment, PfMSX binding were abolished (lane 3–5). When competed with 100-fold, 200-fold, 400-fold excess of the mutant DNA fragment which the TAAT core was mutated to TCCA (mut), PfMSX binding did not disrupted (lane 6–8). These results suggested PfMSX may directly bind to the ACTAATTGG motif, and bind to the TAAT sequence. Since the core sequence of MBS-2 is identical to that of MBS-1, it is reasonable to speculate that PfMSX could bind to MBS-2.The ability of a protein to bind selectively to a particular DNA site in the genome is the foundation upon which transcriptional regulatory pathways are built  complex , lane 9.Pif expression, we cloned regulatory elements in the pearl oyster Pif 5′-flanking region from −1358 to −1 with a series of 5′ deletion promoter-luciferase constructs , P1125LucΔ1 had 2.8-fold increases; Deletion of the MBS-2 (P1125LucΔ2), the increase was 4.1-fold; Deletion of the both binding site (P1125LucΔΔ), the increases was 2.1-fold. There is significant difference was noted in the ability of P1125Luc construct and P1125LucΔ1 or P1125LucΔΔconstruct to enhance this responsiveness but no significant difference between P1125Luc and P1125LucΔ2 construct  or green fluorescent protein (GFP) dsRNA-injected group respectively (PfMSX dsRNA-injected groups (PfMSX caused disorder in CaCO3 crystallization, for normal expression of some matrix protein like Pif was disturbed. These changes may further affect the iridescent color of nacre which depends on the thickness of the last few layers of nacre and the regularity of the tablets at the surface of the shell Double-stranded RNA injection in invertebrate has been shown to be an effective tool for interfering with the function of endogenous genes ectively . The surectively , whereasd groups  and of nd groups . On closd groups . With rePfMSX is the first gene reported here.So far as metazoans, our knowledge of gene regulatory mechanisms involved in biomineralization in invertebrates has been limited compared to that in vertebrates. Up to now, there is no information about transcription factor involved in direct regulation of matrix protein in pearl oyster, and Pif gene harbors two MSX binding sites which is identical to that in mammals in its promoter. Current MSX appears to have retained an ancient MSX binding site during the long course of evolution. Our data showed PfMSX could bind to the MSX binding site in the promoter of Pif gene and then mainly activate its expression through MBS-1 not MBS-2. The MBS-2 seems to be redundancy to Pif promoter, actually it implies more. Even in regulatory sequences with highly conserved function, transcription factor binding sites can be gained and lost over time at a high rate, leading to considerable differences in the composition and arrangement of binding sites between even closely related species. It is likely that the redundancy offered by such a system plays a role in avoiding the deleterious effects of uncontrollable mutations Pif luciferase promoter being markedly activation by the PfMSX vector after deleting both the MSX binding sites (Pif promoter which we are not certain exhibit functioning in Pif gene regulation. We subsequently investigated the function of PfMSX in vivo by the means of RNAi. The injection of PfMSX dsRNA decreased the expression levels of Pif mRNA strikingly and at the same time disordered the nacreous layer. The lamellar sheet in nacre surface showed different pattern comparing to that injected with Pif dsRNA in the literature, implicating that PfMSX may exert function in other unknown matrix protein in the pearl oyster. Collectively, the MSX of P. fucata could activate the expression of Pif gene.Pif is an important component in the nacreous layer and takes part in the initiation of aragonite crystallization as well as subsequent stacking of aragonite tablets in the nacreous layer. Crassostrea gigas was sequenced recently PfMSX has high expression in the mantle tissue.The Pacific oyster and the pearl oyster are in close kinship. The genome of the Pacific oyster Taken together, PfMSX takes part in the regulation of biomineralization in pearl oyster.MSX genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa Highly conserved The DNA binding sites of MSX1 proteins have been defined. The canonical TAAT sequence is the core binding site for MSX1 Strongylocentrotus purpurutus was abundant in blastula stage and prism stage during which the skeleton formed and the related sea urchin Heliocidaris tuberculata injected with a morpholino to HtMSX exhibited reduced skeletal growth The highly conserved homeodomian and highly conserved binding site may share some identical features in the course of evolution. For many homeobox genes involved in developmental processes, more and more comparative data are becoming available. From these data, it is evident that in groups of very diverse animals many genes share the same function. There are accumulating examples of MSX orthologs implicating in regulating biomineralization: vertebrate MSX genes are expressed in limb bud, mandibular process, tooth; Zebrafish MSX genes are expressed strongly in fin tissues


The genome has the ability to respond in a precise and co-ordinated manner to cellular signals. It achieves this through the concerted actions of transcription factors and the chromatin platform, which are targets of the signaling pathways. Our understanding of the molecular mechanisms through which transcription factors and the chromatin landscape each control gene activity has expanded dramatically over recent years, and attention has now turned to understanding the complex, multifaceted interplay between these regulatory layers in normal and disease states. It has become apparent that transcription factors as well as the components and modifiers of the epigenetic machinery are frequent targets of genomic alterations in cancer cells. Through the study of these factors, we can gain unique insight into the dynamic interplay between transcription factors and the epigenome, and how their dysregulation leads to aberrant gene expression programs in cancer. Here, we will highlight how these factors normally co-operate to establish and maintain the transcriptional and epigenetic landscape of cells, and how this is reprogramed in cancer, focusing on the RUNX1 transcription factor and oncogenic derivative RUNX1–ETO in leukemia as paradigms of transcriptional and epigenetic reprograming. However, control of gene expression is exerted by mechanisms involving the interaction of transcriptional complexes with not only the DNA sequence