Demogenomic inference from spatially and temporally heterogeneous samples.

Modern and ancient genomes are not necessarily drawn from homogeneous populations, as they may have been collected from different places and at different times. This heterogeneous sampling can be an issue for demographic inferences and results in biased demographic parameters and incorrect model choice if not properly considered. When explicitly accounted for, it can result in very complex models and high data dimensionality that are difficult to analyse. In this paper, we formally study the impact of such spatial and temporal sampling heterogeneity on demographic inference, and we introduce a way to circumvent this problem. To deal with structured samples without increasing the dimensionality of the site frequency spectrum (SFS), we introduce a new structured approach to the existing program fastsimcoal2. We assess the efficiency and relevance of this methodological update with simulated and modern human genomic data. We particularly focus on spatial and temporal heterogeneities to evidence the interest of this new SFS-based approach, which can be especially useful when handling scattered and ancient DNA samples, as in conservation genetics or archaeogenetics.

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems.

BACKGROUND: Microbial communities in recirculating aquaculture systems (RAS) play a role in system success, nutrient cycling, and water quality. Considering the increasing socio-economic role of fish farming, e.g., regarding food security, an in-depth understanding of aquaculture microbial communities is also relevant from a management perspective, especially regarding the growth, development, and welfare of the farmed animal. However, the current data on the composition of microbial communities within RAS is patchy, which is partly attributable to diverging method choices that render comparative analyses challenging. Therefore, there is a need for accurate, standardized, and user-friendly methods to study microbial communities in aquaculture systems. RESULTS: We compared sequencing approach performances (3 types of 16S short amplicon sequencing, PacBio long-read amplicon sequencing, and amplification-free shotgun metagenomics) in the characterization of microbial communities in two commercial RAS fish farms. Results showed that 16S primer choice and amplicon length affect some values (e.g., diversity measures, number of assigned taxa or distinguishing ASVs) but have no impact on spatio-temporal patterns between sample types, farms and time points. This implies that 16S rRNA approaches are adequate for community studies. The long-read amplicons underperformed regarding the quantitative resolution of spatio-temporal patterns but were suited to identify functional services, e.g., nitrification cycling and the detection of pathogens. Finally, shotgun metagenomics extended the picture to fungi, viruses, and bacteriophages, opening avenues for exploring inter-domain interactions. All sequencing datasets agreed on major prokaryotic players, such as Actinobacteriota, Bacteroidota, Nitrospirota, and Proteobacteria. CONCLUSION: The different sequencing approaches yielded overlapping and highly complementary results, with each contributing unique data not obtainable with the other approaches. We conclude that a tiered approach constitutes a strategy for obtaining the maximum amount of information on aquaculture microbial communities and can inform basic research on community evolution dynamics. For specific and/or applied questions, single-method approaches are more practical and cost-effective and could lead to better farm management practices.

The genomic origins of the world's first farmers.

The precise genetic origins of the first Neolithic farming populations in Europe and Southwest Asia, as well as the processes and the timing of their differentiation, remain largely unknown. Demogenomic modeling of high-quality ancient genomes reveals that the early farmers of Anatolia and Europe emerged from a multiphase mixing of a Southwest Asian population with a strongly bottlenecked western hunter-gatherer population after the last glacial maximum. Moreover, the ancestors of the first farmers of Europe and Anatolia went through a period of extreme genetic drift during their westward range expansion, contributing highly to their genetic distinctiveness. This modeling elucidates the demographic processes at the root of the Neolithic transition and leads to a spatial interpretation of the population history of Southwest Asia and Europe during the late Pleistocene and early Holocene.

Impact of Genetic Variation in Gene Regulatory Sequences: A Population Genomics Perspective.

The unprecedented rise of high-throughput sequencing and assay technologies has provided a detailed insight into the non-coding sequences and their potential role as gene expression regulators. These regulatory non-coding sequences are also referred to as cis-regulatory elements (CREs). Genetic variants occurring within CREs have been shown to be associated with altered gene expression and phenotypic changes. Such variants are known to occur spontaneously and ultimately get fixed, due to selection and genetic drift, in natural populations and, in some cases, pave the way for speciation. Hence, the study of genetic variation at CREs has improved our overall understanding of the processes of local adaptation and evolution. Recent advances in high-throughput sequencing and better annotations of CREs have enabled the evaluation of the impact of such variation on gene expression, phenotypic alteration and fitness. Here, we review recent research on the evolution of CREs and concentrate on studies that have investigated genetic variation occurring in these regulatory sequences within the context of population genetics.

Demographic analyses of a new sample of haploid genomes from a Swedish population of Drosophila melanogaster.

