Intronic Element Research Articles

Intronic DNA elements regulate Nrf2 chemical responsiveness of the human microsomal epoxide hydrolase gene (EPHX1) through a far upstream alternative promoter

In humans, microsomal epoxide hydrolase (mEH) contributes important biological functions that underlie both detoxification and bioactivation fates arising from exposures to foreign chemicals. Previously, we discovered that human mEH gene transcription is initiated from alternative promoters. The respective transcripts are programmed with tissue specificity and the upstream E1b promoter contributes predominantly to mEH expression. The results presented demonstrate that exposures to the Nrf2 activators, sulforaphane (SFN) and tert-butylhydroquinone (tBHQ), markedly activate E1b transcription in human lung and liver cells. Genomic analyses identified two major DNase I hypersensitive regions (HS-1 and HS-2) within the ~15kb intervening sequence separating E1b from the downstream E1 promoter. In BEAS-2B cells, the Nrf2 effectors, SFN and tBHQ, selectively activated the more distal HS-2 through an antioxidant response element (ARE). An activator protein 1/12-O-tetradecanoylphorbol-13-acetate interaction was further identified within the HS-2 enhancer that functioned to additionally contribute to ARE-mediated induction responsiveness of the E1b promoter. The results demonstrate that ARE modulation, integrated with additional transcriptional complexes, regulates the tissue-specific expression of mEH and that these processes likely coordinate both the protective and bioactivation functions contributed by mEH activities in human tissues.

Dual Masking of Specific Negative Splicing Regulatory Elements Resulted in Maximal Exon 7 Inclusion of SMN2 Gene

Spinal muscular atrophy (SMA) is a fatal autosomal recessive disease caused by survival motor neuron (SMN) protein insufficiency due to SMN1 mutations. Boosting SMN2 expression is a potential therapy for SMA. SMN2 has identical coding sequence as SMN1 except for a silent C-to-T transition at the 6th nucleotide of exon 7, converting a splicing enhancer to a silencer motif. Consequently, most SMN2 transcripts lack exon 7. More than ten putative splicing regulatory elements (SREs) were reported to regulate exon 7 splicing. To investigate the relative strength of each negative SRE in inhibiting exon 7 inclusion, antisense oligonucleotides (AONs) were used to mask each element, and the fold increase of full-length SMN transcripts containing exon 7 were compared. The most potent negative SREs are at intron 7 (in descending order): ISS-N1, 3' splice site of exon 8 (ex8 3'ss) and ISS+100. Dual-targeting AONs were subsequently used to mask two nonadjacent SREs simultaneously. Notably, masking of both ISS-N1 and ex8 3'ss induced the highest fold increase of full-length SMN transcripts and proteins. Therefore, efforts should be directed towards the two elements simultaneously for the development of optimal AONs for SMA therapy.

Regulation of HER2 Oncogene Transcription by a Multifunctional Coactivator/Corepressor Complex

Transcription of the HER2 oncogene can be repressed by estrogen (E2). We now show that, a splice isoform of the nuclear receptor coactivator AIB1, AIB1-Δ4, is able to reverse E2 repression of HER2 gene expression in breast cancer cells. The first 224 amino acids of AIB1 that are absent in AIB1-Δ4, bind a co-repressor, ANCO1. Using chromatin immunoprecipitation assay approaches in MCF7 and BT474 cell lines, we demonstrate that AIB1 and AIB1-Δ4 can bind to the E2 regulatory site in the first intron of the HER2 gene, after E2 treatment, but only full-length AIB1 recruits ANCO1. Consistent with E2-induced chromatin repression, the AIB1-ANCO1 complex recruits HDAC3 and HDAC4 to the intronic estrogen response element and the proximal promoter acquires the repressive chromatin mark H3K9me3 and loses H3K4me1. In contrast, AIB1-Δ4 does not recruit ANCO 1, HDAC3, or HDAC4 and the proximal promoter retains activation marks of H3K4me1. In cell lines with low levels of ANCO1 (T47D), E2 does not repress HER2 gene transcription but the repressive response can be restored by overexpression of ANCO1. ANCO1 can also repress other E2-responsive genes, indicating that AIB1, AIB1-Δ4 and ANCO1 are important determinants of endocrine and growth factor responsiveness in breast cancer.

