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Segmental duplications (SDs) constitute a considerable fraction of primate genomes. They contribute to genetic variation and provide raw material for evolution. Groups of SDs are characterized by the presence of shared core duplicons. One of these core duplicons, low copy repeat (lcr)16a, has been shown to be particularly active in the propagation of interspersed SDs in primates. The underlying mechanisms are, however, only partially understood. Alu short interspersed elements (SINEs) are frequently found at breakpoints and have been implicated in the expansion of SDs. Detailed analysis of lcr16a-containing SDs shows that the hominid-specific SVA (SINE-R-VNTR-Alu) retrotransposon is an integral component of the core duplicon in Asian and African great apes. In orang-utan, it provides breakpoints and contributes to both interchromosomal and intrachromosomal lcr16a mobility by inter-element recombination. Furthermore, the data suggest that in hominines (human, chimpanzee, gorilla) SVA recombination-mediated integration of a circular intermediate is the founding event of a lineage-specific lcr16a expansion. One of the hominine lcr16a copies displays large flanking direct repeats, a structural feature shared by other SDs in the human genome. Taken together, the results obtained extend the range of SVAs’ contribution to genome evolution from RNA-mediated transduction to DNA-based recombination. In addition, they provide further support for a role of circular intermediates in SD mobilization.

The corticotropin releasing factor (CRF) system plays a critical role in responses to stressful stimuli, and is expressed in many areas of the brain involved in processing fear, anxiety, and social behaviors. To better understand the mechanisms by which the CRF system modulates responses to stressful events and social stimuli, we employed a mouse model that selectively disrupts NMDA receptor function via NMDA receptor subunit NR1 (Grin1) knockout specifically in Cre-expressing CRF neurons. These animals (Cre+/ fGrin1+ ) were compared with littermates lacking Cre expression (Cre−/ fGrin1+ ). Following cue discrimination fear condition- ing, male Cre+/ fGrin1+ mice showed increased fear expression to the tone paired with a foot shock (CS+) while still discriminating the CS+ from a tone never paired with a foot shock (CS−). In contrast to males, female mice learned and discriminated fear cues equivalently across the genotypes. Similarly, no genotype differences in sociability or social novelty were observed in female mice, but Cre+/ fGrin1+ males displayed greater naive sociability and preference for social novelty than Cre−/ fGrin1+ littermates. Furthermore, the level of social withdrawal exhibited by male Cre+/ fGrin1+ mice susceptible to social defeat stress relative to same genotype controls was significantly more pronounced than that displayed by susceptible Cre−/ fGrin1+ mice compared to control Cre−/ fGrin1+ mice. Together, these results demonstrate increased fear, social, and stress responsiveness specifically in male Cre+/ fGrin1+ mice. Our findings indicate that NMDA- mediated glutamatergic regulation of CRF neurons is important for appropriately regulating fear and social responses, likely functioning to promote survival under aversive circumstances. © 2015 Elsevier Ltd. All rights reserved.

SINEs (Short INterspersed Elements) are widely distributed among eukaryotes. Some SINE families are organized in superfamilies characterized by a shared central domain. These central domains are conserved across species, classes, and even phyla. Here we report the identification of two novel such superfamilies in the genomes of gastropod and bivalve mollusks. The central conserved domain of the first superfamily is present in SINEs in Caenogastropoda and Vetigastropoda as well as in all four subclasses of Bivalvia. We designated the domain MESC (Romanian for MElc-snail and SCoica-mussel) because it appears to be restricted to snails and mussels. The second superfamily is restricted to Caenogastropoda. Its central conserved domain-Snail-is related to the Nin-DC domain. Furthermore, we provide evidence that a 40-bp subdomain of the SINE V-domain is conserved in SINEs in mollusks and arthropods. It is predicted to form a stable stem-loop structure that is preserved in the context of the overall SINE RNA secondary structure in invertebrates. Our analysis also recovered short retrotransposons with a Long INterspersed Element (LINE)-derived 5' end. These share the body and/or the tail with transfer RNA (tRNA)-derived SINEs within and across species. Finally, we identified CORE SINEs in gastropods and bivalves-extending the distribution range of this superfamily.

