Animal Genomes Research Articles

A conserved sequence that sparked the field of evo-devo.

The discovery that homeotic genes in Drosophila are conserved and utilized for embryonic development throughout the animal kingdom, including humans, revolutionized the fields of developmental biology and evolutionary developmental biology (evo-devo). In a pair of back-to-back papers published in Cell in 1984, researchers at the Biozentrum in Basel, Switzerland, showed that the homeobox - previously identified as a sequence shared by homeotic genes in Drosophila - was also present in the genome of diverse animals. The first paper (McGinnis et al., 1984a) showed that genomes of both invertebrates and vertebrates contain sequences that cross-hybridized with Drosophila homeobox probes. The second paper (Carrasco et al., 1984) identified a cross-hybridizing sequence in the model system Xenopus laevis. They then isolated the first vertebrate homeobox-containing gene by cloning and sequencing of the corresponding genomic region. Finally, they showed that this gene (AC1, later renamed HoxC6) was expressed during embryonic development, the first evidence that developmentally expressed Drosophila genes could be used to isolate regulators of vertebrate embryonic development. These findings led to a flurry of activity in the evo-devo field, initially focused on isolating Hox genes across diverse species, and then expanding to isolation of other gene families based on Drosophila orthologs, an approach that continues today. This led to the notion of a conserved genetic toolkit for embryonic development, currently accepted, but unexpected at the time of its discovery. We attempt to provide some context for the sea-change in thinking that these discoveries brought about by referring to Jean Piaget's theories about the sequential acquisition of scientific knowledge.

EPCO-45. COMPLETE CHROMOSOME-SCALE DIPLOID-PHASED ASSEMBLY OF A NORMAL HUMAN ASTROCYTE CELL LINE

Abstract Genomic studies of the malignant brain tumor glioma invariably rely on aligning sequencing reads to a reference genome such as HG38, even though no single reference can capture the diversity of an individual patient or cell line. To begin addressing these biases in genomics research, we sought to construct a chromosome-scale diploid-phased assembly of a normal human astrocyte (NHA) cell line that is frequently used as a basis for developing isogenic glioma cell models. To achieve this, we generated 45x Pacific Biosciences HiFi data (N50: 16kb), 26x Oxford Nanopore Technologies ultra-long data (N50: 80kb), and 2 billion paired-end reads of Hi-C data. The assembler verkko2 was used to integrate these data and build a phased diploid assembly. We achieved an assembly size of 6.01 Gbp, with 17 gapless chromosomes. The two haplotypes contain 214 and 182 contigs, respectively. Our assembly includes 76 fully assembled telomeres and 20 fully assembled centromeres. The contiguity of our assembly is 130 Mbp, which vastly exceeds that of HG38 (N50: 68 Mbp) and approaches that of the recent telomere-to-telomere CHM13 assembly (N50: 155 Mbp). We ran BUSCO on each haplotype of the assembly using the metazoa database to quantify the single-copy genes that are expected to be present in every animal genome. The haplotypes of our assembly contain the complete sequence of 98.6% and 99.9% of these genes respectively. Finally, we used Merqury to perform a k-mer based evaluation of the two haplotypes and found quality values of 59.09 and 59.75 respectively. This diploid genome assembly of NHA will serve as a resource for researchers that wish to develop NHA-derived glioma models. It can be used to call indels or structural variants in a haplotype-specific manner which will further elucidate genotypes underlying glioma phenotypes. Most importantly, it sets the stage for personalized genome assembly and tackling the lack of diversity in genomic studies of glioma to date.