Multiple conserved regulatory domains promote Fezf2 expression in the developing cerebral cortex

BackgroundThe genetic programs required for development of the cerebral cortex are under intense investigation. However, non-coding DNA elements that control the expression of developmentally important genes remain poorly defined. Here we investigate the regulation of Fezf2, a transcription factor that is necessary for the generation of deep-layer cortical projection neurons.ResultsUsing a combination of chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) we mapped the binding of four deep-layer-enriched transcription factors previously shown to be important for cortical development. Building upon this we characterized the activity of three regulatory regions around the Fezf2 locus at multiple stages throughout corticogenesis. We identified a promoter that was sufficient for expression in the cerebral cortex, and enhancers that drove reporter gene expression in distinct forebrain domains, including progenitor cells and cortical projection neurons.ConclusionsThese results provide insight into the regulatory logic controlling Fezf2 expression and further the understanding of how multiple non-coding regulatory domains can collaborate to control gene expression in vivo.

Identification of a New Intronic BMPR2-Mutation and Early Diagnosis of Heritable Pulmonary Arterial Hypertension in a Large Family with Mean Clinical Follow-Up of 12 Years

BackgroundMutations in the bone morphogenetic protein receptor 2 (BMPR2) gene can lead to hereditary pulmonary arterial hypertension (HPAH) and are detected in more than 80% of cases with familial aggregation of the disease. Factors determining disease penetrance are largely unknown.MethodsA mean clinical follow-up of 12 years was accomplished in 46 family members including echocardiography, stress-Dopplerechocardiography and genetic analysis of TGF-β pathway genes. Right heart catheterization and RNA-analysis was performed in members with pathological findings.ResultsManifest HPAH was diagnosed in 8 members, 4 were already deceased, two died during the follow-up, two are still alive. Normal pulmonary artery systolic pressure at rest but hypertensive response to exercise has been identified in 19 family members. Analysis of BMPR2 transcripts revealed aberrant splicing due to an insertion of an intronic Alu element adjacent to exon 6. All HPAH patients and 12 further asymptomatic family members carried this insertion. During follow-up two family members carrying hypertensive response and the Alu insertion developed manifest HPAH.ConclusionThis is the first report of an intronic BMPR2 mutation due to an Alu element insertion causing HPAH in a large family which has been confirmed on RNA-level. Only those members that carried both hypertensive response and the mutation developed manifest HPAH during follow-up. Our findings highlight the importance of including further methods such as RNA analysis into the molecular genetic diagnostic of PAH patients. They suggest that at least in some families hypertensive response may be an additional risk factor for disease manifestation and penetrance.

Context-dependent Cooperation between Nuclear Factor κB (NF-κB) and the Glucocorticoid Receptor at a TNFAIP3 Intronic Enhancer: A MECHANISM TO MAINTAIN NEGATIVE FEEDBACK CONTROL OF INFLAMMATION

TNF expression is elevated in asthma and other inflammatory airway diseases that are commonly treated with glucocorticoid-based therapies, but the impact of glucocorticoids on negative feedback control of TNF is not well understood. We analyzed the effect of dexamethasone, a potent synthetic glucocorticoid, on TNF-regulated gene expression in cultured airway epithelial cells. Although dexamethasone-mediated activation of the glucocorticoid receptor (GR) potently repressed expression of IL1β, IL8, and several other pro-inflammatory TNF targets, the expression of anti-inflammatory TNF targets such as TNFAIP3 (A20) and NFKBIA was selectively spared or augmented by dexamethasone treatment. Despite divergent effects on gene expression, GR and NF-κB occupancy at the TNFAIP3 locus and GR-repressed targets was similar. A co-occupied intronic TNFAIP3 regulatory element mediated cooperative enhancement of transcription by GR and NF-κB that required the presence of a functional GR binding site (GBS). GBS exchanges between reporters for TNFAIP3 and FKBP5, a canonical GR-induced target, revealed substantial latitude in the GBS sequence requirements for GR/NF-κB cooperation, suggesting that the TNFAIP3 GBS acts primarily as a docking site in this context. Supporting this notion, a selective GR ligand with only weak agonist activity for induction of FKBP5 enabled robust GR/NF-κB cooperative induction of a mutant TNFAIP3 reporter harboring the FKBP5 GBS. Taken together, our data support a model in which the expression of anti-inflammatory targets of TNF is maintained during treatment with glucocorticoids through context-dependent cooperation between GR and NF-κB.

Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells

The forkhead box A transcription factors, FOXA1 and FOXA2, function as pioneer factors to open condensed chromatin and facilitate binding of other proteins. We showed previously that these factors are key components of a transcriptional network that drives enhancer function at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in intestinal epithelial cells. The CFTR promoter apparently lacks tissue-specific regulatory elements and expression of the gene is controlled by multiple cis-acting elements, which coordinate gene expression in different cell types. Here we show that concurrent depletion of FOXA1 and FOXA2 represses CFTR expression and alters the three-dimensional architecture of the active locus by diminishing interactions between the promoter and intronic cis-acting elements. Reduction of FOXA1/A2 also modifies the enrichment profile of the active enhancer marks H3K27ac and H3K4me2 across the CFTR locus and alters chromatin accessibility at individual cis-elements. Moreover, loss of FOXA1/A2 suppresses the recruitment of other members of the transcriptional network including HNF1 and CDX2, to multiple cis-elements. These data reveal a complex molecular mechanism underlying the role of FOXA1/A2 in achieving high levels of CFTR expression in intestinal epithelial cells.

Rolling Nagoya mouse strain (PROD-rol/rol) with classic piebald mutation.

ABSTRACT Ataxic rolling Nagoya (PROD-rol/rol) mice, which carry a mutation in the α1 subunit of the Cav2.1 channel (Cacna1a) gene, were discovered in 1969. They show white spots on agouti coat and have a mutation in the piebald spotting (s) locus. However, mutation analysis of the s locus encoding the endothelin receptor type B (Ednrb) gene in PROD-rol/rol mice had not been performed. Here, we examined the genomic and mRNA sequences of the Ednrb gene in PROD-rol/rol and wild-type rolling Nagoya (PROD-s/s) and studied the expression patterns of Ednrb and Cacna1a genes in these mice in comparison with C57BL/6J mice. Polymerase chain reaction analyses revealed two silent nucleotide substitutions in the coding region and insertion of a retroposon-like element in intron 1 of the Ednrb gene. Expression analyses demonstrated similar localizations and levels of Ednrb and Cacna1a expression in the colon between PROD-rol/rol and PROD-s/s mice, but the expression levels of both genes were diminished compared with C57BL/6J mice. Microsatellite genotyping showed that at least particular regions of chromosome 14 proximal to the Ednrb locus of the PROD strain were derived from Japanese fancy piebald mice. These results indicated that PROD-rol/rol mice have two mutant genes, Ednrb and Cacna1a. As no PROD strain had an intact Ednrb gene, using congenic rolling mice would better serve to examine rolling Nagoya-type Cav2.1 channel dysfunctions.

The multiplicity of alternative splicing decisions in Caenorhabditis elegans is linked to specific intronic regulatory motifs and minisatellites.

BackgroundAlternative splicing diversifies the pool of messenger RNA molecules encoded by individual genes. This diversity is particularly high when multiple splicing decisions cause a combinatorial arrangement of several alternate exons. We know very little on how the multiple decisions occurring during the maturation of single transcripts are coordinated and whether specific sequence elements might be involved.ResultsHere, the Caenorhabditis elegans genome was surveyed in order to identify sequence elements that might play a specific role in the regulation of multiple splicing decisions. The introns flanking alternate exons in transcripts whose maturation involves multiple alternative splicing decisions were compared to those whose maturation involves a single decision. Fifty-eight penta-, hexa-, and hepta-meric elements, clustered in 17 groups, were significantly over-represented in genes subject to multiple alternative splicing decisions. Most of these motifs relate to known splicing regulatory elements and appear to be well conserved in the related species Caenorhabditis briggsae. The usage of specific motifs is not linked to the gene product function, but rather depends on the gene structure, since it is influenced by the distance separating the multiple splicing decision sites. Two of these motifs are part of the CeRep25B minisatellite, which is also over-represented at the vicinity of alternative splicing regions. Most of the remaining motifs are not part of repeated sequence elements, but tend to occur in specific heterologous pairs in genes subject to multiple alternative splicing decisions.ConclusionsThe existence of specific intronic sequence elements linked to multiple alternative splicing decisions is intriguing and suggests that these elements might have some specialized regulatory role during splicing.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-364) contains supplementary material, which is available to authorized users.