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

Background VNTR (Variable Number of Tandem Repeats) composite retrotransposons - SVA (SINE-R-VNTR-Alu), LAVA (LINE-1-Alu-VNTR-Alu), PVA (PTGR2-VNTR-Alu) and FVA (FRAM-VNTR-Alu) - are specific to hominoid primates. Their assembly, the evolution of their 5’ and 3’ domains, and the functional significance of the shared 5’ Alu-like region are well understood. The central VNTR domain, by contrast, has long been assumed to represent a more or less random collection of 30-50 bp GC-rich repeats. It is only recently that it attracted attention in the context of regulation of SVA expression. Results Here we provide evidence that the organization of the VNTR is non-random, with conserved repeat unit (RU) arrays at both the 5’ and 3’ ends of the VNTRs of human, chimpanzee and orangutan SVA and gibbon LAVA. The younger SVA subfamilies harbour highly organized internal RU arrays. The composition of these arrays is specific to the human/chimpanzee and orangutan lineages, respectively. Tracing the development of the VNTR through evolution we show for the first time how tandem repeats evolve within the constraints set by a functional, non-autonomous non-LTR retrotransposon in two different families - LAVA and SVA - in different hominoid lineages. Our analysis revealed that a microhomology-driven mechanism mediates expansion/contraction of the VNTR domain at the DNA level. Elements of all four VNTR composite families have been shown to be mobilized by the autonomous LINE1 retrotransposon in trans. In case of SVA, key determinants of mobilization are found in the 5’ hexameric repeat/Alu-like region. We now demonstrate that in LAVA, by contrast, the VNTR domain determines mobilization efficiency in the context of domain swaps between active and inactive elements. Conclusions The central domain of VNTR composites evolves in a lineage-specific manner which gives rise to distinct structures in gibbon LAVA, orangutan SVA, and human/chimpanzee SVA. The differences observed between the families and lineages are likely to have an influence on the expression and mobilization of the elements. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1543-z) contains supplementary material, which is available to authorized users.

Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.

SVA (SINE-R-VNTR-Alu) elements constitute the youngest family of composite non-LTR retrotransposons in hominoid primates. The sequence of their assembly, however, remains unclear. Recently, a second family of VNTR-containing composites, LAVA (L1-Alu-VNTR-Alu), has been identified in gibbons. We now report the existence of two additional VNTR composite families, PVA (PTGR2-VNTR-Alu) and FVA (FRAM-VNTR-Alu), in the genome of Nomascus leucogenys. Like LAVA, they share the 5'-Alu-like region and VNTR with SVA, but differ at their 3'-ends. The 3'-end of PVA comprises part of the PTGR2 gene, whereas FVA is characterized by the presence of a partial FRAM element in its 3'-domain. Splicing could be identified as the mechanism of acquisition of the variant 3'-ends in all four families of VNTR composites. SVAs have been shown to be mobilized by the L1 protein machinery in trans. A critical role in this process has been ascribed to their 5'-hexameric repeat/ Alu-like region. The Alu-like region displays specific features in each of the VNTR composite families/subfamilies with characteristic deletions found in the evolutionary younger subfamilies. Using reciprocal exchanges between SVA_E and PVA/FVA elements, we demonstrate that the structure, not the presence of the (CCCTCT)n/ Alu-like region determines mobilization capacity. Combination of LAVA and SVA_E domains does not yield any active elements-suggesting the use of different combinations of host factors for the two major groups of VNTR composites. Finally, we demonstrate that the LAVA 3'-L1ME5 fragment attenuates mobilization capacity.

Carbone, Lucia et al.-- This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported licence., Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ∼5 million years ago, coincident with major geographical changes in southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.

