Characterization of RNA content in individual phase-separated coacervate microdroplets

Liquid-liquid phase separation or condensation is a form of macromolecular compartmentalization. Condensates formed by complex coacervation were hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of condensation in cells and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and how single condensates differ in their RNA composition. Therefore, we developed an approach to read the RNA content from single condensates using high-throughput sequencing. We find that RNAs which are enriched for specific sequence motifs efficiently accumulate in condensates. These motifs show high sequence similarity to short interspersed elements (SINEs). We observed similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.Competing Interest StatementThe authors have declared no competing interest

The accessible chromatin landscape of the human genome

DNaseI hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers, and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ~2.9 million DHSs that encompass virtually all known experimentally-validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation, and regulatory factor occupancy patterns. We connect ~580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is choreographed with dozens to hundreds of co-activated elements, and the trans-cellular DNaseI sensitivity pattern at a given region can predict cell type-specific functional behaviors. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation

The evolutionary history of Neanderthal and Denisovan Y chromosomes

Ancient DNA has provided new insights into many aspects of human history. However, we lack comprehensive studies of the Y chromosomes of Denisovans and Neanderthals because the majority of specimens that have been sequenced to sufficient coverage are female. Sequencing Y chromosomes from two Denisovans and three Neanderthals shows that the Y chromosomes of Denisovans split around 700 thousand years ago from a lineage shared by Neanderthals and modern human Y chromosomes, which diverged from each other around 370 thousand years ago. The phylogenetic relationships of archaic and modern human Y chromosomes differ from the population relationships inferred from the autosomal genomes and mirror mitochondrial DNA phylogenies, indicating replacement of both the mitochondrial and Y chromosomal gene pools in late Neanderthals. This replacement is plausible if the low effective population size of Neanderthals resulted in an increased genetic load in Neanderthals relative to modern humans.Q.F. was supported by funding from the Chinese Academy of Sciences (XDB26000000) and the National Natural Science Foundation of China (91731303, 41925009, 41630102). A.R. was funded by Spanish government (MICINN/ FEDER) (grant number CGL2016-75109-P). The reassessment of the Spy collection by H.R., I.C., and P.S. was supported by the Belgian Science Policy Office (BELSPO 2004-2007, MO/36/0112). M.V.S., M.B.K., and A.P.D. were supported by the Russian Foundation for Basic Research (RFBR 17-29-04206). This study was funded by the Max Planck Society and the European Research Council (grant agreement number 694707)

An expansive human regulatory lexicon encoded in transcription factor footprints.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency

The evolutionary history of Neandertal and Denisovan Y chromosomes

Ancient DNA has allowed the study of various aspects of human history in unprecedented detail. However, because the majority of archaic human specimens preserved well enough for genome sequencing have been female, comprehensive studies of Y chromosomes of Denisovans and Neandertals have not yet been possible. Here we present sequences of the first Denisovan Y chromosomes (Denisova 4 and Denisova 8), as well as the Y chromosomes of three late Neandertals (Spy 94a, Mezmaiskaya 2 and El Sidrón 1253). We find that the Denisovan Y chromosomes split around 700 thousand years ago (kya) from a lineage shared by Neandertal and modern human Y chromosomes, which diverged from each other around 370 kya. The phylogenetic relationships of archaic and modern human Y chromosomes therefore differ from population relationships inferred from their autosomal genomes, and mirror the relationships observed on the level of mitochondrial DNA. This provides strong evidence that gene flow from an early lineage related to modern humans resulted in the replacement of both the mitochondrial and Y chromosomal gene pools in late Neandertals. Although unlikely under neutrality, we show that this replacement is plausible if the low effective population size of Neandertals resulted in an increased genetic load in their Y chromosomes and mitochondrial DNA relative to modern humans.Q.F. was supported by funding from the Chinese Academy of Sciences (XDB26000000), and the National Natural Science Foundation of China (91731303, 41925009,41630102). A.R. was funded by Spanish government (MICINN/FEDER), grant number CGL2016-75109-P. The reassessment of the Spy collection by H.R., I.C. and P.S. was supported by the Belgian Science Policy Office (BELSPO 2004-2007, MO/36/0112). M.S., M.K. and A.D. were supported by the Russian Foundation for Basic Research (RFBR 17-29-04206). This study was funded by the Max Planck Society and the European Research Council (grant agreement number 694707).N