RNA sequencing of the exercise transcriptome in equine athletes.

The horse is an optimal model organism for studying the genomic response to exercise-induced stress, due to its natural aptitude for athletic performance and the relative homogeneity of its genetic and environmental backgrounds. Here, we applied RNA-sequencing analysis through the use of SOLiD technology in an experimental framework centered on exercise-induced stress during endurance races in equine athletes. We monitored the transcriptional landscape by comparing gene expression levels between animals at rest and after competition. Overall, we observed a shift from coding to non-coding regions, suggesting that the stress response involves the differential expression of not annotated regions. Notably, we observed significant post-race increases of reads that correspond to repeats, especially the intergenic and intronic L1 and L2 transposable elements. We also observed increased expression of the antisense strands compared to the sense strands in intronic and regulatory regions (1 kb up- and downstream) of the genes, suggesting that antisense transcription could be one of the main mechanisms for transposon regulation in the horse under stress conditions. We identified a large number of transcripts corresponding to intergenic and intronic regions putatively associated with new transcriptional elements. Gene expression and pathway analysis allowed us to identify several biological processes and molecular functions that may be involved with exercise-induced stress. Ontology clustering reflected mechanisms that are already known to be stress activated (e.g., chemokine-type cytokines, Toll-like receptors, and kinases), as well as “nucleic acid binding” and “signal transduction activity” functions. There was also a general and transient decrease in the global rates of protein synthesis, which would be expected after strenuous global stress. In sum, our network analysis points toward the involvement of specific gene clusters in equine exercise-induced stress, including those involved in inflammation, cell signaling, and immune interactions.

X-linked sideroblastic anemia due to ALAS2 intron 1 enhancer element GATA-binding site mutations.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA.

Regulatory variation: an emerging vantage point for cancer biology

Transcriptional regulation involves complex and interdependent interactions of noncoding and coding regions of the genome with proteins that interact and modify them. Genetic variation/mutation in coding and noncoding regions of the genome can drive aberrant transcription and disease. In spite of accounting for nearly 98% of the genome comparatively little is known about the contribution of noncoding DNA elements to disease. Genome-wide association studies of complex human diseases including cancer have revealed enrichment for variants in the noncoding genome. A striking finding of recent cancer genome re-sequencing efforts has been the previously underappreciated frequency of mutations in epigenetic modifiers across a wide range of cancer types. Taken together these results point to the importance of dysregulation in transcriptional regulatory control in genesis of cancer. Powered by recent technological advancements in functional genomic profiling, exploration of normal and transformed regulatory networks will provide novel insight into the initiation and progression of cancer and open new windows to future prognostic and diagnostic tools.