itself but also the chromatin proteins associated with the DNA. Access to the DNA by transcription factors is controlled by the packaging of DNA within the nucleus as chromatin. The specific composition of chromatin can dictate gene expression patterns in a cell by regulating the relative accessibility provided to transcription factors and the transcriptional machinery. Although the tight packaging of nucleosomes into heterochromatin inhibits transcription factor access, and transcription, euchromatin, with its associated relaxed chromatin and nucleosome positioning is more conducive to transcriptional activation due to the relative ease of transcription factor access. Chromatin composition is dynamic and is maintained or modified through the concerted actions of transcription factors and chromatin modifiers responding to cellular signaling cascades. Not surprisingly then, mutations to transcription factors and the molecules that comprise and modify the chromatin landscape commonly underlie the altered gene expression profiles that are characteristic of cancer cells. Herein, we will focus on the co-operative actions of these factors to determine the transcriptional and epigenetic landscape of cells, and how this is reprogramed by modifications to key regulatory factors in cancer.The gene expression profile of a cell determines its phenotype and function, and transcription factors play a key role in defining these gene expression profiles in response to cellular signals. Typically they do so by binding to Within the eukaryotic cell, DNA is assembled into chromatin, consisting of repeating units of nucleosomes. Each nucleosome is a complex of eight histone proteins  around which 147 base pairs of DNA are wrapped. This complex forms the basis of the epigenetic landscape of a cell, which in its broadest definition encompasses DNA methylation, histone modifications, histone variants, and nucleosome positioning . TogethePerhaps the most widely investigated mechanism of epigenetic control is DNA methylation, the modification of DNA involving the addition of methyl groups to CpG residues, by DNA methyltransferases. CpG dinucleotides are found throughout the genome in dense clusters, referred to as CpG islands that are associated with a large proportion of gene promoters. CpG methylation is generally, although not always, associated with gene silencing. CpG methylation facilitates gene repression by acting as a recruiting surface for proteins, which possess methyl binding domains and initiate chromatin condensation. Interestingly, DNA methylation alone has been shown to further promote chromatin condensation . To a leHistone tails can be post-translationally modified via the addition or removal of numerous chemical groups, with over a dozen now reported, including acetyl, methyl, and phosphate groups . These mNucleosome structure can also be altered via the inclusion of histone variants within the histone octamer. During chromatin assembly and disassembly, histone variants can be deposited and exchanged by histone chaperones. Histone variants possess unique structural and functional properties. For example, the inclusion of the histone variant H3.3 is associated with transcriptionally active regions but is also deposited in telomeric and pericentric heterochromatin . The hiscis and trans interactions possible  chromosomal translocation found in AML. This translocation fuses the N-terminal region of the RUNX1 gene to the RUNX1T1 gene , generating a RUNX1–ETO chimeric protein   .While early studies characterized RUNX1 as a transcriptional activator, and demonstrated co-operation between RUNX1 and a range of other transcription factors to drive promoter activity , 33. RUNRUNX1 has been found to complex with an array of epigenetic modifiers, with the outcome for both RUNX1 function and target gene activity dependent on the balance of activating and repressive factors associated with RUNX1 at a particular time Figure A. RUNX1 RUNX1 itself is also a target of acetyltransferases as well as other modifiers, with modification of RUNX1 activity through interaction with epigenetic modifiers emerging as a common theme. Acetylation of RUNX1 by p300 was found to enhance its DNA-binding activity . MethylaThese studies thus build a picture of RUNX1 itself as a target of epigenetic modifiers, which post-translationally modify the RUNX1 protein, altering its coactivator/corepressor interactions and thus influencing its transcriptional activity. In addition, RUNX1 recruits these coactivators and corepressors to target genes, resulting in modification of the chromatin environment and thus affecting gene activity at this second level. An elegant example of this multi-layered interaction between RUNX1, epigenetic modifiers and the epigenome is demonstrated in the recently described interaction of RUNX1 with protein arginine methyltransferase 6 (PRMT6). PRMT6 was found to be a component of a repressive RUNX1 complex also containing sin3a and HDAC1 . RecruitThe oncogenic RUNX1–ETO fusion protein contains the RUNX1 DNA binding domain but the transcriptionally active C-terminal domain of RUNX1 is replaced by almost the entire ETO protein. While RUNX1–ETO alone is not sufficient to induced leukemic transformation, it is sufficient to promote self-renewal and disrupt myeloid and erythroid differentiation . The RUNOver recent years, we have gained considerable insight into how chromatin structure is modified and the impact of this on the genome. Attention has now turned to understanding the dynamic and multifaceted interactions between transcription factors and epigenetic modifiers, and how they co-operate to determine gene expression responses to cellular signals. This is providing a better understanding of how alterations to these key regulatory molecules in cancer, as well as other disease processes, impact gene expression profiles. Further, this is offering hope that a better understanding of the interaction between aberrant transcription factors and the epigenome of cancer cells will provide increased opportunities for intervention in the disease. Interest in this field is being stimulated by the promising advances that are being made in cancer treatment with current epigenetic therapies, as highlighted recently . For exaKB conducted literature research and drafted sections of the manuscript and edited the manuscript. PT edited the manuscript and prepared figures. AH conducted literature research, drafted sections of the manuscript and edited the manuscript.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.


