Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the caspase 1 protease. Although genetic data support a critical role for inflammasomes in immune defence and inflammatory diseases, the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing 4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin and PrgJ, a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homologue of NLRC4, NAIP5 (NLR family, apoptosis inhibitory protein 5), has been implicated in activation of NLRC4 (refs 7-11), but is widely assumed to have only an auxiliary role, as NAIP5 is often dispensable for NLRC4 activation. However, Naip5 is a member of a small multigene family, raising the possibility of redundancy and functional specialization among Naip genes. Here we show in mice that different NAIP paralogues determine the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that the NAIP2-NLRC4 complex physically associates with PrgJ but not flagellin, whereas NAIP5-NLRC4 associates with flagellin but not PrgJ. Our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP-NLRC4 inflammasomes.

A major portion of most eukaryotic genomes are transposable elements (TEs). During evolution, TEs have introduced profound changes to genome size, structure, and function. As integral parts of genomes, the dynamic presence of TEs will continue to be a major force in reshaping genomes. Early computational analyses of TEs in genome sequences focused on filtering out "junk" sequences to facilitate gene annotation. When the high abundance and diversity of TEs in eukaryotic genomes were recognized, these early efforts transformed into the systematic genome-wide categorization and classification of TEs. The availability of genomic sequence data reversed the classical genetic approaches to discovering new TE families and superfamilies. Curated TE databases and their accurate annotation of genome sequences in turn facilitated the studies on TEs in a number of frontiers including:(1) TE-mediated changes of genome size and structure,(2) the influence of TEs on genome and gene functions,(3) TE regulation by host,(4) the evolution of TEs and their population dynamics, and (5) genomic scale studies of TE activity. Bioinformatics and genomic approaches have become an integral part of large-scale studies on TEs to extract information with pure in silico analyses or to assist wet lab experimental studies. The current revolution in genome sequencing technology facilitates further progress in the existing frontiers of research and emergence of new initiatives. The rapid generation of large-sequence datasets at record low costs on a routine basis is challenging the computing industry on storage capacity and manipulation speed and the bioinformatics community for improvement in algorithms and their implementations.

H2Av is a versatile histone variant that plays both positive and negative roles in transcription, DNA repair, and chromatin structure in Drosophila. H2Av, and its broader homolog H2A.Z, tend to be enriched toward 5' ends of genes, and exist in both euchromatin and heterochromatin. Its organization around euchromatin genes and other features have been described in many eukaryotic model organisms. However, less is known about H2Av nucleosome organization in heterochromatin. Here we report the properties and organization of individual H2Av nucleosomes around genes and transposable elements located in Drosophila heterochromatic regions. We compare the similarity and differences with that found in euchromatic regions. Our analyses suggest that nucleosomes are intrinsically positioned on inverted repeats of DNA transposable elements such as those related to the "1360" element, but are not intrinsically positioned on retrotransposon-related elements.

ABSTRACT: Transposable elements (TEs) are increasingly being recognized as powerful facilitators of evolution. We propose the TE-Thrust hypothesis to encompass TE-facilitated processes by which genomes self-engineer coding, regulatory, karyotypic or other genetic changes. Although TEs are occasionally harmful to some individuals, genomic dynamism caused by TEs can be very beneficial to lineages. This can result in differential survival and differential fecundity of lineages. Lineages with an abundant and suitable repertoire of TEs have enhanced evolutionary potential and, if all else is equal, tend to be fecund, resulting in species-rich adaptive radiations, and/or they tend to undergo major evolutionary transitions. Many other mechanisms of genomic change are also important in evolution, and whether the evolutionary potential of TE-Thrust is realized is heavily dependent on environmental and ecological factors. The large contribution of TEs to evolutionary innovation is particularly well documented in the primate lineage. In this paper, we review numerous cases of beneficial TE-caused modifications to the genomes of higher primates, which strongly support our TE-Thrust hypothesis.