European and African natural populations of Drosophila melanogaster have been the focus of several studies aiming at inferring demographic and adaptive processes based on genetic variation data. However, in these analyses little attention has been given to gene flow between African and European samples. Here we present a dataset consisting of 14 fully sequenced haploid genomes sampled from a natural population from the northern species range (Umeå, Sweden). We co-analyzed this new data with an African population to compare the likelihood of several competing demographic scenarios for European and African populations and show that gene flow improves the fit of demographic models to data.

Mitochondrial haplotypes affect metabolic phenotypes in the Drosophila Genetic Reference Panel.

The nature and extent of mitochondrial DNA variation in a population and how it affects traits is poorly understood. Here we resequence the mitochondrial genomes of 169 Drosophila Genetic Reference Panel lines, identifying 231 variants that stratify along 12 mitochondrial haplotypes. We identify 1,845 cases of mitonuclear allelic imbalances, thus implying that mitochondrial haplotypes are reflected in the nuclear genome. However, no major fitness effects are associated with mitonuclear imbalance, suggesting that such imbalances reflect population structure at the mitochondrial level rather than genomic incompatibilities. Although mitochondrial haplotypes have no direct impact on mitochondrial respiration, some haplotypes are associated with stress- and metabolism-related phenotypes, including food intake in males. Finally, through reciprocal swapping of mitochondrial genomes, we demonstrate that a mitochondrial haplotype associated with high food intake can rescue a low food intake phenotype. Together, our findings provide new insight into population structure at the mitochondrial level and point to the importance of incorporating mitochondrial haplotypes in genotype-phenotype relationship studies.

The Demographic History of African Drosophila melanogaster.

As one of the most commonly utilized organisms in the study of local adaptation, an accurate characterization of the demographic history of Drosophila melanogaster remains as an important research question. This owes both to the inherent interest in characterizing the population history of this model organism, as well as to the well-established importance of an accurate null demographic model for increasing power and decreasing false positive rates in genomic scans for positive selection. Although considerable attention has been afforded to this issue in non-African populations, less is known about the demographic history of African populations, including from the ancestral range of the species. While qualitative predictions and hypotheses have previously been forwarded, we here present a quantitative model fitting of the population history characterizing both the ancestral Zambian population range as well as the subsequently colonized west African populations, which themselves served as the source of multiple non-African colonization events. We here report the split time of the West African population at 72 kya, a date corresponding to human migration into this region as well as a period of climatic changes in the African continent. Furthermore, we have estimated population sizes at this split time. These parameter estimates thus represent an important null model for future investigations in to African and non-African D. melanogaster populations alike.

The evolution of gene expression and binding specificity of the largest transcription factor family in primates.

The KRAB-containing zinc finger (KRAB-ZF) proteins represent the largest family of transcription factors (TFs) in humans, yet for the great majority, their function and specific genomic target remain unknown. However, it has been shown that a large fraction of these genes arose from segmental duplications, and that they have expanded in gene and zinc finger number throughout vertebrate evolution. To determine whether this expansion is linked to selective pressures acting on different domains, we have manually curated all KRAB-ZF genes present in the human genome together with their orthologous genes in three closely related species and assessed the evolutionary forces acting at the sequence level as well as on their expression profiles. We provide evidence that KRAB-ZFs can be separated into two categories according to the polymorphism present in their DNA-contacting residues. Those carrying a nonsynonymous single nucleotide polymorphism (SNP) in their DNA-contacting amino acids exhibit significantly reduced expression in all tissues, have emerged in a recent lineage, and seem to be less strongly constrained evolutionarily than those without such a polymorphism. This work provides evidence for a link between age of the TF, as well as polymorphism in their DNA-contacting residues and expression levels-both of which may be jointly affected by selection.

Release of human cytomegalovirus from latency by a KAP1/TRIM28 phosphorylation switch.

Human cytomegalovirus (HCMV) is a highly prevalent pathogen that induces life-long infections notably through the establishment of latency in hematopoietic stem cells (HSC). Bouts of reactivation are normally controlled by the immune system, but can be fatal in immuno-compromised individuals such as organ transplant recipients. Here, we reveal that HCMV latency in human CD34(+) HSC reflects the recruitment on the viral genome of KAP1, a master co-repressor, together with HP1 and the SETDB1 histone methyltransferase, which results in transcriptional silencing. During lytic infection, KAP1 is still associated with the viral genome, but its heterochromatin-inducing activity is suppressed by mTOR-mediated phosphorylation. Correspondingly, HCMV can be forced out of latency by KAP1 knockdown or pharmacological induction of KAP1 phosphorylation, and this process can be potentiated by activating NFkB with TNF-α. These results suggest new approaches both to curtail CMV infection and to purge the virus from organ transplants.

