- Results: We have identified spliced lncRNAs that are expressed during development and found that lncRNAs that are expressed in the heart but not in the brain are located close to genes that are important for heart. - Importantly, many of the lncRNAs are divergently transcribed from the promoter of these genes. - The surprising re- producibility of developmental processes is underpinned by the robustness of the genetic program [1]. - However, in spite of the high robustness under normal genetic conditions, the program can be easily collapsed by gen- etic abnormalities. - In the heart, even a slight alter- ation of the program leads to congenital heart diseases (CHDs) and this fact is associated with the high fre- quency of CHDs, which is around one in one hundred births [3]. - Genetic studies have shown that many of the transcription factor genes involved in the heart develop- ment are regulated in a highly spatiotemporal manner [4]. - Comparative genomics have shown that the complex- ity of the body plan and the proportion of non-coding regions in the genome are positively correlated [5].. - While most of the non-coding regions have previously been considered as “junk”, it is now accepted that some. - Full list of author information is available at the end of the article. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Recent advancements in genomics and tran- scriptomics have demonstrated that nearly half of the mammalian genome is actually transcribed into RNAs [8]. - Some lncRNAs are known to recruit epi- genetic regulators to specific loci in the genome to modulate transcription. - Although several lncRNAs that function in mammalian heart development have been reported, the identification and characterization of lncRNAs in the mammalian heart are still insufficient [18 – 23]. - Because many of the currently known functional lncRNAs are spliced and because it is difficult to confirm the existence of non-spliced transcripts unless they are expressed at very high levels, we focused on spliced lncRNA candidates in our analysis. - We found that the direction of a majority of the lncRNAs that are located within 10,000 bp from known genes are in the opposite direction from them (225 vs 86), suggesting that such mis-annotations are rare.. - To omit lncRNAs that are ubiquitously expressed without tissue specificity, we examined the expression of the ob- tained candidates in the mouse brain. - We here just wanted to exclude lncRNAs that are expressed with no tissue specificity and did not intended to find lncRNAs that are exclusively expressed in the heart since many genes are known to func- tion differently according to the context of the tissues. - The comparison revealed that 316 of the identified spliced lncRNA candidates were selectively expressed in the heart (Fig. - We checked the ex- pression of these genes in the kidney and the liver and found that only 34 were expressed in both of them, and 213 of them were expressed only in the heart. - Many of the cardiac transcription factor genes have neighboring lncRNAs. - First, we plotted the distribution of the expression levels of the obtained lncRNAs at E10.5 along with that of mRNAs. - a The flowchart for the identification of lncRNAs expressed in the mouse heart. - The expression levels of lncRNAs are generally lower and lncRNAs with tissue-specificity rarely exceed 10 fpkm (red circle). - We require genes to be expressed in the ventricle at a minimum of one stage.. - The number of genes in each category is shown in the parenthesis. - neighboring genes of the identified lncRNAs. - The distri- bution of the distances from the transcriptional start site (TSS) of lncRNAs to the nearest genes was examined (Fig. - To this end, we conducted a gene enrichment analysis on such protein coding genes using the DAVID bioinformatics tool (http://david.ncifcrf.gov/) [26] and found that transcription factor genes were enriched among genes near lncRNAs in the heart. - Consistent with the result that the distance between heart-selective lncRNAs and their neighboring genes is generally greater than the distance between all lncRNAs and their neighboring genes, both antisense lncRNA and bidirectional lncRNA were enriched among lncRNAs that are expressed both in the heart and the brain (Fig. - Some of the lncRNAs and neighboring genes were judged to be both antisense and bidirectional because of alternative promoter isoforms.. - expression levels of the bidirectional promoter pairs over the course of development. - The distribution of the correl- ation coefficients is plotted in Fig. - Some of these lncRNAs (e.g., those divergent to Irx5, Gata6 and Wt1 ) are expressed in the kidney or in the liver, and in such cases divergent genes are also expressed, suggesting that the expression of bidirectional pairs are correlated not only temporally but spatially. - We examined the conservation of these lncRNAs near transcription factors by searching the RefSeq database and found that at least some lncRNAs were conserved in the human genome ( Tbx5, Nkx2–5, Hand2, Gata6, Wt1 and Nr2f1 ) (Additional file 5) and that the bidirectional lncRNA to Tbx5 ( Lnc125 ) was even con- served in chicken, which diverged from mammals 400 mil- lion years ago. - Table 1 Gene ontology analysis of the genes closest to all lncRNAs that are expressed in the heart. - These findings prompted us to examine the function of the Tbx5 -divergent lncRNA.. - Analysis of the Tbx5 -divergent lncRNA. - Tbx5 is a transcription factor that is known to be essen- tial for the development of the heart and forelimb.. - Holt-Oram syndrome is a dominant disorder caused by a single-allele mutation of TBX5 and is characterized by hypoplasia of the forelimb, abnormalities in the thumb, and atrial and/or ventricular septal defects [31–33]. - Tbx5ua is transcribed from one of the promoters of Tbx5 in the opposite direction and overlaps with the intron of one of the Tbx5 isoforms (Fig. - 3a, Additional file 9). - We first quantified the expression level of the transcript in the heart ventricle, atrium and forelimb during normal. - We found that the expression level of Tbx5ua was increased in the ventricle as development progressed, which was inconsist- ent with the expression pattern of Tbx5 . - We also exam- ined the expression level of the Tbx5 isoform that is also transcribed from the bidirectional promoter (Isoform 2, RefSeq: XM . - The expression level of that isoform was stable during the entire developmental process, which was also different from the expression