Sequencing of rat and human vascular endothelial growth factor (VEGF) cDNA clones has previously identified a 3' untranslated region of approximately 1.9 kb, although the apparent site of polyadenylation differed in the two species, despite a high degree of sequence conservation in the region. Neither site is preceded by a canonical AAUAAA polyadenylation signal, a situation frequently found in genes that are subject to alternative polyadenylation. We have sequenced 2.25 kb of the 3' region of the mouse VEGF gene and mapped the usage of potential polyadenylation sites in fibroblasts cultured under both normoxic and hypoxic conditions. We find that two sites for polyadenylation are present in the mouse VEGF gene but the majority of transcripts contain the longer form of the 3'UTR and that their usage is not effected by environmental oxygen tension.

The endothelial cell-specific mitogen vascular-endothelial growth factor (VEGF) plays a key role in both physiological and pathological angiogenesis. The up-regulation of VEGF expression in response to reduced oxygen tension occurs through transcriptional and post-transcriptional mechanisms. To investigate the molecular mechanisms of transcriptional activation by hypoxia (1% oxygen), fine mapping of a hypoxia-responsive region of the human VEGF promoter was carried out using luciferase reporter-gene constructs in C6 glioma cells. Here, we report that the binding site of hypoxia-inducible factor 1 (HIF1) is crucial for the hypoxic induction of VEGF gene expression. However, an enhancer subfragment containing the HIF1 binding site was not sufficient to confer full hypoxia responsiveness. Addition of upstream sequences restored the full sensitivity to hypoxia induction. This potentiating effect is due to activator protein 1 binding. The ‘potentiating’ sequences are unable to confer hypoxia responsiveness on their own. Our results strongly suggest that in C6 glioma cells a complex array of trans-acting factors facilitates full transcriptional induction of VEGF gene expression by hypoxia.

Since the short intron in the 5′-untranslated region (5′-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5′-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5′-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins1IVS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides +87 and +110 showed a reduction of expression by 30 %. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.

SVA elements are nonautonomous non–long terminal repeat (non-LTR) retrotransposons that originated

During development, hematopoiesis initiates in the yolk sac through a process that depends on VEGF/Flk1 signaling and on the function of the SCL/Tal1 transcription factor. Here we show that VEGF modifies the developmental potential of primitive erythroid progenitors and prolongs their life span. Furthermore, the survival of yolk sac erythrocytes in vivo depends on the dose of VEGF. Interestingly, in Vegflo/lo embryos carrying a hypomorph allele, Flk1-positive cells reach the yolk sac at E8.5,but are severely compromised in their ability to generate primitive erythroid precursors. These observations indicate that during embryonic development,different thresholds of VEGF are required for the migration and clonal expansion of hematopoietic precursors. The near absence of primitive erythroid precursors in Vegflo/lo embryos correlates with low levels of Scl in the yolk sac. Strikingly, gain-of-function of SCL partially complements the hematopoietic defect caused by the hypomorph Vegflo allele, and re-establishes the survival of erythroid cells and the expression of erythroid genes (Gata1 andβH1). This indicates that SCL functions downstream of VEGF to ensure an expansion of the hematopoietic compartment.

Vascular endothelial growth factor A (VEGFA) plays a pivotal role in the first steps of endothelial and haematopoietic development in the yolk sac, as well as in the establishment of the cardiovascular system of the embryo. At the onset of gastrulation, VEGFA is primarily expressed in the yolk sac visceral endoderm and in the yolk sac mesothelium. We report the generation and analysis of a Vegf hypomorphic allele, Vegflo. Animals heterozygous for the targeted mutation are viable. Homozygous embryos, however, die at 9.0 dpc because of severe abnormalities in the yolk sac vasculature and deficiencies in the development of the dorsal aortae. We find that providing ‘Vegf wild-type’ visceral endoderm to the hypomorphic embryos restores normal blood and endothelial differentiation in the yolk sac, but does not rescue the phenotype in the embryo proper. In the opposite situation, however, when Vegf hypomorphic visceral endoderm is provided to a wild-type embryo, the ‘Vegf wild-type’ yolk sac mesoderm is not sufficient to support proper vessel formation and haematopoietic differentiation in this extra-embryonic membrane. These findings demonstrate that VEGFA expression in the visceral endoderm is absolutely required for the normal expansion and organisation of both the endothelial and haematopoietic lineages in the early sites of vessel and blood formation. However, normal VEGFA expression in the yolk sac mesoderm alone is not sufficient for supporting the proper development of the early vascular and haematopoietic system.

