Chromosome Maintenance Research Articles

Loop-extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex.

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC-family Wadjet complexes structurally resemble the widespread MukBEF but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves plasmid DNA; however, the molecular mechanism underlying plasmid recognition remains unclear. Here, we use invitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric loop extruder that simultaneously reels in DNA from both sides of a loop and that this activity requires a dimeric JetABC supercomplex. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44-kb-pair plasmid, stalls, and cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across all kingdoms of life.

Loop Extrusion Machinery Impairments in Models and Disease.

Structural maintenance of chromosomes (SMC) complexes play a crucial role in organizing the three-dimensional structure of chromatin, facilitating key processes such as gene regulation, DNA repair, and chromosome segregation. This review explores the molecular mechanisms and biological significance of SMC-mediated loop extrusion complexes, including cohesin, condensins, and SMC5/6, focusing on their structure, their dynamic function during the cell cycle, and their impact on chromatin architecture. We discuss the implications of impairments in loop extrusion machinery as observed in experimental models and human diseases. Mutations affecting these complexes are linked to various developmental disorders and cancer, highlighting their importance in genome stability and transcriptional regulation. Advances in model systems and genomic techniques have provided deeper insights into the pathological roles of SMC complex dysfunction, offering potential therapeutic avenues for associated diseases.

A novel regulatory motif at the hinge dimer interface of the MksB mediates dimerization and DNA binding activity

The Structural Maintenance of Chromosome (SMC) protein is essential for various cellular processes, including chromosome organization, DNA repair, and genome stability. MksB, an alternative SMC protein present in prokaryotes, comprises a hinge dimerization domain and an ABC ATPase head domain linked by a coiled-coil arm. While hinge dimerization in bacterial and eukaryotic SMCs is attributed to conserved glycines, our study unveils the critical role of a novel KDDR motif located at a loop near the hinge dimer interface in Mycobacterium smegmatis MksB (MsMksB). We demonstrate the regulatory role of this motif in MsMksB dimerization and DNA binding activity. The K600D mutation in the KDDR motif induces MsMksB dimer-to-monomer conversion, highlighting the significance of this motif in MsMksB dimerization. Mass spectrometry-based mapping of the DNA binding site revealed the lysine's involvement in protein-DNA interaction. Monomers of the hinge domain lose DNA binding activity, and MsMksB single-arm mutants exhibit reduced DNA binding and ATPase activity, underscoring the importance of hinge-mediated dimerization in MsMksB function. Notably, the R603D mutant retains dimerization but shows compromised ATPase and DNA binding activities. Mutants with defective ATPase activity exhibit impaired DNA condensation in vivo. These findings provide novel regulatory insight into the mechanism of MksB dimerization and DNA binding, uncovering the fundamental processes of chromosome condensation and segregation.

Deciphering Plasmodium Condensin Core Subunits of Structural Maintenance of Chromosomes 2 (SMC2) as a Putative Drug Target for Antimalarial Drug

Background: The structural maintenance of chromosomes (SMC) proteins plays a noteworthy role in chromosome dynamics. Several recent studies reported that the condensin core subunits of structural maintenance of chromosomes 2 (SMC2) play important roles in the atypical mitosis of the Plasmodium life cycle and may perform different functions during different proliferative stages. For eukaryotes, the structural maintenance of chromosomes (SMC) proteins are divided into six subunits and form three heterodimers of structural maintenance of chromosomes (SMC1/3) cohesion complex, structural maintenance of chromosomes (SMC2/4) condensin complex, and structural maintenance of chromosomes (SMC5/6) complex for chromosome cohesion, condensation, and DNA damage repair, respectively. Objective: The objective of this study was to investigate the structural maintenance of chromosomes 2 (SMC2) protein of P. falciparum as a putative drug target of malariacausing Plasmodium falciparum. Methods: In this study, we investigated the structural maintenance of chromosomes 2 (SMC2) protein of P. falciparum as a putative drug target of malaria-causing P. falciparum by using in-silico approaches like Homology modeling, in-silico evaluation of the modeled structure, molecular docking study to investigate the interaction of receptor-ligand, and molecular dynamic simulation study with MM calculation. Results: We reported the structural maintenance of chromosomes 2 (SMC2) protein of P. falciparum as a potent drug target that can pave the way for novel drug discovery to mitigate malaria. Conclusion: In-silico-based studies play a significant role in understanding any protein for potential drug development.

SMC5/6-Mediated Transcriptional Regulation of Hepatitis B Virus and Its Therapeutic Potential.

