Bidirectional promoters are shared promoter sequences between divergent gene pair , and can regulate the genes in both directions. In the human genome, > 10% of protein-coding genes are arranged head-to-head on opposite strands, with transcription start sites that are separated by < 1,000 base pairs. Many transcription factor binding sites occur in the bidirectional promoters that influence the expression of 2 opposite genes. Recently, RNA polymerase II (RPol II) ChIP-seq data are used to identify the promoters of coding genes and non-coding RNAs. However, a bidirectional promoter with RPol II ChIP-Seq data has not been found.In some bidirectional promoter regions, the RPol II forms a bi-peak shape, which indicates that 2 promoters are located in the bidirectional region. We have developed a computational approach to identify the regulatory regions of all divergent gene pairs using genome-wide RPol II binding patterns derived from ChIP-seq data, based upon the assumption that the distribution of RPol II binding patterns around the bidirectional promoters are accumulated by RPol II binding of 2 promoters. In HeLa S3 cells, 249 promoter pairs and 1094 single promoters were identified, of which 76 promoters cover only positive genes, 86 promoters cover only negative genes, and 932 promoters cover 2 genes. Gene expression levels and STAT1 binding sites for different promoter categories were therefore examined.The regulatory region of bidirectional promoter identification based upon RPol II binding patterns provides important temporal and spatial measurements regarding the initiation of transcription. From gene expression and transcription factor binding site analysis, the promoters in bidirectional regions may regulate the closest gene, and STAT1 is involved in primary promoter. A major class of adjacently located gene pairs that are divergently transcribed on opposite strands, with < 1000 base pairs separating their transcription start site (TSS) has been identified [This organization of protein-coding genes is common with > 10% of genes arranged in this configuration . Recent Bidirectional promoters often regulate DNA repair genes related to cancer and non-DNA housekeeping functions ,2,5. BidBidirectional promoters are GC rich, with a median GC-content of 66% . There iet al. found that active promoters could be identified using H3K4me3 profile [et al. [Attempts to identify regulatory regions in the genome involved many experimental and computational methods. One strategy has been to analyze sequence composition, such as GC content, level of conservation, transcription factor binding sites and expressed sequence tags -15. Some profile . With th profile . RPol II profile -23. Bars [et al.  showed a [et al. . HoweverWe have developed a computational approach to identify the promoter regions of all divergent gene pairs using genome-wide RPol II binding patterns derived from ChIP-seq data, based upon the assumption that the distribution of RPol II binding patterns around the bidirectional promoters are accumulated by RPol II binding of two promoters. We also evaluated the regulatory function of promoters according to the gene expression and STAT1 binding sites.We have examined the RPol II binding pattern around the TSS of common protein coding gene and the bidirectional promoter region. To avoid the RPol II binding effect of a gene's neighbourhood, focus was only on the genes whose transcript lengths were > 10,000 bp and no other genes were presented within 10,000 bp of their TSSs. This identified 4,120 expressed genes and 2,682 unexpressed genes in HeLa cells, based on the gene expression array data. We divided the genomic regions into multiple 20 bp bins and calculated the total number of RPol II derived fragments located in each bins within 2,000 bp upstream and downstream of the TSS, producing a RPol II binding landscape in the regulatory regions of the expressed genes. Not unexpectedly, a significant enrichment of the RPol II signal on top of the TSS was seen Figure , which gRemoving the same distance between the different isoforms of genes, 1564 head-to-head paired genes in which 522 pairs included the non-coding genes were used to analysis. The numbers of RPol II fragments in each 20 bp bins within 2,000 bp upstream and downstream of the centre of bidirectional regions were calculated. Based on the distance between 2 opposite genes, the bidirectional regions were divided into 5 categories, and the average RPol II signals of each category were calculated Figure . RPol IIIn the bidirectional promoter region, RPol II signals usually formed 3 shapes, bi-peak, single peak and no peak. Bi-peak denotes 2 different promoters located in the bidirectional promoter region, and each promoter regulates its closest gene. Single peak had 2 options: (1) the regulatory regions of 2 opposite genes were very close, RPol II bound the same regulatory region; and (2) RPol II bound only one gene promoter, and no RPol II bound the other.To find the exact regulatory regions, it was important to identify the RPol II bi-peak shape between the TSSs of 2 opposite genes. We adopted 3 features to identify bi-peak distribution of RPol II on bidirectional promoters: P1 - the number of RPol II fragments at TSS of the gene on the positive strand, P2 - the number of RPol II fragments at TSS of the gene on the negative strand, and V - the lowest number of RPol II fragments between the TSSs Figure . Two parTo estimate the cutoff of significant ratio and difference ratio for bi-peak distribution of RPol II, we simulated some bi-peak shapes using both expressed genes, and single peak shapes with an expressed gene and an unexpressed gene. The paired genes randomly selected from 4,120 expressed genes and 2,682 unexpressed genes were arranged head-to-head, and RPol II signals around the TSSs of genes were summed. According to the distance between paired genes, we simulated 5 sets, each of which included 10,000 bi-peak shapes and 10,000 single peak shapes.The area under the curve (AUC) in the Receiver Operator Characteristic (ROC) reached 0.78 in differentiating all bi-peak shapes and single peak shapes of RPol II signals Figure , suggestpS, B, T, K, and t Kdetermine the Poisson parameter, λ, associated with the distribution of the number of RPol II binding fragments. In the bidirectional promoter region that showed bi-peak distribution of RPol II signals, 10 parameters were used to describe RPol II binding pattern of 2 different promoters  were identified. RPol II signals located around 2 TSSs tend to completely overlap and form one peak in the middle of bidirectional region, making it difficult to distinguish the 2 promoters and single promoter in these regions.In double promoter and centre promoter categories, > 60% paired genes were expressed. We also found that some genes were expressed, whereas other genes on the other side were unexpressed. This suggests that some other mechanism, such as post-transcriptional regulation, can effect gene expression. The RPol II signal in the gene body or RNA-seq data can be recruited to assess gene regulation by promoters.Between the genomic region of 2 genes, the proportion of STAT1 binding sites of double promoters was higher than other 3 promoter categories, which indicates that these STAT1 binding sites are important in recruiting RPol II for both genes. Otherwise, to avoid the effect on the other side genes in the left and right promoter categories, more STAT1 binds on the genomics region of gene body. Compared to the gene body region, less STAT1 binding sites were found between the TSSs in the centre promoter category, which suggests that some left promoters and right promoters could not be distinguished from the centre promoter category.ChIP-seq data was downloaded from GEO(GSE12783), in which genome-wide binding patterns of transcription factor RPol II and STAT1 in HeLa S3 cells were detected using the GA II platform from Illumina . The reaTo distinguish bi-peak and single peak binding patterns of RPol II on bidirectional promoters, we introduce features associated with RPol II binding pattern at the TSS. As there is a significant enrichment of RPol II signal here of each protein coding gene, the level of RPol II signal at both TSSs should be noticeably higher than those between them if there is a bi-peak distribution of RPol II on bidirectional promoters. Thus, we define the significance of RPol II binding pattern at TSSs as:where P1 is the number of RPol II fragments at TSS of the gene on the positive strand, with P2 being the one on the negative strand. V represents the lowest number of RPol II fragments between the 2 TSSs Figure . In addiWe simulate some "bidirectional promoters" using both expressed genes, and others with an expressed and an unexpressed gene. There is a bi-peak RPol II signal on the former promoters and a single peak signal on the latter. We confirmed the amount of the 2 features, a and b, which can distinguish the 2 patterns in the best degree with the simulated data. These 2 features were used to identify bi-peak and single peak patterns on real bidirectional promoters. If Sig > a and Dif < b, the pattern is a bi-peak, otherwise the pattern would be recognized as a single peak.i of the i-th bin was determined by the following equation.The genomic regions neighboring 2 TSSs were divided into 20-bp bins, which were classified into 3 categories: transcript bins of the gene on the negative strand, bins between the 2 TSSs and transcript bins of the gene on the positive strand. For bidirectional promoter region, we assumed the amount of RPol II was the sum of RPol II fragments at the promoters of the 2 genes. The number of RPol II fragments of 2 single promoters detected in each bin should follow a Poisson distribution. The sum of 2 independent Possion distributions also follows Possion distribution, and the parameter λ of the Possion distribution equals the sum of the 2 parameters that belong to the 2 single promoters. The expected RPol II quantity λi, Bi and Ti denote the number of RPol II fragments in the bin of TSS, inter-genetic region and transcription region, respectively . Kpi and Kti denote the decay rate of RPol II (Figure i1 denotes the distance between the i-th bin and TSS of the gene on the positive strand, whereas Di2 denotes the distance between the i-th bin and TSS of the gene on the negative strand.Where SI Figure . Di1 den1, B2, T1, T2, S1, S2, Kp1, Kp2, Kt1, Kt2 }, each follows a gamma distribution in the genome