Fine-Mapping Complex Inversion Breakpoints and Investigating Somatic Pairing in the Anopheles gambiae Species Complex Using Proximity-Ligation Sequencing

Chromosomal inversions are fundamental drivers of genome evolution. In the main Afrotropical malaria vector species, belonging to the Anopheles gambiae species complex, inversions play an important role in local adaptation and have a rich history of cytological study. Despite the importance and ubiquity of some chromosomal inversions across the species complex, inversion breakpoints are often challenging to map molecularly due to the presence of large repetitive regions. Here, we develop an approach that uses Hi-C sequencing data to molecularly fine-map the breakpoints of inversions. We demonstrate that this approach is robust and likely to be widely applicable for both identification and fine-mapping inversion breakpoints in species whose inversions have heretofore been challenging to characterize. We apply our method to interrogate the previously unknown inversion breakpoints of 2Rbc and 2Rd in An. coluzzii. We found that inversion breakpoints occur in large repetitive regions, and, strikingly, among three inversions analyzed, two breakpoints appear to be reused in two separate inversions. These breakpoint-adjacent regions are strongly enriched for the presence of a 30 bp satellite repeat sequence. Because low frequency inversion breakpoints are not correlated with genomic regions containing this satellite, we suggest that interrupting this particular repeat may result in arrangements with higher relative fitness. Additionally, we use heterozygous individuals to quantitatively investigate the impacts of somatic pairing in the regions immediately surrounding inversion breakpoints. Finally, we discuss important considerations for possible applications of this approach for inversion breakpoint identification in a range of organisms.

Genes in Tumor Formation

With the definition of four gene classes, all differences between tumor cells and normal cells can be explained. Proliferative mutations induce a shortcut, forcing the cell to divide. They allow replication without control, induce somatic pairing defects of chromosomes and genome instability. Intact Tumor Supressors or mutant Switch Functions can inhibit this process. Oncogene mutations optimize the growth of the cells.

Recurrent Losses and Rapid Evolution of the Condensin II Complex in Insects

Condensins play a crucial role in the organization of genetic material by compacting and disentangling chromosomes. Based on studies in a few model organisms, the condensins I and II complexes are considered to have distinct functions, with the condensin II complex playing a role in meiosis and somatic pairing of homologous chromosomes in Drosophila. Intriguingly, the Cap-G2 subunit of condensin II is absent in Drosophila melanogaster, and this loss may be related to the high levels of chromosome pairing seen in flies. Here, we find that all three non-SMC subunits of condensin II (Cap-G2, Cap-D3, and Cap-H2) have been repeatedly and independently lost in taxa representing multiple insect orders, with some taxa lacking all three. We also find that all non-Dipteran insects display near-uniform low-pairing levels regardless of their condensin II complex composition, suggesting that some key aspects of genome organization are robust to condensin II subunit losses. Finally, we observe consistent signatures of positive selection in condensin subunits across flies and mammals. These findings suggest that these ancient complexes are far more evolutionarily labile than previously suspected, and are at the crossroads of several forms of genomic conflicts. Our results raise fundamental questions about the specific functions of the two condensin complexes in taxa that have experienced subunit losses, and open the door to further investigations to elucidate the diversity of molecular mechanisms that underlie genome organization across various life forms.

Fine-mapping complex inversion breakpoints and investigating somatic pairing in the Anopheles gambiae species complex using proximity-ligation sequencing

Chromosomal inversions are fundamental drivers of genome evolution. In the main afro-tropical malaria vector species, belonging to the Anopheles gambiae species complex, inversions play an important role in local adaptation and have a rich history of cytological study. Despite the importance and ubiquity of some chromosomal inversions across the species complex, inversion breakpoints are often challenging to map molecularly due to the presence of large repetitive regions. Here, we develop an approach that uses Hi-C sequencing data to molecularly fine-map the breakpoints of inversions 2Rbc and 2Rd in A. coluzzii. We found that inversion breakpoints occur in large repetitive regions, and strikingly among three inversions analyzed, two breakpoints appear to be reused in two separate inversions. Additionally, we use heterozygous individuals to quantitatively investigate somatic pairing disruption in the regions immediately surrounding inversion breakpoints, and we find that pairing disruption is undetectable beyond approximately 250 Kb from the inversion breakpoints.

Maintenance of the active and inactive states

The maintenance of a gene in an active or inactive state is carried out by epigenetic modifications of the histones and of the DNA itself. Two major classes of complexes (PRC1 and PRC2), containing Polycomb group (PcG) proteins mediate transcriptional repression. PRC2 trimethylates histone H3 at lysine 27, a modification that attracts PRC1 leading to the ubiquitination of histone H2A. Variant PRC1 complexes can be targeted first, and mono-ubiquitinated histone H2A recruits PRC2 complexes that serve as the target for canonical PRC1 complexes. PRC2 can be targeted to sites of repression by associating with long non-coding RNAs. Trithorax group (TrxG) proteins form complexes that counteract PcG-mediated repression. Some subunits of these complexes maintain and enhance transcription by carrying out different lysine methylations (H3K4me, H3K36me and H3K79me) that are associated with active gene function; other subunits remodel chromatin by displacing and repositioning nucleosomes. Additional effects on transcription are transvections, whereby somatic pairing allows the regulatory region of one allele of a gene to influence the activity of the promoter of the allele on the homologous chromosome

Condensins promote chromosome individualization and segregation during mitosis, meiosis, and amitosis in Tetrahymena thermophila

Condensin is a protein complex with diverse functions in chromatin packaging and chromosome condensation and segregation. We studied condensin in the evolutionarily distant protist model Tetrahymena, which features noncanonical nuclear organization and divisions. In Tetrahymena, the germline and soma are partitioned into two different nuclei within a single cell. Consistent with their functional specializations in sexual reproduction and gene expression, condensins of the germline nucleus and the polyploid somatic nucleus are composed of different subunits. Mitosis and meiosis of the germline nucleus and amitotic division of the somatic nucleus are all dependent on condensins. In condensin-depleted cells, a chromosome condensation defect was most striking at meiotic metaphase, when Tetrahymena chromosomes are normally most densely packaged. Live imaging of meiotic divisions in condensin-depleted cells showed repeated nuclear stretching and contraction as the chromosomes failed to separate. Condensin depletion also fundamentally altered chromosome arrangement in the polyploid somatic nucleus: multiple copies of homologous chromosomes tended to cluster, consistent with a previous model of condensin suppressing default somatic pairing. We propose that failure to form discrete chromosome territories is the common cause of the defects observed in the absence of condensins.