Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

Kinetochores assemble on centromeres (CENs) via histone H3 variant CENP-A and low levels of CEN transcripts. RNA polymerase II (RNAPII) activity is restrained by the CEN histone code, while CEN RNA concentrations are reduced by the nuclear exosome. Using S. cerevisiae, we add kinase Rio1 to this scheme as it downregulates RNAPII, and promotes CEN RNA turnover via exoribonuclease Rat1. Transcription factor Cbf1 and the assembled kinetochore further restrain CEN transcription. CEN transcripts exist as long (up to 11,000nt) and short RNAs (119±40nt), which may underlie CEN identity and kinetochore recruitment. While also curtailed by Rio1, Rat1, and the exosome, periCEN RNAs (<200nt) accumulate at levels that are one order of magnitude higher than the CEN transcripts. Depleting Rio1 causes CEN and periCEN RNA buildup, kinetochore malformation, and chromosome loss. Depleting human orthologue RioK1 leads to CEN RNA accumulation and micronuclei formation, suggesting that Rio1/RioK1 activity at centromeres is conserved.

Autophagy-related protein Atg11 preserves asymmetric inheritance of spindle microtubule-organizing centers

Asymmetric spindle pole body (SPB) inheritance requires a cascade of events that involve kinases, phosphatases and structural scaffold proteins including molecular motors and microtubule-associated proteins present in the nucleus and/or the cytoplasm. Higher levels of an SPB component Spc72 and the spindle positioning factor Kar9 at the old SPB, which migrates to the daughter cell, ensure asymmetric SPB inheritance. Timely SPB duplication followed by its asymmetric inheritance is a key to correct spindle alignment leading to high-fidelity chromosome segregation. By combining in silico analysis of known protein-protein interactions of autophagy (Atg)-related proteins with those that constitute the chromosome segregation machinery, and growth dynamics of 35 atg mutants in the presence of a microtubule poison, we identified Atg11 as a potential regulator of chromosome transmission. Cells lacking Atg11 did not show any kinetochore defects but displayed a high rate of chromosome loss and delayed anaphase onset. Atg11 positively interacted with Kar9 and Kip2 and negatively with Dyn1 and Kar3 in mediating proper chromosome segregation suggesting a role of Atg11 in spindle positioning. Indeed, atg11∆ cells displayed an inverted SPB inheritance. We further show that Atg11 promotes asymmetric localization of Spc72 and Kar9 on the old SPB. Atg11 physically interacted with Spc72 and transiently localized close to the old SPB during metaphase-to-anaphase progression. Taken together, our study uncovers an autophagy-independent role of Atg11 in spindle alignment and emphasizes the importance of unbiased screens to identify factors mediating the complex and intricate crosstalk between processes fundamental to genomic integrity.

Enhanced chromatin accessibility contributes to X chromosome dosage compensation in mammals

Background Precise gene dosage of the X chromosomes is critical for normal development and cellular function. In mice, XX female somatic cells show transcriptional X chromosome upregulation of their single active X chromosome, while the other X chromosome is inactive. Moreover, the inactive X chromosome is reactivated during development in the inner cell mass and in germ cells through X chromosome reactivation, which can be studied in vitro by reprogramming of somatic cells to pluripotency. How chromatin processes and gene regulatory networks evolved to regulate X chromosome dosage in the somatic state and during X chromosome reactivation remains unclear. Results Using genome-wide approaches, allele-specific ATAC-seq and single-cell RNA-seq, in female embryonic fibroblasts and during reprogramming to pluripotency, we show that chromatin accessibility on the upregulated mammalian active X chromosome is increased compared to autosomes. We further show that increased accessibility on the active X chromosome is erased by reprogramming, accompanied by erasure of transcriptional X chromosome upregulation and the loss of increased transcriptional burst frequency. In addition, we characterize gene regulatory networks during reprogramming and X chromosome reactivation, revealing changes in regulatory states. Our data show that ZFP42/REX1, a pluripotency-associated gene that evolved specifically in placental mammals, targets multiple X-linked genes, suggesting an evolutionary link between ZFP42/REX1, X chromosome reactivation, and pluripotency. Conclusions Our data reveal the existence of intrinsic compensatory mechanisms that involve modulation of chromatin accessibility to counteract X-to-Autosome gene dosage imbalances caused by evolutionary or in vitro X chromosome loss and X chromosome inactivation in mammalian cells.