wide among all the expressed genes. The probability density function is modeled by the following equation:A mixture model was established to describe the probability of RPol II fragments neighboring a certain gene pair, where X denotes the observed number of RPol II fragments in each bins, and Y={ BB, βB, αT, βT, αS, βS, αKp, βKp, αKt, βKt} is the gamma distribution parameters.where the vector θ = {α1, B2, T1, T2, S1, S2, Kp1, Kp2, Kt1, Kt2 } as a result. Two regulatory regions were recognized when a certain gene pair was characterized as a bi-peak.Following a similar strategy as previously , we usedA previous method  was usedThe authors declare that they have no competing interests.GW, KQ, and YW contributed to the design of the study. GW and KQ designed and performed the computational modeling and drafted the manuscript. YZ, YL, LJ, MT and YW participated in coordination, discussions related to result interpretation and revision of the manuscript. All the authors read and approved the final manuscript.Table of 249 bidirectional gene pairs with bi-peak RPol II signal and their regulatory regions.Click here for fileTable of 1094 bidirectional gene pairs with single peak Rpol II signal and their regulatory regions.Click here for fileStat1 binding sites of bidirectional promoters with single peak in the middle.Click here for fileStat1 binding sites of bidirectional promoters with bi-peak.Click here for fileStat1 binding sites of bidirectional promoters with single peak on the right.Click here for fileStat1 binding sites of bidirectional promoters with single peak on the left.Click here for file


Beside its use in biotechnology, in vitro shoot regeneration is also an important system to study de novo organogenesis. Phytohormones and transcription factor WUSCHEL (WUS) play critical roles in this process but whether and how epigenetic modifications are involved is unknown. Here, we report that epigenetic marks of DNA methylation and histone modifications regulate de novo shoot regeneration of Arabidopsis through modulating WUS expression and auxin signaling. First, functional loss of key epigenetic genes—including METHYLTRANSFERASE1 (MET1) encoding for DNA methyltransferase, KRYPTONITE (KYP) for the histone 3 lysine 9 (H3K9) methyltransferase, JMJ14 for the histone 3 lysine 4 (H3K4) demethylase, and HAC1 for the histone acetyltransferase—resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Second, we showed that regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications through bisulfite sequencing and chromatin immunoprecipitation. Third, DNA methylation in the regulatory regions of WUS was lost in the met1 mutant, thus leading to increased WUS expression and its localization. Fourth, we did a genome-wide transcriptional analysis and found out that some of differentially expressed genes between wild type and met1 were involved in signal transduction of the phytohormone auxin. We verified that the increased expression of AUXIN RESPONSE FACTOR3 (ARF3) in met1 indeed was due to DNA demethylation, suggesting DNA methylation regulates de novo shoot regeneration by modulating auxin signaling. We propose that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role in this process.Plants have a profound capacity to regenerate organs from differentiated somatic tissues, based on which propagating plants  in vitro has been used as an important way for producing whole plants in agriculture and biotechnology. Phytohormones and the transcription factor WUSCHEL (WUS) are essential for reprogramming during de novo shoot regeneration. Epigenetic modifications are also critical for mammalian cell differentiation and organogenesis. Here, we show that epigenetic modifications mediate the de novo shoot regeneration in Arabidopsis. Mutations of key epigenetic genes resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Bisulfite sequencing and chromatin immunoprecipitation revealed that the regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications. By transcriptome analysis, we identified that some differentially expressed genes between wild type and met1 are involved in signal transduction of the phytohormone auxin. Our results suggest that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role during de novo organogenesis.Plants have a strong ability to generate organs from differentiated somatic tissues. Due to this feature, shoot regeneration  This feature is not only critical for in vitro plant propagation and application of biotechnology, but also provides a good experimental system for understanding regulatory mechanisms underlying organogenesis.Differentiated somatic tissues of plants can be reprogrammed to generate various organs, a process called de novo shoot regeneration in Arabidopsisde novo shoot regeneration. During de novo shoot regeneration in Arabidopsis, expression of WUS is sufficient to specify the organizing center, which is required for stem cell induction and subsequent shoot regeneration WUS induction is also essential for shoot formation during de novo somatic embryogenesis WUS expression during de novo shoot regeneration was regulated by the master phytohormone auxin WUS expression in the organizing center of the Arabidopsis plant SAM was shown to be regulated by epigenetic modifications Recent studies have revealed some molecular mechanisms underlying de novo DNA methyltransferase 3 (DNMT3) family and maintained by methyltransferase DNMT1 Epigenetic modifications, including DNA methylation and histone modifications, occur extensively during cellular differentiation and development in mammals de novo shoot regeneration is unknown. Here we showed that mutations of key epigenetic genes altered WUS expression and developmental rates of regenerated shoots in vitro. In addition, epigenetic marks of DNA methylation and histone modifications in the regions of WUS underwent dynamic changes during de novo shoot regeneration, correlating with dynamic WUS expression levels. Genome-wide transcriptional analysis indicated that some genes involved in auxin signaling and meristem development were methylated within the callus, but were demethylated following an induction treatment. Based on these results, we propose that dynamic DNA methylation and histone modifications mediate de novo shoot regeneration in Arabidopsis through WUS and auxin signaling.In plants, pattern changes of DNA methylation and histone modifications leading to changes in chromatin state occur in plant cells undergoing dedifferentiation de novo shoot regeneration, we first compared the capacity and rates of shoot regeneration between wild type and various epigenetic mutants after calli being transferred onto a shoot induction medium (SIM) from a callus induction medium (CIM) Arabidopsis METHYLTRANSFERASE1 (MET1), KRYPTONITE (KYP), JMJ14 and HISTONE ACETYLTRANSFERASE1 (HAC1), among diverse genes involved in epigenetic modifications, have been well characterized MET1 is an ortholog of DNMT1, which maintains DNA methylation directly at CpG motif and indirectly at non-CG motif MET1 resulted in delayed transition from vegetative phase to reproductive phase KYP encodes histone H3K9 methyltransferase, and mutation of which resulted in abnormal number of floral organs JMJ14 encodes histone H3K4 demethylase that inhibited flowering under long-day condition HAC1 encodes histone acetyltransferase, regulating flowering time through histone acetylation de novo shoot regeneration, whereas the timely appearance of shoot primordia to reflect their developmental rates.To find out whether DNA methylation and histone modifications played roles in met1, kyp, jmj14 and hac1  and 20 days , and similar results were obtained in the prolonged time, such as calli on CIM for 24 days (C24) and 26 days (C26). We further determined the expression patterns of WUS by pWUS::GUS reporter and in situ hybridization, and the results demonstrated that local distribution of WUS transcripts occurred in wild-type calli on SIM  and those on SIM (S6) by bisulfite genomic sequencing. Three regions within the WUS genomic sequences were hyper-methylated in S0 calli but substantially decreased in S6 calli  and on SIM (S6) showed much lower level of DNA methylation in the WUS genomic region than those of wild type under the same condition  was shown to occur in euchromatin undergoing active transcription We analyzed several histone modifications for the WUS genomic sequences during de novo shoot regeneration. Compared with S0, S6 showed an increase in the levels of H3K4me3 at region a and d, but not at b and c  to do in situ hybridization analysis. The results showed that localization of WUS signals in kyp-2 calli on SIM occurred early comparing to that in wild-type calli on SIM  was more than that in wild-type calli at the same time points  and to SIM for 6 days (S6). Because met1 calli showed significantly different developmental rate from wild-type calli, we also analyzed transcriptomes of met1 calli being transferred to CIM for 20 days (M0) for comparison. Significance Analysis of Microarrays software package analysis was conducted for three biological samples replicates between the Ws and met1. The q value≤0.05 and fold change ≥2 were used as the threshold for candidate gene selection , AUXIN RESPONSE FACTOR4 (ARF4), INDOLE-3-ACETIC ACID INDUCIBLE18 (IAA18) and BELL1-LIKE HOMEODOMAIN7 (BLH7) , ARABIDOPSIS RESPONSE REGULATOR15 (ARR15), CYTOKININ OXIDASE/DEHYDROGENASE1 (CKX1), INDOLE-3-ACETIC ACID27 (IAA27) and PINOID2 (PID2), but they displayed great changes in their transcriptional levels upon SIM-induction, implying that those genes might not be directly regulated by MET1  . A loss 7 (BLH7) . Their e by MET1 .ArabidopsisFLOWERING LOCUS T (FT) in Arabidopsis, and distant regulatory regions are required for FT transcription WUS transcription is regulated through a fairly complicated chromatin remodeling mechanism in the SAM of the Arabidopsis plant WUS expression was positively correlated with FASCIATA1 (FAS1)/FAS2, subunits of ASSEMBLY FACTOR-1 (CAF-1), and BRUSHY1 (BRU1), both of which regulate post-replicative stabilization of chromatin structure WUS to maintain proper WUS transcript levels in its spatial expression domain WUS is required for its spatiotemporal expression during plant development WUS were regulated by SIM-induced changes of DNA methylation and histone modifications. Because the met1-3 kyp-7 double mutant displayed more severe phenotypes than each single mutant WUS by DNA methylation and histone