BACE1 gene encodes for β-Site amyloid β precursor protein (APP)-cleaving enzyme1, which is required for generating amyloid β protein(Aβ). Deposition of Aβ in brain plays an essential role in Alzheimer's Disease (AD) pathogenesis. BACE1 gene has a tissue-specific expression pattern and its expression is tightly regulated at transcriptional level. Core promoter is a minimal DNA sequence to direct transcription initiation and serves as a converging platform for the vast network of regulatory events. Here we identified the core promoter of human BACE1 gene, which is a 71 nucleotides region absent of typical known core promoter elements and is sufficient to initiate a basal transcription. Two novel DNA motifs, designated TCE1 and TCE2, were found to be involved in activating the transcription of human BACE1 gene in a synergistic way. Two single nucleotide mutations in these motifs completely abolished the promoter activity. In conclusion, our studies have demonstrated that novel DNA motif TCE1 and TCE2 in human BACE1 gene promoter are two essential cis-acting elements for BACE1 gene transcription. Studies on how these two motifs being regulated by different stimuli could provide insights into the molecular mechanisms underlying AD pathogenesis and pharmaceutical potentials of targeting these motifs for AD treatment. BACE1 gene encodes for β-Site APP cleaving enzyme1 , a type 1 membrane-associated aspartyl protease of 501 amino acids345in vivo2789101314217Amyloid β protein (Aβ) is the major component of amyloid neuritic plaques in the brain of AD and its deposition in the brain plays an essential role in AD pathogenesisBACE1 gene has a tissue and stimuli-specific expression pattern2BACE1 mRNA has been found mainly in neuronal cells and in the pancreas3BACE1 expression is tightly regulated at the level of transcriptionBACE1 gene contains many putative transcription factor binding sites1920BACE1 gene and to positively regulate its expression. Transcription factor specificity protein 1 (Sp1) was the first one discovered to play an important role in regulating human BACE1 expressionBACE1 promoter and mediated the increases of BACE1 transcription induced by hypoxia222324BACE1 constitutively expression in neurons and inducible expression by interferon γ (IFNγ) in astrocytes26BACE1 transcription2829BACE1 gene transcription. Two GATA-1 sites located at -106 to -97 and -209 to -200 from authentic ATG codon of rat BACE1 were found to act as suppressors of BACE1 transcriptionBACE1 promoter and suppress BACE1 transcription, and decreasing PPARγ level by proinflammatory cytokines is responsible for activation of BACE1 promoter31BACE1 promoter region2133BACE1 gene transcription, which depends on cell-type or cell-condition2335BACE1 gene expression, while it activates BACE1 in higher Aβ concentrations or in activated astrocytes. The majority of cis-acting elements of BACE1 gene promoter are located far from transcriptional start site (TSS). There is little research on the function and structure of the proximal region of BACE1 promoter or core promoter near TSS.The BACE1 gene promoter lacks the typical CAAT and TATA boxes within 2 kb upstream region of the TSSBACE1 gene core promoter will greatly advance our understanding the transcriptional mechanisms of TATA-less core promoter and may benefit for developing new strategies in inhibiting BACE1 expression in prevention and treatment of Alzheimer's disease.Core promoter is a minimal DNA sequence to direct transcription initiation and serves as a converging platform for the vast network of regulatory events by interaction of transcription factors and proximal or distal regulatory DNA motifs in the promoter373737BACE1 promoter, which is a 104 base pairs in length and sufficient for high levels of expression in cells. We identified the core promoter region of 71 nucleotides from −550 to −480 bp relative to ATG of the BACE1 gene and this core promoter region is able to initiate a basal transcription of downstream gene. BACE1 gene core promoter lacks known core promoter elements. Two novel DNA motifs, designated as TCE1 and TCE2, were found to be involved in activating the transcription of BACE1 gene in a synergistic way. Reducing the spacing between TCE1 and TCE2 decrease the promoter activity and two single nucleotide mutations in these motifs are able to completely abolish promoter activity of BACE1 gene. Collectively, our studies have demonstrated that novel DNA motif TCE1 and TCE2 identified in human BACE1 gene promoter are essential for controlling transcription of BACE1 gene.In this study, we defined the boundary of the proximal region of human BACE1 gene has promoter activityBACE1 gene inserted in pGL3-Basic vector. DNA sequencing confirmed that the sequence of inserted BACE1 –1273 to -400 in p3BU-1273/-400 is identical to 117187784-117186911 of chromosome 11 (GRCh37/hg19), except for one SNP rs536817 (chr11:117187441), which is G in our sequence. The pGL3-Basic vector lacks eukaryotic promoter and enhancer sequences upstream of a reporter luciferase gene. Luciferase activity in cells transfected with reporter plasmid depends on the expression of luciferase gene, and the expression level depends on insertion and proper orientation of a functional promoter upstream from the reporter luciferase gene.Our previous study demonstrated that the region from −1311 to −400 bp upstream of the translation start codon (as +1) of the human BACE1 promoter activity, several deletion plasmids were generated and transfected into HEK293 cell. Luciferase activity measured from cells transfected with the original pGL3-Basic vector served as control. The results of luciferase assay indicated that p3BU-1273/-400, p3BU-1149/-400, p3BU-919/-400, p3BU-800/-400, p3BU-700/-400, p3BU-583/-400 have similar promoter activity and are significantly higher than pGL3-Basic control  had no significant effects on luciferase activity . FurtherBACE1 minimal promoter activity in the different context of 5′ end, more 5′ end deletion plasmids were generated and transfected into HEK293 cell. Luciferase activity showed that a series of plasmids ended with -480 as 3′end have similar pattern of luciferase activity as a series of plasmids 3′ ended with -400  showed similar activity as p3BU-570/-480 and continuing to delete 10 bp from 5′end of -550 to -480 caused further