Concepts and Consequences of a Core Gut Microbiota for Animal Growth and Development

Animal microbiomes are occasionally considered as an extension of host anatomy, physiology, and even their genomic architecture. Their compositions encompass variable and constant portions when examined across multiple hosts. The latter, termed the core microbiome, is viewed as more accommodated to its host environment and suggested to benefit host fitness. Nevertheless, discrepancies in its definitions, characteristics, and importance to its hosts exist across studies. We survey studies that characterize the core microbiome, detail its current definitions and available methods to identify it, and emphasize the crucial need to upgrade and standardize the methodologies among studies. We highlight ruminants as a case study and discuss the link between the core microbiome and host physiology and genetics, as well as potential factors that shape it. We conclude with main directives of action to better understand the host–core microbiome axis and acquire the necessary insights into its controlled modulation. Expected final online publication date for the Annual Review of Animal Biosciences, Volume 10 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

A chromosomal inversion may facilitate adaptation despite periodic gene flow in a freshwater fish

Genomic architecture, such as chromosomal inversions, may play an important role in facilitating adaptation despite opportunities for gene flow. One system where chromosomal inversions may be important for eco-evolutionary dynamics are in freshwater fish, which often live in heterogenous environments characterized by varying levels of connectivity and varying opportunities for gene flow. In the present study, reduced representation sequencing was used to study possible adaptation in n=345 walleye (Sander vitreus) from three North American waterbodies: Cedar Bluff Reservoir (Kansas, USA), Lake Manitoba (Manitoba, Canada), and Lake Winnipeg (Manitoba, Canada). Haplotype and outlier-based tests revealed a putative chromosomal inversion that contained three expressed genes and was nearly fixed for alternate genotypes in each Canadian lake. These patterns exist despite several opportunities for gene flow between these proximate Canadian lakes, suggesting that the inversion may be important for facilitating adaptative divergence between the two lakes despite gene flow. Our study illuminates the importance of genomic architecture for facilitating local adaptation in freshwater fish and provides additional evidence that inversions may facilitate local adaptation in many organisms that inhabit connected but heterogenous environments.

Genomic architecture controls spatial structuring in Amazonian birds

Large rivers are ubiquitously invoked to explain the distributional limits and speciation of the Amazon Basin's mega-diversity. However, inferences on the spatial and temporal origins of Amazonian species have narrowly focused on evolutionary neutral models, ignoring the potential role of natural selection and intrinsic genomic processes known to produce heterogeneity in differentiation across the genome. To test how these factors may influence evolutionary inferences across multiple taxa, we sequenced whole genomes of populations for three bird species that co-occur in southeastern Amazonian and exhibit different life histories linked to their propensity to maintain gene flow across the landscape. We found that phylogenetic relationships within species and demographic parameters varied across the genome in predictable ways. Genetic diversity was positively associated with recombination rate and negatively associated with the species tree topology weight. Gene flow was less pervasive in regions of low recombination, making these windows more suitable for commonly used phylogenetic methods that assume a bifurcating-branching model. To corroborate that these associations were attributable to selection, we modeled the signature of adaptive alleles across the genome taking demographic history into account, and found that on average 31.6 percent of the genome showed high probability for patterns consistent with selective sweeps and linked selection directly affecting the estimation of evolutionary parameters. By implementing a comparative genomic approach we were able to disentangle the effects of intrinsic genomic characteristics and selection from the neutral processes and show how speciation hypotheses are sensitive to genomic architecture.

A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers

Background The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. Results This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. Conclusions The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Changes in Clonal Architecture Inform MPN Disease Course in Advance of Phenotypic Manifestations

