The Genomic History of the Bronze Age Southern Levant.

We report genome-wide DNA data for 73 individuals from five archaeological sites across the Bronze and Iron Ages Southern Levant. These individuals, who share the "Canaanite" material culture, can be modeled as descending from two sources: (1) earlier local Neolithic populations and (2) populations related to the Chalcolithic Zagros or the Bronze Age Caucasus. The non-local contribution increased over time, as evinced by three outliers who can be modeled as descendants of recent migrants. We show evidence that different "Canaanite" groups genetically resemble each other more than other populations. We find that Levant-related modern populations typically have substantial ancestry coming from populations related to the Chalcolithic Zagros and the Bronze Age Southern Levant. These groups also harbor ancestry from sources we cannot fully model with the available data, highlighting the critical role of post-Bronze-Age migrations into the region over the past 3,000 years.

Reconstructing Denisovan Anatomy Using DNA Methylation Maps.

Denisovans are an extinct group of humans whose morphology remains unknown. Here, we present a method for reconstructing skeletal morphology using DNA methylation patterns. Our method is based on linking unidirectional methylation changes to loss-of-function phenotypes. We tested performance by reconstructing Neanderthal and chimpanzee skeletal morphologies and obtained >85% precision in identifying divergent traits. We then applied this method to the Denisovan and offer a putative morphological profile. We suggest that Denisovans likely shared with Neanderthals traits such as an elongated face and a wide pelvis. We also identify Denisovan-derived changes, such as an increased dental arch and lateral cranial expansion. Our predictions match the only morphologically informative Denisovan bone to date, as well as the Xuchang skull, which was suggested by some to be a Denisovan. We conclude that DNA methylation can be used to reconstruct anatomical features, including some that do not survive in the fossil record.

Reconstructing DNA methylation maps of ancient populations.

Studying premortem DNA methylation from ancient DNA (aDNA) provides a proxy for ancient gene activity patterns, and hence valuable information on evolutionary changes in gene regulation. Due to statistical limitations, current methods to reconstruct aDNA methylation maps are constrained to high-coverage shotgun samples, which comprise a small minority of available ancient samples. Most samples are sequenced using in-situ hybridization capture sequencing which targets a predefined set of genomic positions. Here, we develop methods to reconstruct aDNA methylation maps of samples that were not sequenced using high-coverage shotgun sequencing, by way of pooling together individuals to obtain a DNA methylation map that is characteristic of a population. We show that the resulting DNA methylation maps capture meaningful biological information and allow for the detection of differential methylation across populations. We offer guidelines on how to carry out comparative studies involving ancient populations, and how to control the rate of falsely discovered differentially methylated regions. The ability to reconstruct DNA methylation maps of past populations allows for the development of a whole new frontier in paleoepigenetic research, tracing DNA methylation changes throughout human history, using data from thousands of ancient samples.

LINADMIX: evaluating the effect of ancient admixture events on modern populations.

MOTIVATION: The rise in the number of genotyped ancient individuals provides an opportunity to estimate population admixture models for many populations. However, in models describing modern populations as mixtures of ancient ones, it is typically difficult to estimate the model mixing coefficients and to evaluate its fit to the data. RESULTS: We present LINADMIX, designed to tackle this problem by solving a constrained linear model when both the ancient and the modern genotypes are represented in a low-dimensional space. LINADMIX estimates the mixing coefficients and their standard errors, and computes a P-value for testing the model fit to the data. We quantified the performance of LINADMIX using an extensive set of simulated studies. We show that LINADMIX can accurately estimate admixture coefficients, and is robust to factors such as population size, genetic drift, proportion of missing data and various types of model misspecification. AVAILABILITY AND IMPLEMENTATION: LINADMIX is available as a python code at https://github.com/swidler/linadmix. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lead in Archeological Human Bones Reflecting Historical Changes in Lead Production.

