Chromosome Diversity and Evolution in Helicoide a (Gastropoda: Stylommatophora): A Synthesis from Original and Literature Data

We performed a molecular and a comparative cytogenetic analysis on different Helicoidea species and a review of all the available chromosome data on the superfamily to provide an updated assessment of its karyological diversity. Standard karyotyping, banding techniques, and Fluorescence in situ hybridization of Nucleolus Organizer Region loci (NOR-FISH) were performed on fifteen species of three families: two Geomitridae, four Hygromiidae and nine Helicidae. The karyotypes of the studied species varied from 2n = 44 to 2n = 60, highlighting a high karyological diversity. NORs were on a single chromosome pair in Cernuella virgata and on multiple pairs in four Helicidae, representing ancestral and derived conditions, respectively. Heterochromatic C-bands were found on pericentromeric regions of few chromosomes, being Q- and 4′,6-diamidino-2-phenylindole (DAPI) negative. NOR-associated heterochromatin was C-banding and chromomycin A3 (CMA3) positive. Considering the available karyological evidence on Helicoidea and superimposing the chromosome data gathered from different sources on available phylogenetic inferences, we describe a karyotype of 2n = 60 with all biarmed elements as the ancestral state in the superfamily. From this condition, an accumulation of chromosome translocations led to karyotypes with a lower chromosome number (2n = 50–44). This process occurred independently in different lineages, while an augment of the chromosome number was detectable in Polygyridae. Chromosome inversions were also relevant chromosome rearrangements in Helicoidea, leading to the formation of telocentric elements in karyotypes with a relatively low chromosome count.

A universal karyotypic system for hexaploid and diploid Avena species brings oat cytogenetics into the genomics era

Background The identification of chromosomes among Avena species have been studied by C-banding and in situ hybridization. However, the complicated results from several cytogenetic nomenclatures for identifying oat chromosomes are often contradictory. A universal karyotyping nomenclature system for precise chromosome identification and comparative evolutionary studies would be essential for genus Avena based on the recently released genome sequences of hexaploid and diploid Avena species. Results Tandem repetitive sequences were predicted and physically located on chromosomal regions of the released Avena sativa OT3098 genome assembly v1. Eight new oligonucleotide (oligo) probes for sequential fluorescence in situ hybridization (FISH) were designed and then applied for chromosome karyotyping on mitotic metaphase spreads of A. brevis, A. nuda, A. wiestii, A. ventricosa, A. fatua, and A. sativa species. We established a high-resolution standard karyotype of A. sativa based on the distinct FISH signals of multiple oligo probes. FISH painting with bulked oligos, based on wheat-barley collinear regions, was used to validate the linkage group assignment for individual A. sativa chromosomes. We integrated our new Oligo-FISH based karyotype system with earlier karyotype nomenclatures through sequential C-banding and FISH methods, then subsequently determined the precise breakage points of some chromosome translocations in A. sativa. Conclusions This new universal chromosome identification system will be a powerful tool for describing the genetic diversity, chromosomal rearrangements and evolutionary relationships among Avena species by comparative cytogenetic and genomic approaches.

Classical, Molecular, and Genomic Cytogenetics of the Pig, a Clinical Perspective

The chromosomes of the domestic pig (Sus scrofa domesticus) are known to be prone to reciprocal chromosome translocations and other balanced chromosome rearrangements with concomitant fertility impairment of carriers. In response to the remarkable prevalence of chromosome rearrangements in swine herds, clinical cytogenetics laboratories have been established in several countries in order to screen young boars for chromosome rearrangements prior to service. At present, clinical cytogenetics laboratories typically apply classical cytogenetics techniques such as giemsa-trypsin (GTG)-banding to produce high-quality karyotypes and reveal large-scale chromosome ectopic exchanges. Further refinements to clinical cytogenetics practices have led to the implementation of molecular cytogenetics techniques such as fluorescent in-situ hybridization (FISH), allowing for rearrangements to be visualized and breakpoints refined using fluorescently labelled painting probes. The next-generation of clinical cytogenetics include the implementation of DNA microarrays, and next-generation sequencing (NGS) technologies such as DNA sequencing to better explore tentative genome architecture changes. The implementation of these cytogenomics techniques allow the genomes of rearrangement carriers to be deciphered at the highest resolution, allowing rearrangements to be detected; breakpoints to be delineated; and, most importantly, potential gene implications of those chromosome rearrangements to be interrogated. Clinical cytogenetics has become an integral tool in the livestock industry, identifying rearrangements and allowing breeders to make informed breeding decisions.

RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair

R-loops, consisting of ssDNA and DNA-RNA hybrids, are potentially vulnerable unless they are appropriately processed. Recent evidence suggests that R-loops can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. Yet, how the vulnerability of R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops and chromosome translocations and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end-joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

Structure, function and inhibition of critical protein–protein interactions involving mixed lineage leukemia 1 and its fusion oncoproteins

Mixed lineage leukemia 1 (MLL1, also known as MLL or KMT2A) is an important transcription factor and histone-H3 lysine-4 (H3K4) methyltransferase. It is a master regulator for transcription of important genes (e.g., Hox genes) for embryonic development and hematopoiesis. However, it is largely dispensable in matured cells. Dysregulation of MLL1 leads to overexpression of certain Hox genes and eventually leukemia initiation. Chromosome translocations involving MLL1 cause ~ 75% of acute leukemia in infants and 5–10% in children and adults with a poor prognosis. Targeted therapeutics against oncogenic fusion MLL1 (onco-MLL1) are therefore needed. Onco-MLL1 consists of the N-terminal DNA-interacting domains of MLL1 fused with one of > 70 fusion partners, among which transcription cofactors AF4, AF9 and its paralog ENL, and ELL are the most frequent. Wild-type (WT)- and onco-MLL1 involve numerous protein–protein interactions (PPI), which play critical roles in regulating gene expression in normal physiology and leukemia. Moreover, WT-MLL1 has been found to be essential for MLL1-rearranged (MLL1-r) leukemia. Rigorous studies of such PPIs have been performed and much progress has been achieved in understanding their structures, structure–function relationships and the mechanisms for activating gene transcription as well as leukemic transformation. Inhibition of several critical PPIs by peptides, peptidomimetic or small-molecule compounds has been explored as a therapeutic approach for MLL1-r leukemia. This review summarizes the biological functions, biochemistry, structure and inhibition of the critical PPIs involving MLL1 and its fusion partner proteins. In addition, challenges and perspectives of drug discovery targeting these PPIs for the treatment of MLL1-r leukemia are discussed.

CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy

Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.

A universal karyotypic system for hexaploid and diploid Avena species brings oat cytogenetics into the genomics era

Background The identification of chromosomes among Avena species have been studied by C-banding and in situ hybridization. However, the complicated results from several cytogenetic nomenclatures for identifying oat chromosomes are often contradictory. A universal karyotyping nomenclature system for precise chromosome identification and comparative evolutionary studies would be essential for genus Avena based on the recently released genome sequences of hexaploid and diploid Avena species. Results Tandem repetitive sequences were predicted and physically located on chromosomal regions of the Avena sativa genomes. Thirteen new oligonucleotide (oligo) probes for sequential fluorescence in situ hybridization (FISH) were designed and then applied for chromosome karyotyping on mitotic metaphase spreads of eleven hexaploid and diploid Avena accessions. We established a high resolution standard karyotype of A. sativa based on the distinct FISH signals of multiple oligo probes. FISH painting with bulked oligos, based on wheat-barley collinear regions, was used to validate the linkage group assignment for individual A. sativa chromosomes. We integrated our new Oligo-FISH based karyotype system with earlier karyotype nomenclatures through sequential C-banding and FISH methods, then subsequently determined the precise breakage points of some chromosome translocations. Conclusion This new universal chromosome identification system will be a powerful tool for describing the genetic diversity, chromosomal rearrangements and evolutionary relationships among Avena species by comparative cytogenetic and genomic approaches.

Challenges and Opportunities in NUT Carcinoma Research

NUT carcinoma (NC) is a type of aggressive cancer driven by chromosome translocations. Fusion genes between a DNA-binding protein, such as bromodomain and extraterminal domain (BET) proteins, and the testis-specific protein NUTM1 generated by these translocations drive the formation of NC. NC can develop in very young children without significant accumulation of somatic mutations, presenting a relatively clean model to study the genetic etiology of oncogenesis. However, after 20 years of research, a few challenging questions still remain for understanding the mechanism and developing therapeutics for NC. In this short review, we first briefly summarize the current knowledge regarding the molecular mechanism and targeted therapy development of NC. We then raise three challenging questions: (1) What is the cell of origin of NC? (2) How does the germline analogous epigenetic reprogramming process driven by the BET-NUTM1 fusion proteins cause NC? and (3) How will BET-NUTM1 targeted therapies be developed? We propose that with the unprecedented technological advancements in genome editing, animal models, stem cell biology, organoids, and chemical biology, we have unique opportunities to address these challenges.

Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.