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murineFlk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5′-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5′-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a mitogen and chemotactic factor for endothelial cells in vitro and an angiogenesis and vascular permeability factor in vivo. Due to its properties, VEGF is a candidate for both angiogenesis and vascular permeability/oedema induction which typically occur in glioblastomas. In this study we test the hypothesis that the antioedema effect of dexamethasone is mediated by downregulation of VEGF or VEGF receptor expression. VEGF mRNA and protein levels of two rat glioma cells lines, C6 and GS-9L, were determined after incubation with dexamethasone under normoxic and hypoxic conditions. In normoxic C6 and GS9L cells, we observed 50-60% downregulation of VEGF mRNA by dexamethasone (P=0.015 and P=0. 01, respectively). This effect was dependent on glucocorticoid-receptor (GR) function. The inhibitory effect of dexamethasone on VEGF gene expression by tumour cells was markedly reduced by hypoxia which suggests that the upregulation of VEGF driven by hypoxia overcomes the effect of the dexamethasone. Dexamethasone did not alter VEGFR-2 mRNA levels in human umbilical endothelial cells. In a subcutaneous glioma tumour model, we observed only a 15% decrease in VEGF mRNA expression in dexamethasone treated animals (n = 12) compared with controls animals (P = 0.24). We conclude that dexamethasone may decrease brain tumour-associated oedema by reduction of VEGF expression in tumour cells. However, the highly reduced activity on hypoxic tumour cells suggests that dexamethasone efficacy may be limited by hypoxia in rapidly growing tumours.

Vascular endothelial growth factor (VEGF), an essential regulator of angiogenesis during early development as well as during the growth of solid tumours, bears an unusually large 5′ untranslated region (5′-UTR) in the mRNA of over 1000 nucleotides. We found that the VEGF 5′-UTR, despite being GC-rich and containing an upstream short open reading frame, promotes efficient translation of a luciferase reporter. The VEGF 5′-UTR also allowed translation of luciferase from a dicistronic mRNA when placed between the two cistrons, demonstrating that it contains an internal ribosome entry site. Deletion analysis indicated that the IRES resides towards the 3′ end of the 5′-UTR.

Up-regulation of vascular endothelial growth factor (VEGF) expression is a major event leading to neovascularization in malignant gliomas. Hypoxia is believed to be the crucial environmental stimulus for this up-regulation. To critically assess this hypothesis, we asked whether the mechanisms defined previously for hypoxia-induced VEGF expression in vitro are similarly involved and sufficient for up-regulation of VEGF gene expression in vivo, using a lacZ reporter gene under the control of VEGF regulatory sequences in an experimental glioma model. Inclusion of the binding site for hypoxia-inducible factor 1 (HIF 1) in the 5' regulatory sequences used in the hybrid gene produced weak beta-galactosidase staining in a special tumor cell subtype, the so-called perinecrotic palisading (PNP) cells that flank necrotic regions within the tumor. Deletion of the HIF 1 binding site abolished reporter gene expression in the PNP cells, indicating that transcriptional activation of VEGF expression in gliomas is mediated by HIF 1. Inclusion of 3' untranslated sequences from the VEGF gene in the reporter constructs resulted in an increased beta-galactosidase staining in the PNP cells, suggesting that mRNA stabilization also contributes to VEGF up-regulation in glioblastoma cells growing as solid tumors. Combination of the 5' flanking region including the HIF 1 site along with 3' untranslated sequences produced increased levels of beta-galactosidase expression in PNP cells. EF 5 immunostaining for regions of low oxygen partial pressure covered the same PNP cells that were stained for beta-galactosidase. Collectively, the data provide experimental evidence that VEGF gene expression is activated in a distinct tumor cell subpopulation, the perinecrotic palisading cells of gliomas, by two distinct hypoxia-driven regulatory mechanisms.