significant decrease of luciferase activity in p3BU-540/-480. It suggested that 71 bp region of -550 to -480 still has significant promoter activity. Inserting the 104 bp fragment of -583 to -480 bp in reverse direction in plasmid p3BU-583/-480R abolished luciferase activity  had no negative effects, but mutation -578G/A, -510A/C or -482T/A negatively affected luciferase activity of plasmid p3BU-583/-400  or 580C/T (p3BU-583/-400M580T), double nucleotides mutation of 578G/A and 580C/T (p3BU-583/-400M580T578A) showed similar luciferase activity as single nucleotide mutation of 578G/A, suggesting that mutation of 580C/T has no additional effects on luciferase activity of plasmid p3BU-583/-400M578A  as a reporter gene were constructed by replacing the luciferase reporter gene with EGFP gene in plasmids pGL3-Basic, pGL3-Promoter, p3BU-1149/-400 and p3BU-1149/-400M578A510C. Empty control plasmid pGL3basic-EGFP didn't show florescence 24 hours after transfection into HEK293 cell, positive plasmid pGL3promoter-EGFP, containing a strong SV-40 promoter, showed very strong florescence. Plasmid p3BU-1149/-400EGFP displayed significant brighter florescence than pGL3basicEGFP. As expected, plasmid p3BU-1149/-400M578A510CEGFP displayed much weaker florescence than p3BU-1149/-400EGFP  or 510A/C (p3BU-1149/-400M510C), double mutant 578G/A and 510A/C (p3BU-1149/-400M578A510C) drastically reduced luciferase activity of wildtype plasmid p3BU-1149/-400 and had much lower luciferase activity than each single mutant 578G/A or 510A/C . To furt-400EGFP . These rBACE1-588/-568 (CACCTCGGCAGAGGGCATCCC). The MIZ1-binding sequence identified by MatInspector is gatgcCCTCtg , which is located in antisense strand of BACE1-580 to -570 (CAGAGGGCATC). Since the candidate binding site of MIZ1 is located in antisense strand of BACE1 gene, a reporter plasmid (p3BU-1149/-400MIZ) with candidate binding site of MIZ1 at sense strand was constructed and transfected into HEK cells. The luciferase activity of p3BU-1149/-400MIZ was significantly lower than wildtype p3BU-1149/-400(BACE1 gene promoter sequence of -520/-500 (TACACTTCCCAGCGATCCCAG) with program MatInspector resulted in no hit.In order to identify possible proteins binding to surrounding sequences of nucleotide -578G or -510A, a 21-nucleotide sequence corresponding to 10 nucleotides flanking each side of -578G or -510A was used for transcriptional factor analysis using MatInspector1149/-400, suggestcis-acting element and the sequence of CGGCAGAGGGC was named as BACE1 Transcription Critical Element 1 (TCE1), which may bind to unknown transcriptional factor. The nucleotide replacement at -584T with A or G has the similar luciferase activity as wildtype and the nucleotide replacement at -584T with C and at -572A with G even increased the promoter activity. The nucleotide replacement at -580C with T also significantly increase the promoter activity, suggesting that the wildtype sequence of TCE1 may not be the ideal sequence for binding with a transcriptional factor.Since there is no known transcriptional factor bound to these two important regions, next we defined the boundaries of both regions and investigated the effects of nucleotide substitution on promoter activity. We first generated p3BU-1149/-400 mutant plasmids carrying mutations surrounding -578G or -510A, and each nucleotide was replaced with three other nucleotides, respectively. The luciferase assay results showed that the majority of nucleotide replacements in region between -583 and -573 had negative effects on promoter activity , indicatcis-acting element and the sequence of TTCCCAG was named as BACE1 Transcription Critical Element 2 (TCE2). Comparing the effects between TCE1 and TCE2 on BACE1 promoter activity, many nucleotide replacements in TCE2 caused 70% reduction and nucleotide replacements in TCE1 only caused 40-50% reduction, suggesting that TCE2 has stronger effects than TCE1 on BACE1 transcription.For the functional region surrounding -510A, the boundary seems to be -515 and -509, since almost every replacement between -515 and -509 significantly reduce the promoter activity . HoweverBACE1 transcription.Next we analysed evolutionary conservation of 100 vertebrate species within the region of -583/-480. Three highly conserved regions were identified and they are -583/-570 , -551/-544  and -528/-509  . NucleotBACE1 core promoter  is a recognition site for the binding of transcriptional factor TFIID and has a consensus sequence of YYANWYY in human. Two putative Inr elements are located at -569/-563(CCAGACC) and -521/-515(CTACACT) respectively. Because upstream putative Inr site -569/-563 is located between-570 and -550 in which the sequence are not functional as shown before, and deleting region -570/-550 has no effects on BACE1 basal transcription, upstream putative Inr is most likely not functional. Downstream putative Inr site is located at -521/-515 and is not consistent with published and reference gene TSS at GenBank, which are located between nucleotide 453 to 461 upstream of translational start site ATG. To verify whether it is functional Inr site, first we constructed plasmid p3BU-1149/-400InrM by replacing Inr site with an reverse Inr sequence (-521/-515 CTACACT was changed to tcACAtc) , the TATA box (tatawaar), BRE , DPE , MTE , and XCPE1tcACAtc) . The luctcACAtc) , even thtcACAtc) . Because521/-515 . Taken tBACE1 gene, but similarity between rat and human in the region -1000 bp to -600 bp from ATG drops down to 39% and the similarity is lost beyond -1000 bpBACE1 expression, consistent with our results that deleting the upstream region of -583 has no effects on promoter activity. These results also suggest that human and mouse may have similar regulatory mechanism with 600 bp upstream of the ATG codon and have different regulatory mechanism beyond 600 bp region. Studies on promoter structure and transcriptional regulation of BACE1 have been mainly focused on the rat and human genes. The cis-acting elements identified and verified experimentally in the rat and human BACE1 gene need to be carefully considered in term of the location. The ones located over-600 bp from ATG, especially over-1000 bp region may play different roles in the transcriptional regulation of BACE1 gene between rat and human. The cis-acting elements identified experimentally in human BACE1 