Dedicated transcriptomics combined with power analysis lead to functional understanding of genes with weak phenotypic changes in knockout lines.

Systematic knockout studies in mice have shown that a large fraction of the gene replacements show no lethal or other overt phenotypes. This has led to the development of more refined analysis schemes, including physiological, behavioral, developmental and cytological tests. However, transcriptomic analyses have not yet been systematically evaluated for non-lethal knockouts. We conducted a power analysis to determine the experimental conditions under which even small changes in transcript levels can be reliably traced. We have applied this to two gene disruption lines of genes for which no function was known so far. Dedicated phenotyping tests informed by the tissues and stages of highest expression of the two genes show small effects on the tested phenotypes. For the transcriptome analysis of these stages and tissues, we used a prior power analysis to determine the number of biological replicates and the sequencing depth. We find that under these conditions, the knockouts have a significant impact on the transcriptional networks, with thousands of genes showing small transcriptional changes. GO analysis suggests that A930004D18Rik is involved in developmental processes through contributing to protein complexes, and A830005F24Rik in extracellular matrix functions. Subsampling analysis of the data reveals that the increase in the number of biological replicates was more important that increasing the sequencing depth to arrive at these results. Hence, our proof-of-principle experiment suggests that transcriptomic analysis is indeed an option to study gene functions of genes with weak or no traceable phenotypic effects and it provides the boundary conditions under which this is possible.

Dealing with the adaptive immune system during de novo evolution of genes from intergenic sequences.

BACKGROUND: The adaptive immune system of vertebrates has an extraordinary potential to sense and neutralize foreign antigens entering the body. De novo evolution of genes implies that the genome itself expresses novel antigens from intergenic sequences which could cause a problem with this immune system. Peptides from these novel proteins could be presented by the major histocompatibility complex (MHC) receptors to the cell surface and would be recognized as foreign. The respective cells would then be attacked and destroyed, or would cause inflammatory responses. Hence, de novo expressed peptides have to be introduced to the immune system as being self-peptides to avoid such autoimmune reactions. The regulation of the distinction between self and non-self starts during embryonic development, but continues late into adulthood. It is mostly mediated by specialized cells in the thymus, but can also be conveyed in peripheral tissues, such as the lymph nodes and the spleen. The self-antigens need to be exposed to the reactive T-cells, which requires the expression of the genes in the respective tissues. Since the initial activation of a promotor for new intergenic transcription of a de novo gene could occur in any tissue, we should expect that the evolutionary establishment of a de novo gene in animals with an adaptive immune system should also involve expression in at least one of the tissues that confer self-recognition. RESULTS: We have studied this question by analyzing the transcriptomes of multiple tissues from young mice in three closely related natural populations of the house mouse (M. m. domesticus). We find that new intergenic transcription occurs indeed mostly in only a single tissue. When a second tissue becomes involved, thymus and spleen are significantly overrepresented. CONCLUSIONS: We conclude that the inclusion of de novo transcripts in the processes for the induction of self-tolerance is indeed an important step in the evolution of functional de novo genes in vertebrates.

Segmental duplications and evolutionary acquisition of UV damage response in the SPATA31 gene family of primates and humans.

BACKGROUND: Segmental duplications are an abundant source for novel gene functions and evolutionary adaptations. This mechanism of generating novelty was very active during the evolution of primates particularly in the human lineage. Here, we characterize the evolution and function of the SPATA31 gene family (former designation FAM75A), which was previously shown to be among the gene families with the strongest signal of positive selection in hominoids. The mouse homologue for this gene family is a single copy gene expressed during spermatogenesis. RESULTS: We show that in primates, the SPATA31 gene duplicated into SPATA31A and SPATA31C types and broadened the expression into many tissues. Each type became further segmentally duplicated in the line towards humans with the largest number of full-length copies found for SPATA31A in humans. Copy number estimates of SPATA31A based on digital PCR show an average of 7.5 with a range of 5-11 copies per diploid genome among human individuals. The primate SPATA31 genes also acquired new protein domains that suggest an involvement in UV response and DNA repair. We generated antibodies and show that the protein is re-localized from the nucleolus to the whole nucleus upon UV-irradiation suggesting a UV damage response. We used CRISPR/Cas mediated mutagenesis to knockout copies of the gene in human primary fibroblast cells. We find that cell lines with reduced functional copies as well as naturally occurring low copy number HFF cells show enhanced sensitivity towards UV-irradiation. CONCLUSION: The acquisition of new SPATA31 protein functions and its broadening of expression may be related to the evolution of the diurnal life style in primates that required a higher UV tolerance. The increased segmental duplications in hominoids as well as its fast evolution suggest the acquisition of further specific functions particularly in humans.