Mobile genetic elements feature prominently in mammalian genome evolution. Several transposition-competent retrotransposon families (L1, Alu, SVA) remain active in the human germ line, leading to pathogenesis as well as genome structural variation across the global population. High-throughput screening approaches have recently been developed to detect retrotransposon insertion polymorphisms. Evidence produced by these and other genome-scale technologies indicates an expanded role for retrotransposition in human biology, including somatic mobilisation in the developing embryo and in neural cells.

Malignancy results from a complex combination of genetic and epigenetic changes, the full effects of which are still largely unknown. Here we summarize current knowledge of the origin, retrotranspositional activity, epigenetic state, and transcription of human endogenous retroviruses (HERVs), and then discuss the potential effects of their deregulation in cancer. Evidence suggests that cancer-associated epigenetic changes most likely underlie potential HERV-mediated effects on genome and transcriptome instability and may play a role in malignancy. Despite our currently limited understanding of the importance of HERVs or other transposable elements in cancer development, we believe that the emerging era of high-throughput sequencing of cancer genomes, epigenomes, and transcriptomes will provide unprecedented opportunities to investigate these roles in the future.

Non-protein-coding DNA comprises the majority of animal genomes but its functions are largely unknown. We identified over 17,000 different tetranucleotide pairs in the Drosophila melanogaster genome that are over-represented at distances up to 100nt in conserved non-exonic sequences. Those exhibiting the highest information content in surrounding nucleotides were classified into five groups: tRNAs, motifs associated with histone genes, Suppressor-of-Hairy-wing binding sites, and two sets of previously unrecognized motifs (DLM3 and DLM4). There are hundreds to thousands of copies of DLM3 and DLM4, respectively, in the genome, located almost exclusively in non-coding regions. They have similar copy numbers among drosophilids, but are largely absent in other insects. DLM3 is likely a cis-regulatory element, whereas DLM4 sequences are capable of forming a short hairpin structure and are expressed as approximately 80nt RNAs. This work reports the existence of Drosophila genus-specific sequence motifs, and suggests that many more novel functional elements may be discovered in genomes using the general approach outlined herein.

Scientific history has had a profound effect on the theories of evolution. At the beginning of the 21st century, molecular cell biology has revealed a dense structure of information-processing networks that use the genome as an interactive read-write (RW) memory system rather than an organism blueprint. Genome sequencing has documented the importance of mobile DNA activities and major genome restructuring events at key junctures in evolution: exon shuffling, changes in cis-regulatory sites, horizontal transfer, cell fusions and whole genome doublings (WGDs). The natural genetic engineering functions that mediate genome restructuring are activated by multiple stimuli, in particular by events similar to those found in the DNA record: microbial infection and interspecific hybridization leading to the formation of allotetraploids. These molecular genetic discoveries, plus a consideration of how mobile DNA rearrangements increase the efficiency of generating functional genomic novelties, make it possible to formulate a 21st century view of interactive evolutionary processes. This view integrates contemporary knowledge of the molecular basis of genetic change, major genome events in evolution, and stimuli that activate DNA restructuring with classical cytogenetic understanding about the role of hybridization in species diversification.

Due to an almost complete absence of fossil record, the evolutionary history of chimpanzees has only been studied recently on the basis of genetic data. Although the general topology of the chimpanzee phylogeny is well established, uncertainties remain concerning the size of current and past populations, the occurrence of bottlenecks or population expansions, or about divergence times and migrations rates between subspecies. Here, we present a novel attempt at globally inferring the detailed evolution of the Pan genus based on approximate Bayesian computation, an approach preferentially applied to complex models where the likelihood cannot be computed analytically. Based on two microsatellite and DNA sequence data sets and adjusting simulated data for local levels of inbreeding and patterns of missing data, we find support for several new features of chimpanzee evolution as compared with previous studies based on smaller data sets and simpler evolutionary models. We find that the central chimpanzees are certainly the oldest population of all P. troglodytes subspecies and that the other two P. t. subspecies diverged from the central chimpanzees by founder events. We also find an older divergence time (1.6 million years [My]) between common chimpanzee and Bonobos than previous studies (0.9-1.3 My), but this divergence appears to have been very progressive with the maintenance of relatively high levels of gene flow between the ancestral chimpanzee population and the Bonobos. Finally, we could also confirm the existence of strong unidirectional gene flow from the western into the central chimpanzee. These results show that interesting and innovative features of chimpanzee history emerge when considering their whole evolutionary history in a single analysis, rather than relying on simpler models involving several comparisons of pairs of populations.

