Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes.

We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism.

Evolution of human-specific neural SRGAP2 genes by incomplete segmental duplication.

Gene duplication is an important source of phenotypic change and adaptive evolution. We leverage a haploid hydatidiform mole to identify highly identical sequences missing from the reference genome, confirming that the cortical development gene Slit-Robo Rho GTPase-activating protein 2 (SRGAP2) duplicated three times exclusively in humans. We show that the promoter and first nine exons of SRGAP2 duplicated from 1q32.1 (SRGAP2A) to 1q21.1 (SRGAP2B) ∼3.4 million years ago (mya). Two larger duplications later copied SRGAP2B to chromosome 1p12 (SRGAP2C) and to proximal 1q21.1 (SRGAP2D) ∼2.4 and ∼1 mya, respectively. Sequence and expression analyses show that SRGAP2C is the most likely duplicate to encode a functional protein and is among the most fixed human-specific duplicate genes. Our data suggest a mechanism where incomplete duplication created a novel gene function-antagonizing parental SRGAP2 function-immediately "at birth" 2-3 mya, which is a time corresponding to the transition from Australopithecus to Homo and the beginning of neocortex expansion.

The Atlantic salmon genome provides insights into rediploidization.

The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.

Deficiency in ZMPSTE24 and resulting farnesyl-prelamin A accumulation only modestly affect mouse adipose tissue stores.

Zinc metallopeptidase STE24 (ZMPSTE24) is essential for the conversion of farnesyl-prelamin A to mature lamin A, a key component of the nuclear lamina. In the absence of ZMPSTE24, farnesyl-prelamin A accumulates in the nucleus and exerts toxicity, causing a variety of disease phenotypes. By ∼4 months of age, both male and female Zmpste24-/- mice manifest a near-complete loss of adipose tissue, but it has never been clear whether this phenotype is a direct consequence of farnesyl-prelamin A toxicity in adipocytes. To address this question, we generated a conditional knockout Zmpste24 allele and used it to create adipocyte-specific Zmpste24-knockout mice. To boost farnesyl-prelamin A levels, we bred in the "prelamin A-only" Lmna allele. Gene expression, immunoblotting, and immunohistochemistry experiments revealed that adipose tissue in these mice had decreased Zmpste24 expression along with strikingly increased accumulation of prelamin A. In male mice, Zmpste24 deficiency in adipocytes was accompanied by modest changes in adipose stores (an 11% decrease in body weight, a 23% decrease in body fat mass, and significantly smaller gonadal and inguinal white adipose depots). No changes in adipose stores were detected in female mice, likely because prelamin A expression in adipose tissue is lower in female mice. Zmpste24 deficiency in adipocytes did not alter the number of macrophages in adipose tissue, nor did it alter plasma levels of glucose, triglycerides, or fatty acids. We conclude that ZMPSTE24 deficiency in adipocytes, and the accompanying accumulation of farnesyl-prelamin A, reduces adipose tissue stores, but only modestly and only in male mice.

Gibbon genome and the fast karyotype evolution of small apes.

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.

Insights into bilaterian evolution from three spiralian genomes.

Current genomic perspectives on animal diversity neglect two prominent phyla, the molluscs and annelids, that together account for nearly one-third of known marine species and are important both ecologically and as experimental systems in classical embryology. Here we describe the draft genomes of the owl limpet (Lottia gigantea), a marine polychaete (Capitella teleta) and a freshwater leech (Helobdella robusta), and compare them with other animal genomes to investigate the origin and diversification of bilaterians from a genomic perspective. We find that the genome organization, gene structure and functional content of these species are more similar to those of some invertebrate deuterostome genomes (for example, amphioxus and sea urchin) than those of other protostomes that have been sequenced to date (flies, nematodes and flatworms). The conservation of these genomic features enables us to expand the inventory of genes present in the last common bilaterian ancestor, establish the tripartite diversification of bilaterians using multiple genomic characteristics and identify ancient conserved long- and short-range genetic linkages across metazoans. Superimposed on this broadly conserved pan-bilaterian background we find examples of lineage-specific genome evolution, including varying rates of rearrangement, intron gain and loss, expansions and contractions of gene families, and the evolution of clade-specific genes that produce the unique content of each genome.

The Norway spruce genome sequence and conifer genome evolution.

Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels.

A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines.

Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼ 80% of mutants showed specific staining in one or more tissues, while ∼ 20% showed no specific staining, ∼ 13% had staining in only one tissue, and ∼ 25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼ 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known.

Mutating a conserved cysteine in GPIHBP1 reduces amounts of GPIHBP1 in capillaries and abolishes LPL binding.

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease-chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein.

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9-FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.

A conditional knockout resource for the genome-wide study of mouse gene function.

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.

The genome of the green anole lizard and a comparative analysis with birds and mammals.

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse-more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.

Farnesylation of lamin B1 is important for retention of nuclear chromatin during neuronal migration.

The role of protein farnesylation in lamin A biogenesis and the pathogenesis of progeria has been studied in considerable detail, but the importance of farnesylation for the B-type lamins, lamin B1 and lamin B2, has received little attention. Lamins B1 and B2 are expressed in nearly every cell type from the earliest stages of development, and they have been implicated in a variety of functions within the cell nucleus. To assess the importance of protein farnesylation for B-type lamins, we created knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. Mice expressing nonfarnesylated lamin B2 developed normally and were free of disease. In contrast, mice expressing nonfarnesylated lamin B1 died soon after birth, with severe neurodevelopmental defects and striking nuclear abnormalities in neurons. The nuclear lamina in migrating neurons was pulled away from the chromatin so that the chromatin was left "naked" (free from the nuclear lamina). Thus, farnesylation of lamin B1--but not lamin B2--is crucial for brain development and for retaining chromatin within the bounds of the nuclear lamina during neuronal migration.