The accessible chromatin landscape of the human genome

DNaseI hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers, and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ~2.9 million DHSs that encompass virtually all known experimentally-validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation, and regulatory factor occupancy patterns. We connect ~580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is choreographed with dozens to hundreds of co-activated elements, and the trans-cellular DNaseI sensitivity pattern at a given region can predict cell type-specific functional behaviors. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation

Patterns of Archaic Hominin DNA in Modern Human Genomes

Thesis (Ph.D.)--University of Washington, 2015In this dissertation I describe the development of a method for identifying introgressed archaic haplotypes. I then present the application of this method to several populations. In 15 African hunter-gatherer genomes, I identify signatures of introgression from an unknown archaic hominin with an apparent divergence time with modern humans that is similar to the divergence time of Neanderthals. In a sample of 379 European and 279 East Asian genomes, I identify on average 1/4 of each individual's introgressed Neanderthal sequence, composing a total of 600Mb of the Neanderthal genome. I use characteristics of this sequence to estimate demographic parameters, including ancestral effective population size (Ne), and the complexity of the introgression event. I also present signatures of both purifying selection against Neanderthal sequence in modern humans, and selection for other, beneficial Neanderthal alleles. In a separate analysis, I show that the inferred parameters of the introgression event are not influenced by differing efficiency of selection between Europeans and East Asians, and explore alternative models for the complexity of the introgression event. In a sample of 35 Melanesian individuals from Papua New Guinea, I identify both Neanderthal and Denisovan introgression, and use this map of archaic introgression to identify regions of the genome that are depleted of archaic sequence from two independent introgression events, implying the presence of non-random forces such as selection in the creation of such regions

Resurrecting Surviving Neandertal Lineages from Modern Human Genomes

Neandertal Shadows in Us Non-African modern humans carry a remnant of Neandertal DNA from interbreeding events that have been postulated to have occurred as humans migrated out of Africa. While the total amount of Neandertal sequence is estimated to be less than 3% of the modern genome, the specific retained sequences vary among individuals. Analyzing the genomes of more than 600 Europeans and East Asians, Vernot and Akey (p. 1017 , published online 29 January) identified Neandertal sequences within modern humans that taken together span approximately 20% of the Neandertal genome. Some Neandertal-derived sequences appear to be under positive selection in humans, including several genes associated with skin phenotypes. </jats:p

Complex History of Admixture between Modern Humans and Neandertals

Recent analyses have found that a substantial amount of the Neandertal genome persists in the genomes of contemporary non-African individuals. East Asians have, on average, higher levels of Neandertal ancestry than do Europeans, which might be due to differences in the efficiency of purifying selection, an additional pulse of introgression into East Asians, or other unexplored scenarios. To better define the scope of plausible models of archaic admixture between Neandertals and anatomically modern humans, we analyzed patterns of introgressed sequence in whole-genome data of 379 Europeans and 286 East Asians. We found that inferences of demographic history restricted to neutrally evolving genomic regions allowed a simple one-pulse model to be robustly rejected, suggesting that differences in selection cannot explain the differences in Neandertal ancestry. We show that two additional demographic models, involving either a second pulse of Neandertal gene flow into the ancestors of East Asians or a dilution of Neandertal lineages in Europeans by admixture with an unknown ancestral population, are consistent with the data. Thus, the history of admixture between modern humans and Neandertals is most likely more complex than previously thought

Human Evolution: Genomic Gifts from Archaic Hominins

SummaryThe dispersal of humans throughout the world was accompanied by adaptations to local environments. New research shows that a previously identified haplotype of the EPAS1 gene, which allows Tibetans to live at high altitude, was inherited from archaic hominin ancestors