Forty years ago, in a seminal paper published in Science, Settle and Patterson used archeological and historical data to estimate the rates of worldwide lead production since the discovery of cupellation, approximately 5000 years ago. Here, we record actual lead exposure of a human population by direct measurements of the concentrations of lead in petrous bones of individuals representing approximately 12 000 years of inhabitation in Italy. This documentation of lead pollution throughout human history indicates that, remarkably, much of the estimated dynamics in lead production is replicated in human exposure. Thus, lead pollution in humans has closely followed anthropogenic lead production. This observation raises concerns that the forecasted increase in the production of lead and other metals might affect human health in the near future.

Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay.

Primate-specific Alu short interspersed elements (SINEs) as well as rodent-specific B and ID (B/ID) SINEs can promote Staufen-mediated decay (SMD) when present in mRNA 3'-untranslated regions (3'-UTRs). The transposable nature of SINEs, their presence in long noncoding RNAs, their interactions with Staufen, and their rapid divergence in different evolutionary lineages suggest they could have generated substantial modification of posttranscriptional gene-control networks during mammalian evolution. Some of the variation in SMD regulation produced by SINE insertion might have had a similar regulatory effect in separate mammalian lineages, leading to parallel evolution of the Staufen network by independent expansion of lineage-specific SINEs. To explore this possibility, we searched for orthologous gene pairs, each carrying a species-specific 3'-UTR SINE and each regulated by SMD, by measuring changes in mRNA abundance after individual depletion of two SMD factors, Staufen1 (STAU1) and UPF1, in both human and mouse myoblasts. We identified and confirmed orthologous gene pairs with 3'-UTR SINEs that independently function in SMD control of myoblast metabolism. Expanding to other species, we demonstrated that SINE-directed SMD likely emerged in both primate and rodent lineages >20-25 million years ago. Our work reveals a mechanism for the convergent evolution of posttranscriptional gene regulatory networks in mammals by species-specific SINE transposition and SMD.

Small RNA sequences derived from pre-microRNAs in the supraspliceosome.

MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate the expression and translation of genes in healthy and diseased tissues. Herein, we characterize short RNAs from human HeLa cells found in the supraspliceosome, a nuclear dynamic machine in which pre-mRNA processing occurs. We sequenced small RNAs (<200 nt) extracted from the supraspliceosome, and identified sequences that are derived from 200 miRNAs genes. About three quarters of them are mature miRNAs, whereas the rest account for various defined regions of the pre-miRNA, and its hairpin-loop precursor. Out of these aligned sequences, 53 were undetected in cellular extract, and the abundance of additional 48 strongly differed from that in cellular extract. Notably, we describe seven abundant miRNA-derived sequences that overlap non-coding exons of their host gene. The rich collection of sequences identical to pre-miRNAs at the supraspliceosome suggests overlooked nuclear functions. Specifically, the abundant hsa-mir-99b may affect splicing of LINC01129 primary transcript through base-pairing with its exon-intron junction. Using suppression and overexpression experiments, we show that hsa-mir-7704 negatively regulates the level of the lncRNA HAGLR. We claim that in cases of extended base-pairing complementarity, such supraspliceosomal pre-miRNA sequences might have a role in transcription attenuation, maturation and processing.

Assessing predictions of the impact of variants on splicing in CAGI5.

Precision medicine and sequence-based clinical diagnostics seek to predict disease risk or to identify causative variants from sequencing data. The Critical Assessment of Genome Interpretation (CAGI) is a community experiment consisting of genotype-phenotype prediction challenges; participants build models, undergo assessment, and share key findings. In the past, few CAGI challenges have addressed the impact of sequence variants on splicing. In CAGI5, two challenges (Vex-seq and MaPSY) involved prediction of the effect of variants, primarily single-nucleotide changes, on splicing. Although there are significant differences between these two challenges, both involved prediction of results from high-throughput exon inclusion assays. Here, we discuss the methods used to predict the impact of these variants on splicing, their performance, strengths, and weaknesses, and prospects for predicting the impact of sequence variation on splicing and disease phenotypes.