Primate segmental duplication creates novel promoters for the LRRC37 gene family within the 17q21.31 inversion polymorphism region.

The LRRC37 gene family maps to a complex region of the human genome and has been subjected to multiple rounds of segmental duplication. We investigate the expression and regulation of this gene family in multiple tissues and organisms and show a testis-specific expression of this gene family in mouse but a more ubiquitous pattern of expression among primates. Evolutionary and phylogenetic analyses support a model in which new alternative promoters have been acquired during primate evolution. We identify two promoters, Cl8 and particularly Cl3, both of which are highly active in the cerebellum and fetal brain in human and have been duplicated from a promoter region of two unrelated genes, BPTF and DND1, respectively. Two of these more broadly expressed gene family members, LRRC37A1 and A4, define the boundary of a common human inversion polymorphism mapping to chromosome 17q21.31 (the MAPT locus)-a region associated with risk for frontal temporal dementia, Parkinsonism, and intellectual disability. We propose that the regulation of the LRRC37 family occurred in a stepwise manner, acquiring foreign promoters from BPTF and DND1 via segmental duplication. This unusual evolutionary trajectory altered the regulation of the LRRC37 family, leading to increased expression in the fetal brain and cerebellum.

Whole genome sequencing of Turkish genomes reveals functional private alleles and impact of genetic interactions with Europe, Asia and Africa.

BACKGROUND: Turkey is a crossroads of major population movements throughout history and has been a hotspot of cultural interactions. Several studies have investigated the complex population history of Turkey through a limited set of genetic markers. However, to date, there have been no studies to assess the genetic variation at the whole genome level using whole genome sequencing. Here, we present whole genome sequences of 16 Turkish individuals resequenced at high coverage (32×-48×). RESULTS: We show that the genetic variation of the contemporary Turkish population clusters with South European populations, as expected, but also shows signatures of relatively recent contribution from ancestral East Asian populations. In addition, we document a significant enrichment of non-synonymous private alleles, consistent with recent observations in European populations. A number of variants associated with skin color and total cholesterol levels show frequency differentiation between the Turkish populations and European populations. Furthermore, we have analyzed the 17q21.31 inversion polymorphism region (MAPT locus) and found increased allele frequency of 31.25% for H1/H2 inversion polymorphism when compared to European populations that show about 25% of allele frequency. CONCLUSION: This study provides the first map of common genetic variation from 16 western Asian individuals and thus helps fill an important geographical gap in analyzing natural human variation and human migration. Our data will help develop population-specific experimental designs for studies investigating disease associations and demographic history in Turkey.

Death and resurrection of the human IRGM gene.

Immunity-related GTPases (IRG) play an important role in defense against intracellular pathogens. One member of this gene family in humans, IRGM, has been recently implicated as a risk factor for Crohn's disease. We analyzed the detailed structure of this gene family among primates and showed that most of the IRG gene cluster was deleted early in primate evolution, after the divergence of the anthropoids from prosimians ( about 50 million years ago). Comparative sequence analysis of New World and Old World monkey species shows that the single-copy IRGM gene became pseudogenized as a result of an Alu retrotransposition event in the anthropoid common ancestor that disrupted the open reading frame (ORF). We find that the ORF was reestablished as a part of a polymorphic stop codon in the common ancestor of humans and great apes. Expression analysis suggests that this change occurred in conjunction with the insertion of an endogenous retrovirus, which altered the transcription initiation, splicing, and expression profile of IRGM. These data argue that the gene became pseudogenized and was then resurrected through a series of complex structural events and suggest remarkable functional plasticity where alleles experience diverse evolutionary pressures over time. Such dynamism in structure and evolution may be critical for a gene family locked in an arms race with an ever-changing repertoire of intracellular parasites.

Human core duplicon gene families: game changers or game players?

Illuminating the role of specific gene duplications within the human lineage can provide insights into human-specific adaptations. The so-called human core duplicon gene families have received particular attention in this respect, due to special features, such as expansion along single chromosomes, newly acquired protein domains and signatures of positive selection. Here, we summarize the data available for 10 such families and include some new analyses. A picture emerges that suggests broad functions for these protein families, possibly through modification of core cellular pathways. Still, more dedicated studies are required to elucidate the function of core-duplicons gene families and how they have shaped adaptations and evolution of humans.