modifications may function in a partially redundant manner during de novo shoot regeneration. To understand mechanism of the in vitro organogeneis mediated by the factors involved in both DNA methylation and histone modifications, knocking out both DNA methylation and histone modifications remains to be investigated in the future.DNA methylation and histone modifications are critical epigenetic processes that control chromatin structure and gene expression during development and differentiation Arabidopsis ptstils as explants on CIM to obtain the callus, a mass of pluripotent cells WUS was induced in a group of cells termed the organizing center as a self-renewing source of stem cells within calli. The induced organizing center and stem cells were responsible for subsequent shoot regeneration. Here, we showed that expression of many genes was induced by SIM-induction  background was a kind gift from Dr. J. Bender (The MCB Department of Brown University) kyp-2er) background, jmj14-1, jmj14-2hac1-3, and hac1-5The Arabidopsis seeds were surface sterilized and plated on germination medium Arabidopsis plants and transferred onto callus induction medium . An antisense probe from a full-length 2HPO4, 50 mmol/L KH2PO4, pH 7.2, 10 mmol/L Na2EDTA, 0.5 mmol/L K3Fe(CN)6, 0.5 mmol/L K4Fe(CN)6, 1% Triton X-100 and 2 mmol/L X-Gluc ) at 37°C for 12 h. To further investigate WUS expression pattern, some GUS-stained tissues were embedded in paraffin (Sigma) and sectioned. To display the outline of cells clearly, ruthenium red (200 mg/L) was used to stain cell walls.Plant tissues were incubated in GUS assay solution  and on SIM for 6 days (S6) were vacuum-infiltrated with formaldehyde crosslinking solution. Chromatin immunoprecipitation was performed according to manufactures' instructions . Chromatin samples were immunoprecipitated with antibodies against a negative control normal mouse IgG and H3 dimethyl Lys 9 (both included in EpiQuik™ Plant ChIP Kit), or with antibodies against H3 trimethyl Lys 4  and H3 acetyl Lys 9 . PCR amplification was performed in 25 µL volumes for 32 to 37 cycles to determine the appropriate conditions for the PCR products of each region. Primer sequences are shown in The TUBULIN2. Each reaction was carried out in three biological replicates. The relative expression level of each gene, corresponding to the expression level of TUBULIN2, was calculated using the comparative CT method Total RNAs were isolated from callus tissues 2 to 3 mm deep from the surface. Quantitative real-time PCRs (qRT-PCRs) were performed as described previously met1 mutant calli cultured on CIM for 20 days (M0). RNA purification, probe labeling, chip hybridization, probe array scanning and data pre-processing normalization were performed by the Affymetrix custom service . Significance Analysis of Microarrays software package analysis was conducted for three biological samples replicates between the Ws and met1. When all replicates clustered together, further analysis was performed based on mean values. A two-fold change in the gene expression levels between one versus another samples with a q value≤0.05 was set as the threshold for altered gene expression. Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-3120.RNA of three plant samples was prepared from each of the following tissue types: the wild-type calli cultured on CIM for 20 days (S0), and on SIM for 6 days (S6); the Figure S1met1 mutant and the mutants defective in histone modifications. Frequency of shoot regeneration of the wild type (Col) and the mutants jmj14-2 and hac1-5 was shown, using pistils as explants. Frequency of shoot regeneration of the wild type  and the mutants met1, kyp-2, jmj14-1, jmj14-2, hac1-3 and hac1-5 was shown, using roots as explants. Standard errors were calculated from three sets of biological replicates. In each replicate, at least 60 calli were examined.Frequency of shoot regeneration of (TIF)Click here for additional data file.Figure S2MET1 mutation promotes shoot regeneration in Arabidopsis using roots as explants. Calli of the wild type (Ws) and the met1 mutant were cultured on SIM for 6 to 18 days. Scale bars, 1 mm.(TIF)Click here for additional data file.Figure S3WUS were changed in met1 and kyp-2 mutants. In situ hybridization of WUS expression in calli of the wild type (Ws) cultured on SIM for A) 0 day, B) 4 days and C) 6 days, and that of met1 mutant cultured on SIM for D) 0 day, E) 4 days and F) 6 days. In situ hybridization of WUS expression in calli of the wild type (Ler) cultured on SIM for G) 0 day, H) 4 days and I) 6 days, and that of kyp-2 mutant cultured on SIM for J) 0 day, K) 4 days and L) 6 days. Scale bars, 50 µm.Expression patterns of (TIF)Click here for additional data file.Figure S4met1 cultured on SIM at the indicated time points, and the transcripts of genes ARF3, ARF4, IAA18, BLH7, ANT, AS1, CKX1, and ARR15 were measured by qRT-PCR. Three independent RNA preparations were analyzed for each time point. Mean values were calculated from triplicate qRT-PCR analysis with standard errors. The relative expression level of each gene, corresponding to the expression level of TUBULIN2, was calculated using the comparative C(T) method.Expression patterns of candidate genes validated by qRT-PCR. Total RNAs were isolated from calli of wild type and (TIF)Click here for additional data file.Table S1WUS expressing signals detected by in situ hybridization.The percentage of the calli with (DOC)Click here for additional data file.Table S2pWUS::GUS signal distribution detected in each callus.The number of (DOC)Click here for additional data file.Table S3List of 1334 up-regulated genes and 501 down-regulated genes in S6 as compared to S0.(XLS)Click here for additional data file.Table S4List of 768 genes showing more than two-fold difference between M0 and S0.(XLS)Click here for additional data file.Table S5List of 308 genes showing more than two fold difference both between S6 and S0 and between M0 and S0.(XLS)Click here for additional data file.Table S6Sequences of primers used in this study.(XLS)Click here for additional data file.


PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.The PITX3  Mutations in PITX3 result in congenital cataracts, anterior segment mesenchymal dysgenesis (ASMD), Peter's anomaly, and microphthalmia in humans Pitx3 promoter region in mice produce the aphakia phenotype, which is characterized by small eyes lacking a lens pitx3 was shown to be essential to normal lens and retina formation Pitx3/pitx3 is first detected in the lens placode and then the lens vesicle; early expression is observed in the lens epithelial cells and primary fibers while later expression is restricted to the equator regions of the developing lens The PITX3 PITX3/Pitx3 and its downstream targets. Expression of several genes/proteins was found to be altered in the lenses of Pitx3-deficient mice. Some early reports demonstrated that expression of β- and γ -crystallins is completely absent at developmental stages 10–18 days as well as in newborn aphakia mice Pitx3–knockout mice aphakia mice with α- and β- crystallin expression being reduced at both transcript and protein levels and γ –crystallin expression being downregulated at the protein level Foxe3Prox1p57KIP2Pitx3-deficient animals, which seems more likely to be related to the overall abnormal lens development in aphakia mice rather than direct involvement of Pitx3 in transcriptional regulation of these genes Despite its vital importance for eye development, little is currently known about the ocular function of bicoid-type homeodomain-containing proteins that regulate expression of other genes during development and, possibly, in adult organisms. Other members of this family were shown to be involved in developmental disorders such as idiopathic clubfoot bicoid-type DNA sequences and to regulate downstream gene expression through these interactions bicoid sequences located in the regulatory regions of these downstream genes were shown to mediate these interactions; two or more bicoid sites were found in some promoters bicoid element was demonstrated to be sufficient in several other cases bicoid sequences has been reported for some promoters PITX3 belongs to the PITX family of bicoid sites and located in proximity to known genes, therefore potentially interacting with PITX3 to regulate expression of that gene. As a result, we identified two bicoid sites located in the promoter of Major Intrinsic Protein of lens fiber (MIP) or Aquaporin 0 (AQP0) that are conserved between human, mouse, zebrafish and several other species. We further demonstrated that PITX3 is able to specifically interact with the identified sequences both in vitro and in vivo and to transactivate gene expression as a result of this interaction. In addition to this, expression of mip1 was found to be altered in pitx3 deficient zebrafish morphants. Our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and provide new insight into the PITX3 pathway as well as mechanisms of lens development.In order to isolate downstream targets of the PITX3 homeodomain transcription factor we performed a search for evolutionarily conserved non-coding sequences containing The study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin (protocol number AUA00000352).http://ecrbrowser.dcode.org) was used to identify conserved paired bicoid sites in the promoters/intronic regions of genes with known expression/function. Paired comparison of human and mouse genomes was performed using the following parameters: presence of two conserved bicoid sites with distance between the sites not to exceed 650-bp. Secondary analysis of identified regions for sequence conservation was performed using the UCSC Genome Brower multiple alignment module (http://genome.ucsc.edu) as well as the BLAST tool (http://blast.ncbi.nlm.nih.gov), including examination of the corresponding genes in lower vertebrates when available.ECR Browser web-based tool  and human embryonic kidney cells (293HEK) were obtained from ATCC . B3 cells were cultured in MEM medium  supplemented with heat-inactivated 20% fetal calf serum (FBS), glutamine, sodium pyruvate to a final concentration of 1 mM, non-essential amino acids and antibiotic-antimycotic . 