A mechanism for survival of prostate cancer cells in an androgen-deprived environment remains elusive. Here, we find that expression of neuronal apoptosis inhibitory protein (NAIP) was significantly increased in vivo and in vitro in response to androgen deprivation therapy (ADT). Increased expression of NAIP corresponded to increased DNA-binding activity of NF-kappaB that physically associated to previously uncharacterized kappaB-like sites in the NAIP locus. Importantly, expression of NAIP was significantly increased (p=0.04) in clinical samples of prostate cancer from patients receiving ADT. Expression of NAIP may be associated with enhanced survival of prostate cancer in response to castration.

The inbred mouse is an invaluable model for human biology and disease. Nevertheless, when considering genetic mechanisms of variation and disease, it is important to appreciate the significant differences in the spectra of spontaneous mutations that distinguish these species. While insertions of transposable elements are responsible for only approximately 0.1% of de novo mutations in humans, the figure is 100-fold higher in the laboratory mouse. This striking difference is largely due to the ongoing activity of mouse endogenous retroviral elements. Here we briefly review mouse endogenous retroviruses (ERVs) and their influence on gene expression, analyze mechanisms of interaction between ERVs and the host cell, and summarize the variety of mutations caused by ERV insertions. The prevalence of mouse ERV activity indicates that the genome of the laboratory mouse is presently behind in the "arms race" against invasion.

Eight percent of the human genome is derived from endogenous retrovirus (ERV) insertions. ERV long terminal repeats (LTRs) contain strong promoters that are known to contribute to the transcriptional regulation of certain human genes. While some LTRs are known to possess bidirectional promoter activity in vitro, only sense orientation LTR promoters have previously been shown to regulate human gene expression. Here we demonstrate that an ERV1 LTR acts as a bidirectional promoter for the human Down syndrome critical region 4 (DSCR4) and DSCR8 genes. We show that while DSCR4 and DSCR8 are essentially co-expressed, their shared LTR promoter is more active in the sense than the antisense orientation. Through deletion analysis of the LTR we have identified positive and negative regulatory elements, and defined a core region of the promoter that is required for transcriptional activity in both orientations. Finally, we show that the ERV LTR also exists in the genomes of several non-human primates, and present evidence that potential transcription factor binding sites in the core region have been maintained throughout primate evolution.

Throughout the course of vertebrate evolution, germline retroviral infections have resulted in heritable provirus insertions into host DNA. These endogenous retroviruses (ERVs) contain long terminal repeat (LTR) promoters that can be adopted for use by nearby host genes. It is not known whether the transcription factor (TF) binding sites and tissue-specificities of modern LTR gene promoters have been retained since the time of ERV insertion, or if these features evolved later as the LTR became involved in host gene regulation. To address this issue, we have conducted a case study of the ERV-L LTR promoter of human beta1,3-galactosyltransferase 5 (beta3GAL-T5). We have previously shown that the human beta3GAL-T5 LTR promoter is responsible for the majority of gene transcripts in the colon. The murine beta3gal-t5 gene is also expressed primarily in the colon, despite the absence of an orthologous ERV-L LTR in the mouse genome. We therefore hypothesized that both the ERV-L LTR and the non-retroviral ancestral beta3GAL-T5 promoter were active in the colon at the time of ERV insertion. In support of this hypothesis, we have shown that the orthologous LTRs of four non-human primates are also active in a human colorectal cell line, and that the baboon LTR is active in primary baboon colon tissue. We also present evidence that the functional TF binding sites of the human beta3GAL-T5 LTR promoter were present in the original consensus sequence for this class of LTRs. Upon similar analysis of other ERV sequences, we have concluded that this evolutionary history is shared by certain other LTR gene promoters, and may be a general phenomenon.