Gene ORGANizer: linking genes to the organs they affect.

One of the biggest challenges in studying how genes work is understanding their effect on the physiology and anatomy of the body. Existing tools try to address this using indirect features, such as expression levels and biochemical pathways. Here, we present Gene ORGANizer (geneorganizer.huji.ac.il), a phenotype-based tool that directly links human genes to the body parts they affect. It is built upon an exhaustive curated database that links >7000 genes to ∼150 anatomical parts using >150 000 gene-organ associations. The tool offers user-friendly platforms to analyze the anatomical effects of individual genes, and identify trends within groups of genes. We demonstrate how Gene ORGANizer can be used to make new discoveries, showing that chromosome X is enriched with genes affecting facial features, that positive selection targets genes with more constrained phenotypic effects, and more. We expect Gene ORGANizer to be useful in a variety of evolutionary, medical and molecular studies aimed at understanding the phenotypic effects of genes.

Inferring Past Environments from Ancient Epigenomes.

Analyzing the conditions in which past individuals lived is key to understanding the environments and cultural transitions to which humans had to adapt. Here, we suggest a methodology to probe into past environments, using reconstructed premortem DNA methylation maps of ancient individuals. We review a large body of research showing that differential DNA methylation is associated with changes in various external and internal factors, and propose that loci whose DNA methylation level is environmentally responsive could serve as markers to infer about ancient daily life, diseases, nutrition, exposure to toxins, and more. We demonstrate this approach by showing that hunger-related DNA methylation changes are found in ancient hunter-gatherers. The strategy we present here opens a window to reconstruct previously inaccessible aspects of the lives of past individuals.

Archaic Adaptive Introgression in TBX15/WARS2.

A recent study conducted the first genome-wide scan for selection in Inuit from Greenland using single nucleotide polymorphism chip data. Here, we report that selection in the region with the second most extreme signal of positive selection in Greenlandic Inuit favored a deeply divergent haplotype that is closely related to the sequence in the Denisovan genome, and was likely introgressed from an archaic population. The region contains two genes, WARS2 and TBX15, and has previously been associated with adipose tissue differentiation and body-fat distribution in humans. We show that the adaptively introgressed allele has been under selection in a much larger geographic region than just Greenland. Furthermore, it is associated with changes in expression of WARS2 and TBX15 in multiple tissues including the adrenal gland and subcutaneous adipose tissue, and with regional DNA methylation changes in TBX15.

Alu exaptation enriches the human transcriptome by introducing new gene ends.

In mammals, transposable elements are largely silenced, but under fortuitous circumstances may be co-opted to play a functional role. Here, we show that when Alu elements are inserted within or nearby genes in sense orientation, they may contribute to the transcriptome diversity by forming new cleavage and polyadenylation sites. We mapped these new gene ends in human onto the Alu sequence and identified three hotspots of cleavage and polyadenylation site formation. Interestingly, the native Alu sequence does not contain any canonical polyadenylation signal. We therefore studied what evolutionary processes might explain the formation of these specific hotspots of novel gene ends. We show that two of the three hotspots might have emerged from mutational processes that turned sequences that resemble polyadenylation signals into full-blown canonical signals, whereas one hotspot is tightly linked to the process of Alu insertion into the genome. Overall, Alu elements may lie behind the formation of 302 new gene end variants, affecting a total of 243 genes. Intergenic Alu elements may elongate genes by creating a downstream cleavage site, intronic Alu elements may lead to gene variants which code for truncated proteins, and 3'UTR Alu elements may result in gene variants with alternative 3'UTR.

JuncDB: an exon-exon junction database.