Cells have developed various mechanisms to counteract viral infections. In an evolutionary arms race, cells mobilize cellular restriction factors to fight off viruses, targeted by viral factors to facilitate their own replication. The hepatitis B virus (HBV) is a small dsDNA virus that causes acute and chronic infections of the liver. Its genome persists in the nuclei of infected hepatocytes as a covalently closed circular DNA (cccDNA) minichromosome, thus building up an episomal persistence reservoir. The chromosomal maintenance complex SMC5/6 acts as a restriction factor hindering cccDNA transcription, whereas the viral regulatory protein HBx targets SMC5/6 for proteasomal degradation, thus relieving transcriptional suppression of the HBV minichromosome. To date, no curative therapies are available for chronic HBV carriers. Knowledge of the factors regulating the cccDNA and the development of therapies involving silencing the minichromosome or specifically interfering with the HBx-SMC5/6 axis holds promise in achieving sustained viral control. Here, we summarize the current knowledge of the mechanism of SMC5/6-mediated HBV restriction. We also give an overview of SMC5/6 cellular functions and how this compares to the restriction of other DNA viruses. We further discuss the therapeutic potential of available and investigational drugs interfering with the HBx-SMC5/6 axis.

Discovery of Potent Covalent CRM1 Inhibitors Via a Customized Structure-Based Virtual Screening Pipeline and Bioassays.

CRM1 (chromosomal region maintenance 1, also referred to as exportin 1 or XPO1) plays a crucial role in maintaining the appropriate nuclear levels of tumor suppressor proteins (TSPs), growth regulatory proteins (GRPs), and antiapoptotic proteins, thereby contributing significantly to their anticancer effects. Dysregulation of CRM1-mediated nuclear transport, observed in a range of cancers such as colon cancer as well as autoimmune diseases, highlights its significance in various disease processes. In this paper, we employed a customized structure-based virtual screening campaign to search for novel covalent CRM1 inhibitors and purchased 50 potentially active compounds for in vitro bioassays. Among these candidates, AN-988 displayed a notably higher binding affinity (KD = 615 nM) toward CRM1, as determined by the biolayer interferometry (BLI) assay. Furthermore, AN-988 exhibited a strong suppression of colorectal cancer cell proliferation and remarkable anti-inflammatory effects. Notably, AN-988 induced cell apoptosis and cell cycle arrest in a time- and dose-dependent manner by effectively inhibiting the translocation of FOXO3a from the nucleus to the cytosol, thereby preserving the activity of FOXO3a. Collectively, our study identified AN-988 as a promising CRM1 inhibitor, underscoring its potential as a preclinical colon cancer therapy candidate.

HMGB2-induced calreticulin translocation required for immunogenic cell death and ferroptosis of cancer cells are controlled by the nuclear exporter XPO1

Cisplatin and oxaliplatin cause the secretion of high mobility group box 1 (HMGB1) protein from cancer cells, which is necessary for initiation of immunogenic cell death (ICD). Calreticulin (CRT) translocation from the endoplasmic reticulum to the plasma membrane is also required; oxaliplatin induces this translocation but cisplatin does not. We have discovered that oxaliplatin causes the secretion of both HMGB1 and HMGB2 from the cell nucleus into the extracellular milieu. We previously showed that cisplatin-mediated secretion of HMGB1 is controlled by the nuclear exporter XPO1 (chromosomal maintenance 1; CRM1). We now find that XPO1 regulates oxaliplatin-mediated secretion of both HMGB1 and HMGB2. XPO1 inhibition causes nuclear accumulation of both proteins, inhibition of oxaliplatin-mediated ferroptosis of colon cancer cells, and inhibition of CRT translocation to the plasma membrane of lung and colon cancer cells. Incubation of cancer cells with cell targeted (CT)-HMGB2 confirmed that HMGB2 is required for the CRT translocation. Furthermore, CT-HMGB2 is three orders of magnitude more potent at inducing CRT translocation than oxaliplatin.

Symmetry of loop extrusion by dimeric SMC complexes is DNA-tension-dependent.