A comprehensive molecular cytogenetic analysis of chromosome rearrangements in gibbons.

Chromosome rearrangements in small apes are up to 20 times more frequent than in most mammals. Because of their complexity, the full extent of chromosome evolution in these hominoids is not yet fully documented. However, previous work with array painting, BAC-FISH, and selective sequencing in two of the four karyomorphs has shown that high-resolution methods can precisely define chromosome breakpoints and map the complex flow of evolutionary chromosome rearrangements. Here we use these tools to precisely define the rearrangements that have occurred in the remaining two karyomorphs, genera Symphalangus (2n = 50) and Hoolock (2n = 38). This research provides the most comprehensive insight into the evolutionary origins of chromosome rearrangements involved in transforming small apes genome. Bioinformatics analyses of the human-gibbon synteny breakpoints revealed association with transposable elements and segmental duplications, providing some insight into the mechanisms that might have promoted rearrangements in small apes. In the near future, the comparison of gibbon genome sequences will provide novel insights to test hypotheses concerning the mechanisms of chromosome evolution. The precise definition of synteny block boundaries and orientation, chromosomal fusions, and centromere repositioning events presented here will facilitate genome sequence assembly for these close relatives of humans.

Mouse lipin-1 and lipin-2 cooperate to maintain glycerolipid homeostasis in liver and aging cerebellum.

The three lipin phosphatidate phosphatase (PAP) enzymes catalyze a step in glycerolipid biosynthesis, the conversion of phosphatidate to diacylglycerol. Lipin-1 is critical for lipid synthesis and homeostasis in adipose tissue, liver, muscle, and peripheral nerves. Little is known about the physiological role of lipin-2, the predominant lipin protein present in liver and the deficient gene product in the rare disorder Majeed syndrome. By using lipin-2-deficient mice, we uncovered a functional relationship between lipin-1 and lipin-2 that operates in a tissue-specific and age-dependent manner. In liver, lipin-2 deficiency led to a compensatory increase in hepatic lipin-1 protein and elevated PAP activity, which maintained lipid homeostasis under basal conditions, but led to diet-induced hepatic triglyceride accumulation. As lipin-2-deficient mice aged, they developed ataxia and impaired balance. This was associated with the combination of lipin-2 deficiency and an age-dependent reduction in cerebellar lipin-1 levels, resulting in altered cerebellar phospholipid composition. Similar to patients with Majeed syndrome, lipin-2-deficient mice developed anemia, but did not show evidence of osteomyelitis, suggesting that additional environmental or genetic components contribute to the bone abnormalities observed in patients. Combined lipin-1 and lipin-2 deficiency caused embryonic lethality. Our results reveal functional interactions between members of the lipin family in vivo, and a unique role for lipin-2 in central nervous system biology that may be particularly important with advancing age. Additionally, as has been observed in mice and humans with lipin-1 deficiency, the pathophysiology in lipin-2 deficiency is associated with dysregulation of lipid intermediates.

Efficient de novo assembly of large and complex genomes by massively parallel sequencing of Fosmid pools.

BACKGROUND: Sampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality. RESULTS: In order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with ~40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS. CONCLUSIONS: By exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process.We have made public the input data (FASTQ format) for the set of pools used in this study:ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/.

Clcn4-2 genomic structure differs between the X locus in Mus spretus and the autosomal locus in Mus musculus: AT motif enrichment on the X.

In Mus spretus, the chloride channel 4 gene Clcn4-2 is X-linked and dosage compensated by X up-regulation and X inactivation, while in the closely related mouse species Mus musculus, Clcn4-2 has been translocated to chromosome 7. We sequenced Clcn4-2 in M. spretus and identified the breakpoints of the evolutionary translocation in the Mus lineage. Genetic and epigenetic differences were observed between the 5'ends of the autosomal and X-linked loci. Remarkably, Clcn4-2 introns have been truncated on chromosome 7 in M. musculus as compared with the X-linked loci from seven other eutherian mammals. Intron sequences specifically preserved in the X-linked loci were significantly enriched in AT-rich oligomers. Genome-wide analyses showed an overall enrichment in AT motifs unique to the eutherian X (except for genes that escape X inactivation), suggesting a role for these motifs in regulation of the X chromosome.

The amphioxus genome and the evolution of the chordate karyotype.

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

Oral versus intravenous antibiotic treatment of moderate-to-severe community-acquired pneumonia: a propensity score matched study.

Community-acquired Pneumonia (CAP) guidelines generally recommend to admit patients with moderate-to-severe CAP and start treatment with intravenous antibiotics. This study aims to explore the clinical outcomes of oral antibiotics in patients with moderate-to-severe CAP. We performed a nested cohort study of an observational study including all adult patients presenting to the emergency department of the Haga Teaching Hospital, the Netherlands, between April 2019 and May 2020, who had a blood culture drawn. We conducted propensity score matching with logistic and linear regression analysis to compare patients with moderate-to-severe CAP (Pneumonia Severity Index class III-V) treated with oral antibiotics to patients treated with intravenous antibiotics. Outcomes were 30-day mortality, intensive care unit admission, readmission, length of stay (LOS) and length of antibiotic treatment. Of the original 314 patients, 71 orally treated patients were matched with 102 intravenously treated patients. The mean age was 73 years and 58% were male. We found no significant differences in outcomes between the oral and intravenous group, except for an increased LOS of + 2.6 days (95% confidence interval 1.2-4.0, p value < 0.001) in those treated intravenously. We conclude that oral antibiotics might be a safe and effective treatment for moderate-to-severe CAP for selected patients based on the clinical judgement of the attending physician.