In vitro cytotoxic and genotoxic effects of Cissus verticillata and Sphagneticola trilobata used for treatment of Diabetes Mellitus in Brazilian folk medicine

Cissus verticillata and Sphagneticola trilobata have been used in Brazilian folk medicine for Diabetes Mellitus treatment, although their pharmacological and toxicological profile has not been clearly established. Thus, the aim of this study was to evaluate the preclinical toxicity of the aqueous extracts of C. verticillata and S. trilobata. The main groups of secondary metabolites were investigated, and the species differed by the presence of coumarins in C. verticillata and by tannins in S. trilobata extracts. The highest contents of phenolic compounds and flavonoids were quantified in C. verticillata infusion with 2.594 ± 0.04 mg equivalents of gallic acid g-1 of extract and 1.301 ± 0.015 mg equivalents of catechin g-1 of extract, respectively. While the extract of S. trilobata showed minimum values of these compounds, with 0.002 ± 0.001 mg equivalents of gallic acid g-1 extract and 0.005 ± 0.0004 mg equivalents of catechin g-1 of extract, respectively. These differences implied the results of in vitro antioxidant activity evaluated using ferric reducing antioxidant power (FRAP), in which the sample of C. verticillata at 5 mg mL-1 showed a value of 122 µM ferrous sulfate equivalents (FSE), while S. trilobata showed 0.93 µM FSE at the same concentration. With respect to cytotoxic assay with murine fibroblast cell line (3T3) only S. trilobata exhibited cytotoxic effects measured by MTT and Sulforhodamine B assays, evidenced by the cell viability value of approximately 16%, in both tests after 24 and 72 hours of exposure of the cells to 5 mg mL-1 of the extract. Comparatively, at 5 mg mL-1 the C. verticillata extract showed cell viability of 142% and 95%, respectively, after 24 hours of cell exposure. On the other hand, both species showed genotoxic profiles evidenced by chromosomal aberrations by Allium cepa bioassay, observed by the higher percentage values of chromosome bridges, chromosome loss, and disturbed anaphase for all concentrations of both extracts than those of the negative control. The results support the characterization of the toxicological profile for both species and create an alert regarding the use of S. trilobata, which should be avoided.

Haplotyping-based preimplantation genetic testing reveals parent-of-origin specific mechanisms of aneuploidy formation

Chromosome instability is inherent to human IVF embryos, but the full spectrum and developmental fate of chromosome anomalies remain uncharacterized. Using haplotyping-based preimplantation genetic testing for monogenic diseases (PGT-M), we mapped the parental and mechanistic origin of common and rare genomic abnormalities in 2300 cleavage stage and 361 trophectoderm biopsies. We show that while single whole chromosome aneuploidy arises due to chromosome-specific meiotic errors in the oocyte, segmental imbalances predominantly affect paternal chromosomes, implicating sperm DNA damage in segmental aneuploidy formation. We also show that postzygotic aneuploidy affects multiple chromosomes across the genome and does not discriminate between parental homologs. In addition, 6% of cleavage stage embryos demonstrated signatures of tripolar cell division with excessive chromosome loss, however hypodiploid blastomeres can be excluded from further embryo development. This observation supports the selective-pressure hypothesis in embryos. Finally, considering that ploidy violations may constitute a significant proportion of non-viable embryos, using haplotyping-based approach to map these events might further improve IVF success rate.

Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing

Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.

Systems approaches identify the consequences of monosomy in somatic human cells

Chromosome loss that results in monosomy is detrimental to viability, yet it is frequently observed in cancers. How cancers survive with monosomy is unknown. Using p53-deficient monosomic cell lines, we find that chromosome loss impairs proliferation and genomic stability. Transcriptome and proteome analysis demonstrates reduced expression of genes encoded on the monosomes, which is partially compensated in some cases. Monosomy also induces global changes in gene expression. Pathway enrichment analysis reveals that genes involved in ribosome biogenesis and translation are downregulated in all monosomic cells analyzed. Consistently, monosomies display defects in protein synthesis and ribosome assembly. We further show that monosomies are incompatible with p53 expression, likely due to defects in ribosome biogenesis. Accordingly, impaired ribosome biogenesis and p53 inactivation are associated with monosomy in cancer. Our systematic study of monosomy in human cells explains why monosomy is so detrimental and reveals the importance of p53 for monosomy occurrence in cancer.

Targeting chromosome trisomy for chromosome editing

A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.