The fact that transposable elements (TEs) can influence host gene expression was first recognized more than 50 years ago. However, since that time, TEs have been widely regarded as harmful genetic parasites-selfish elements that are rarely co-opted by the genome to serve a beneficial role. Here, we survey recent findings that relate to TE impact on host genes and remind the reader that TEs, in contrast to other noncoding parts of the genome, are uniquely suited to gene regulatory functions. We review recent studies that demonstrate the role of TEs in establishing and rewiring gene regulatory networks, and discuss the overall ubiquity of exaptation. We suggest that although individuals within a population can be harmed by the deleterious effects of new TE insertions, the presence of TE sequences in a genome is of overall benefit to the population. Expected final online publication date for the Annual Review of Genetics Volume 46 is November 2, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Comprising nearly half of the human and mouse genomes, transposable elements (TEs) are found within most genes. Although the vast majority of TEs in introns are fixed in the species and presumably exert no significant effects on the enclosing gene, some markedly perturb transcription and result in disease or a mutated phenotype. Factors determining the likelihood that an intronic TE will affect transcription are not clear. In this study, we examined intronic TE distributions in both human and mouse and found several factors that likely contribute to whether a particular TE can influence gene transcription. Specifically, we observed that TEs near exons are greatly underrepresented compared to random distributions, but the size of these "underrepresentation zones" differs between TE classes. Compared to elsewhere in introns, TEs within these zones are shorter on average and show stronger orientation biases. Moreover, TEs in extremely close proximity (<20 bp) to exons show a strong bias to be near splice-donor sites. Interestingly, disease-causing intronic TE insertions show the opposite distributional trends, and by examining expressed sequence tag (EST) databases, we found that the proportion of TEs contributing to chimeric TE-gene transcripts is significantly higher within their underrepresentation zones. In addition, an analysis of predicted splice sites within human long terminal repeat (LTR) elements showed a significantly lower total number and weaker strength for intronic LTRs near exons. Based on these factors, we selectively examined a list of polymorphic mouse LTR elements in introns and showed clear evidence of transcriptional disruption by LTR element insertions in the Trpc6 and Kcnh6 genes. Taken together, these studies lend insight into the potential selective forces that have shaped intronic TE distributions and enable identification of TEs most likely to exert transcriptional effects on genes.

Gene regulatory changes are thought to be major factors driving species evolution, with creation of new regulatory regions likely being instrumental in contributing to diversity among vertebrates. There is growing appreciation for the role of transposable elements (TEs) in gene regulation and, indeed, laboratory investigations have confirmed many specific examples of mammalian genes regulated by promoters donated by endogenous retroviruses (ERVs) or other TEs. Bioinformatics studies have revealed hundreds of additional instances where this is likely to be the case. Since the long terminal repeats (LTRs) of retroviruses naturally contain abundant transcriptional regulatory signals, roles for ERV LTRs in regulating mammalian genes are eminently plausible. Moreover, it seems reasonable that exaptation of an LTR regulatory module provides opportunities for evolution of new gene regulatory patterns. In this Review we summarize known examples of LTRs that function as human gene alternative promoters, as well as the evidence that LTR exaptation has resulted in a pattern of novel gene expression significantly different from the pattern before LTR insertion or from that of gene orthologs lacking the LTR. Available data suggest that, while new expression patterns can arise as a result of LTR usage, this situation is relatively rare and is largely restricted to the placenta. In many cases, the LTR appears to be a minor, alternative promoter with an expression pattern similar to that of the native promoter(s) and hence likely exerts a subtle overall effect on gene expression. We discuss these findings and offer evolutionary models to explain these trends.