The genetic mechanism of B chromosome drive in rye illuminated by chromosome-scale assembly

The genomes of many plants, animals, and fungi frequently comprise dispensable B chromosomes that rely upon various chromosomal drive mechanisms to counteract the tendency of non-essential genetic elements to be purged over time. The B chromosome of rye – a model system for nearly a century – undergoes targeted nondisjunction during first pollen mitosis, favouring segregation into the generative nucleus, thus increasing their numbers over generations. However, the genetic mechanisms underlying this process are poorly understood. Here, using a newly-assembled, ~430 Mb-long rye B chromosome pseudomolecule, we identify five candidate genes whose role as trans-acting moderators of the chromosomal drive is supported by karyotyping, chromosome drive analysis and comparative RNA-seq. Among them, we identify DCR28, coding a microtubule-associated protein related to cell division, and detect this gene also in the B chromosome of Aegilops speltoides. The DCR28 gene family is neo-functionalised and serially-duplicated with 15 B chromosome-located copies that are uniquely highly expressed in the first pollen mitosis of rye.

Systematic screening of enhancer-blocking insulators in Drosophila identifies their DNA sequence determinants.

Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes calls for mechanisms to limit inappropriate regulation. DNA elements called insulators serve this purpose by shielding promoters from an enhancer when interposed. Unlike promoters and enhancers, insulators have not been systematically characterized due to lacking high-throughput screening assays, and questions regarding how insulators are distributed and encoded in the genome remain. Here, we establish "insulator-seq" as a plasmid-based massively parallel reporter assay in Drosophila cultured cells to perform a systematic insulator screen of selected genomic loci. Screening developmental gene loci showed that not all insulator protein binding sites effectively block enhancer-promoter communication. Deep insulator mutagenesis identified sequences flexibly positioned around the CTCF insulator protein binding motif that are critical for functionality. The ability to screen millions of DNA sequences without positional effect has enabled functional mapping of insulators and provided further insights into the determinants of insulators.

Accelerating de novo SINE annotation in plant and animal genomes

Genome annotation is an important but challenging task. Accurate identification of short interspersed nuclear elements (SINEs) is particularly difficult due to their lack of highly conserved sequences. AnnoSINE is state-of-the-art software for annotating SINEs in plant genomes, but it is computationally inefficient for large genomes. Moreover, its applicability to animals is limited due to the absence of animal pHMMs in its HMM library. Therefore, we propose AnnoSINE_v2, which extends accurate SINE annotation for animal genomes with greatly optimized computational efficiency. Our results show that AnnoSINE_v2’s annotation of SINEs has over 20% higher F1-score compared to the existing tools on animal genomes and enables the processing of complicated genomes, like human and zebrafish, which were beyond the capabilities of AnnoSINE_v1. AnnoSINE_v2 is freely available on Conda and GitHub: https://github.com/liaoherui/AnnoSINE_v2.

The Highly Repetitive Genome of Myxobolus rasmusseni, an Emerging Myxozoan Parasite of Fathead Minnows.

Myxozoans are a monophyletic taxon of approximately 2,400 described species of parasites from the phylum Cnidaria. The recent focus on their negative impacts on fisheries, on their evolution from free-living ancestors, and on their emergence into new fish host populations has stressed the critical need for genomic resources for this parasitic group. Here, we describe the genome assembly and annotation of Myxobolus rasmusseni, an emerging parasite of fathead minnows in Alberta, Canada. The assembly is 174.6 Mb in size, 68% of which is made up of repetitive elements, making it one of the most repetitive animal genomes sequenced to date. Through comparisons to other myxozoans, we show that widespread gene loss, a known phenomenon of this group of parasites, is consistent with closely related species. Additionally, we assembled the M. rasmusseni mitochondrial genome, which is nearly twice the size of the typical animal mitochondrial genome yet contains only five of the canonical mitochondrial protein-coding genes and open reading frames not found in other myxozoans. These results add to our understanding of the gene- and genome-level diversity observed in myxozoans.

Cohesin organizes 3D DNA contacts surrounding active enhancers in C. elegans.