include NF-κB binding site33BACE1 include NF-κB binding siteBACE1 gene454647BACE1 promoter is located at -575/-560(ACTCCATCTCGGCAG) upstream of rat authentic ATG, same as the human sequence at -592/-578 (ACTCCACCTCGGCAG) except for one nucleotide difference at -586. Although putative YY1 in human gene overlap partially with TCE1, TCE1 is not like to be YY1 binding site based on following three reasons. First, only six nucleotides out of 15 in the 3′end of human putative YY1 binding site overlap with 5′end of -583 bp/-578 bp in TCE1. Secondly, deleting 9 nucleotides out of 15 at 5′ end in p3BU-583/-400 has similar luciferase activity as p3BU-700/-400, which include full YY1 binding sequence. It suggests that 5′ end 9 nucleotides of human putative YY1 binding site are not functional. Thirdly, according to reported YYI consensus sequence CGCCATnTTBACE1 gene.Phylogenetic analysis of DNA sequences is a useful strategy to identify DNA sequence motifs that have been strongly conserved through evolution and well-conserved sequence can provide much stronger evidence of functional relevance. A conservation analysis revealed 92% similarity between rat and mouse and 81% similarity between rat and human within the -600 bp upstream of the authentic ATG codon of cis-acting transcriptional control sequences, promoters and distal regulatory elements3951BACE1 promoter, our results demonstrated that BACE1 gene core promoter lacks typical known core promoter elements including Inr, TATA box, BRE, DPE, MTE, and XCPE1. The newly identified TCE2 from our study is most likely to be a novel unknown core promoter element, which plays an important role in initiating a basal transcription and may be involved in different transcriptional mechanism.RNAPII-transcribed genes typically contain two distinct families of BACE1 gene minimal and core promoter and found two important DNA motifs, TCE1 and TCE2. TCE1 is likely to be a promoter proximal element and TCE2 is a core promoter element. TCE1 and TCE2 function in a synergistic way and these two novel DNA motifs are essential for transcription of human BACE1 gene. Future study on how these two motifs being affected by different stimuli to regulate BACE1 processing of APP could provide insights into the molecular mechanisms underlying Alzheimer pathogenesis and pharmaceutical potentials of targeting these motifs for AD treatment.In this study, we defined the boundary of human Human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 mmol/L of sodium pyruvate, 2 mmol/L of L-glutamine and 1% penicillin-streptomycin at 37°C in a humidified incubator containing 5% CO2. Cells were grown to approximately 70 to 80% confluence and plasmid DNA were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the supplier's protocol., 3.2 μg of total plasmid DNA were transfected into the cells cultured on a 35-mm-diameter dish for 5′-RACE and RNA extraction and 200 ng of total plasmid DNA per well were transfected into the cells cultured on a 48-well plate for luciferase reporter assay.BACE1 promoter firefly luciferase reporter plasmid p3BU-1273/-400 contains human genomic sequence from –1273 to -400 relative to BACE1 gene translating start site as +1. BACE1 genomic fragment B1U-1273/-400 was amplified with primer B1U1273F (CCGCTCGAGAACCATACCGGCTTTTCTCCT)/B1U400R (CACAAGCTTCCACCATAATCCAGCTCG) and BAC DNA(RP11-970G21) as template. PCR product of B1U-1273/-400 was inserted in front of the firefly luciferase reporter gene in the promoterless reporter plasmid, pGL3-Basic (Promega) at XhoI/HindIII sites and inserted sequence was confirmed by DNA sequencing. The methods for constructing other plasmids are described in BACE1 in p3BU-1149/-400.The plasmid p3BU-1149/-400 was transfected into the cells and total RNA was extracted from cells with TRI reagent following the manufacturer's protocol (Sigma). The 5′-RACE was carried out according to the FirstChoice RLM-RACE Instruction Manual . Briefly, 10 μg RNA was treated with Calf Intestine Alkaline Phosphatase (CIP) and Tobacco Acid Pyrophosphatase (TAP), resulted RNA was ligated with 5′RACE Adapter (5′-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA). The RNA with adapter was reverse transcripted into cDNA with SuperScript III First-Strand Synthesis System (Invitrogen) and amplified with outerprimer (5′outer primer: 5′-GCTGATGGCGATGAATGAACACTG/3′specific outer primer: 5′-TTCCAGGAACCAGGGCGTATCTCT). 3′ specific outer reverse primer matches luciferase coding region and start from minus strand +115, which is relative to luciferase gene translating start site as+1. Above PCR product was re-amplified with inner primer (5′inner primer: 5′-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG/3′specific inner primer:5′-GTGAAGCTTGGTTCCATCTTCCAGCGGATAGAAT). 3′ specific inner reverse primer matches luciferase coding region and start from minus strand +66. Resulted PCR products were digested with BamHI/HindIII and cloned into plasmid pGL3-Basic at Bgl II/HindIII sites. Positive clone was sequenced with pGL3-Basic sequencing primer 5′-CTAGCAAAATAGGCTGTCCC to determine the transcription initiation site of Renilla luciferase gene and serves as transfection control to normalize for the transfection efficiency of various luciferase reporter constructs. Twenty-four hours after transfection, cells were harvested and luciferase activities were measured using the dual-luciferase reporter assay system according to the supplier's protocol (Promega). The firefly luciferase activity was normalized to the Renilla luciferase activity and expressed as relative luciferase units (RLUs) to reflect the promoter activity.Cells cultured in 48-well plates were co-transfected with 180 ng of BACE1-luciferase reporter plasmids and 20 ng of p3PRluc plasmid per well. P3PRluc has W.Z. designed the research, performed experiments and wrote the manuscript; W.S. planned the research and wrote the manuscript; Y.X. and S.M. performed experiments; J.W., S.L. and J.L. constructed plasmids; H.L. constructed plasmids and analyzed data; T.L. discussed the project and provided reagents.Supplementary Methods