Structural maintenance of chromosome (SMC) complexes organize and regulate genomes via DNA loop extrusion. During this process, the complexes increase the loop size by reeling in DNA from one or both sides of the loop. The factors governing this symmetry remain unclear. Here, we combine single-molecule analysis and molecular dynamic simulations to investigate the symmetry of loop extrusion of various SMC complexes. We find that whereas monomeric condensin and cohesin are one-sided extruders, the symmetry of dimeric SMCs, such as Smc5/6 and Wadjet, is DNA tension dependent. At low DNA tension (< 0.1pN), Smc5/6 and Wadjet extrude DNA from both sides of the loop. At higher tension, however, they transition to a behavior akin to one-sided extruders, yet still capable of extruding from one or the other side thereby switching the direction of extrusion. Our simulations further reveal that thermal fluctuations significantly influence loop extrusion symmetry, causing variations in DNA reeling rates between the two motors in the dimeric complexes and their direction switching at stalling tensions. Our findings challenge the previous view of loop extrusion symmetry as a fixed characteristic, revealing its dynamic nature and regulation by both intrinsic protein properties and extrinsic factors.

Positive Selection Drives the Evolution of the Structural Maintenance of Chromosomes (SMC) Complexes.

Structural Maintenance of Chromosomes (SMC) complexes are an evolutionary conserved protein family. In most eukaryotes, three SMC complexes have been characterized, as follows: cohesin, condensin, and SMC5/6 complexes. These complexes are involved in a plethora of functions, and defects in SMC genes can lead to an increased risk of chromosomal abnormalities, infertility, and cancer. To investigate the evolution of SMC complex genes in mammals, we analyzed their selective patterns in an extended phylogeny. Signals of positive selection were identified for condensin NCAPG, for two SMC5/6 complex genes (SMC5 and NSMCE4A), and for all cohesin genes with almost exclusive meiotic expression (RAD21L1, REC8, SMC1B, and STAG3). For the latter, evolutionary rates correlate with expression during female meiosis, and most positively selected sites fall in intrinsically disordered regions (IDRs). Our results support growing evidence that IDRs are fast evolving, and that they most likely contribute to adaptation through modulation of phase separation. We suggest that the natural selection signals identified in SMC complexes may be the result of different selective pressures: a host-pathogen arms race in the condensin and SMC5/6 complexes, and an intragenomic conflict for meiotic cohesin genes that is similar to that described for centromeres and telomeres.

The physical chemistry of interphase loop extrusion.

Loop extrusion constitutes a universal mechanism of genome organization, whereby structural maintenance of chromosomes (SMC) protein complexes load onto the chromatin fiber and generate DNA loops of increasingly-larger sizes until their eventual release. In mammalian interphase cells, loop extrusion is mediated by the cohesin complex, which is dynamically regulated by the interchange of multiple accessory proteins. Although these regulators bind the core cohesin complex only transiently, their disruption can dramatically alter cohesin dynamics, gene expression, chromosome morphology and contact patterns. Still, a theory of how cohesin regulators and their molecular interplay with the core complex modulate genome folding remains at large. Here we derive a model of cohesin loop extrusion from first principles, based on in vivo measurements of the abundance and dynamics of cohesin regulators. We systematically evaluate potential chemical reaction networks that describe the association of cohesin with its regulators and with the chromatin fiber. Remarkably, experimental observations are consistent with only a single biochemical reaction cycle, which results in a unique minimal model that may be fully parameterized by quantitative protein measurements. We demonstrate how distinct roles for cohesin regulators emerge simply from the structure of the reaction network, and how their dynamic exchange can regulate loop extrusion kinetics over time-scales that far exceed their own chromatin residence times. By embedding our cohesin biochemical reaction network within biophysical chromatin simulations, we evidence how variations in regulatory protein abundance can alter chromatin architecture across multiple length- and time-scales. Predictions from our model are corroborated by biophysical and biochemical assays, optical microscopy observations, and Hi-C conformation capture techniques. More broadly, our theoretical and numerical framework bridges the gap between in vitro observations of extrusion motor dynamics at the molecular scale and their structural consequences at the genome-wide level.

BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during Caenorhabditis elegans meiosis.

The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Using genetic and cytological methods to monitor resolution of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5 repress intersister crossover recombination events. Sequencing analysis of conversion tracts from homolog-independent DSB repair events further indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal functional interactions of BRC-1 and SMC-5/6 in regulating repair pathway engagement: BRC-1 is required for localization of recombinase proteins to DSBs in smc-5 mutants and enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). These results are consistent with a model in which some functions of BRC-1 act upstream of SMC-5/6 to promote recombination and inhibit error-prone DSB repair, while SMC-5/6 acts downstream of BRC-1 to regulate the formation or resolution of recombination intermediates. Taken together, our study illuminates the coordinated interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.

Amplified Cell Cycle Genes Identified in High-Grade Serous Ovarian Cancer.