In mammals, cohesin and CTCF organize the 3D genome into topologically associated domains (TADs) to regulate communication between cis-regulatory elements. Many organisms, including S. cerevisiae, C. elegans, and A. thaliana contain cohesin but lack CTCF. Here, we used C. elegans to investigate the function of cohesin in 3D genome organization in the absence of CTCF. Using Hi-C data, we observe cohesin-dependent features called "fountains", which are also reported in zebrafish and mice. These are population average reflections of DNA loops originating from distinct genomic regions and are ~20-40 kb in C. elegans. Hi-C analysis upon cohesin and WAPL depletion support the idea that cohesin is preferentially loaded at NIPBL occupied sites and loop extrudes in an effectively two-sided manner. ChIP-seq analyses show that cohesin translocation along the fountain trajectory depends on a fully intact complex and is extended upon WAPL-1 depletion. Hi-C contact patterns at individual fountains suggest that cohesin processivity is unequal on each side, possibly due to collision with cohesin loaded from surrounding sites. The putative cohesin loading sites are closest to active enhancers and fountain strength is associated with transcription. Compared to mammals, average processivity of C. elegans cohesin is ~10-fold shorter and NIPBL binding does not depend on cohesin. We propose that preferential loading and loop extrusion by cohesin is an evolutionarily conserved mechanism that regulates the 3D interactions of enhancers in animal genomes.

Homologous mapping yielded a comprehensive predicted protein–protein interaction network for peanut (Arachis hypogaea L.)

BackgroundProtein–protein interactions are the primary means through which proteins carry out their functions. These interactions thus have crucial roles in life activities. The wide availability of fully sequenced animal and plant genomes has facilitated establishment of relatively complete global protein interaction networks for some model species. The genomes of cultivated and wild peanut (Arachis hypogaea L.) have also been sequenced, but the functions of most of the encoded proteins remain unclear.ResultsWe here used homologous mapping of validated protein interaction data from model species to generate complete peanut protein interaction networks for A. hypogaea cv. ‘Tifrunner’ (282,619 pairs), A. hypogaea cv. ‘Shitouqi’ (256,441 pairs), A. monticola (440,470 pairs), A. duranensis (136,363 pairs), and A. ipaensis (172,813 pairs). A detailed analysis was conducted for a putative disease-resistance subnetwork in the Tifrunner network to identify candidate genes and validate functional interactions. The network suggested that DX2UEH and its interacting partners may participate in peanut resistance to bacterial wilt; this was preliminarily validated with overexpression experiments in peanut.ConclusionOur results provide valuable new information for future analyses of gene and protein functions and regulatory networks in peanut.

PSLBI-4 Influence of gender on animal science research funding from the National Institute of Food and Agriculture

Abstract According to the National Academies of Sciences, Engineering, and Medicine, females and males in science disciplines obtain bachelor’s degrees at or near parity, yet females are not equally represented in academic faculty positions. This is true for agricultural fields including the animal sciences. The USDA’s National Institute of Food and Agriculture (NIFA) administers the Agriculture and Food Research Initiative (AFRI), a flagship competitive grants program that funds research, education, and extension efforts with a total appropriation of $445 million in 2024. Our objective was to determine the difference between genders for submission rates (i.e., number of standard and seed grant applications submitted), peer reviewers of grant applications, and success rates (i.e., number of grant awards/number of applications submitted) in the main animal related AFRI program area priorities. All applications submitted to AFRI programs in animal genomics, reproduction, health, nutrition and growth, and welfare during yr 2013-2022 were evaluated. Gender was assigned to Project Directors (PD) and peer reviewers using first names as input into Gender API software (Gender-API.com; Passau, Germany) with ≥ 95% accuracy and ≥ 20 database occurrences used as quality filters and binary outcomes only (i.e., male or female). Effect of gender on submission rates, peer review panel compositions, and success rates were then analyzed using Pearson’s Chi-squared tests with P ≤ 0.05 considered significant. Results: A total of 3,757 applications were submitted, of which 2,761 (73%) PD genders were assigned and analyzed further. Overall, the proportional submission rate for females was 28% and for males was 72%. Submission rates for females increased (P = 0.0614) over time ranging from 22% in 2013 to 33% in 2021. Submission rates differed by gender across programs (P < 0.0001) and ranged from 23% female in animal reproduction to 48% female in animal welfare. Across 48 panels, 41% of panelists were female; no differences were identified for the proportion of female panelists over time (P = 0.9509) or by program (P = 0.9298). Overall, the funding success rate for females was 23% and for males was 22%. Success rates did not differ between genders over time (P = 0.9080) or by program (P = 0.9094). Females submitted less than one-half as many applications as did males, but the proportion of submissions by females increased over time. This trend remained consistent despite challenges of relocation of NIFA and COVID-19 during this time. Overall, the proportion of NIFA panelists who were female exceeded the proportion of applications submitted by females. Funding success rates did not differ by gender and increased for both genders over time. These results indicate that although differences in gender existed for application submission rate and panel composition, these differences did not impact funding success rate of applications in animal science programs at NIFA, which did not differ between genders.