pat- tern of Tbx5ua (Additional file 10). - Next, we compared the expression level of the lncRNA in both of the ventri- cles at E11.5 because it is well-known that the expression level of Tbx5 is higher in the left ventricle than in the right ventricle and that the steep gradient is crucial for estab- lishing a proper ventricular septum [37, 38]. - Tbx5ua -knockdown (KD) mice were embryonic lethal with severe abnormalities in the heart. - Although the expression levels of Tbx5 and Tbx5ua seemed to be anticorrelated in the heart during development (Fig. - 3c), KD of Tbx5ua did not result in the increase of Tbx5 expression level. - We also showed that the expression levels of the different Tbx5 isoforms that are transcribed from all three promoters were not significantly changed (Additional file 11).. - Table 2 Gene ontology analysis of the genes closest to heart-selective lncRNAs. - Genes associated with heart development are strongly enriched among genes closest to lncRNAs that are selectively expressed in the heart. - Hematoxylin and eosin (HE) staining of the cryosections. - showed that the ventricular walls of E9.5 KD mice were irregular and lacked trabeculae at some parts in the ven- tricle (Fig. - None of the. - a Classification of the lncRNA candidates found in the screen.. - b Distribution of the Pearson correlation coefficients between the bidirectional promoter pairs over the course of development. - d The proportion of genes that possess bidirectional lncRNAs in the mouse were examined by referring to RefSeq and a paper that identified haploinsufficient genes.. - embryos showed a visible abnormality in the fore- limbs, which is observed in Tbx5 -deficient embryos.. - By E13.5, all of the KD embryos were dead with a pale body (Fig. - which was probably the cause of the lethality. - The forelimbs seemed com- pletely normal even at this stage, which was a signifi- cant difference between the phenotype of the Tbx5ua KD mice and that of the mouse model of Holt-Oram syndrome (i.e., Tbx5 heterozygous knockout) (Fig. - revealed normal mRNA expression in the KD ven- tricle (Additional file 14A). - By gene ontology analysis, we found that the genes. - c The expression levels of Tbx5 and Tbx5ua during development as determined by qRT-PCR. - In the ventricle, the expression level of Tbx5ua is increased with the progression of development while that of Tbx5 is decreased ( n = 3). - d The expression levels of Tbx5 and Tbx5ua in the left and right ventricles at E11.5 as determined by qRT-PCR. - e Schematic diagram of the Tbx5ua knockdown experiment. - However, none of the structural genes that are im- portant for cardiomyocyte contraction were changed (Additional file 15C), suggesting the possibility that Tbx5ua has a critical role in morphogenesis rather than in cell differentiation. - The clear correlation of the ex- pression levels of some bidirectional pairs suggests their regulatory roles. - It is possible that divergent lncRNAs are enriched among dose-sensitive genes to stabilize the expression level of adjacent genes.. - Comparison of the sequence of Tbx5ua be- tween mouse and chicken showed less similarity, but it does not mean that the function is not conserved as the previous studies have shown that precise conserva- tion at the sequence level is not necessarily required for the functional conservation of lncRNAs [42, 43].. - Tbx5ua was not found in the NCBI genomic annota- tions of reptiles, amphibians or fish at the correspond- ing loci. - In fact, by conducting the reanalysis on the publicly available RNA-seq data, including RNA-seq of the adult heart of chicken, anole and frog (GSE we could confirm that Tbx5ua is expressed only in chicken among these species at the adult stages (Additional file 16). - Although preliminary, our data suggested that the expression pattern of Tbx5 protein is altered in the KD mice (Additional file 17). - This study revealed that many genes involved in the heart development, particularly transcription factor genes, are associated with spliced lncRNAs that are de- rived from nearby genomic regions. - Gene-specific primers are listed in the Additional file 18: Materials and Methods.. - We only took into account genes with their exon number 12 or less since the exon numbers of more than 98.5% of known lncRNAs expressed in the heart fall under the category. - We found that many of the mouse lncRNAs divergent to important cardiac transcription factor genes have conserved transcripts at the corresponding loci in human genome. - List of bidirectional lncRNA candidates that were identified from the analysis of the NCBI RefSeq database (GRCm38.p3). - The expression levels are not significantly changed for all the isoforms. - Additional file 13: The thickness of the ventricular wall around the interventricular zone was measured for WT and KD embryos and KD embryos tended to have thinner wall. - The expression pattern of Tbx5 at the mRNA level appeared to be not changed. - (C) The scatter plot of log2-transformed expression levels shows that the expression pattern of KD embryos did not change drastically. - Note that we only quantified cells that are not in the outermost layer of the ventricle because speckle-like back- ground was observed in the region. - Tbx5 expression is vanished in the interventricular zone and diminished in the left ventricle in KD embryos. - We also thank the animal centers of the University of Tokyo and the University of Tsukuba.. - The funding body had no role in the design of the study and collection, analysis, and interpretation of data.. - YH performed all of the other experiments and analyses.. - All authors read and approved the final version of the manuscript.. - All experimental procedures and animal care were performed according to the animal ethics committee of the University of Tokyo (2806).. - The transcriptional landscape of the mammalian genome. - Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development. - The tissue- specific lncRNA Fendrr is an essential regulator of heart and Body Wall development in the mouse. - accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. - The T-box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. - Haploinsufficiency of the cardiac transcription factor Nkx2-5 variably affects the expression of putative target genes. - A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. - Holt- Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. - Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB
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