The human neuronal apoptosis inhibitory protein (NAIP) gene is no longer principally considered a member of the Inhibitor of Apoptosis Protein (IAP) family, as its domain structure and functions in innate immunity also warrant inclusion in the Nod-Like Receptor (NLR) superfamily. NAIP is located in a region of copy number variation, with one full length and four partly deleted copies in the reference human genome. We demonstrate that several of the NAIP paralogues are expressed, and that novel transcripts arise from both internal and upstream transcription start sites. Remarkably, two internal start sites initiate within Alu short interspersed element (SINE) retrotransposons, and a third novel transcription start site exists within the final intron of the GUSBP1 gene, upstream of only two NAIP copies. One Alu functions alone as a promoter in transient assays, while the other likely combines with upstream L1 sequences to form a composite promoter. The novel transcripts encode shortened open reading frames and we show that corresponding proteins are translated in a number of cell lines and primary tissues, in some cases above the level of full length NAIP. Interestingly, some NAIP isoforms lack their caspase-sequestering motifs, suggesting that they have novel functions. Moreover, given that human and mouse NAIP have previously been shown to employ endogenous retroviral long terminal repeats as promoters, exaptation of Alu repeats as additional promoters provides a fascinating illustration of regulatory innovations adopted by a single gene.

Endogenous retroviral elements (ERVs) in mice are significant genomic mutagens, causing approximately 10% of all reported spontaneous germ line mutations in laboratory strains. The majority of these mutations are due to insertions of two high copy ERV families, the IAP and ETn/MusD elements. This significant level of ongoing retrotranspositional activity suggests that inbred mice are highly variable in content of these two ERV groups. However, no comprehensive genome-wide studies have been performed to assess their level of polymorphism. Here we compared three test strains, for which sufficient genomic sequence is available, to each other and to the reference C57BL/6J genome and detected very high levels of insertional polymorphism for both ERV families, with an estimated false discovery rate of only 0.4%. Specifically, we found that at least 60% of IAP and 25% of ETn/MusD elements detected in any strain are absent in one or more of the other three strains. The polymorphic nature of a set of 40 ETn/MusD elements found within gene introns was confirmed using genomic PCR on DNA from a panel of mouse strains. For some cases, we detected gene-splicing abnormalities involving the ERV and obtained additional evidence for decreased gene expression in strains carrying the insertion. In total, we identified nearly 700 polymorphic IAP or ETn/MusD ERVs or solitary LTRs that reside in gene introns, providing potential candidates that may contribute to gene expression differences among strains. These extreme levels of polymorphism suggest that ERV insertions play a significant role in genetic drift of mouse lines.

BACKGROUND Endogenous retroviruses (ERVs) and solitary long terminal repeats (LTRs) have a significant antisense bias when located in gene introns, suggesting strong negative selective pressure on such elements oriented in the same transcriptional direction as the enclosing gene. It has been assumed that this bias reflects the presence of strong transcriptional regulatory signals within LTRs but little work has been done to investigate this phenomenon further. RESULTS In the analysis reported here, we found significant differences between individual human ERV families in their prevalence within genes and degree of antisense bias and show that, regardless of orientation, ERVs of most families are less likely to be found in introns than in intergenic regions. Examination of density profiles of ERVs across transcriptional units and the transcription signals present in the consensus ERVs suggests the importance of splice acceptor sites, in conjunction with splice donor and polyadenylation signals, as the major targets for selection against most families of ERVs/LTRs. Furthermore, analysis of annotated human mRNA splicing events involving ERV sequence revealed that the relatively young human ERVs (HERVs), HERV9 and HERV-K (HML-2), are involved in no human mRNA splicing events at all when oriented antisense to gene transcription, while elements in the sense direction in transcribed regions show considerable bias for use of strong splice sites. CONCLUSION Our observations suggest suppression of splicing among young intronic ERVs oriented antisense to gene transcription, which may account for their reduced mutagenicity and higher fixation rate in gene introns.

Insertion of transposable elements is a major cause of genomic expansion in eukaryotes. Less is understood, however, about mechanisms underlying contraction of genomes. In this study, we show that retroelements can, in rare cases, be precisely deleted from primate genomes, most likely via recombination between 10- to 20-bp target site duplications (TSDs) flanking the retroelement. The deleted loci are indistinguishable from pre-integration sites, effectively reversing the insertion. Through human-chimpanzee-Rhesus monkey genomic comparisons, we estimate that 0.5%-1% of apparent retroelement "insertions" distinguishing humans and chimpanzees actually represent deletions. Furthermore, we demonstrate that 19% of genomic deletions of 200-500 bp that have occurred since the human-chimpanzee divergence are associated with flanking identical repeats of at least 10 bp. A large number of deletions internal to Alu elements were also found flanked by homologies. These results suggest that illegitimate recombination between short direct repeats has played a significant role in human genome evolution. Moreover, this study lends perspective to the view that insertions of retroelements represent unidirectional genetic events.