PSVII-1 Widespread allele-specific expression across tissues and cells of the chicken genome

Abstract Chickens are a valuable livestock species, providing an affordable and nutritious food source worldwide through eggs and meat. Additionally, chickens are extensively used in scientific research as model organisms in virology, immunology, epigenetics, development, and conservation biology. Considering this, there are increasing efforts to deepen our understanding of their genome complexity, such as efforts by the Functional Annotation of Animal Genomes (FAANG) Consortium. Toward this goal, we are working to contribute to the annotation of the chicken transcriptome. Allele-specific expression (ASE) is an imbalance of allelic gene expression often driven by genetic and epigenetic changes in cis-regulatory regions. We, therefore, aimed to determine allelic imbalances in gene expression across 20 tissues and cells to shed light on mechanisms regulating gene expression. Using replicate paired-end (PE) RNA-seq samples, we identified around 7 million variants across 20 tissues and cells (e.g., reproductive tissues, intestine tissues, muscle, and immune tissues and cells). We applied quality control measures and filtered out monoallelic and homozygous variants. After that, we calculated read counts per allele to determine allelic expression. We found 365,894 significant ASE SNPs and 11,530 ASE genes in at least one tissue or cell type (FDR ≤ 0.05). We discovered that ASE SNPs are widely distributed throughout the chicken genome, and ASE SNPs and ASE genes showed a varied distribution across tissues and cells. Primary macrophage exhibited the most abundant, while the pectoralis major (breast muscle) had the lowest ASE genes and ASE SNPs, ranging from 7,592 to 32,505 ASE SNPs and 773 to 2,387 ASE genes. Our findings reveal several important pathways affected by allelic imbalance of expression. These pathways included fatty acid biosynthesis and regulation, cell proliferation and differentiation, cell metabolism, adipocytokine signaling, proteolysis and autophagy, and focal adhesion. In summary, our study provides insight into relevant biological processes critical to chickens and can contribute to improving genome annotation, helping to expand the transcriptomic reference source.

Distribution rules of 8-mer spectra and characterization of evolution state in animal genome sequences

BackgroundStudying the composition rules and evolution mechanisms of genome sequences are core issues in the post-genomic era, and k-mer spectrum analysis of genome sequences is an effective means to solve this problem.ResultWe divided total 8-mers of genome sequences into 16 kinds of XY-type due to XY dinucleotides number in 8-mers. Previous works explored that the independent unimodal distributions observed only in three CG-type 8-mer spectra, while non-CG type 8-mer spectra have not the universal phenomenon from prokaryotes to eukaryotes. On this basis, we analyzed the distribution variation of non-CG type 8-mer spectra across 889 animal genome sequences. Following the evolutionary order of animals from primitive to more complex, we found that the spectrum distributions gradually transition from unimodal to tri-modal. The relative distance from the average frequency of each non-CG type 8-mers to the center frequency is different within a species and among different species. For the 8-mers contain CG dinucleotides, we further divided these into 16 subsets, where each 8-mer contains both CG and XY dinucleotides, called XY1_CG1 subsets. We found that the separability values of XY1_CG1 spectra are closely related to the evolution and specificity of animals. Considering the constraint of Chargaff’s second parity rule, we finally obtained 10 separability values as the feature set to characterize the evolution state of genome sequences. In order to verify the rationality of the feature set, we used 14 common classification algorithms to perform binary classification tests. The results showed that the accuracy (Acc) ranged between 98.70% and 83.88% among birds, other vertebrates and mammals.ConclusionWe proposed a credible feature set to characterizes the evolution state of genomes and obtained satisfied results by the feature set on large scale classification of animals.