293HEK cells were maintained in DMEM medium containing 10% FBS, glutamine and antibiotic-antimycotic solution.2, 10 mM KCl) with Protease inhibitor cocktail . After 15 minutes of incubation on ice, Igepal CA-630 was added to a final concentration 0.6%, then the cells were vortexed and spun down for 30 seconds at 10000 g. Crude nuclear pellet was extracted with about 2/3 of the original packed cell volume of Extraction buffer  in the presence of protease inhibitors for 30 minutes on ice. 32-mer TAATCCCTGGGAACTAAAG5′-GGAGAAAGGCTTC oligonucleotide spanning region −533/−502 from transcriptional start site (tss) of MIP/AQP0 promoter, 32-mer GGATTAAGAGTCCTCTATA5′-CTGCCCCTCCCAG corresponding to the promoter sequence −71/−40 and their complement oligonucletides as well as both sets of oligonucleotides with TAATCC (GGATTA)bicoid sites replaced by TAATTT (AAATTA) (see above) were labeled with Biotin 3′ End DNA Labeling kit (PIERCE) and annealed. Electrophoretic mobility shift assays (EMSA) were performed with LightShift Chemiluminescent EMSA kit  in accordance with the manufacturer's protocol and using 50 ng/µl of Poly(dI-dC), 20 fmol of labeled DNA and 2 µl of nuclear extracts. After 20 minutes of incubation at room temperature, reactions were either diluted with 5× Loading buffer or further incubated for 30 minutes in presence of 1 µg of polyclonal Pitx3 antibody for supershift assay. Binding reactions and free probe were run on 5% native polyacrylamide gel in 0.5× TBE buffer.Nuclear extracts were prepared from B3 cells transiently transfected with PITX3-pcDNA3.1 vector with CelLytic NuCLEAR extraction kit . Cells were harvested after 48 hours with a cell scraper and the compact cellular pellet was re-suspended in 5 volumes of Hypotonic lysis buffer bicoid sites replaced by TAATTT (AAATTA) to be used as a control for bicoid site binding specificity. Nuclear extracts were generated from sixty 48-hpf zebrafish pitx3 morphant or wild-type embryos that demonstrated normal body length and morphology; the preparation was carried out as described above except for that the embryos were first grinded with glass tissue homogenizer equipped with type B pestle in hypotonic detergent-less lysis buffer to assist nuclei release. Binding reaction was performed in the presence of 2.5% glycerol, 5 mM MgCl2 and 0.05% NP-40 in addition to the buffer composition described above; 3 µl of extract was used in each binding reaction. Five embryos from each group were analyzed for pitx3 transcript presence to verify the degree of morpholino-mediated knockdown.For EMSA experiments performed using whole zebrafish embryo nuclear extracts, the 32-mer and its compliment corresponding to the region from −44 to −76 of zebrafish ChIP was performed with ChIP-IT enzymatic kit or ChIP-IT Express enzymatic kit  according to manufacturer recommendations.MIP/AQP0 promoter, the following primers were utilized: set 1 (spanning region −110/+95) forward, 5′-GCTGTGAAGGGGTTAAGAGG-3′ and reverse 5′-GAGGGTGGCAAAGAACTCAG-3′ and set 2 (spanning region −473/−275) forward, 5′-CTGAACCCCACTCCTTACCA-3′ and reverse, 5′- TCTGCCCTTCTGTGTGTGTC-3′. For control experiments, the following primers were used: GAPDH forward 5′- TACTAGCGGTTTTACGGGCG-3′ and reverse 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′, product = 166 bp ; forward 5′-ATGGTTGCCACTGGGGATCT-3′ and reverse 5′-TGCCAAAGCCTAGGGGAAGA-3′, product = 174 bp . The PCR reactions were repeated at least three times using precipitated DNA from independent chromatin immunoprecipitation experiments. Quantification of ChIP PCR products was performed by densitometry using the ImageJ program developed by Dr. Rasband, NIH (http://rsbweb.nih.gov/ij/). The measurements obtained for ChIP PCR results were normalized by input DNA and expressed as percent of its value. Data from at least three independent experiments were combined to calculate mean and standard deviation. Statistical significance was determined using the homoscedastic Student's t-test with two-tailed distribution.B3 human lens epithelial (HLE) cells were grown in 100 mm tissue culture dish to 90–95% confluence and utilized for ChIP assays; experiments were performed using native untransfected cells as well as cells transfected with PITX3 expression plasmids. Cells were transfected with 7.5–10 µg of pcDNA3.1_PITX3_FLAG or empty pcDNA3.1 plasmid and cross-linked after 48 hours with 1% formaldehyde for 10 minutes at room temperature with agitation. Following this, the monolayers were washed with 125 mM of glycine and lysed. The nuclear pellet was resuspended in digestion buffer and DNA was sheared with Enzymatic shearing cocktail for 10 minutes at 37°C in a water bath. The resulting fragments ranged between 200- and 1000-bp in size. The quality of chromatin was verified in a control experiment of immunoprecipitation with the Polymerase II antibody followed by PCR with primers specific for the GADPH promoter. Only those chromatin preparations that demonstrated significant enrichment in these control experiments were used in further analysis. Immunoprecipitation was performed with 2 µg of Pitx3, FLAG or control IgG antibody overnight in a cold room and, after washing and de-crosslinking, the precipitated DNA was analyzed by PCR. Goat polyclonal PITX3 (N-20) and normal goat IgG were purchased from Santa Cruz Biotechnology, Inc.  and FLAG-M2 mouse monoclonal antibody from Sigma . For PCR amplification of PITX3 wild type, PITX3-WT, and mutant expression constructs, PITX3-K111E, PITX3-S13N and PITX3- G219f, were previously described MIP656-bcd1,2 reporter plasmid, a 656-bp fragment containing 597-bp of upstream and 59-bp of downstream sequence from the transcriptional start site (tss) of MIP/AQP0 was amplified by PCR and cloned into the pCRII-TOPO vector  and then subcloned into basic pGL3 luciferase reporter vector . The transcriptional start site (tss) of MIP/AQP0 was designated based on the ENST00000257979 entry in Ensemble Database. Site-directed mutagenesis was performed with QuikChange II Site-Directed mutagenesis kit , oligonucleotide AAATTAAGAGTCCTCTATAAA-3′5′-CTCAGCCTGCCCCTCCCAG and its complement for the proximal bicoid site and oligonucleotide TAATTTCTGGGAACTAAAGAATT-3′5′-CTAGCCAATGGGAGAAAGGCTTC and its compliment for the distal site on MIP/AQP0 promoter. Therefore, in both bicoid sites the consensus recognition sequence TAATCC was replaced by TAATTT and three additional constructs were produced: MIP656-bcd1 (carrying mutant bcd2 site), MIP656-bcd2 (carrying mutant bcd1 site) and MIP656-bcd0 (carrying mutations in both bicoid sites). All constructs were verified by sequencing.MIP656 wild-type and mutant reporters (114 ng) were used. Each cotransfection included 300 ng of effector (PITX3 wild type and mutant expression constructs) and 60 ng of β-galactosidase in pcDNA3.1 vector ; the total DNA amount was kept the same in all transfections by adding empty pcDNA3.1 vector when needed. Cells were harvested after 48 hours; luciferase and β-galactosidase activities were determined using Luciferase assay and Enzyme Assay systems , respectively. Every experiment was performed at least three times in triplicate. Student's paired t-Test with a one-tailed distribution was utilized to determine the statistical significance of any differences in activity level.Human embryonic kidney cells (293HEK) were plated in 24-well plates and transfected using Lipofectamine 2000  according to the manufacturer's protocol. Equimolar amounts of basic pGL3 reporter plasmid (100 ng) and Danio rerio) were maintained on a 14-hour light/ 10-hour dark cycle. The embryos were obtained by natural spawning and maintained at 28.5°C. The pitx3 morpholino, 5′-AGGTTAAAATCCATCACCTCTACCG-3′, that was previously reported 4, 1.8 mM Ca(NO3)2 and 1.5 mM HEPES, pH 7.6) and 19.2 ng was injected into zebrafish embryos immediately after fertilization at the 1–2 cell stage. Microinjections were performed using the Nanoject II injector . Embryos were incubated at 28.5°C in 0.2 mM 1-phenyl-2-thiourea (PTU) to inhibit pigment formation and anesthetized with 0.05% Tricane before imaging. The developmental stage was determined by time (hours post fertilization (hpf)) and by morphological criteria Zebrafish ; for pitx3, exon_1F, 5′-CTCCACTAGACCGGGATTCA-3′, and exon_3R, 5′- AAAGGTGGCTTCCAGTTCCT-3′ (PCR product = 276 bp); and for β-actin, exon2F, 5′-GAGAAGATCTGGCATCACAC-3′ and exon_3R, 5′-ATCAGGTAGTCTGTCAGGTC-3′ (PCR product = 323 bp). The PCR conditions were as follows: initial denaturation at 94°C for 3 min followed by 23–32 cycles of 94°C for 20 seconds, 59°C for 30 seconds and 72°C for 30 seconds and final extension at 72°C for 7 min. The number of cycles was optimized to maintain PCR reaction in linear range.For RNA isolation, the embryos were homogenized in TRI reagent (Sigma) in the presence of glycogen and processed using a standard extraction protocol. The cDNA was generated using equal amounts of RNA for every sample and SuperScript III First-Strand Synthesis system  according to manufacturer recommendations. Semi-quantitative PCR was performed using gene-specific oligonucleotides for in situ hybridization experiments, a 368-bp fragment specific to zebrafish mip1 transcript was generated using the following primers, forward, 5′-CTGCAGGACATGCTCATCAC-3′ and reverse, 5′-GGCTGCAAAAAGTCAACAGA-3′, and inserted into pCRII-TOPO plasmid vector. An antisense RNA probe was generated using DIG RNA Labeling Kit  following manufacturer recommendation; in situ hybridization was performed as previously described To construct an antisense riboprobe for bicoid sites across multiple species- the promoter region of the gene encoding for the major intrinsic protein of lens fiber or aquaporin 0 (MIP/AQP0).Examination of the ECR Browser web-based tool for clusters of PITX3 binding sites conserved between different species yielded a total of 976 genomic regions: 511 sequences were found inside of intergenic regions, 454 elements were located within genes , and only 11 were positioned within 1500 bp from a transcriptional start site. The eleven identified promoter regions were subjected to a secondary analysis of sequence conservation that included examination of the corresponding genes in lower vertebrates when available. One sequence demonstrated the strongest level of conservation of MIP/AQP0 promoter region contains two bicoid sites separated by 456 base pairs at positions −58 (bcd1) and −520 (bcd2) from the transcriptional start site. Alignment of MIP/AQP0 promoters from different species demonstrates high conservation of both bicoid sites in nine mammalian/vertebrate species from human to zebrafish (Danio rerio) has two orthologs of the human MIP/AQP0 gene designated mip1 and mip2mip1 appears to be more similar to mammalian species