The objective of this study was to identify differentially expressed genes and their potential influence on the carcinogenesis of serous-type ovarian cancer tumors. Serous cancer is an epithelial ovarian cancer subtype and is the most common type of ovarian cancer. Transcriptomic profiles of serous cancer and non-cancerous datasets were obtained from the Gene Expression Omnibus (GEO-NCBI). Differentially expressed genes were then derived from those profiles; the identified genes were consistently upregulated in three or more transcriptomic profiles. These genes were considered as the serous ovarian cancer gene set for further study. The serous gene set derived from the transcriptomic profiles was then evaluated for ontological functional analysis using the Molecular Signatures Database. Next, we examined the mutational impact of this serous gene set on the transcriptomic profile of high-grade serous ovarian (HGSO) adenocarcinoma using the cBioPortal database. Results from OncoPrint revealed that 26 genes were amplified in more than 5% of HGSO cancer patients. Interestingly, several of these genes are involved in cell cycle processes, including genes ATPase family AAA domain containing 2 (ATAD2), recQ-like helicase 4 (RECQL4), cyclin E1 (CCNE1), anti-silencing function 1B histone chaperone (ASF1B), ribonuclease H2 subunit A (RNASEH2A), structural maintenance of chromosome 4 (SMC4), cell division cycle associated 20 (CDC20), and cell division cycle associated 8 (CDCA8). The receiver operating characteristic (ROC) curve results also revealed higher specificity and sensitivity for this subtype of tumors. Furthermore, these genes may affect the recurrence of serous ovarian carcinogenesis. Overall, our analytical study identifies cell cycle-related genes that can potentially be targeted as diagnostic and prognostic markers for serous ovarian cancer.

Condensin I folds the Caenorhabditis elegans genome.

The structural maintenance of chromosome (SMC) complexes-cohesin and condensins-are crucial for chromosome separation and compaction during cell division. During the interphase, mammalian cohesins additionally fold the genome into loops and domains. Here we show that, in Caenorhabditis elegans, a species with holocentric chromosomes, condensin I is the primary, long-range loop extruder. The loss of condensin I and its X-specific variant, condensin IDC, leads to genome-wide decompaction, chromosome mixing and disappearance of X-specific topologically associating domains, while reinforcing fine-scale epigenomic compartments. In addition, condensin I/IDC inactivation led to the upregulation of X-linked genes and unveiled nuclear bodies grouping together binding sites for the X-targeting loading complex of condensin IDC. C. elegans condensin I/IDC thus uniquely organizes holocentric interphase chromosomes, akin to cohesin in mammals, as well as regulates X-chromosome gene expression.

Genome-wide CRISPR screenings identified SMCHD1 as a host-restricting factor for AAV transduction.

AAV-mediated gene therapy typically requires a high dose of viral transduction, risking acute immune responses and patient safety, part of which is due to limited understanding of the host-viral interactions, especially post-transduction viral genome processing. Here, through a genome-wide CRISPR screen, we identified SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1), an epigenetic modifier, as a critical broad-spectrum restricting host factor for post-entry AAV transgene expression. SMCHD1 knock-down by RNAi and CRISPRi or knock-out by CRISPR all resulted in significantly enhanced transgene expression across multiple viral serotypes, as well as for both single-strand and self-complementary AAV genome types. Mechanistically, upon viral transduction, SMCHD1 effectively repressed AAV transcription by the formation of an LRIF1-HP1-containing protein complex and directly binding with the AAV genome to maintain a heterochromatin-like state. SMCHD1-KO or LRIF1-KD could disrupt such a complex and thus result in AAV transcriptional activation. Together, our results highlight the host factor-induced chromatin remodeling as a critical inhibitory mechanism for AAV transduction and may shed light on further improvement in AAV-based gene therapy.

Cryo-EM structures of Smc5/6 in multiple states reveal its assembly and functional mechanisms.

Smc5/6 is a member of the eukaryotic structural maintenance of chromosomes (SMC) family of complexes with important roles in genome maintenance and viral restriction. However, limited structural understanding of Smc5/6 hinders the elucidation of its diverse functions. Here, we report cryo-EM structures of the budding yeast Smc5/6 complex in eight-subunit, six-subunit and five-subunit states. Structural maps throughout the entire length of these complexes reveal modularity and key elements in complex assembly. We show that the non-SMC element (Nse)2 subunit supports the overall shape of the complex and uses a wedge motif to aid the stability and function of the complex. The Nse6 subunit features a flexible hook region for attachment to the Smc5 and Smc6 arm regions, contributing to the DNA repair roles of the complex. Our results also suggest a structural basis for the opposite effects of the Nse1-3-4 and Nse5-6 subcomplexes in regulating Smc5/6 ATPase activity. Collectively, our integrated structural and functional data provide a framework for understanding Smc5/6 assembly and function.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

NSE5 subunit interacts with distant regions of the SMC arms in the Physcomitrium patens SMC5/6 complex.