Intron positions upon the mRNA transcript are sometimes remarkably conserved even across distantly related eukaryotic species. This has made the comparison of intron-exon architectures across orthologous transcripts a very useful tool for studying various evolutionary processes. Moreover, the wide range of functions associated with introns may confer biological meaning to evolutionary changes in gene architectures. Yet, there is currently no database that offers such comparative information. Here, we present JuncDB (http://juncdb.carmelab.huji.ac.il/), an exon-exon junction database dedicated to the comparison of architectures between orthologous transcripts. It covers nearly 40,000 sets of orthologous transcripts spanning 88 eukaryotic species. JuncDB offers a user-friendly interface, access to detailed information, instructive graphical displays of the comparative data and easy ways to download data to a local computer. In addition, JuncDB allows the analysis to be carried out either on specific genes, or at a genome-wide level for any selected group of species.

The role of nucleotide composition in premature termination codon recognition.

BACKGROUND: It is not fully understood how a termination codon is recognized as premature (PTC) by the nonsense-mediated decay (NMD) machinery. This is particularly true for transcripts lacking an exon junction complex (EJC) along their 3' untranslated region (3'UTR), and thus degrade through the EJC-independent NMD pathway. RESULTS: Here, we analyzed data of transcript stability change following NMD repression and identified over 200 EJC-independent NMD-targets. We examined many features characterizing these transcripts, and compared them to NMD-insensitive transcripts, as well as to a group of transcripts that are destabilized following NMD repression (destabilized transcripts). CONCLUSIONS: We found that none of the known NMD-triggering features, such as the presence of upstream open reading frames, significantly characterizes EJC-independent NMD-targets. Instead, we saw that NMD-targets are strongly enriched with G nucleotides upstream of the termination codon, and even more so along their 3'UTR. We suggest that high G content around the termination codon impedes translation termination as a result of mRNA folding, thus triggering NMD. We also suggest that high G content in the 3'UTR helps to activate NMD by allowing for the accumulation of UPF1, or other NMD-promoting proteins, along the 3'UTR.

Combined mineralocorticoid and glucocorticoid deficiency is caused by a novel founder nicotinamide nucleotide transhydrogenase mutation that alters mitochondrial morphology and increases oxidative stress.

BACKGROUND: Familial glucocorticoid deficiency (FGD) reflects specific failure of adrenocortical glucocorticoid production in response to adrenocorticotropic hormone (ACTH). Most cases are caused by mutations encoding ACTH-receptor components (MC2R, MRAP) or the general steroidogenesis protein (StAR). Recently, nicotinamide nucleotide transhydrogenase (NNT) mutations were found to cause FGD through a postulated mechanism resulting from decreased detoxification of reactive oxygen species (ROS) in adrenocortical cells. METHODS AND RESULTS: In a consanguineous Palestinian family with combined mineralocorticoid and glucocorticoid deficiency, whole-exome sequencing revealed a novel homozygous NNT_c.598 G>A, p.G200S, mutation. Another affected, unrelated Palestinian child was also homozygous for NNT_p.G200S. Haplotype analysis showed this mutation is ancestral; carrier frequency in ethnically matched controls is 1/200. Assessment of patient fibroblasts for ROS production, ATP content and mitochondrial morphology showed that biallelic NNT mutations result in increased levels of ROS, lower ATP content and morphological mitochondrial defects. CONCLUSIONS: This report of a novel NNT mutation, p.G200S, expands the phenotype of NNT mutations to include mineralocorticoid deficiency. We provide the first patient-based evidence that NNT mutations can cause oxidative stress and both phenotypic and functional mitochondrial defects. These results directly demonstrate the importance of NNT to mitochondrial function in the setting of adrenocortical insufficiency.

Computational identification of functional introns: high positional conservation of introns that harbor RNA genes.

An appreciable fraction of introns is thought to have some function, but there is no obvious way to predict which specific intron is likely to be functional. We hypothesize that functional introns experience a different selection regime than non-functional ones and will therefore show distinct evolutionary histories. In particular, we expect functional introns to be more resistant to loss, and that this would be reflected in high conservation of their position with respect to the coding sequence. To test this hypothesis, we focused on introns whose function comes about from microRNAs and snoRNAs that are embedded within their sequence. We built a data set of orthologous genes across 28 eukaryotic species, reconstructed the evolutionary histories of their introns and compared functional introns with the rest of the introns. We found that, indeed, the position of microRNA- and snoRNA-bearing introns is significantly more conserved. In addition, we found that both families of RNA genes settled within introns early during metazoan evolution. We identified several easily computable intronic properties that can be used to detect functional introns in general, thereby suggesting a new strategy to pinpoint non-coding cellular functions.