showing conservation for both bicoid sites (positions −59 (bcd1) and −442 (bcd2)) and surrounding sequence  to examine whether these putative ed cells . The PITed cells . In addiractions .bicoid sites located in the human MIP/AQP0 promoter in vitro.These results demonstrate that PITX3 is capable of binding to both MIP/AQP0 promoter in vivo, we performed chromatin immunoprecipitation (ChIP) assays. Native untransfected human lens epithelial (HLE) cells or HLE cells following transfection with pcDNA3.1_PITX3_FLAG expression plasmid or pcDNA3.1 empty vector were used in these experiments.To investigate whether PITX3 interacts with the MIP/AQP0 promoter sequences in the precipitated DNA in comparison to ChIP samples produced with control antibody (IgG) (MIP/AQP0 promoter sequences in the precipitated DNA in comparison to ChIP samples produced with control antibody (IgG)/same nuclear extracts as well as precipitations that utilized the same antibody (FLAG) but employed mock-transfected cells (MIP/AQP0 promoter sequences in the precipitated DNA was demonstrated by semi-quantitative PCR using specific MIP/AQP0 and control primers (described in Immunoprecipitations with PITX3 antibody that used nuclear extracts from native untransfected HLE cells resulted in enrichment of dy (IgG) . Immunoped cells . This enMIP/AQP0 promoter region in vivo.These experiments demonstrated the specific association of PITX3 with the bicoid sites within the MIP/AQP0 promoter are involved in regulation of its activity, we created several reporter constructs: MIP656-bcd1,2, which contained a 656-bp fragment of the MIP/AQP0 wild-type promoter encompassing both bicoid sites and nucleotides from positions −597 to +59 in relation to the MIP/AQP0 transcriptional start site inserted into a basic pGL3 plasmid containing the luciferase reporter gene; MIP656-bcd1, which contained a mutation in the bcd2 site that changed the 5′-TAATCC-3′ sequence to 5′-TAATTT-3′, thus abolishing its interaction with wild-type PITX3 MIP656-bcd2, which contained a similar mutation in the bcd1 site changing the 5′-GGATTA-3′ sequence into 5′-AAATTA-3′; and MIP656-bcd0, which included both of the above described mutations.To examine whether the conserved MIP656-bcd1,2 promoter fragment in comparison to the empty vector in human embryonic kidney cells  when the bcd1 site was mutated, to 4.3-fold  when the bcd2 site was abolished and to 3.4-fold  when both sites were disrupted.Reporter assays demonstrated a 5.2-fold upregulation of luciferase expression in the presence of the ey cells . Mutatiobicoid sites appear to be involved in regulation of MIP/AQP0 expression with the proximal site, bcd1, playing a more significant role in its activation.Based upon these results, both MIP/AQP0 promoter, we performed cotransfection assays using the above described MIP656-bcd1,2, MIP656-bcd1, MIP656-bcd2 and MIP656-bcd0 reporter constructs and a PITX3 expression plasmid.In order to investigate the effect of PITX3 on the transcriptional activity of the PITX3-WT expression plasmid with the MIP656-bcd1,2 luciferase reporter into human embryonic kidney cells resulted in a ∼4.8-fold normalized activation in comparison to the cotransfection of the PITX3-WT plasmid with the promoter-less reporter , ∼3.1-fold for S13N  and no transactivation was observed for the K111E mutant. These results are consistent with the previously reported data on the residual activities of the corresponding mutant PITX3 forms Cotransfection of the reporter . In contbicoid sites present in the MIP/AQP0 promoter for their role in this observed transactivation. The PITX3-WT expression plasmid and mutant MIP/AQP0 promoter constructs, MIP656-bcd1 (carrying mutant bcd2 site), MIP656-bcd2 (carrying mutant bcd1 site) and MIP656-bcd0 (carrying mutations in both bicoid sites) were cotransfected into human embryonic kidney cells and the resultant luciferase activities were compared to values observed in experiments involving cotransfection of wild-type MIP/AQP0 promoter  and PITX3-WT. The transactivation of the MIP656-bcd1,2 promoter by PITX3-WT decreased to ∼2-fold (42% of PITX3 induced wild-type promoter transactivation) when the mutation in the proximal bicoid site, bcd1, was introduced; increased to ∼6-fold  when the distal bicoid site, bcd2, was mutated; and produced ∼2.2-folds  when both bicoid sites were disrupted  was observed in ∼20% of pitx3-mo and control-mo injected embryos. The pitx3-mo displayed a misshapen smaller head, jaw abnormalities and reduction in eye size due to progressive lens degeneration and retinal defects leading to a complete loss of lens by 7-dpf consistent with the previous reports pitx3 is seen in lens vesicle at 24-hpf and it continues to be highly expressed during all stages of lens development pitx3 transcripts are also detected in the developing brain, craniofacial region and trunk musculature as previously described An abnormal phenotype was detected in ∼95% of MIP/AQP0 promoter in vivo, further experiments were performed to establish if knockdown of pitx3 would affect formation of protein-mip1 promoter complexes in zebrafish. We injected zebrafish embryos with above described pitx3 morpholino oligonucleotides that result in abnormal splicing of pitx3 transcript. Embryos were harvested at 48-hpf and nuclear extracts from wild-type embryos and pitx3 morphants were tested for their ability to bind a biotinylated DNA fragment containing the proximal bicoid site and corresponding to zebrafish −44/−76 mip1 promoter region  in normal pitx3 transcript based on RT-PCR analysis performed using RNA extracted from the lower trunk region of the same embryos , while a complete absence (9/14 or 64.3%) or a very low level (5/14 or 35.7%) of mip1 expression was seen in pitx3-mo embryos at 29-hpf  demonstrated similar levels of expression between pitx3-mo and control-mo injected embryos , the expression of mip1 in pitx3 morphants appears to be largely unaffected, suggesting that regulation of mip1 activity at these stages may be mainly controlled by other, pitx3 pathway-independent, factors; pitx3 may also contribute to the recovery of mip1 expression since increasing amounts of normal pitx3 transcript can be observed in zebrafish embryos starting at 48-hpf due to weakening of the effects of morpholino injections (pitx3 mutant lines is needed to allow more careful evaluation of the relationship between these factors.In this manuscript, we present evidence that the two evolutionarily conserved sequences containing jections . IdentifMIP/AQP0 gene were shown to underlie various dominant forms of cataracts MIP/AQP0 mutations were explored for functional defects and a dominant-negative mechanism was suggested MIP/AQP0 mutations may also be explained by haploinsufficiency which would suggest that lens development is highly sensitive to dosage/timely expression of MIP/AQP0. The later possibility is further supported by the phenotype reported in the mouse carrying a null allele of Aqp0Aqp0 was shown to result in cataracts at 3 weeks of age and at 24 weeks of age in homozygous and heterozygous mice, respectively. In heterozygous animals, the lens osmotic water permeability value was reduced to around 46% and the lens focusing power was significantly decreased in comparison to wild-type Aqp0 allele, which presumably leads to reduced Aqp0 expression, can be associated with lens abnormalities. Therefore, since MIP/AQP0 represents an apparent transcriptional target of PITX3, the alteration of the MIP/AQP0 expression in patients affected with PITX3 mutations is likely to contribute to the lens phenotype observed in these individuals.Mutations in the human MIP/AQP0 promoter will not only yield important insight into the transcriptional regulation of this critical lens differentiation factor but will also provide better understanding of the function of PITX3 and its interacting partners and allow for more specific identification of additional downstream targets of this ocular factor. Also, genetic screening of both PITX3 and MIP/AQP0 in human patients affected with ocular conditions may lead to an identification of synergistic or compensatory mutations/variants that may help to explain the considerable intra- and interfamilial phenotypic variability associated with mutations in either gene.Further studies of the


Glutamate neurotransmission and the N-methyl-D-aspartate receptor (NMDAR) are central to photic signaling to the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). NMDARs also play important roles in brain development including visual input circuits. The functional NMDAR is comprised of multiple subunits, but each requiring the NR1 subunit for normal activity. The NR1 can be alternatively spliced to produce isoforms that confer different functional properties on the NMDAR. The SCN undergoes extensive developmental changes during postnatal life, including synaptogenesis and acquisition of photic signaling. These changes are especially important in the highly photoperiodic Siberian hamster, in which development of sensitivity to photic cues within the SCN could impact early physiological programming. In this study we examined the expression of NR1 isoforms in the hamster at different developmental ages. Gene expression in the forebrain was quantified by in situ hybridization using oligonucleotide probes specific to alternatively spliced regions of the NR1 heteronuclear mRNA, including examination of anterior hypothalamus, piriform cortex, caudate-putamen, thalamus and hippocampus. Gene expression analysis within the SCN revealed the absence of the N1 cassette, the presence of the C2 cassette alone and the combined absence of C1 and C2 cassettes, indicating that the dominant splice variants are NR1-2a and NR1-4a. Whilst we observe changes at different developmental ages in levels of NR1 isoform probe hybridization in various forebrain structures, we find no significant changes within the SCN. This suggests that a switch in NR1 isoform does not underlie or is not produced by developmental changes within the hamster SCN. Consistency of the NR1 isoforms would ensure that the response of the SCN cells to photic signals remains stable throughout life, an important aspect of the function of the SCN as a responder to environmental changes in quality/quantity of light over the circadian day and annual cycle. In situ hybridization and Reverse transcription polymerase chain reaction (RT-PCR) studies indicate that several glutamate receptor genes are expressed in the