Transcription factors of the bHLH-PAS protein family are important regulators of developmental processes such as neurogenesis and tracheal development in invertebrates. Recently a bHLH-PAS protein, named trachealess (trl) was identified as a master regulator of tracheogenesis. Hypoxia-inducible factor, HIF-1 alpha, is a vertebrate relative of trl which is likely to be involved in growth of blood vessels by the induction of vascular endothelial growth factor (VEGF) in response to hypoxia. In the present study we describe mRNA cloning and mRNA expression pattern of mouse HIF-related factor (HRF), a novel close relative of HIF-1 alpha which is expressed most prominently in brain capillary endothelial cells and other blood vessels as well as in bronchial epithelium in the embryo and the adult. In addition, smooth muscle cells of the uterus, neurons, brown adipose tissue and various epithelial tissues express HRF mRNA as well. High expression levels of HRF mRNA in embryonic choroid plexus and kidney glomeruli, places where VEGF is highly expressed, suggest a role of this factor in VEGF gene activation similar to that of HIF-1 alpha. Given the similarity between morphogenesis of the tracheal system and the vertebrate vascular system, the expression pattern of HRF in the vasculature and the bronchial tree raises the possibility that this family of transcription factors may be involved in tubulogenesis.

To determine the function of VEGF-A in nervous system development, we have utilized the Nestin promoter-driven Cre recombinase transgene, in conjunction with a conditional and hypomorphic VEGF-A allele, to lower VEGF-A activity in neural progenitor cells. Mice with intermediate levels of VEGF-A activity showed decreased blood vessel branching and density in the cortex and retina, resulting in a thinner retina and aberrant structural organization of the cortex. Severe reductions in VEGF-A led to decreases in vascularity and subsequent hypoxia, resulting in the specific degeneration of the cerebral cortex and neonatal lethality. Decreased neuronal proliferation and hypoxia was evident at E11.5, leading to increased neuronal apoptosis in the cortex by E15.5. In order to address whether the observed changes in the structural organization of the nervous system were due to a direct and autocrine role of VEGF-A on the neural population, we conditionally inactivated the main VEGF-A receptor, Flk1, specifically in neuronal lineages, by using the Nestin Cre transgene. The normality of these mice ruled out the possibility that VEGF-A/Flk1 signaling has a significant autocrine role in CNS development. VEGF-A dosage is therefore a critical parameter regulating the density of the vascular plexus in the developing CNS that is in turn a key determinant in the development and architectural organization of the nervous system.

Angiogenic growth factors and their endothelial receptors are thought to function as major regulators of blood vessel formation. Vascular endothelial growth factor (VEGF) and its receptors, Flt-1 (VEGFR-1) and Flk-1 (VEGFR-2), as well as Angiopoietin-1 and its receptor, Tie-2, represent key signal transduction systems involved in the regulation of embryonic vascular development. The expression of these molecules correlates with phases of blood vessel formation during embryogenesis. Inactivation of any of the genes encoding these molecules in mouse embryos results in defective vascular development and embryonic lethality around mid-gestation. In addition, the VEGF signal transduction system has been implicated in the regulation of pathological blood vessel growth during certain angiogenesis-dependent diseases that are often associated with tissue ischemia, such as proliferative retinopathy or solid tumor growth. This hypothesis is substantiated by experiments, in which the inhibition of VEGF signal transduction resulted in the the inhibition of neovascularization in these diseases. Thus, the VEGF signal transduction system represents a useful target for an anti-angiogenic therapy.