A de novo evolved gene in the house mouse regulates female pregnancy cycles.

The de novo emergence of new genes has been well documented through genomic analyses. However, a functional analysis, especially of very young protein-coding genes, is still largely lacking. Here, we identify a set of house mouse-specific protein-coding genes and assess their translation by ribosome profiling and mass spectrometry data. We functionally analyze one of them, Gm13030, which is specifically expressed in females in the oviduct. The interruption of the reading frame affects the transcriptional network in the oviducts at a specific stage of the estrous cycle. This includes the upregulation of Dcpp genes, which are known to stimulate the growth of preimplantation embryos. As a consequence, knockout females have their second litters after shorter times and have a higher infanticide rate. Given that Gm13030 shows no signs of positive selection, our findings support the hypothesis that a de novo evolved gene can directly adopt a function without much sequence adaptation.

Involvement of SPATA31 copy number variable genes in human lifespan.

The SPATA31 (alias FAM75A) gene family belongs to the core duplicon families that are thought to have contributed significantly to hominoid evolution. It is also among the gene families with the strongest signal of positive selection in hominoids. It has acquired new protein domains in the primate lineage and a previous study has suggested that the gene family has expanded its function into UV response and DNA repair. Here we show that over-expression of SPATA31A1 in fibroblast cells leads to premature senescence due to interference with aging-related transcription pathways. We show that there are considerable copy number differences for this gene family in human populations and we ask whether this could influence mutation rates and longevity in humans. We find no evidence for an influence on germline mutation rates, but an analysis of long-lived individuals (> 96 years) shows that they carry significantly fewer SPATA31 copies in their genomes than younger individuals in a control group. We propose that the evolution of SPATA31 copy number is an example for antagonistic pleiotropy by providing a fitness benefit during the reproductive phase of life, but negatively influencing the overall life span.

Critical role for the tapasin-docking site of TAP2 in the functional integrity of the MHC class I-peptide-loading complex.

The transporter associated with Ag processing (TAP) translocates antigenic peptides into the endoplasmic reticulum for binding onto MHC class I (MHC I) molecules. Tapasin organizes a peptide-loading complex (PLC) by recruiting MHC I and accessory chaperones to the N-terminal regions (N domains) of the TAP subunits TAP1 and TAP2. To investigate the function of the tapasin-docking sites of TAP in MHC I processing, we expressed N-terminally truncated variants of TAP1 and TAP2 in combination with wild-type chains, as fusion proteins or as single subunits. Strikingly, TAP variants lacking the N domain in TAP2, but not in TAP1, build PLCs that fail to generate stable MHC I-peptide complexes. This correlates with a substantially reduced recruitment of accessory chaperones into the PLC demonstrating their important role in the quality control of MHC I loading. However, stable surface expression of MHC I can be rescued in post-endoplasmic reticulum compartments by a proprotein convertase-dependent mechanism.

The interferon-inducible p47 (IRG) GTPases in vertebrates: loss of the cell autonomous resistance mechanism in the human lineage.

BACKGROUND: Members of the p47 (immunity-related GTPases (IRG) family) GTPases are essential, interferon-inducible resistance factors in mice that are active against a broad spectrum of important intracellular pathogens. Surprisingly, there are no reports of p47 function in humans. RESULTS: Here we show that the p47 GTPases are represented by 23 genes in the mouse, whereas humans have only a single full-length p47 GTPase and an expressed, truncated presumed pseudo-gene. The human full-length gene is orthologous to an isolated mouse p47 GTPase that carries no interferon-inducible elements in the promoter of either species and is expressed constitutively in the mature testis of both species. Thus, there is no evidence for a p47 GTPase-based resistance system in humans. Dogs have several interferon-inducible p47s, and so the primate lineage that led to humans appears to have lost an ancient function. Multiple p47 GTPases are also present in the zebrafish, but there is only a tandem p47 gene pair in pufferfish. CONCLUSION: Mice and humans must deploy their immune resources against vacuolar pathogens in radically different ways. This carries significant implications for the use of the mouse as a model of human infectious disease. The absence of the p47 resistance system in humans suggests that possession of this resistance system carries significant costs that, in the primate lineage that led to humans, are not outweighed by the benefits. The origin of the vertebrate p47 system is obscure.