SCN Circadian rhythms are an integral component of physiology and behavior, and the master circadian oscillator in the mammal is located within the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN clock is entrained to the light-dark (LD) cycle by photic signals from the retina via a monosynaptic retinohypothalamic tract (RHT) input to the SCN. Glutamate is the predominant neurotransmitter of the RHT and mediator of light entrainment cues in the adult mammal e.g. . MoreoveThe NMDA receptors (NMDARs) are glutamate-gated ion channels composed of heterodimeric subunits, and the NMDAR1 (NR1) sub-unit is present in all functional receptor complexes Phodopus sungorus) was studied because the early development and photic regulation of the circadian system is necessary for the generation of rhythms of melatonin secretion from the pineal gland. This pattern of melatonin secretion provides the key seasonal time cue regulating somatic and reproductive development in this species via maternal melatonin secretion The aim of the current study was to investigate the developmental regulation of NR1 mRNA isoforms in the Siberian hamster SCN. The Siberian hamster  and NR1000 (NR1-4a). The fact that the SCN showed a consistent expression of NR1001 and NR1000 at all ages examined suggests that the ontogeny of the postnatal hamster SCN is not associated with a switch in NR1 subclass or a change in its level its expression. This is different to other brain structures such as the thalamus and hippocampus where we do observe developmental changes in the levels of NR1 isoforms. Whilst the conserved nature of high expression does suggest that the NR1 subunit is important in the regulation of SCN function throughout postnatal development, we provide no evidence that postnatal development events are regulated by an alteration of NR1 isoform.Phodopus sungorus) were obtained from the breeding colony in the Department of Anatomy, University of Cambridge Rattus rattus) were obtained from Harlan UK Ltd., Bicester, Oxford, UK, and maintained on a 16∶8 or 12∶12 light∶dark photoschedule, respectively . The day of birth was assigned as postnatal day 1, PD1 Siberian hamsters (In situ hybridization (ISH) was carried out on 16 µm serial coronal sections, cut at the level of the SCN and caudate-putamen and as detailed previously 35S labeled oligonucleotide probes (36 mer–48 mer) complimentary to the rat NMDAR1 gene Relative grey scale values were calculated by dividing the specific region measure by the corpus callosum measure taken from the same section. Emulsion-dipped sections were analyzed by a silver grain counting procedure using Image J. Sections were stained by cresyl violet to define brain structures of interest. Grains were counted over cells within a 120 µm2 area of brain section in the regions of the SCN and piriform cortex. Grains were also counted in the corpus callosum, but throughout the 120 µm2 area and not specifically over the few cells present. Mean values were determined for each animal from at least 3 sections, and at least 3 animals were used to derive a group mean.Autoradiographic films were scanned with a Hamamatsu CCD camera  and analyzed using Image J software. For each section the mean pixel density  was determined for each specific forebrain region in 2–4 sections per animal. Values were background corrected against the mean pixel density value for the corpus callosum. The corpus callosum was chosen as the control area because it was assumed that it contains little NMDA receptor mRNA Immunohistochemical analysis (IHC) of NR1-immunoreactivity was performed as previously described post-hoc t-tests. Significance was set at the P<0.05 level.Data were analyzed by one-factor ANOVA to determine age-related changes in NR1 splice variant expression, followed by Dunnett's et al.Regional binding of the probes within the brain of both rat and Siberian hamster was conserved and consistent with the observations of Standaert 2,10 = 8.2, P<0.01), anterior hypothalamus , piriform cortex , caudate-putamen  and thalamus . Only in the hippocampus was there no significant age related change in relative binding. Post hoc Dunnett's t-tests indicated significant reductions (P<0.05) between PD2 and PD6 in the SCN and caudate-putamen, between PD6 and adult in the anterior hypothalamus, piriform cortex, caudate-putamen and thalamus, and between PD2 and adult in all regions. Levels of hybridization were highest in the piriform cortex and hippocampus at all ages. Comparing between age matched brain regions, the SCN, caudate-putamen and thalamus showed similar levels of hybridization, and with comparable age related declines in signals.The PAN probe hybridized ubiquitously in the hamster and rat forebrain , and in 2,6 = 85.6, P<0.001).2,6 = 30.9, P<0.001). The relative binding in all other brain regions was conserved across ages. Regions showing greatest binding density were the piriform cortex and hippocampus . Relative grey scale values for C2 hybridization in striatum and thalamus did not decline significantly between neonatal and adult ages .Hybridization of the C2 probe was relatively high within the SCN at all ages . This co2,6 = 7.7, P<0.05) and hippocampus . An incremental decline in expression occurred in the thalamus during development, whilst the hippocampus showed an elevated hybridization signal at PD6.2,10 = 3.48, n.s.; piriform cortex, F2,10 = 0.77, n.s.), which did not show a significant age-related change in the density of grains  and piriform cortex , but not in the corpus callosum . Post hoc t-tests indicated reductions between PD2 and PD6 in the SCN, between PD6 and adult in the piriform cortex, and between PD2 and adult in both the SCN and piriform cortex.Examination of emulsion-dipped sections revealed hybridization of the PAN and C2 probes throughout the SCN as defined by cresyl violet staining of the same sections . Silver f grains . Levels f grains . Howeverf grains . One-facISH controls were carried out to ascertain the specificity of hybridization by the C2 probe in the hamster, because of its high levels of binding within the SCN. Competition of the labeled C2 with 100 times excess cold probe completely blocked all binding in the SCN and all other brain forebrain regions. A labeled sense probe failed to bind to any regions, and prior treatment of labeled C2 hybridized sections with RNAse diminished binding .001 (NR1-2a) and NR1000 (NR1-4a)  .Immunohistochemical analysis revealed NR1-ir cell perikarya throughout the SCN and surrounding hypothalamus . NR1-ir The expression of NR1 subunit in the adult rat SCN is well described et al.001 (NR1-2a) but not the NR1000 (NR1-4a) splice variant (see below).It cannot always be assumed that translation of mRNA invariably follows transcription. However, our immunohistochemical results revealed widespread distribution of NR1-ir cells in the SCN. This is consistent with other immunohistochemical studies in the Siberian hamster 001 (NR1-2a) and NR1000 (NR1-4a).The regional binding of the splice variant probes was similar between the Siberian hamster and rat, which suggests that the specificity of the probe is maintained between the two species. The results indicate the presence of the C2 (exon 22) cassette, but not the N1 (exon 5) or C1 (exon 21) cassettes in the SCN of both species. Thus, the predominant splice variants in the adult Siberian hamster and rat SCN would appear to be NR1Examination of relative levels of hybridization of the PAN probe that identifies all NR1 mRNA variants, reveals an age-related decrease in NR1 expression in the SCN, anterior hypothalamus, caudate-putamen, thalamus and piriform cortex. It might then be expected that a decrease in the mRNA containing one or more of the splice-specific regions would underlie this observation and thus a developmental reduction in the binding of one or more of the region-specific probes would be apparent. However, apart from the reduction in NR1-4 probe . Similar to the DELL-II probe, NR1-2 hybridization signal in the SCN region was strong, and higher than the NR1-4 probe.Our data in the hamster SCN reveals a high level of relative hybridization signal for the C2 probe and a relatively lower signal for the NR1-4 probe. Whilst it would be tempting to suggest that the ratio expression of NR1001 (NR1-2a) and NR1000 (NR1-4a) isoforms is conserved throughout postnatal and adult life. The conserved nature of the regional expression of an isoform, as demonstrated by the SCN, is consistent with observations of other forebrain regions in the rat, such as the basal ganglia. This suggests that whilst there may be developmental changes in relative levels of the hybridization signal, there is little change in their regional distributions. Moreover, these patterns appear to be established around birth The presence of C2 and NR1-4, but not N1 or C1 probe hybridization within the SCN throughout the postnatal period, strongly suggests that the dominance of the NR1001 isoform would render the receptor subunit relatively insensitive to phosphorylation by protein kinase C X00 (NR1-4a and NR1-4b) isoforms and NR2B subunit have been shown to be colocalized with neuronal nitric oxide synthase (nNOS), and shown to be physically coupled with nNOS X00 isoform possesses the shortest C-terminal tail, is not retained in the endoplasmic reticulum and is translocated directly to the cell membrane surface X00 subunit in the rat is rhythmically expressed, it has been suggested that this might facilitate the transfer of NR2B subunit and nNOS to the cell surface and contribute to the night photosensitivity of cell membranes in the SCN It is interesting to note that the NR1001  and NR1000  are ubiquitously expressed throughout postnatal stages and in the adult. However, in contrast to the Siberian hamster SCN, Bendová et al.011 isoform (probes NR1a and NR1-1) in the rat that persists at constant levels throughout development and in the adult, and report developmental changes in the NR1010 (probes NR1a and NR1-3) and NR1-E isoforms, specifically their elevation during embryonic and postnatal stages but low/absent expression in the adult. As the C1 cassette possesses regulatory and signaling elements, its absence in the Siberian hamster and its presence in the rat indicates that there may be functional differences in the glutamatergic signaling in the SCN between the two species.These data are generally consistent with the study of NR1 splice variants in a recent study of adult and developing rat SCN Whilst