Long interspersed nuclear element 1 (LINE-1; L1) retrotransposons are the most common retroelements in mammalian genomes. Unlike individual families of endogenous retroviruses (ERVs), they have remained active throughout the mammalian radiation and are responsible for most of the retroelement movement and much genome rearrangement within mammals. They can be viewed as occupying a substantial niche within mammalian genomes. Our previous demonstration that L1s and B1 short interspersed nuclear elements (SINEs) are inactive in a group of South American rodents led us to ask if other elements have amplified to fill the empty niche. We identified a novel and highly active family of ERVs (mysTR). To determine whether loss of L1 activity was correlated with expansion of mysTR, we examined mysTR activity in four South American rodent species that have lost L1 and B1 activity and four sister species with active L1s. The copy number of recent mysTR insertions was extremely high, with an average of 4,200 copies per genome. High copy numbers exist in both L1-active and L1-extinct species, so the mysTR expansion appears to have preceded the loss of both SINE and L1 activity rather than to have filled an empty niche created by their loss. It may be coincidental that two unusual genomic events--loss of L1 activity and massive expansion of an ERV family--occur in the same group of mammals. Alternatively, it is possible that this large ERV expansion set the stage for L1 extinction.

Haploinsufficiency, wherein a single functional copy of a gene is insufficient to maintain normal function, is a major cause of dominant disease. Human disease studies have identified several hundred haploinsufficient (HI) genes. We have compiled a map of 1,079 haplosufficient (HS) genes by systematic identification of genes unambiguously and repeatedly compromised by copy number variation among 8,458 apparently healthy individuals and contrasted the genomic, evolutionary, functional, and network properties between these HS genes and known HI genes. We found that HI genes are typically longer and have more conserved coding sequences and promoters than HS genes. HI genes exhibit higher levels of expression during early development and greater tissue specificity. Moreover, within a probabilistic human functional interaction network HI genes have more interaction partners and greater network proximity to other known HI genes. We built a predictive model on the basis of these differences and annotated 12,443 genes with their predicted probability of being haploinsufficient. We validated these predictions of haploinsufficiency by demonstrating that genes with a high predicted probability of exhibiting haploinsufficiency are enriched among genes implicated in human dominant diseases and among genes causing abnormal phenotypes in heterozygous knockout mice. We have transformed these gene-based haploinsufficiency predictions into haploinsufficiency scores for genic deletions, which we demonstrate to better discriminate between pathogenic and benign deletions than consideration of the deletion size or numbers of genes deleted. These robust predictions of haploinsufficiency support clinical interpretation of novel loss-of-function variants and prioritization of variants and genes for follow-up studies.

4.5S(I) RNA is a small nuclear RNA synthesized by RNA polymerase III and detected in rodents of only four families. Hundreds of copies of this RNA retropseudogenes are interspersed throughout the mouse (Mus musculus) and rat (Rattus norvegicus) genomes. We found a single locus containing 4.5S(I) RNA genes in the genomes of these rodents. The locus harbors three genes and occupies 80 kb on M. musculus chromosome 6 and 44 kb on R. norvegicus chromosome 4. Two long duplications seem to have taken place during evolution of this locus. Two mouse 4.5S(I) RNA genes were used for a study of the role of 5'-flanking sequences in transcription in vitro and ex vivo. We found that removal of these DNA sequences resulted in a dramatic reduction of transcription though an internal promoter for RNA polymerase III was preserved in 4.5S(I) RNA genes. Thus, 5'-flanking sequences (from -1 to -90) containing conserved regions are important for 4.5S(I) RNA gene expression.