XX ovarian dysgenesis is caused by a PSMC3IP/HOP2 mutation that abolishes coactivation of estrogen-driven transcription.

XX female gonadal dysgenesis (XX-GD) is a rare, genetically heterogeneous disorder characterized by lack of spontaneous pubertal development, primary amenorrhea, uterine hypoplasia, and hypergonadotropic hypogonadism as a result of streak gonads. Most cases are unexplained but thought to be autosomal recessive. We elucidated the genetic basis of XX-GD in a highly consanguineous Palestinian family by using homozygosity mapping and candidate-gene and whole-exome sequencing. Affected females were homozygous for a 3 bp deletion (NM_016556.2, c.600_602del) in the PSMC3IP gene, leading to deletion of a glutamic acid residue (p.Glu201del) in the highly conserved C-terminal acidic domain. Proteasome 26S subunit, ATPase, 3-Interacting Protein (PSMC3IP)/Tat Binding Protein Interacting Protein (TBPIP) is a nuclear, tissue-specific protein with multiple functions. It is critical for meiotic recombination as indicated by the known role of its yeast ortholog, Hop2. Through the C terminus (not present in yeast), PSMC3IP also coactivates ligand-driven transcription mediated by estrogen, androgen, glucocorticoid, progesterone, and thyroid nuclear receptors. In cell lines, the p.Glu201del mutation abolished PSMC3IP activation of estrogen-driven transcription. Impaired estrogenic signaling can lead to ovarian dysgenesis both by affecting the size of the follicular pool created during fetal development and by failing to counteract follicular atresia during puberty. PSMC3IP joins previous genes known to be mutated in XX-GD, the FSH receptor, and BMP15, highlighting the importance of hormonal signaling in ovarian development and maintenance and suggesting a common pathway perturbed in isolated XX-GD. By analogy to other XX-GD genes, PSMC3IP is also a candidate gene for premature ovarian failure, and its role in folliculogenesis should be further investigated.

The role of reverse transcriptase in intron gain and loss mechanisms.

Intron density is highly variable across eukaryotic species. It seems that different lineages have experienced considerably different levels of intron gain and loss events, but the reasons for this are not well known. A large number of mechanisms for intron loss and gain have been suggested, and most of them have at least some level of indirect support. We therefore figured out that the variability in intron density can be a reflection of the fact that different mechanisms are active in different lineages. Quite a number of these putative mechanisms, both for intron loss and for intron gain, postulate that the enzyme reverse transcriptase (RT) has a key role in the process. In this paper, we lay out three predictions whose approval or falsification gives indication for the involvement of RT in intron gain and loss processes. Testing these predictions requires data on the intron gain and loss rates of individual genes along different branches of the eukaryotic phylogenetic tree. So far, such rates could not be computed, and hence, these predictions could not be rigorously evaluated. Here, we use a maximum likelihood algorithm that we have devised in the past, Evolutionary Reconstruction by Expectation Maximization, which allows the estimation of such rates. Using this algorithm, we computed the intron loss and gain rates of more than 300 genes in each branch of the phylogenetic tree of 19 eukaryotic species. Based on that we found only little support for RT activity in intron gain. In contrast, we suggest that RT-mediated intron loss is a mechanism that is very efficient in removing introns, and thus, its levels of activity may be a major determinant of intron number. Moreover, we found that intron gain and loss rates are negatively correlated in intron-poor species but are positively correlated for intron-rich species. One explanation to this is that intron gain and loss mechanisms in intron-rich species (like metazoans) share a common mechanistic component, albeit not a RT.