the role of the NMDAR including NR1 is accepted as important in the regulation of retino-recipient development in the vertebrate optic tectum and mammalian visual thalamus and superior colliculus 011 and NR1010 isoforms in the rat The NMDA receptor can also contribute to non-glutamatergic pathways involved in the modulation of photic entrainment, such as those utilizing pituitary adenylyl cyclase activating peptide (PACAP), neuropeptide Y (NPY) and 5-hydroxytryptophan (5-HT) in vivoIt is perhaps not surprising that the NR1 isoforms are conserved throughout postnatal life to adult if it is important in the regulation of the circadian clock by light. The early onset of a cellular response to light in the Siberian hamster SCN suggests that the circadian clock can be light entrained early in the postnatal period in utero, thereby indicating the direction of change of photoperiod The biological significance of a response to photic cues for a burrowing rodent is questionable in the first few days of life, but Siberian hamster pups do become ambulatory prior to opening of the eye lids and may therefore become exposed to photic cues in the environment. Certainly by PD14 Siberian hamster pups are capable of independent survival, and neuroanatomical development of the SCN and its stimulation by photic signals suggest that hamsters are capable of responding to the environmental photoperiod by this age 001 (NR1-2a) and NR1000 (NR1-4a), and that this pattern of gene expression is conserved throughout postnatal development. Therefore we conclude that a switch in NR1 isoform does not underlie or is not produced by the dramatic changes in that occur within the developing SCN. This consistency in the presence of the NR1 isoforms highlights the importance of having a stable response to synaptic glutamatergic activation. As the SCN contains the master circadian pacemaker that is reset by light, and is critical in photoperiodic time measurement, this would allow for a stable response to photic signals from the environment that are intrinsically variable over the 24 hr day and calendar year.In conclusion, these data reveal that the predominant splice variants expressed within the Siberian hamster SCN are NR1Figure S1Hybridization of NR1 variable region probes in rat forebrain. Shown are the PAN (top), N1, C1 and C2 (bottom) probes in the adult rat brain. Autoradiograph of alternate coronal sections taken through the forebrain at the level of the SCN from a representative animal. a, anterior hypothalamus; cp, caudate putamen; p, piriform cortex; s, suprachiasmatic nucleus; t, thalamus. Scale bar = 1000 µm.(PDF)Click here for additional data file.Figure S2Control studies for in situ hybridization of C2 probe in PD6 Siberian hamster. A, B, labeled C2 probe; C, D, labeled C2 probe with excess cold C2 probe; E, F, RNAse treatment and labeled C2 probe; G, H, labeled C2 sense probe. Autoradiographs of alternate coronal sections taken through the forebrain at the level of the SCN from a representative animal. Whole section (left) and higher magnification of the hypothalamus including SCN (right). Arrow indicates position of SCN. Scale bars = 500 µm.(PDF)Click here for additional data file.Figure S3Confirmation of the identity of the NR1 splice variants expressed in the adult Siberian hamster SCN. (A) Oligonucleotide probes were used designed to discriminate between splice variant isoforms but hybridizing to alternative regions of the NR1 rat gene sequence. NR1 mRNA structure with the position of three alternatively spliced exons in the mRNA (upper) and the complementary positions of the additional splice-specific oligonucleotide probes (lower) (see wer) see . The NR1wer) see . NR1-1 p(PDF)Click here for additional data file.Figure S4NR1-immunoreactive cells in the adult Siberian hamster brain. Coronal sections through A, the SCN and anterior hypothalamus; B, caudate-putamen; C, cerebral cortex; D, piriform cortex; E, cerebellum (arrow indicates a Purkinje cell); and F, hippocampus. DG, dentate gyrus; OC, optic chiasm; 3 V, third ventricle. Scale bar for A to E = 100 µm, scale bar for F = 200 µm.(PDF)Click here for additional data file.Table S1Oligonucleotide probes used in the Siberian hamster and rat.(PDF)Click here for additional data file.


This review summarizes the current understanding of the role of nuclear bodies in regulating gene expression. The compartmentalization of cellular processes, such as ribosome biogenesis, RNA processing, cellular response to stress, transcription, modification and assembly of spliceosomal snRNPs, histone gene synthesis and nuclear RNA retention, has significant implications for gene regulation. These functional nuclear domains include the nucleolus, nuclear speckle, nuclear stress body, transcription factory, Cajal body, Gemini of Cajal body, histone locus body and paraspeckle. We herein review the roles of nuclear bodies in regulating gene expression and their relation to human health and disease.  Gene expression is a multistep process that is vital for the development, adaptation and survival of all living organisms. Regulation of gene expression occurs at the level of transcription, RNA processing, RNA export, translation and protein degradation ,2,3. Thehttp://npd.hgu.mrc.ac.uk). The diverse mechanisms of gene regulation associated with nuclear bodies add another dimension to our understanding of gene regulation. Part of the answer lies in the compartmentalization of the nuclear space into nuclear bodies of specific functions. Nuclear bodies are broadly defined as morphologically distinct regions within the nucleus; they are distinguishable from their surroundings by techniques, such as transmission electron microscopy, differential interference contrast microscopy and immunofluorescent detection of proteins that localize to a nuclear body of interest. Similar to conventional cytoplasmic organelles, nuclear bodies are distinct local environments of unique functions . NuclearIn this review, we focus on the role of nuclear bodies in gene regulation and discuss human diseases associated with mutations in genes encoding for key components. A review of the perinucleolar compartment is deferred, since it has so far only been identified in cancerous cells, and its relevance to general biology remains unclear. Additionally, although the promyelocytic leukemia (PML) body, the clastosome, the cleavage body, the Oct1/PTF/transcription (OPT) domain, the polymorphic interphase karyosomal association (PIKA) domain and the polycomb group (PcG) body contribute to the regulation of gene expression ,5, theseThe nucleolus is one of the most prominent and clearly visible structures in the nucleus. Described by the anatomist Rudolf Wagner in 1835 and by the physiologist Gabriel Valentin the following year, the nucleolus has fascinated scientists for decades ,7. Thoughttp://www.lamondlab.com/NOPdb/). RNA members include the box C/D and H/ACA small nucleolar RNAs (snoRNAs); these are incorporated into the snoRNPs that process precursor ribosomal RNA (pre-rRNA)  metabolism, as well as ribosomal proteins, chaperones, translation factors and many others of unknown function ,13. Thesre-rRNA) .Most proteins in the nucleolus participate in ribosomal RNA (rRNA) transcription and pre-rRNA processing ,13 Tabl. NucleopThe nucleolus regulates gene expression by modulating protein production via ribosome biogenesis. Ribosome biogenesis is critical to cellular function, growth and response to stimuli. The multifunctional phosphoproteins, nucleophosmin and nucleolin, regulate ribosome biogenesis at several levels. Nucleophosmin specifically interacts with ribosomal DNA (rDNA) and regulates the transcription of rDNA as a histone chaperone . FurtherIn addition to the modulation of gene expression through ribosome biogenesis, the nucleolus modulates protein post-translational modifications, such as ubiquitination and SUMOylation. Ubiquitination of transcription termination factor 1 (TTF1) and p53 by ubiquitin ligase MDM2 regulates ribosome biogenesis and p53 regulation, respectively ,137, andSequestration of proteins is another mechanism by which the nucleolus modulates expression of gene products. Proteins may be stabilized through sequestration of degradation factors to the nucleolus. For example, sequestration of ubiquitin ligases MDM2, and von Hippel-Lindau disease tumor suppressor (VHL) enhances the stability of their targets p53 and hypoxia-inducible factor 1 alpha , respectively ,142. DeaWRN and BLM, which encode nucleolar DNA helicases, cause Werner syndrome (OMIM 277700) and Bloom syndrome (OMIM 210900), respectively. Several human disorders associate with mutations in genes encoding nucleolar proteins . Mutatiogrumos hialinas or transparent lumps by Santiago Ramón y Cajal in 1910 [First described as  in 1910  and subs in 1910 ,188, nuc in 1910 ,190,191  in 1910 . MALAT1) and polyadenylated (poly(A)+) RNA [Most constituents in the nuclear speckle are components of the spliceosome and function in pre-mRNA splicing; these include the spliceosomal snRNAs and several associated protein factors that comprise the snRNPs ,34 Tabl. Other cA)+) RNA ,39.SRSF1 or SRSF2 decreases localization of other pre-mRNA processing factors to nuclear speckles, enlarges nuclear speckles and decreases the transcription of some genes [Members of the serine (S)/arginine (R)-rich splicing factor (SRSF) family, which are the predominant splicing factors found in the nuclear speckle , particime genes  , eukaryotic initiation factor 4A III (eIF4AIII), RNA-binding protein S1 (RNPS1), Y14 and Aly/REF, which are members of the EJC, function in RNA surveillance, NMD and, in conjunction with nuclear speckle protein, RAE1, the nuclear export of mRNA  Table 1Table 1. MALAT1, which is retained in the nucleus, binds and regulates SRSF proteins and, thereby, modulates alternative splicing [Malat1-null mice are viable and fertile, and the cells and tissues tested from these animals have appropriately localized nuclear speckle proteins [MALAT1 in HeLa cells causes aberrant alternative splicing, decreased localization of pre-mRNA processing factors to nuclear speckles and a distorted ratio of dephosphorylated to phosphorylated pools of SRSF proteins [The long ncRNA splicing . Despiteproteins . In contproteins . FurtherThe nuclear speckle regulates gene expression by possibly regulating transcription directly and via post-transcriptional mechanisms. Observations supporting a direct effect on transcription