Structural maintenance of chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P.patens Ppnse5KO1 mutant line with an N-terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5-PpSMC6 interaction in SMC5/6 loading to DNA lesions.

Structural maintenance of chromosomes and associated genetic disorders

Structural maintenance of chromosomes (SMC), including cohesin, condensin and the SMC5/6 complex, are protein complexes which maintain the higher structure and dynamic stability of chromatin. Such circular complexes, with similar structures, play pivotal roles in chromatid cohesion, chromosomal condensation, DNA replication and repair, as well as gene transcription. Despite extensive research on the functions of the SMCs, our understanding of the SMC5/6 complex has remained limited compared with the other two complexes. This article has reviewed the architecture and crucial physiological roles of the SMCs, and explored the associated phenotypes resulting from mutations of the SMC components such as Cornelia de Lange syndrome (CdLS) and microcephaly, with an aim to provide insights into their functions in eukaryotic cells and implications for human diseases.

Identification of SMC2 and SMC4 as prognostic markers in breast cancer through bioinformatics analysis.

Breast cancer (BRCA) is one of the most common malignant tumors. The structural maintenance of chromosome (SMC) gene family has been shown to play an important role in human cancers. However, the role of SMC families in BRCA is unclear. This study aimed to explore the role and potential clinical value of whole SMCs in BRCA. TIMER and UALCAN database were used to analysis the expression level. Genetic variations were analyzed by cBioPortal. Promoter methylation and protein level were analyzed by UCLCAN. GO and KEGG were analyzed by Metascape database. Prognostic value of SMCs was analyzed by Kaplan-Meier and multivariate cox regression analyses. Immune infiltration analysis was conducted by CIBERSORT. Immunotherapy outcome prediction was conducted by Cancer Immunome Atlas. Targeted drug therapy outcome prediction was taken by GDSC and R language. The cell viability was tested by CCK8 and migration was tested by wound healing assay. Xenograft model was used to investigate the in vivo role of SMC2. Expression levels of SMC1A, SMC2, SMC4, SMC5 and SMC6 mRNA were increased in BRCA tissues, and negatively correlated with promoter methylation. Overexpression of SMC2 and SMC4 was negatively correlated with survival. Function of SMCs family regulatory genes was mainly related to ATPase activity. Expression of most SMCs was negatively correlated with immunotherapy and drug therapy outcomes. Interfere SMC2 and SMC4 decreased IC50 values of 5-fluorouracil and oxaliplatin and inhibited the migration of MCF7 cells. Tumor growth and weights were significantly decreased in si-SMC2 groups. Combined bioinformatics and clinical specimen analysis verified SMC2 and SMC4 as independent prognostic factors in BRCA, suggesting their significance for the diagnosis and treatment of BRCA.

The role of satellite DNAs in the chromosomal rearrangements and the evolution of the rare XY1Y2 sex system in Harttia (Siluriformes: Loricariidae).

The underlying processes behind the formation, evolution, and long-term maintenance of multiple sex chromosomes have been largely neglected. Among vertebrates, fishes represent the group with the highest diversity of multiple sex chromosome systems and, with six instances, the Neotropical fish genus Harttia stands out by presenting the most remarkable diversity. However, although the origin mechanism of their sex chromosome systems is well discussed, little is known about the importance of some repetitive DNA classes in the differentiation of multiple systems. In this work, by employing a combination of cytogenetic and genomic procedures, we evaluated the satellite DNA composition of H. carvalhoi with a focus on their role in the evolution, structure, and differentiation process of the rare XY1Y2 multiple sex chromosome system. The genome of H. carvalhoi contains a total of 28 satellite DNA families, with the A+T content ranging between 38,1 and 68,1% and the predominant presence of long satellites. The in situ hybridization experiments detected 15 satellite DNAs with positive hybridization signals mainly on centromeric and pericentromeric regions of almost all chromosomes or clustered on a few pairs. Five of them presented clusters on X, Y1, and/or Y2 sex chromosomes which were therefore selected for comparative hybridization in the other three congeneric species. We found several conserved satellites accumulated on sex chromosomes and also in regions that were involved in chromosomal rearrangements. Our results provide a new contribution of satellitome studies in multiple sex chromosome systems in fishes and represent the first satellitome study for a Siluriformes species.