The recent advent of high-throughput approaches has revealed widespread transcription of the human genome, leading to a new appreciation of transcription regulation, especially from noncoding regions. Distinct from most coding and small noncoding RNAs, long noncoding RNAs (lncRNAs) are generally expressed at low levels, are less conserved and lack protein-coding capacity. These intrinsic features of lncRNAs have not only hampered their full annotation in the past several years, but have also generated controversy concerning whether many or most of these lncRNAs are simply the result of transcriptional noise. Here, we assess these intrinsic features that have challenged lncRNA discovery and further summarize recent progress in lncRNA discovery with integrated methodologies, from which new lessons and insights can be derived to achieve better characterization of lncRNA expression regulation. Full annotation of lncRNA repertoires and the implications of such annotation will provide a fundamental basis for comprehensive understanding of pervasive functions of lncRNAs in biological regulation. It is well known that DNA is transcribed into messenger RNA (mRNA), which is then translated to protein(s) with the help of housekeeping noncoding RNAs (ncRNAs) such as transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs). Messenger RNAs serve as intermediate carriers, forwarding genetic information (as coding genes) from DNA to protein. Characterization of coding genes and their protein products has been of great importance in our goal to understand gene expression regulation. While early expectations were to find about 100,000 genes in the human genome, the current estimate stands at 20,000-25,000 ,4, despiNew small regulatory ncRNAs were first identified by exogenous RNA interference in plants and nematodes, and later found to exist endogenously. These small ncRNAs, including but not limited to microRNAs (about 22 nt long), function as posttranscriptional repressors . ThroughXist) [Air, Kcnq1ot1) [NORN) [HOTAIR [NEAT1 and MALAT1 [trans-acting gene regulation (HOTAIR) [NEAT1) [MALAT1) [sno-lncRNAs) [Beyond the small regulatory ncRNAs, the multifaceted transcriptome has become even more complex with the discovery of the pervasive transcription of long noncoding RNAs . LncRNAs are known to play important roles in both biological and pathological events ,12,13,14Xist) , genomiccnq1ot1) ,17 and n) [NORN) . The app [HOTAIR , NEAT1 a(HOTAIR) , providi [NEAT1) ,22,23,24[MALAT1) , althoug[MALAT1)  and mous[MALAT1) . Detaile[MALAT1)  and huma[MALAT1) . In addi[MALAT1) , to acti[MALAT1) , to perf[MALAT1) , and to [MALAT1)  and embr[MALAT1) . In addi[MALAT1) . In somelncRNAs) . As up to 70% of the human genome can be transcribed  and onlya). However, several intrinsic features of lncRNAs have posed challenges for their discovery as well as their functional study, as discussed below.In the first decade of this century, whole genome sequencing revealed approximately 20,000 protein coding genes in humans, which is comparable to estimates in the fly and worm, although humans exhibit much more complexity through alternative splicing ,2. With Figure 1H19 in NHEK cells [sno-lncRNAs in hES cells [RNA-seq (deep sequencing from reverse-transcribed RNAs) datasets revealed that the human genome is pervasively transcribed . HoweverEK cells  and sno-ES cells ). Accumulated results suggested that most lncRNAs exhibit a low level of expression but high tissue-/cell-specific patterns ,38,43. AHomologous sequence comparison is an efficient method for identifying genes that exhibit similar functions between species and for discovering novel coding regions , howeverUsually, evolutionary constraint can be estimated from the nucleotide substitution rate in functional sequences . NucleotNEAT1 RNA and its mouse homolog Menε/β have low sequence similarity [The aforementioned studies suggested that low evolutionary conservation might be a natural feature of noncoding transcripts, which is consistent with their rather poor genome-wide annotations in early studies ,2,4. Howmilarity  but are milarity ,22,23,24milarity . In factmilarity ,53.Xist [Xist [Exclusion of protein-encoding capacity is a fundamental requirement for lncRNA definition. In the post-genomic era, this capacity can be predicted genome wide using computational approaches, mainly based on the length and conservation of ORFs . CutoffsXist , have reXist  containsXist . AlthougXist  and thisst [Xist . A phylost [Xist , and sucst [Xist  and humast [Xist . NEAT1 [DEB-T [in situ hybridization (FISH) is an alternative way to examine the subcellular localization. A growing list of well-characterized lncRNAs do localize in the nucleus and within specific subnuclear structures as illuminated by RNA FISH and are associated with nuclear proteins as revealed by RNA-protein double FISH [Besides computational judgments based on critical features of putative ORFs, several other crucial criteria, such as the subcellular localization and the accessibility to the translation machinery, could also be used to evaluate whether a given transcript is a true lncRNA or not. RNA transcripts localized in the nucleus principally suggest functions that are primarily non-coding. This can be estimated experimentally by RNA fractionation from nuclear homogenates , as exemNEAT1  and DEB-1 [DEB-T , despiteble FISH . Furtherble FISH . Moreoveble FISH ,66.in vivo.The best way to distinguish between coding and non-coding sequences is to integrate computational and experimental approaches that enhance understanding of lncRNA expression regulation and biological function With technological improvements and the application of integrated methodologies, significant progress has been achieved in uncovering new lncRNA molecules. Some of these practical strategies can be further applied to achieve new insights into lncRNA functions. b). In addition, other stringent criteria have also been taken into account for lncRNA characterization, including the identification of poly(A) sites, transcription initiation signals, expression patterns among tissues and potential coding capacity. Loss-of-function and gain-of-function of certain conserved lncRNAs demonstrated crucial biological roles of lncRNAs in zebrafish [Several individual studies have applied a systematic and integrative strategy with multiple biological features to identify lncRNAs, mainly in intergenic regions (lincRNAs), first in mouse  and thenFigure 1ebrafish , indicatebrafish , Rinn [1ebrafish  and theib). In addition, with the same separation strategy for poly(A)− transcripts