TRIM28 represses transcription of endogenous retroviruses in neural progenitor cells.

TRIM28 is a corepressor that mediates transcriptional silencing by establishing local heterochromatin. Here, we show that deletion of TRIM28 in neural progenitor cells (NPCs) results in high-level expression of two groups of endogenous retroviruses (ERVs): IAP1 and MMERVK10C. We find that NPCs use TRIM28-mediated histone modifications to dynamically regulate transcription and silencing of ERVs, which is in contrast to other somatic cell types using DNA methylation. We also show that derepression of ERVs influences transcriptional dynamics in NPCs through the activation of nearby genes and the expression of long noncoding RNAs. These findings demonstrate a unique dynamic transcriptional regulation of ERVs in NPCs. Our results warrant future studies on the role of ERVs in the healthy and diseased brain.

Evolutionally dynamic L1 regulation in embryonic stem cells.

Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1, also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution, with, at any given time, a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage, likely under pressure of host restriction factors, which act notably by silencing L1 expression during early embryogenesis. Here, we demonstrate that in human embryonic stem (hES) cells, KAP1 (KRAB [Krüppel-associated box domain]-associated protein 1), the master cofactor of KRAB-containing zinc finger proteins (KRAB-ZFPs) previously implicated in the restriction of endogenous retroviruses, represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 million and 7.6 million years ago. In mice, we documented a similar chronologically conditioned pattern, albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP, suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hES cells induced the expression of KAP1-bound L1 elements, but their younger, human-specific counterparts (L1Hs) were unaffected. Instead, they were stimulated by depleting DNA methyltransferases, consistent with recent evidence demonstrating that the PIWI-piRNA (PIWI-interacting RNA) pathway regulates L1Hs in hES cells. Altogether, these data indicate that the early embryonic control of L1 is an evolutionarily dynamic process and support a model in which newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms before KAP1-recruiting protein repressors are selected.

Loss of transcriptional control over endogenous retroelements during reprogramming to pluripotency.

Endogenous retroelements (EREs) account for about half of the mouse or human genome, and their potential as insertional mutagens and transcriptional perturbators is suppressed by early embryonic epigenetic silencing. Here, we asked how ERE control is maintained during the generation of induced pluripotent stem cells (iPSCs), as this procedure involves profound epigenetic remodeling. We found that all EREs tested were markedly up-regulated during the reprogramming of either mouse embryonic fibroblasts, human CD34(+) cells, or human primary hepatocytes. At the iPSC stage, EREs of some classes were repressed, whereas others remained highly expressed, yielding a pattern somewhat reminiscent of that recorded in embryonic stem cells. However, variability persisted between individual iPSC clones in the control of specific ERE integrants. Both during reprogramming and in iPS cells, the up-regulation of specific EREs significantly impacted on the transcription of nearby cellular genes. While transcription triggered by specific ERE integrants at highly precise developmental stages may be an essential step toward obtaining pluripotent cells, the broad and unspecific unleashing of the repetitive genome observed here may contribute to the inefficiency of the reprogramming process and to the phenotypic heterogeneity of iPSCs.

Interplay of TRIM28 and DNA methylation in controlling human endogenous retroelements.

Reverse transcription-derived sequences account for at least half of the human genome. Although these retroelements are formidable motors of evolution, they can occasionally cause disease, and accordingly are inactivated during early embryogenesis through epigenetic mechanisms. In the mouse, at least for endogenous retroviruses, important mediators of this process are the tetrapod-specific KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor TRIM28. The present study demonstrates that KRAB/TRIM28-mediated regulation is responsible for controlling a very broad range of human-specific endogenous retroelements (EREs) in human embryonic stem (ES) cells and that it exerts, as a consequence, a marked effect on the transcriptional dynamics of these cells. It further reveals reciprocal dependence between TRIM28 recruitment at specific families of EREs and DNA methylation. It finally points to the importance of persistent TRIM28-mediated control of ERE transcriptional impact beyond their presumed inactivation by DNA methylation.

Global and stage specific patterns of Krüppel-associated-box zinc finger protein gene expression in murine early embryonic cells.