The human neuronal apoptosis inhibitory protein (NAIP) gene is no longer principally considered a member of the Inhibitor of Apoptosis Protein (IAP) family, as its domain structure and functions in innate immunity also warrant inclusion in the Nod-Like Receptor (NLR) superfamily. NAIP is located in a region of copy number variation, with one full length and four partly deleted copies in the reference human genome. We demonstrate that several of the NAIP paralogues are expressed, and that novel transcripts arise from both internal and upstream transcription start sites. Remarkably, two internal start sites initiate within Alu short interspersed element (SINE) retrotransposons, and a third novel transcription start site exists within the final intron of the GUSBP1 gene, upstream of only two NAIP copies. One Alu functions alone as a promoter in transient assays, while the other likely combines with upstream L1 sequences to form a composite promoter. The novel transcripts encode shortened open reading frames and we show that corresponding proteins are translated in a number of cell lines and primary tissues, in some cases above the level of full length NAIP. Interestingly, some NAIP isoforms lack their caspase-sequestering motifs, suggesting that they have novel functions. Moreover, given that human and mouse NAIP have previously been shown to employ endogenous retroviral long terminal repeats as promoters, exaptation of Alu repeats as additional promoters provides a fascinating illustration of regulatory innovations adopted by a single gene.

A failure of autoreactive T cells to undergo apoptosis may contribute to the pathogenesis of multiple sclerosis (MS). The role of the inhibitor of apoptosis (IAP) family of anti-apoptotic proteins such as X-linked IAP (XIAP), human inhibitor of apoptosis-1 (HIAP-1), human inhibitor of apoptosis-2 (HIAP-2), neuronal apoptosis inhibitory protein (NAIP) and Survivin in relapsing-remitting, secondary-progressive, primary-progressive or benign forms of MS is unclear. We report here that expression of the IAP family of genes in peripheral blood samples and brain tissues from MS cases support a role for differential regulation of these potent anti-apoptotic proteins in the pathology of MS. XIAP mRNA and protein levels were elevated in peripheral blood mononuclear cells from patients with active disease relative to normal subjects. In patients with active MS, HIAP-1 and HIAP-2 mRNA levels were elevated in resting T cells while NAIP mRNA was increased in whole blood. In post-mortem MS brain tissue, XIAP and HIAP-1 in myelin lesions were co-localized with microglia and T cells, respectively. Only in primary-progressive patients was Survivin expression elevated suggestive of a distinct pathological basis for this subtype of MS. Taken together, these results suggest that patterns of inhibitor of apoptosis expression in immune cells may have value in distinguishing between MS subtypes and offer insight into the mechanisms responsible for their distinct clinical courses.

The neuronal apoptosis inhibitory protein (NAIP) gene, also known as the baculovirus inhibitor of apoptosis repeat-containing protein 1 (BIRC1) gene, is a member of the inhibitors of apoptosis (IAP) family and was first characterized as a candidate gene for spinal muscular atrophy (SMA). The expression of NAIP has been thoroughly studied in the central nervous system and overlaps the pattern of neurodegeneration in SMA. Recent studies have pointed to a role for NAIP in non-neuronal cells. We report here the production of a specific anti-NAIP antibody and the profile of NAIP expression in human adult tissues by Western blot and immunohistochemical detection methods. NAIP was detected in a number of tissues by Western blot analysis, but immunohistochemistry revealed that NAIP's presence in certain tissues, such as liver, lung, and spleen, is most likely due to macrophage infiltration. In the small intestine, the expression of NAIP coincides with the expression of p21(WAF1). This observation, coupled with findings from other groups, suggests a role for NAIP in increasing the survival of cells undergoing terminal differentiation as well as the possibility that the protein serves as an intestinal pathogen recognition protein. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Proteins of the Caspr family are involved in cell contacts and communication in the nervous system. We identified and, by in silico reconstruction, compiled three orthologues of the human CASPR5 gene from the mouse genome, four from the rat genome, and one each from the chimpanzee, dog, opossum, and chicken genomes. Obviously, Caspr5 gene duplications have taken place during evolution of the rodent lineage. In the rat, the four paralogues are located in one chromosome arm, Chr 13p. In the mouse, however, the three Caspr5 genes are located in two chromosomes, Chr 1 and Chr 17. RT-PCR shows that all three mouse paralogues are being expressed. Common expression is found in brain tissue but different expression patterns are seen in other organs during fetal development and in the adult stage. Tissue specificity of expression has diverged during evolution of this young rodent gene family.