The genomes of all lungfish inform on genome expansion and tetrapod evolution.

The genomes of living lungfishes can inform on the molecular-developmental basis of the Devonian sarcopterygian fish-tetrapod transition. We de novo sequenced the genomes of the African (Protopterus annectens) and South American lungfishes (Lepidosiren paradoxa). The Lepidosiren genome (about 91 Gb, roughly 30 times the human genome) is the largest animal genome sequenced so far and more than twice the size of the Australian (Neoceratodus forsteri)1 and African2 lungfishes owing to enlarged intergenic regions and introns with high repeat content (about 90%). All lungfish genomes continue to expand as some transposable elements (TEs) are still active today. In particular, Lepidosiren's genome grew extremely fast during the past 100 million years (Myr), adding the equivalent of one human genome every 10 Myr. This massive genome expansion seems to be related to a reduction of PIWI-interacting RNAs and C2H2 zinc-finger and Krüppel-associated box (KRAB)-domain protein genes that suppress TE expansions. Although TE abundance facilitates chromosomal rearrangements, lungfish chromosomes still conservatively reflect the ur-tetrapod karyotype. Neoceratodus' limb-like fins still resemble those of their extinct relatives and remained phenotypically static for about 100 Myr. We show that the secondary loss of limb-like appendages in the Lepidosiren-Protopterus ancestor was probably due to loss of sonic hedgehog limb-specific enhancers.

Annelid Comparative Genomics and the Evolution of Massive Lineage-Specific Genome Rearrangement in Bilaterians.

The organization of genomes into chromosomes is critical for processes such as genetic recombination, environmental adaptation, and speciation. All animals with bilateral symmetry inherited a genome structure from their last common ancestor that has been highly conserved in some taxa but seemingly unconstrained in others. However, the evolutionary forces driving these differences and the processes by which they emerge have remained largely uncharacterized. Here, we analyze genome organization across the phylum Annelida using 23 chromosome-level annelid genomes. We find that while many annelid lineages have maintained the conserved bilaterian genome structure, the Clitellata, a group containing leeches and earthworms, possesses completely scrambled genomes. We develop a rearrangement index to quantify the extent of genome structure evolution and show that, compared to the last common ancestor of bilaterians, leeches and earthworms have among the most highly rearranged genomes of any currently sampled species. We further show that bilaterian genomes can be classified into two distinct categories-high and low rearrangement-largely influenced by the presence or absence, respectively, of chromosome fission events. Our findings demonstrate that animal genome structure can be highly variable within a phylum and reveal that genome rearrangement can occur both in a gradual, stepwise fashion, or rapid, all-encompassing changes over short evolutionary timescales.

Global Variation in Escherichia coli mcr-1 Genes and Plasmids from Animal and Human Genomes Following Colistin Usage Restrictions in Livestock.

Antimicrobial resistance (AMR) is a significant global health threat, with multidrug-resistant (MDR) bacterial clones becoming a major concern. Polymyxins, especially colistin, have reemerged as last-resort treatments for MDR Gram-negative infections. However, colistin use in livestock has spread mobile colistin resistance (mcr) genes, notably mcr-1, impacting human health. In consequence, its livestock use was banned in 2017, originating a natural experiment to study bacterial adaptation. The aim of this work was to analyse the changes in the mcr-1 genetic background after colistin restriction across the world. This study analyses 3163 Escherichia coli genomes with the mcr-1 gene from human and livestock hosts, mainly from Asia (n = 2621) and Europe (n = 359). Genetic characterisation identifies IncI2 (40.4%), IncX4 (26.7%), and multidrug-resistant IncHI2 (18.8%) as the most common plasmids carrying mcr-1. There were differences in plasmids between continents, with IncX4 (56.6%) being the most common in Europe, while IncI2 (44.8%) was predominant in Asia. Promoter variants related to reduced fitness costs and ISApl1 showed a distinct pattern of association that appears to be associated with adaptation to colistin restriction, which differed between continents. Thus, after the colistin ban, Europe saw a shift to specialised mcr-1 plasmids as IncX4, while ISApl1 decreased in Asia due to changes in the prevalence of the distinct promoter variants. These analyses illustrate the evolution of mcr-1 adaptation following colistin use restrictions and the need for region-specific strategies against AMR following colistin restrictions.