Highly coordinated transcription networks orchestrate the self-renewal of pluripotent stem cell and the earliest steps of mammalian development. KRAB-containing zinc finger proteins represent the largest group of transcription factors encoded by the genomes of higher vertebrates including mice and humans. Together with their putatively universal cofactor KAP1, they have been implicated in events as diverse as the silencing of endogenous retroelements, the maintenance of imprinting and the pluripotent self-renewal of embryonic stem cells, although the genomic targets and specific functions of individual members of this gene family remain largely undefined. Here, we first generated a list of Ensembl-annotated KRAB-containing genes encoding the mouse and human genomes. We then defined the transcription levels of these genes in murine early embryonic cells. We found that the majority of KRAB-ZFP genes are expressed in mouse pluripotent stem cells and other early progenitors. However, we also identified distinctively cell- or stage-specific patterns of expression, some of which are pluripotency-restricted. Finally, we determined that individual KRAB-ZFP genes exhibit highly distinctive modes of expression, even when grouped in genomic clusters, and that these cannot be correlated with the presence of prototypic repressive or activating chromatin marks. These results pave the way to delineating the role of specific KRAB-ZFPs in early embryogenesis.

A KRAB/KAP1-miRNA cascade regulates erythropoiesis through stage-specific control of mitophagy.

During hematopoiesis, lineage- and stage-specific transcription factors work in concert with chromatin modifiers to direct the differentiation of all blood cells. We explored the role of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor KAP1 in this process. In mice, hematopoietic-restricted deletion of Kap1 resulted in severe hypoproliferative anemia. Kap1-deleted erythroblasts failed to induce mitophagy-associated genes and retained mitochondria. This was due to persistent expression of microRNAs (miRNAs) targeting mitophagy transcripts, itself secondary to a lack of repression by stage-specific KRAB-ZFPs. The KRAB/KAP1-miRNA regulatory cascade is evolutionarily conserved, as it also controls mitophagy during human erythropoiesis. Thus, a multilayered transcription regulatory system is present, in which protein- and RNA-based repressors are superimposed in combinatorial fashion to govern the timely triggering of an important differentiation event.

TRIM28 repression of retrotransposon-based enhancers is necessary to preserve transcriptional dynamics in embryonic stem cells.

TRIM28 is critical for the silencing of endogenous retroviruses (ERVs) in embryonic stem (ES) cells. Here, we reveal that an essential impact of this process is the protection of cellular gene expression in early embryos from perturbation by cis-acting activators contained within these retroelements. In TRIM28-depleted ES cells, repressive chromatin marks at ERVs are replaced by histone modifications typical of active enhancers, stimulating transcription of nearby cellular genes, notably those harboring bivalent promoters. Correspondingly, ERV-derived sequences can repress or enhance expression from an adjacent promoter in transgenic embryos depending on their TRIM28 sensitivity in ES cells. TRIM28-mediated control of ERVs is therefore crucial not just to prevent retrotransposition, but more broadly to safeguard the transcriptional dynamics of early embryos.

The KRAB-ZFP/KAP1 system contributes to the early embryonic establishment of site-specific DNA methylation patterns maintained during development.

De novo DNA methylation is an essential aspect of the epigenetic reprogramming that takes place during early development, yet factors responsible for its instatement at particular genomic loci are poorly defined. Here, we demonstrate that the KRAB-ZFP-mediated recruitment of KAP1 to DNA in embryonic stem cells (ESCs) induces cytosine methylation. This process is preceded by H3K9 trimethylation, and genome-wide analyses reveal that it spreads over short distances from KAP1-binding sites so as to involve nearby CpG islands. In sharp contrast, in differentiated cells, KRAB/KAP1-induced heterochromatin formation does not lead to DNA methylation. Correspondingly, the methylation status of CpG islands in the adult mouse liver correlates with their proximity to KAP1-binding sites in ESCs, not in hepatocytes. Therefore, KRAB-ZFPs and their cofactor KAP1 are in part responsible for the establishment during early embryogenesis of site-specific DNA methylation patterns that are maintained through development.

KAP1 regulates gene networks controlling T-cell development and responsiveness.

Chromatin remodeling at specific genomic loci controls lymphoid differentiation. Here, we investigated the role played in this process by Kruppel-associated box (KRAB)-associated protein 1 (KAP1), the universal cofactor of KRAB-zinc finger proteins (ZFPs), a tetrapod-restricted family of transcriptional repressors. T-cell-specific Kap1-deleted mice displayed a significant expansion of immature thymocytes, imbalances in CD4(+)/CD8(+) cell ratios, and altered responses to TCR and TGFß stimulation when compared to littermate KAP1 control mice. Transcriptome and chromatin studies revealed that KAP1 binds T-cell-specific cis-acting regulatory elements marked by the H3K9me3 repressive mark and enriched in Ikaros/NuRD complexes. Also, KAP1 directly controls the expression of several genes involved in TCR and cytokine signaling. Among these, regulation of FoxO1 seems to play a major role in this system. Likely responsible for tethering KAP1 to at least part of its genomic targets, a small number of KRAB-ZFPs are selectively expressed in T-lymphoid cells. These results reveal the so far unsuspected yet important role of KAP1-mediated epigenetic regulation in T-lymphocyte differentiation and activation.