Hmga2 Is a RUNX1 Target Gene In Hematopoietic Stem and Progenitor Cells

RUNX1, also known as AML1, is a DNA binding transcription factor that is expressed in hematopoietic stem and progenitor cells (HSPCs). As demonstrated by several mouse models, RUNX1 is necessary for definitive hematopoiesis and proper homeostasis of HSPCs. Furthermore, mutations of RUNX1have been implicated in patients with a variety of blood-related malignancies and disorders. These findings have established RUNX1 as a master regulator of hematopoiesis. As a transcription factor, RUNX1 exerts its function in hematopoiesis by binding to regulatory regions in order to guide the expression of its direct target genes. Most confirmed RUNX1 target genes are mainly expressed in differentiated blood cells. Direct targets of RUNX1 in HSPCs, however, have largely remained unexplored. Identifying direct target genes of RUNX1 offers an insightful view of how this master regulator influences HSPC function. To elucidate RUNX1 target genes in HSPCs, we have analyzed gene expression signatures from wildtype and RUNX1-deficient HSPCs (Lineage-/cKit+/Sca1+) in a previous report (Matsuura et al., Blood, 2012). With the goal of continuing the characterization of RUNX1 target genes, in this current study, we performed genome-occupancy analysis with chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) using RUNX1 antibodies and a murine HSPC cell line. Bioinformatics analysis of the ChIP-seq data revealed 6370 significant RUNX1 binding peaks (<1% FDR). The majority of these peaks were located in areas outside of promoter regions. The top de novo generated sequence motif from these peaks corresponds with the known RUNX binding consensus motif TG(T/C)GGT, suggesting that our ChIP-seq dataset is highly reliable. The combination of differential gene expression and RUNX1 genome occupancy data has revealed a list of candidate RUNX1-regulated target genes. We hypothesize that RUNX1 directly modulates the expression of these genes in normal hematopoiesis.One of the genes identified is Hmga2. We observed three RUNX1 binding peaks in the upstream, intron, and downstream regions relative to the Hmga2 gene locus. Furthermore, we confirmed strong up-regulation of Hmga2 in RUNX1-deficient HSPCs using reverse transcription coupled with quantitative polymerase chain reaction. HMGA2, also known as High Mobility Group AT-hook 2, is a non-histone chromatin protein. Its expression is highest during embryonic development and in undifferentiated cells. Over-expression of HMGA2 in transgenic mice or in bone marrow transplantation models have been reported to cause expansion of HSPCs. These reports indicate that HMGA2 is a significant mediator of HSPC proliferation. Interestingly, a major characteristic of mice without RUNX1 in their hematopoietic cells is the expansion of HSPCs, suggesting that HMGA2 may contribute to this phenotype. To further validate Hmga2 as a RUNX1 target gene, we cloned the Hmga2 promoter sequence and DNA fragments corresponding to the three RUNX1 binding peaks into luciferase reporter constructs and performed transfection studies using K562 and 293T cells. Interestingly, while these studies demonstrated strong responses to RUNX1 in promoter-luciferase assays, the effect of RUNX1 on Hmga2 promoter activity in these two cell lines was opposite. In addition, eliminating two RUNX binding motifs in the Hmga2 promoter did not affect RUNX1-mediated promoter-luciferase activity, indicating that there are additional regulatory mechanisms that may be important for RUNX1's effect on the Hmga2 promoter. To examine the function of the three regions containing RUNX1 binding peaks in the Hmga2 gene locus, we also used luciferase reporter constructs including these regions in transfection studies. Increase of transcriptional activity was detected in the presence of the two regions that were upstream and downstream of the Hmga2 gene, suggesting that RUNX1 can act as a positive regulator through these regions. In contrast, the RUNX1 binding fragment in the intron region of Hmga2 reduced promoter-luciferase activity. This outcome indicates that RUNX1 acts as a suppressor through the Hmga2 intron element. In summary, these results establish Hmga2 as a novel RUNX1 target gene in HSPCs and mark the first study of the transcriptional regulation of the Hmga2 gene by RUNX1 through differential control regions. Disclosures:No relevant conflicts of interest to declare.

Evidence for regulation of columnar habit in apple by a putative 2OG‐Fe(II) oxygenase

Understanding the genetic mechanisms controlling columnar-type growth in the apple mutant 'Wijcik' will provide insights on how tree architecture and growth are regulated in fruit trees. In apple, columnar-type growth is controlled by a single major gene at the Columnar (Co) locus. By comparing the genomic sequence of the Co region of 'Wijcik' with its wild-type 'McIntosh', a novel non-coding DNA element of 1956 bp specific to Pyreae was found to be inserted in an intergenic region of 'Wijcik'. Expression analysis of selected genes located in the vicinity of the insertion revealed the upregulation of the MdCo31 gene encoding a putative 2OG-Fe(II) oxygenase in axillary buds of 'Wijcik'. Constitutive expression of MdCo31 in Arabidopsis thaliana resulted in compact plants with shortened floral internodes, a phenotype reminiscent of the one observed in columnar apple trees. We conclude that MdCo31 is a strong candidate gene for the control of columnar growth in 'Wijcik'.

Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element

The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a β-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.

The Transcription Factors Sox10 and Myrf Define an Essential Regulatory Network Module in Differentiating Oligodendrocytes

Myelin is essential for rapid saltatory conduction and is produced by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system. In both cell types the transcription factor Sox10 is an essential component of the myelin-specific regulatory network. Here we identify Myrf as an oligodendrocyte-specific target of Sox10 and map a Sox10 responsive enhancer to an evolutionarily conserved element in intron 1 of the Myrf gene. Once induced, Myrf cooperates with Sox10 to implement the myelination program as evident from the physical interaction between both proteins and the synergistic activation of several myelin-specific genes. This is strongly reminiscent of the situation in Schwann cells where Sox10 first induces and then cooperates with Krox20 during myelination. Our analyses indicate that the regulatory network for myelination in oligodendrocytes is organized along similar general principles as the one in Schwann cells, but is differentially implemented.

Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron

Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5′ splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5′ splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.

Small RNA profiling of Xenopus embryos reveals novel miRNAs and a new class of small RNAs derived from intronic transposable elements

Small RNA control of gene expression is critical for developmental processes in vertebrate embryos. To determine the dynamics of small RNA expression and to uncover novel small RNAs in the early vertebrate embryo, we performed high-throughput sequencing of all small RNAs in Xenopus tropicalis embryos at three developmental time points and in dissected halves of gastrula embryos. This analysis allowed us to identify novel microRNAs and we show that microRNA expression is highly dynamic and spatially localized in early embryos. In addition, we have developed a microRNA prediction pipeline and demonstrate that it has the power to predict new miRNAs that are experimentally detectable in frogs, mice, and humans. By combining the small RNA sequencing with mRNA profiling at the different developmental stages, we identify a new class of small noncoding RNAs that we name siteRNAs, which align in clusters to introns of protein-coding genes. We show that siteRNAs are derived from remnants of transposable elements present in the introns. We find that genes containing clusters of siteRNAs are transcriptionally repressed as compared with all genes. Furthermore, we show that this is true for individual genes containing siteRNA clusters, and that these genes are enriched in specific repressive histone modifications. Our data thus suggest a new mechanism of siteRNA-mediated gene silencing in vertebrates, and provide an example of how mobile elements can affect gene regulation.

Cis‐regulatory underpinnings of human GLI3 expression in embryonic craniofacial structures and internal organs

The zinc finger transcription factor Gli3 is an important mediator of Sonic hedgehog (Shh) signaling. During early embryonic development Gli3 participates in patterning and growth of the central nervous system, face, skeleton, limb, tooth and gut. Precise regulation of the temporal and spatial expression of Gli3 is crucial for the proper specification of these structures in mammals and other vertebrates. Previously we reported a set of human intronic cis-regulators controlling almost the entire known repertoire of endogenous Gli3 expression in mouse neural tube and limbs. However, the genetic underpinning of GLI3 expression in other embryonic domains such as craniofacial structures and internal organs remain elusive. Here we demonstrate in a transgenic mice assay the potential of a subset of human/fish conserved non-coding sequences (CNEs) residing within GLI3 intronic intervals to induce reporter gene expression at known regions of endogenous Gli3 transcription in embryonic domains other than central nervous system (CNS) and limbs. Highly specific reporter expression was observed in craniofacial structures, eye, gut, and genitourinary system. Moreover, the comparison of expression patterns directed by these intronic cis-acting regulatory elements in mouse and zebrafish embryos suggests that in accordance with sequence conservation, the target site specificity of a subset of these elements remains preserved among these two lineages. Taken together with our recent investigations, it is proposed here that during vertebrate evolution the Gli3 expression control acquired multiple, independently acting, intronic enhancers for spatiotemporal patterning of CNS, limbs, craniofacial structures and internal organs.

The Arabidopsis thaliana MHX gene includes an intronic element that boosts translation when localized in a 5' UTR intron.

The mechanisms that underlie the ability of some introns to increase gene expression, a phenomenon called intron-mediated enhancement (IME), are not fully understood. It is also not known why introns localized in the 5′-untranslated region (5′ UTR) are considerably longer than downstream eukaryotic introns. It was hypothesized that this extra length results from the presence of some functional intronic elements. However, deletion analyses studies carried out thus far were unable to identify specific intronic regions necessary for IME. Using deletion analysis and a gain-of-function approach, an internal element that considerably increases translational efficiency, without affecting splicing, was identified in the 5′ UTR intron of the Arabidopsis thaliana MHX gene. Moreover, the ability of this element to enhance translation was diminished by a minor downstream shift in the position of introns containing it from the 5′ UTR into the coding sequence. These data suggest that some of the extra length of 5′ UTR introns results from the presence of elements that enhance translation, and, moreover, from the ability of 5′ UTR introns to provide preferable platforms for such elements over downstream introns. The impact of the identified intronic element on translational efficiency was augmented upon removal of neighbouring intronic elements. Interference between different intronic elements had not been reported thus far. This interference may support the bioinformatics-based idea that some of the extra sequence of 5′ UTR introns is also necessary for separating different functional intronic elements.