Combinatorial regulatory states define cell fate diversity during embryogenesis

Cell fate specification occurs along invariant species-specific trajectories that define the animal body plan. This process is controlled by gene regulatory networks that regulate the expression of the limited set of transcription factors encoded in animal genomes. Here we globally assess the spatial expression of ~90% of expressed transcription factors during sea urchin development from embryo to larva to determine the activity of gene regulatory networks and their regulatory states during cell fate specification. We show that >200 embryonically expressed transcription factors together define >70 cell fates that recapitulate the morphological and functional organization of this organism. Most cell fate-specific regulatory states consist of ~15–40 transcription factors with similarity particularly among functionally related cell types regardless of developmental origin. Temporally, regulatory states change continuously during development, indicating that progressive changes in regulatory circuit activity determine cell fate specification. We conclude that the combinatorial expression of transcription factors provides molecular definitions that suffice for the unique specification of cell states in time and space during embryogenesis.

CRISPR-Cpf1 system and its applications in animal genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein (Cas) system is a gene editing technology guided by RNA endonuclease. The CRISPR-Cas12a (also known as CRISPR-Cpf1) system is extensively utilized in genome editing research due to its accuracy and high efficiency. In this paper, we primarily focus on the application of CRISPR-Cpf1 technology in the construction of disease models and gene therapy. Firstly, the structure and mechanism of the CRISPR-Cas system are introduced. Secondly, the similarities and differences between CRISPR-Cpf1 and CRISPR-Cas9 technologies are compared. Thirdly, the main focus is on the application of the CRISPR-Cpf1 system in cell and animal genome editing. Finally, the challenges faced by CRISPR-Cpf1 technology and corresponding strategies are analyzed. Although CRISPR-Cpf1 technology has certain off-target effects, it can effectively and accurately edit cell and animal genomes, and has significant advantages in the preclinical research.

Endogenous Bornavirus-like Elements in Bats: Evolutionary Insights from the Conserved Riboviral L-Gene in Microbats and Its Antisense Transcription in Myotis daubentonii.

Bats are ecologically diverse vertebrates characterized by their ability to host a wide range of viruses without apparent illness and the presence of numerous endogenous viral elements (EVEs). EVEs are well preserved, expressed, and may affect host biology and immunity, but their role in bat immune system evolution remains unclear. Among EVEs, endogenous bornavirus-like elements (EBLs) are bornavirus sequences integrated into animal genomes. Here, we identified a novel EBL in the microbat Myotis daubentonii, EBLL-Cultervirus.10-MyoDau (short name is CV.10-MyoDau) that shows protein-level conservation with the L-protein of a Cultervirus (Wuhan sharpbelly bornavirus). Surprisingly, we discovered a transcript on the antisense strand comprising three exons, which we named AMCR-MyoDau. The active transcription in Myotis daubentonii tissues of AMCR-MyoDau, confirmed by RNA-Seq analysis and RT-PCR, highlights its potential role during viral infections. Using comparative genomics comprising 63 bat genomes, we demonstrate nucleotide-level conservation of CV.10-MyoDau and AMCR-MyoDau across various bat species and its detection in 22 Yangochiropera and 12 Yinpterochiroptera species. To the best of our knowledge, this marks the first occurrence of a conserved EVE shared among diverse bat species, which is accompanied by a conserved antisense transcript. This highlights the need for future research to explore the role of EVEs in shaping the evolution of bat immunity.