followed by deep sequencing analyses, additional poly(A)− lncRNAs from intronic regions were also found in various human cell lines [X. tropicalis [Most RNA polymerase II transcripts, including mRNAs and lncRNAs, are polyadenylated (poly(A)+) at their 3’ ends. The application of transcriptome analysis of poly(A)+ RNA by high-throughput deep sequencing (mRNA-seq) has revealed a digital map of poly(A)+ transcripts from both known and previously un-annotated genes . HoweverFigure 1ll lines . Interesopicalis . As mostMALAT1 and Menε/β lncRNAs, their 3' end maturation depends on RNase P cleavage [S. pombe, incomplete splicing, but not the complete splicing, generates a functional TER1 transcript [et al recently demonstrated that intron-derived sno-lncRNAs depend on the snoRNA machinery at both ends for their processing and on snoRNP complexes at both ends to protect intronic sequences from exonucleotic trimming [What mechanism(s) can generate RNA transcripts without canonical poly(A) tails at their 3' ends? For most of the replication-dependent histone pre-mRNAs, evolutionarily conserved stem-loop structures in their 3’ UTRs direct U7 snRNA-mediated 3’ end formation to stabilize mature mRNAs and confer cell cycle dependent regulation of their accumulation . For MALcleavage ,69, stabcleavage ,74. For anscript . Howevertrimming . Genome-trimming ,38; howein vivo. Accumulated lines of evidence suggest that very often lncRNAs function by recruiting and assembling other co-factors, which are usually proteins but possibly other RNAs [It’s now clear that lncRNAs play important roles in a variety of biological processes ,14,63. Sher RNAs ,76,77 orher RNAs . ClearlyXist is capable of recruiting Polycomb Repressive Complex 2 (PRC2) to remodel chromatin modifications [Air and Kcnq1ot1 lncRNAs achieve transcriptional silencing by recruiting chromatin-remodeling complexes during genomic imprinting [Evf-2 binds the Dlx-2 protein, which in turn increases the activity of the Dlx-5/6 enhancer [The lncRNA ications , resultiprinting ,81. Indeprinting ,82. In aenhancer . Interesenhancer  or Trithenhancer . NEAT1 lncRNA is crucial for the integrity of paraspeckles [NEAT1 is capable of initiation of paraspeckle de novo formation [In addition, lncRNAs can act as molecular scaffolds. For example, telomerase RNA component (TERC) acts as a flexible scaffold for bridging protein subunits together to promote telomerase activity . NEAT1 lspeckles ,22,23,24ormation . Gas5 lncRNA binds the glucocorticoid receptor (GR) to compete against the association of the GR with other glucocorticoid response DNA elements, resulting in functional repression of GR [sno-lncRNAs trap Fox family members to alter local Fox protein concentration and, subsequently, modulate Fox-regulated alternative splicing events [Moreover, lncRNAs can also function as molecular sponges or decoys to affect gene regulation mediated by protein cofactors. For example, on of GR . PWS regg events . Meanwhig events ,51,76,77g events .b), or using labeled lncRNAs as baits to pull down protein partners. Taken together, these studies suggest that the functional specificity of a given lncRNA is largely dependent on the association with its co-factors, mainly protein partners. Hence, it is important to find associated protein co-factors in order to fully understand the functional roles of lncRNAs. While the potential binding capacity can be predicted by computationally searching for consensus RNA sequences/motifs, direct lncRNA-protein interactomes can also be retrieved from cross-linking immuno-precipitation coupled with high-throughput sequencing (CLIP-seq) b, or usiXist contains at least two distinct domains. One is the RepC domain, which is bound by YY1 and hnRNP U for the localization; the other one is the RepA domain, which recruits PRC2 for in-cis gene expression regulation [Xist, the PWS region sno-lncRNAs contain multiple consensus hexamer motifs for Fox family splicing regulators [NEAT1 and mouse Menε/β share low primary sequence similarity, but both are associated with DBSH proteins [How do lncRNAs bind to their protein co-factors? There are a variety of known mechanisms for this. gulation ,90. Diffgulators , which lgulators . Interesproteins ,22,23,24proteins . SimilarIn the era of post-genomics, elucidating the full spectrum of RNA molecules by a given cell is important for understanding gene expression and functional regulation. Largely from the previously imagined “dark matter” of the genome, a variety of lncRNAs have been systematically revealed from different tissues and species with clear characteristics distinguishing them from coding RNAs. The characteristics of lncRNAs are (1) low expression but with a pattern of tissue-specificity, (2) decreased conservation in primary sequence but with a likelihood of functional conservation, and (3) restrained coding capacity but with a probability of ancestral ORF relics. Transcriptome analyses by high-throughput technologies (including tiling arrays and RNA-seq) with high coverage, high sensitivity, and high efficiency represent an evolutionary leap in our methodology for lncRNA characterization. Recent studies have inspired new insights into the study of lncRNAs, and in turn, these insights have prompted further application of novel methodologies for lncRNA study.Despite recent and rapid progress in our understanding of lncRNAs, a number of important features remain to be further addressed. For example, what are the landscapes of lncRNA expression in specific tissues/species and what are the connections of specific expression repertoires with specific tissue/species function? What are the distinct mechanisms for regulation of lncRNAs in specific tissues/species? What secondary structures are associated with lncRNA functions? Furthermore, existing computational algorithms are not sufficiently robust to deal with these sequence analyses. For example, they are less efficient for the accurate alignment of sequencing reads to lncRNAs in repetitive regions as well as for the precise transcript alignment of multiple lncRNA molecules from the same genomic segments.Clearly, the integration of not only new computational pipelines, but also further experimental approaches, will be required to further our ability to discover new lncRNAs and how they function in gene regulation.