Background Disease progression of chronic phase myeloproliferative neoplasms (MPNs) to myelofibrosis (MF) and acute myeloid leukaemia (AML) occurs in 5-10% of patients and is associated with poor prognosis. Disease classification currently uses histology, clinical and laboratory parameters and does not incorporate genomic parameters. Whilst disease evolution can be associated with genetic events , little is known about the timing of additional genetic events or clonal dynamics prior to phenotypic change. Here, we studied the longitudinal clonal dynamics and genomic architecture of MPN and related these to clinical parameters and disease status. Method Longitudinal whole-genome sequencing (WGS) was undertaken in 31 MPN patients with a median interval between WGS of 10.5 years. Tools to identify somatic mutations despite the presence of tumour-in-normal contamination were developed. Clonal trajectories of disease were mapped by reconstructing the subclonal genomic architecture at each timepoint. Mutation burden within individual clones was used to infer the timing of acquisition of clones. Laboratory and clinical information including serial blood count parameters were used to correlate changes in underlying clonal architecture with clinically apparent changes in disease phenotype. Results Disease transformation to MF or AML occurred in 18 patients between sequencing timepoints. In all patients that progressed to AML, clonal evolution was observed with the emergence and expansion of subclones that dominated the clonal landscape at the time of AML. Two patients developed JAK2-negative AML with the genomic features of de novo AML arising independently of the MPN clone. In contrast, progression to MF was not always associated with genomic evolution between sequencing timepoints. Twelve patients had clinically stable disease between sequencing timepoints (median sampling interval 11 years), Stable MPN clones with no evidence of clonal evolution were observed in 6 patients, and these patients remained clinically stable during extended follow up (median 8 years) beyond the last sequencing timepoint. In contrast, the remaining 6 patients had evidence of clonal evolution with a new clonal expansion by the second WGS timepoint despite still having a chronic phase MPN diagnosis. All 6 of these patients subsequently progressed during extended follow up (median 30 months, range 17-55 months), suggesting that genomic progression predates clinical progression by several years. Blood count changes were found to be a late manifestation of clonal evolution. One patient with WHO-defined MPN was not found to have evidence of a clear clonal expansion harbouring a driver mutation. WGS of single cell derived haematopoietic colonies was used to reconstruct the phylogenetic tree of haematopoiesis and did not reveal evidence of an underlying clonal disorder. Using the burden of somatic mutations, we found that in the majority of patients, we were able to time the chronic MPN clones to have been acquired within the first half of the patients' lives. In several patients, clones that had expanded at the time of progression were detectable at the first timepoint and were timed to before diagnosis of their MPN. Conclusions Changes in the underlying clonal composition of MPN predate clinical recognition of a change in disease status by many years. In such patients, the drivers of subsequent disease transformation may have already been acquired years in advance of the initial MPN diagnosis. Serial genetic monitoring of mutant clones in MPN patients could allow prediction of the future disease course of MPN patients and offer opportunities for earlier intervention prior to deterioration in clinical status. Disclosures Campbell: Mu Genomics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Intronic Architecture Links DNA Methylation to Gene Expression and Helps Drive Subtype-Specific Transcriptional Landscapes in DNMT3A- and IDH1/2-Mutant Acute Myeloid Leukemias (AML)

Mutations in DNMT3A and IDH1/2 are each found in ~20% of AML patients. 10-15% of AMLs carry mutations in both genes (herein, double mutants), resulting in a unique methylation landscape and upregulation of a signaling signature. In murine models, the presence of both mutations results in greater leukemogenic potential. However, the specific mechanism through which DNA methylation (DNAme) drives gene expression programs in double mutants remains unclear. We hypothesized that the link between DNAme and gene expression would be explained by more than simple proximity, and that the genomic architecture of the affected genes would play a key role. To test this, we first performed an unbiased correlation analysis of gene expression with DNAme at all CpG sites (mCs) located within the same topologically associated domain (TAD). We identified 406 genes with significant (FDR> 5% and absolute rho > 0.5) expression-methylation correlations with mCs proximal to the respective genes (herein the E-M gene set). In addition, another 2,088 genes (the L E-M set) were identified with long-range correlations (>2Kb from the gene body) with mCs in the respective TAD (median distance = 451 Kb). As a set, the E-M genes significantly overlapped (P < 10 -2) with genes identified as either differentially expressed (DE; n=890) or differentially methylated (DM; n= 4,006) between IDH1/2 and DNMT3A mutant AMLs. Notably, a simple overlap analysis of DE and DM genes showed no significant overlap between them, thus demonstrating that correlation analysis performed better in bridging the epigenome with the transcriptome. DAVID and Gene Set Enrichment Analysis on the genes ranked by correlation strength revealed that signaling, fructose and lipid metabolism pathways are enriched in the E-M gene set (FDR < 5%) but not in the L E-M set. Analysis of transcription factor (TF) binding profiles did not reveal a common set of TF(s) binding to the mCs proximal to the genes of the identified pathways. Thus, we hypothesized that the E-M genes have other structural characteristics in common that drive regulation through DNAme, for which we focused on their genomic architecture. This analysis revealed that introns of genes in both the E-M and L E-M sets are significantly denser in Mammalian Interspersed Repeats (MIR) than expected by random chance (P < 10 -2). Additionally, E-M genes were significantly sparser in endogenous retroviruses (ERVL) and primate-specific Alu elements. mCs with significant correlations were also enriched at MIR and depleted from Alu elements (P < 10 -2), thus creating a regulatory network between mCs and genes with MIR sequences as the common denominator. Genome-wide, CpGs within retrotransposons that were differentially methylated among the three AML subtypes were enriched at enhancer regions or coding genes, particularly the E-M genes. Furthermore, the Dnmt3a knock-out (KO) or Idh2 R140Q knock-in mouse models display the same architectural biases at genes correlated with DNAme as the E-M genes identified in the human samples. Next, we sought to put our findings in the context of normal hematopoiesis and found that genes upregulated during normal hematopoietic differentiation are significantly denser in MIR elements and sparser of Alu elements than expected (P < 10 -2). Alignment of the leukemic samples within normal differentiation trajectories revealed that double mutants resembled differentiated cell types more closely, while DNMT3A and IDH1/2 single mutants resembled hematopoietic stem cells. The E-M and L E-M sets significantly overlapped (P < 10 -2) with those genes upregulated during myeloid but not erythroid or lymphoid differentiation, demonstrating that genes regulated by DNAme are at the core of the biology of these AMLs. In summary, our integrative work sheds light on a novel mechanism in which epigenetic modifications can regulate gene expression through MIR sequences within introns of hematopoietic-relevant genes and we posit that overlapping CpG dinucleotides may act as recruiters or substrates of DNMT3A and/or TET proteins. This mechanism seems to also be active in normal hematopoiesis and thus, is hijacked by leukemic cells. Therefore, our findings identify retrotransposons as a missing link in the understanding of epigenetic regulation of gene expression, reveal a previously uncharacterized role for these elements in leukemogenesis, and point to different cells of origin for each AML subtype. Disclosures No relevant conflicts of interest to declare.