A broad survey of choanoflagellates revises the evolutionary history of the Shaker family of voltage-gated K+ channels in animals

The Shaker family of voltage-gated K+ channels has been thought of as an animal-specific ion channel family that diversified in concert with nervous systems. It comprises four functionally independent gene subfamilies (Kv1-4) that encode diverse neuronal K+ currents. Comparison of animal genomes predicts that only the Kv1 subfamily was present in the animal common ancestor. Here, we show that some choanoflagellates, the closest protozoan sister lineage to animals, also have Shaker family K+ channels. Choanoflagellate Shaker family channels are surprisingly most closely related to the animal Kv2-4 subfamilies which were believed to have evolved only after the divergence of ctenophores and sponges from cnidarians and bilaterians. Structural modeling predicts that the choanoflagellate channels share a T1 Zn2+ binding site with Kv2-4 channels that is absent in Kv1 channels. We functionally expressed three Shakers from Salpingoeca helianthica (SheliKvT1.1-3) in Xenopus oocytes. SheliKvT1.1-3 function only in two heteromultimeric combinations (SheliKvT1.1/1.2 and SheliKvT1.1/1.3) and encode fast N-type inactivating K+ channels with distinct voltage dependence that are most similar to the widespread animal Kv1-encoded A-type Shakers. Structural modeling of the T1 assembly domain supports a preference for heteromeric assembly in a 2:2 stoichiometry. These results push the origin of the Shaker family back into a common ancestor of metazoans and choanoflagellates. They also suggest that the animal common ancestor had at least two distinct molecular lineages of Shaker channels, a Kv1 subfamily lineage predicted from comparison of animal genomes and a Kv2-4 lineage predicted from comparison of animals and choanoflagellates.

Range-limited Heaps’ law for functional DNA words in the human genome

Heaps’ or Herdan-Heaps’ law is a linguistic law describing the relationship between the vocabulary/dictionary size (type) and word counts (token) to be a power-law function. Its existence in genomes with certain definition of DNA words is unclear partly because the dictionary size in genome could be much smaller than that in a human language. We define a DNA word as a coding region in a genome that codes for a protein domain. Using human chromosomes and chromosome arms as individual samples, we establish the existence of Heaps’ law in the human genome within limited range. Our definition of words in a genomic or proteomic context is different from other definitions such as over-represented k-mers which are much shorter in length. Although an approximate power-law distribution of protein domain sizes due to gene duplication and the related Zipf’s law is well known, their translation to the Heaps’ law in DNA words is not automatic. Several other animal genomes are shown herein also to exhibit range-limited Heaps’ law with our definition of DNA words, though with various exponents. When tokens were randomly sampled and sample sizes reach to the maximum level, a deviation from the Heaps’ law was observed, but a quadratic regression in log–log type-token plot fits the data perfectly. Investigation of type-token plot and its regression coefficients could provide an alternative narrative of reusage and redundancy of protein domains as well as creation of new protein domains from a linguistic perspective.

Targeted mutagenesis of specific genomic DNA sequences in animals for the in vivo generation of variant libraries.

Understanding how the number, placement and affinity of transcription factor binding sites dictates gene regulatory programs remains a major unsolved challenge in biology, particularly in the context of multicellular organisms. To uncover these rules, it is first necessary to find the binding sites within a regulatory region with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively parallel reporter assays (MPRAs), where the gene expression stemming from 10,000s of in vitro-generated regulatory sequences is measured, have made this feat possible in high-throughput in single cells in culture. However, because of lack of technologies to incorporate DNA libraries, MPRAs are limited in whole organisms. To enable MPRAs in multicellular organisms, we generated tools to create a high degree of mutagenesis in specific genomic loci in vivo using base editing. Targeting GFP integrated in genome of Drosophila cell culture and whole animals as a case study, we show that the base editor AIDevoCDA1 stemming from sea lamprey fused to nCas9 is highly mutagenic. Surprisingly, longer gRNAs increase mutation efficiency and expand the mutating window, which can allow the introduction of mutations in previously untargetable sequences. Finally, we demonstrate arrays of >20 gRNAs that can efficiently introduce mutations along a 200bp sequence, making it a promising tool to test enhancer function in vivo in a high throughput manner.