Cyclin D1 locus to find multiple distal interactions that contribute to the complex cis-regulation of this cell cycle gene. Furthermore, we characterize a novel distal interaction upstream of the Cyclin D1 gene which provides mechanistic evidence for the abundant overexpression of Cyclin D1 occurring in multiple myeloma cells harboring a pathogenic translocation event. Through use of mapped retroviral integrations and translocation breakpoints, our studies highlight the importance of chromatin looping in oncogene expression, elucidate the epigenetic mechanisms crucial for distal cis-regulation, and in one particular instance, explain how a translocation event drives tumorigenesis through upregulation of a proto-oncogene.The majority of the genome consists of intergenic and non-coding DNA sequences shown to play a major role in different gene regulatory networks. However, the specific potency of these distal elements as well as how these regions exert function across large genomic distances remains unclear. To address these unresolved issues, we closely examined the chromatin architecture around proto-oncogenic loci in the mouse and human genomes to demonstrate a functional role for chromatin looping in distal gene regulation. Using cell culture models, we show that tumorigenic retroviral integration sites within the mouse genome occur near existing large chromatin loops and that this chromatin architecture is maintained within the human genome as well. Significantly, as mutagenesis screens are not feasible in humans, we demonstrate a way to leverage existing screens in mice to identify disease relevant human enhancers and expose novel disease mechanisms. For instance, we characterize the epigenetic landscape upstream of the human  Interestingly, despite previous ideas that non-coding DNA implied non-functional elements, there is a striking amount of recent evidence to show that these non-coding regions play an active role in gene regulation. It is well-established that many of these intergenic regions of DNA are highly conserved sequences among species and contain important elements . Furtherromoters , 3. The The mapping of retroviral integration sites within the mouse genome was originally employed as a powerful means to identify oncogenes and oncogene cooperation, particularly in hematopoietic cancers –7. Throucis-regulation is critical for gene expression. For example, the large intergenic region upstream of the human Cyclin D1 (CCND1) locus on 11q13 is a hotspot for chromosomal translocations, particularly in multiple myelomas. Multiple breakpoints have been mapped within this region, from 47 kb to 300 kb centromeric to the CCND1 TSS in hematologic cancer cells [IGH locus next to 11q13, CCND1 becomes highly overexpressed [CCND1 becomes aberrantly controlled by the IGH locus, the two loci on the rearranged chromosome remain genomically distal to one another; thus how the IGH locus could exert transcriptional activation over such an enormous genomic distance remains elusive. In our study, we provide a mechanism through which the potent IGH locus acts on CCND1 in a translocation context by focusing on the higher order chromatin structures present. In total, our mouse and human studies demonstrate that distal intergenic retroviral integration sites map to functional non-coding elements that are important for cancer pathogenesis. We reveal how utilizing data from a mutagenesis screen in mice can highlight and identify potential oncogenic regulators important in human cancer.Notably, these retroviral integration sites correspond to common distal chromosomal translocation breakpoints mapped in the human genome around proto-oncogenic loci in B- and T-cell malignancies. This overlap further supports the concept that distal er cells –17. As axpressed –21, an uxpressed , 23. Althttp://variation.osu.edu/rtcgd/about_us.html and http://genome.ucsc.edu/.The database is publicly available and was mined using the UCSC genome browser. URLs include The mouse T-cell hybridoma line, 49100.2, was cultured in Dulbecco’s Modified Eagle’s Medium with 10% serum and 1% penicillin-streptomycin under standard conditions. The 49100.2 hybridoma line was produced from the fusion of lymph node cells of B6C2D MHV-68 infected mice with BWZ.36 cells and was a generous gift from EJ Usherwood (Dartmouth College) . The estThe 3C technique was used to test for distal interactions and chromatin looping occurrences. 3C was performed using established procedures , 28. BriGraphed values are presented as mean ± standard deviation (S.D.).Ccnd1 locus, there is a cluster of integration sites within 2 kb from the Ccnd1 promoter, which we will refer to as a proximal cluster. Interestingly, there are also two other distinct clusters of integration sites mapped to the 5’ flanking region of Ccnd1 that reside 100 kb and 170 kb from the gene itself. These distal integration clusters map within a gene desert, and Ccnd1 is the only nearby gene with potential oncogenic function. While it has been assumed that Ccnd1 is the mediator of oncogenic activity for these distal viral integration sites, the mechanism has never been resolved. Given the linear distance between the distal retroviral integration sites and the Ccnd1 promoter, we explored the possibility that there are chromatin loops present that bring distal sites of retroviral integration clusters into close proximity of oncogene promoters throughout the genome.Retroviral integration into the mouse genome causes mainly T and B cell lymphomas through the transcriptional activation of specific oncogenes. At the murine Ccnd1 that link the gene promoter region of DNA 90 kb and 170 kb upstream, regions corresponding to common sites of retroviral integration . Given that these intergenic elements are physically linked to proto-oncogene promoters within the murine genome, we hypothesized that the higher order chromatin structures and presumptive enhancers are also conserved within the human genome. We first mapped conserved DNA elements from the murine Ccnd1 locus to the human genome and found that these regions correspond to unique chromosomal sites in a similar gene desert upstream of the human CCND1 locus . The translocation breakpoints mapped in different cell lines and patients vary within the IGH locus on chromosome 14 and extend between 47 kb and 300 kb in the region upstream of CCND1. A known consequence of these translocations is overexpression of CCND1  in the IGH locus, which is 65 kb from the potent enhancers that have been characterized 3’ of the IGHA2 gene. Numerous probes were designed for the CCND1 upstream region as well as for sequences within the IGH locus. The majority of the probes showed no interaction with the CCND1 promoter, including those within the gene desert upstream of CCND1, which form intrachromosomal loops in other cells. The probes previously used to map the largest loop upstream of CCND1 are physically separated onto a different chromosome due to the reciprocal translocation, and we found no evidence of any residual interaction that would require an interchromosomal interaction. However, we found a very strong interaction indicative of a chromatin loop between the CCND1 promoter and the powerful immunoglobulin enhancers located downstream of the translocation breakpoint within the IGH locus  translocation event provides insight into how these large chromosomal rearrangements are leading to deregulation of important oncogenes. Only one other study has demonstrated the formation of a novel long-range on event . In a lyus cells . This evIn understanding the chromatin architecture surrounding potent oncogenes and how this higher order structure connects distal regulatory sites, we shed more light on the complex regulation of oncogenes to determine how this regulation goes awry during disease pathogenesis. We propose a novel model whereby the most potent retroviral insertions within the mouse genome promote oncogenesis by benefiting from their integration near preexistent chromatin loops and higher order architecture. These integration sites can therefore be used to predict additional long range chromosomal interactions and to reveal distal gene regulatory elements that control genes involved in growth and oncogenic transformation in humans. This is a powerful tool since mutagenesis screens are not feasible in humans, and thus, by utilizing the data from existing screens, it is possible to identify disease relevant human enhancers and expose novel disease mechanisms. Specifically, through a look into a chromosomal translocation event, we begin to understand the natural inherent looping capabilities of particular loci within the genome. This allows for further comprehension of genome organization and the functional output and significance of this higher order chromatin structure. These two genomic events, namely mapped retroviral integrations and translocation breakpoints, predict the presence and highlight the importance of the chromatin architecture within non-coding regions of DNA that greatly contribute to regulation of proto-oncogenes.