Previous studies have suggested that amplification of genes, notably the TOP2A gene, on chromosome arm 17q may be important for the development of malignant peripheral nerve sheath tumour (MPNST). In order to study the frequency, distribution, and chromosomal organization of rearrangements at 17q, interphase and metaphase fluorescence in situ hybridization (FISH) were used to evaluate copy number changes at 17q in 28 MPNSTs. Increased copy numbers were seen for the ERBB2 and TOP2A genes in eight and nine cases, respectively, supporting a potential role for these two genes in MPNST tumourigenesis. Net gain of distal 17q material was observed in 16 of the 28 MPNSTs, with high-level gain in three cases, and was associated with poor outcome. Among the 26 patients for whom follow-up data were available, gain of distal 17q was present in 11 of 12 tumours that had metastasized, compared with 4 of 14 of those that had not metastasized. Detailed FISH mapping analysis of metaphase spreads identified a 2 Mb commonly gained/amplified region at 17q25. Among the genes mapping to this region, BIRC5, which encodes the baculoviral IAP repeat-containing protein 5/survivin protein, is a strong candidate target gene for amplification, as it has been previously shown to be overexpressed in neurofibromatosis type 1-associated MPNST. Three other genes that co-amplified with BIRC5 represent other potential candidate genes: PTDSR involved in apoptosis; SEPT9 overexpressed in human malignant brain tumours; and SOCS3 involved in cell survival and differentiation of neurons. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The TSPY gene is conserved in placental mammals and encodes the testis-specific protein, Y encoded. Within the testis, TSPY expression is restricted to germ cells, and it is assumed that TSPY plays a role in the proliferation of germ cells. Since it was first discovered in humans, TSPY orthologous gene families have been subsequently characterized in many mammalian lineages. In contrast to the situation in cattle and primates, in which TSPY is organized in a moderately repetitive cluster, including functional members and pseudogenes, a peculiar situation is observed in rodents, in which Tspy has been become low or single copy and degenerated to a pseudogene in some species of the subgenus Mus. We have extended this approach and investigated Tspy gene evolution in the Syrian hamster (Mesocricetus auratus) and the Mongolian gerbil (Meriones unguiculatus). Whereas the Syrian hamster Tspy is functionally conserved, organized in multiple copies, and expressed only in testis, the closely related Mongolian gerbil possesses a single-copy pseudogene that is unable to generate a functional transcript. Thus, the Tspy locus has degenerated at least twice at different points of rodent evolution, strongly supporting the hypothesis that the decay of Y-chromosomal genes is an intrinsic evolutionary process. TSPY is the first example of a Y-chromosomal tandem repetitive gene whose decay could be studied in two independent mammalian lineages.

Survivin, a member of the inhibitor of apoptosis (IAP) gene family, overexpresses in various human tumors. Recently this protein has attracted strong interest as a potential prognostic marker because it promotes malignancy through anti-apoptotic activity and is associated with a more aggressive phenotype. To explore the utility of survivin as a veterinary marker of tumor malignancy, we performed molecular cloning of dog survivin cDNA and studied survivin mRNA expression in a variety of naturally occurring dog tumors. The dog cDNA contains a 426-bp open reading frame encoding 142 amino acids of polypeptide, in which a structure termed the baculovirus IAP repeat (BIR) domain, commonly observed in IAPs, is found, as it is in other mammalian survivin protein. The transcript was detected in many adult normal organs including heart, lung, liver, stomach, duodenum, colon, spleen, kidney and testis. As a result of quantitative expression analysis by real-time PCR undertaken for benign and malignant tumors, overexpression of the survivin gene was found in 3 of 18 malignant tumors and in none of the benign tumors, suggesting that survivin overexpression is associated with tumor malignancy in dog.