include (1) the enrichment of the periphery of nuclear speckles for the elongating form of RNA Pol II ,196, (2)Nuclear speckles may also directly modulate gene expression through interaction with the RNA Pol II complex , even thDisorders associated with mutations in genes encoding nuclear speckle components include retinitis pigmentosa , mandibulofacial dysostosis with microcephaly (OMIM 610536) and thrombocytopenia-absent radius syndrome (OMIM 274000) . SeveralDrosophila melanogaster and Caenorhabditis elegans [First identified as foci of heat shock factor 1 in heat-stressed cells ,201,202, elegans ,205  and heat shock factor 2 (HSF2), scaffold attachment factor B (SAF-B), Src-associated in mitosis 68 kDa protein (Sam68) and the SRSF family members, SRSF1, SRSF7 and SRSF9 ,201,206 HSF1 impeded heat induction of nuclear stress bodies, HSF2 binding to DNA and satellite III transcription, whereas knockdown of HSF2 had no effect on HSF1 localization to nuclear stress bodies and increased satellite III transcription [The heat shock transcription factors, HSF1 and HSF2, participate in the cellular response to stress. Present as inactive monomers in the cytoplasm, these transcription factors trimerize and translocate to the nucleus upon cellular stress . They bicription  , and hypomyelinating leukodystrophy is associated with mutations of POLR3A and POLR3B (OMIM 607694 and OMIM 614381). No disorders have been associated with mutations of genes encoding RNA Pol II subunits. Mutations in genes encoding the mediator complex and general transcription factors cause congenital malformations, intellectual disability and features of defective DNA repair. Mediator complex mutations have been associated with Lujan-Fryns (OMIM 309520) and Opitz-Kaveggia (OMIM 305450) syndromes, postnatal progressive microcephaly with seizures and brain atrophy (OMIM 613668), mental retardation autosomal recessive 18 (OMIM 614249) and Charcot-Marie-Tooth disease type 2B2 (OMIM 605589). Mutations of the general transcription factor II H complex cause xeroderma pigmentosum group D (OMIM 278730) and group B (OMIM 610651), cerebrooculofacioskeletal syndrome 2 (OMIM 610756) and trichothiodystrophy (OMIM 601675). Several disorders have been described for mutations in genes encoding transcription factory components . TreacheCajal bodies, also known as coiled bodies, are named after their discoverer, Santiago Ramón y Cajal , and areThe Cajal body is a site for the modification of small nuclear RNAs (snRNAs) and snoRNAs, as well as for the assembly and trafficking of RNPs. In addition to coilin, the Cajal body is enriched in spliceosomal small nuclear RNPs (snRNPs), snoRNPs, the telomerase RNP and in the factors that assemble and mature RNPs, as well as the survival of the motor neurons (SMN) complex ,242,245 TERC) RNA [Coilin, an abundant Cajal body protein of unknown function, interacts with several Cajal body components ,249,250 ERC) RNA ,52,53,54ERC) RNA ,184,256 U2 snRNA primary transcript in vitro, and knockdown of coilin abrogates this activity [snRNP biogenesis is one mechanism by which the Cajal body regulates gene expression. The Cajal body mediates snRNP biogenesis at many levels, including snRNA post-transcriptional modification, snRNA transcription and snRNP assembly. The scaRNPs of the Cajal body mediate the post-transcriptional modifications of 2'-O-methylation and pseudouridylation of snRNA . The funactivity . Finallyactivity ,262 and,TCAB1 mutations to lengthen telomeres [The Cajal body also facilitates the biogenesis and localization of another RNP, the telomerase complex. The telomerase complex is required for telomere synthesis. Evidence suggesting a role for the Cajal body in telomere maintenance includes the Cajal body-dependent assembly and localization of the telomerase complex to the telomeres during the S phase ,265,266,elomeres  and the elomeres .Although Cajal body dysfunction has not been definitively implicated in human disease, mutations of several Cajal body components have been . FunctioIn a study of the subcellular localization of the SMN1 protein, Liu and colleagues observed a novel nuclear structure similar in size, number, response to metabolic conditions and cell cycle behavior to Cajal bodies . HoweverThe identified components of gems include the SMN complex and ZPR1 . The SMNThe SMN complex is required for spliceosomal snRNP biogenesis , as wellZpr1 have motor defects, progressive motor neuron degeneration and mislocalization of the SMN protein [ZPR1, a highly conserved protein ,274, is  protein ,118  with a highly conserved RNA hairpin element in lieu of a poly(A) tail. The histone locus body components contribute to the transcription, RNA processing, export, translation and degradation of the replication-dependent histone genes by linking their expression to the cell cycle. Cyclin E/Cdk2, which regulates the G1/S transition, phosphorylates the histone body component, NPAT, following its localization within the histone locus body by a ZPR1-dependent process ,75. PhosCell cycle regulation of SLBP expression also contributes to the cell cycle-dependent expression of the replication-dependent histone genes; SLBP is increased in expression shortly prior to the S phase, highly expressed during the S phase and degraded at the end of the S phase ,277. ProDrosophila, respectively, have no effect on the formation and function of histone locus bodies [Coilin, the classical marker of Cajal bodies, is also found in histone locus bodies . Its funs bodies ,124.Drosophila embryos deficient for the histone locus [The invariant co-localization of histone locus bodies with the histone locus is a defining feature of this nuclear body. The histone locus itself, therefore, may be required for its structural integrity, and in fact, smaller histone locus bodies have been observed in ne locus   [Ctn) [Established paraspeckle component proteins include the aforementioned NONO, PSP1 and PSP2, as well as splicing factor proline/glutamine-rich (SFPQ)  and mammNEAT1) ,82,83 an1) [Ctn)  Table 1NEAT1 [CtDrosophila behavior/human splicing (DBHS) family of RNA-binding proteins that possess two tandem RNA recognition motifs (RRMs). The DBHS family proteins are involved in nuclear processes, such as transcription, pre-mRNA processing and DNA repair [NONO or SFPQ leads to a loss of paraspeckles [Paraspeckle proteins participate in several biological processes. PSP1, NONO and SFPQ are members of the A repair ,289,290.speckles . Animal speckles ,130,291 PSP2 mediates transcription and splicing in a promoter-preferential manner . CFIm68,NEAT1 RNA is a mammal-specific ncRNA that is developmentally regulated. It is widely expressed in adult mouse tissues, but not earlier in development [NEAT1 knockdown in cultured human cells and of a knockout Neat1 mouse show that these RNAs are essential for paraspeckle formation [Neat1 RNA is not needed for mouse viability, health or fertility [The elopment . Studiesormation ,83,133  and subsequent binding of the modified RNAs to the paraspeckle components, NONO and SFPQ, as well as matrin 3 [NEAT1 RNA, depletion of Ctn RNA by antisense oligonucleotide (ASO) knockdown has no effect on paraspeckle formation [The mouse-specific at2 mRNA . The nucmatrin 3 ,295. In ormation   site, resulting in a longer 3' UTR [Ctn RNA is cleaved at its 3' UTR, exported from the nucleus and translated into the Cat2 protein [A function particular to paraspeckles is nuclear RNA retention ,295. ExeCtn RNA . While nr 3' UTR . Followi protein . Cat2 is protein . Nuclear protein , it is l protein , and num protein ,299,300.No human diseases have been described for mutations in genes encoding for paraspeckle proteins or RNAs. As proposed by Rabl in 1885 , interphBesides potentially regulating gene expression based on concentration or sequestration of factors, nuclear bodies provide a structure for cells to couple and integrate sequential processes in order to increase the efficiency and tuning of gene expression. Although our understanding of this integration is primitive, traces of this integration can be observed in the way in which nuclear bodies share factors and interact with other nuclear bodies and nuclear components in time and space. Many factors are not exclusive to one subnuclear domain and have functions in several domains, e.g., coilin and the SMN complex. This sharing of factors not only allows the cell to maximize the function for any given protein, but also potentially provides a mechanism by which different nuclear bodies can communicate with one another. For instance, the transfer of SMN between the Cajal body and the Gemini of Cajal body might be a mechanism of communication and of modulating their respective functions. Finally, the histone locus body and its role in the transcription of the replication-dependent histone genes and the seamless coordination with the cell cycle exemplify the interaction of a nuclear body with temporal nuclear functions. Several observations also suggest that this integration extends beyond the modulation of gene expression to that of a fundamental role in global nuclear homeostasis. Many recent studies have uncovered intertwining of the processes of DNA transcription, replication, repair and recombination. Although partially attributable to the common substrate DNA and some proteins functioning in multiple processes, nuclear bodies frequently act as nexi contributing to each or many of these processes ,334,335.As might be predicted for structures mediating responses to stimuli, nuclear bodies are highly dynamic ,337. Thede novo [Drosophila melanogaster histone H3 and H4 genes, as well as the transcription of the histone H3 and H4 genes facilitate histone locus body formation [Reinforcing the vital nature of nuclear bodies, they are conserved across species that have been well studied . Howeverde novo . In agreormation . Since sFurther work on these intriguing aspects of nuclear bodies will no doubt provide more knowledge regarding the function and regulation of nuclear bodies, as well as their contribution to the regulation of gene expression.Nuclear bodies utilize a vast array of gene regulatory mechanisms for efficient and controlled gene expression . FurtherThe study of nuclear bodies and the mechanisms by which they regulate gene expression is an exciting, but complex and challenging area of cell biology. There are likely more nuclear bodies, as well as components and functions of existing nuclear bodies to be discovered and elucidated.