Methods in comparative genomics: genome correspondence, gene identification and regulatory motif discovery.

In Kellis et al. (2003), we reported the genome sequences of S. paradoxus, S. mikatae, and S. bayanus and compared these three yeast species to their close relative, S. cerevisiae. Genomewide comparative analysis allowed the identification of functionally important sequences, both coding and noncoding. In this companion paper we describe the mathematical and algorithmic results underpinning the analysis of these genomes. (1) We present methods for the automatic determination of genome correspondence. The algorithms enabled the automatic identification of orthologs for more than 90% of genes and intergenic regions across the four species despite the large number of duplicated genes in the yeast genome. The remaining ambiguities in the gene correspondence revealed recent gene family expansions in regions of rapid genomic change. (2) We present methods for the identification of protein-coding genes based on their patterns of nucleotide conservation across related species. We observed the pressure to conserve the reading frame of functional proteins and developed a test for gene identification with high sensitivity and specificity. We used this test to revisit the genome of S. cerevisiae, reducing the overall gene count by 500 genes (10% of previously annotated genes) and refining the gene structure of hundreds of genes. (3) We present novel methods for the systematic de novo identification of regulatory motifs. The methods do not rely on previous knowledge of gene function and in that way differ from the current literature on computational motif discovery. Based on genomewide conservation patterns of known motifs, we developed three conservation criteria that we used to discover novel motifs. We used an enumeration approach to select strongly conserved motif cores, which we extended and collapsed into a small number of candidate regulatory motifs. These include most previously known regulatory motifs as well as several noteworthy novel motifs. The majority of discovered motifs are enriched in functionally related genes, allowing us to infer a candidate function for novel motifs. Our results demonstrate the power of comparative genomics to further our understanding of any species. Our methods are validated by the extensive experimental knowledge in yeast and will be invaluable in the study of complex genomes like that of the human.     

Comparative 3D Genome Structure Analysis of the Fission and the Budding Yeast

We studied the 3D structural organization of the fission yeast genome, which emerges from the tethering of heterochromatic regions in otherwise randomly configured chromosomes represented as flexible polymer chains in an nuclear environment. This model is sufficient to explain in a statistical manner many experimentally determined distinctive features of the fission yeast genome, including chromatin interaction patterns from Hi-C experiments and the co-locations of functionally related and co-expressed genes, such as genes expressed by Pol-III. Our findings demonstrate that some previously described structure-function correlations can be explained as a consequence of random chromatin collisions driven by a few geometric constraints (mainly due to centromere-SPB and telomere-NE tethering) combined with the specific gene locations in the chromosome sequence. We also performed a comparative analysis between the fission and budding yeast genome structures, for which we previously detected a similar organizing principle. However, due to the different chromosome sizes and numbers, substantial differences are observed in the 3D structural genome organization between the two species, most notably in the nuclear locations of orthologous genes, and the extent of nuclear territories for genes and chromosomes. However, despite those differences, remarkably, functional similarities are maintained, which is evident when comparing spatial clustering of functionally related genes in both yeasts. Functionally related genes show a similar spatial clustering behavior in both yeasts, even though their nuclear locations are largely different between the yeast species.     

The regulatory content of intergenic DNA shapes genome architecture

Background  

Factors affecting the organization and spacing of functionally unrelated genes in metazoan genomes are not well understood. Because of the vast size of a typical metazoan genome compared to known regulatory and protein-coding regions, functional DNA is generally considered to have a negligible impact on gene spacing and genome organization. In particular, it has been impossible to estimate the global impact, if any, of regulatory elements on genome architecture.     

Global chromatin domain organization of the Drosophila genome

In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 50% of all fly genes are organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and tend to have similar biological functions. Furthermore, during evolution fewer chromosomal rearrangements occur inside chromatin domains than outside domains. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution.     

Relaxed 3D genome conformation facilitates the pluripotent to totipotent-like state transition in embryonic stem cells

The 3D genome organization is crucial for gene regulation. Although recent studies have revealed a uniquely relaxed genome conformation in totipotent early blastomeres of both fertilized and cloned embryos, how weakened higher-order chromatin structure is functionally linked to totipotency acquisition remains elusive. Using low-input Hi-C, ATAC-seq and ChIP-seq, we systematically examined the dynamics of 3D genome and epigenome during pluripotent to totipotent-like state transition in mouse embryonic stem cells (ESCs). The spontaneously converted 2-cell-embryo-like cells (2CLCs) exhibited more relaxed chromatin architecture compared to ESCs, including global weakening of both enhancer-promoter interactions and TAD insulation. While the former correlated with inactivation of ESC enhancers and down-regulation of pluripotent genes, the latter might facilitate contacts between the putative new enhancers arising in 2CLCs and neighboring 2C genes. Importantly, disruption of chromatin organization by depleting CTCF or the cohesin complex promoted the ESC to 2CLC transition. Our results thus establish a critical role of 3D genome organization in totipotency acquisition.