Patterns of Genomic Instability in Interspecific Yeast Hybrids With Diverse Ancestries

The genomes of hybrids often show substantial deviations from the features of the parent genomes, including genomic instabilities characterized by chromosomal rearrangements, gains, and losses. This plastic genomic architecture generates phenotypic diversity, potentially giving hybrids access to new ecological niches. It is however unclear if there are any generalizable patterns and predictability in the type and prevalence of genomic variation and instability across hybrids with different genetic and ecological backgrounds. Here, we analyzed the genomic architecture of 204 interspecific Saccharomyces yeast hybrids isolated from natural, industrial fermentation, clinical, and laboratory environments. Synchronous mapping to all eight putative parental species showed significant variation in read depth indicating frequent aneuploidy, affecting 44% of all hybrid genomes and particularly smaller chromosomes. Early generation hybrids with largely equal genomic content from both parent species were more likely to contain aneuploidies than introgressed genomes with an older hybridization history, which presumably stabilized the genome. Shared k-mer analysis showed that the degree of genomic diversity and variability varied among hybrids with different parent species. Interestingly, more genetically distant crosses produced more similar hybrid genomes, which may be a result of stronger negative epistasis at larger genomic divergence, putting constraints on hybridization outcomes. Mitochondrial genomes were typically inherited from the species also contributing the majority nuclear genome, but there were clear exceptions to this rule. Together, we find reliable genomic predictors of instability in hybrids, but also report interesting cross- and environment-specific idiosyncrasies. Our results are an important step in understanding the factors shaping divergent hybrid genomes and their role in adaptive evolution.

Whole genome copy number analyses reveal a highly aberrant genome in TP53 mutant lung adenocarcinoma tumors

Background Genetic alterations are common in non-small cell lung cancer (NSCLC), and DNA mutations and translocations are targets for therapy. Copy number aberrations occur frequently in NSCLC tumors and may influence gene expression and further alter signaling pathways. In this study we aimed to characterize the genomic architecture of NSCLC tumors and to identify genomic differences between tumors stratified by histology and mutation status. Furthermore, we sought to integrate DNA copy number data with mRNA expression to find genes with expression putatively regulated by copy number aberrations and the oncogenic pathways associated with these affected genes. Methods Copy number data were obtained from 190 resected early-stage NSCLC tumors and gene expression data were available from 113 of the adenocarcinomas. Clinical and histopathological data were known, and EGFR-, KRAS- and TP53 mutation status was determined. Allele-specific copy number profiles were calculated using ASCAT, and regional copy number aberration were subsequently obtained and analyzed jointly with the gene expression data. Results The NSCLC tumors tissue displayed overall complex DNA copy number profiles with numerous recurrent aberrations. Despite histological differences, tissue samples from squamous cell carcinomas and adenocarcinomas had remarkably similar copy number patterns. The TP53-mutated lung adenocarcinomas displayed a highly aberrant genome, with significantly altered copy number profiles including gains, losses and focal complex events. The EGFR-mutant lung adenocarcinomas had specific arm-wise aberrations particularly at chromosome7p and 9q. A large number of genes displayed correlation between copy number and expression level, and the PI(3)K-mTOR pathway was highly enriched for such genes. Conclusions The genomic architecture in NSCLC tumors is complex, and particularly TP53-mutated lung adenocarcinomas displayed highly aberrant copy number profiles. We suggest to always include TP53-mutation status when studying copy number aberrations in NSCLC tumors. Copy number may further impact gene expression and alter cellular signaling pathways.