Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation

Background RAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear. Methods To investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomics approach. Results Here, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion. Conclusions Our study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

Sequencing Micronuclei Reveals the Landscape of Chromosomal Instability

Genome instability (GIN) is a main contributing factor to congenital and somatic diseases, but its sporadic occurrence in individual cell cycles makes it difficult to study mechanistically. One profound manifestation of GIN is the formation of micronuclei (MN), the engulfment of chromosomes or chromosome fragments in their own nuclear structures separate from the main nucleus. Here, we developed MN-seq, an approach for sequencing the DNA contained within micronuclei. We applied MN-seq to mice with mutations in Mcm4 and Rad9a, which disrupt DNA replication, repair, and damage responses. Data analysis and simulations show that centromere presence, fragment length, and a heterogenous landscape of chromosomal fragility all contribute to the patterns of DNA present within MN. In particular, we show that long genes, but also gene-poor regions, are associated with chromosome breaks that lead to the enrichment of particular genomic sequences in MN, in a genetic background-specific manner. Finally, we introduce single-cell micronucleus sequencing (scMN-seq), an approach to sequence the DNA present in MN of individual cells. Together, sequencing micronuclei provides a systematic approach for studying GIN and reveals novel molecular associations with chromosome breakage and segregation.

Highly precise breakpoint detection of chromosome balanced translocation in a Chronic Myelogenous Leukemia patient

Chronic Myelogenous Leukemia (CML) has a special phenomenon of chromosome translocation, which is called Philadelphia chromosome translocation. However, the detailed connection of this structure is troublesome and expensive to be identified. Low-coverage whole genome sequencing (LCWGS) could not only detect the chromosomal translocation which does not be known in advance, but also provide the breakpoint candidate small region (with an accuracy of ±200 bases). Importantly, the sequencing cost of LCWGS is about US$300. Then, with the Sanger DNA sequencing, the precise breakpoint can be determined at a single base level. In our project, with LCWGS, BCR and ABL1 are successfully identified and were disrupted at chr22:23,632,356 and chr9:133,590,450, respectively. Due to the reconnection after chromosome breakage, classical fusion gene (BCR-ABL1) was found in bone marrow and peripheral blood. The precise breakpoints were helpful to study the pathogenic mechanism of CML and could better guide the classification of CML subtypes. This LCWGS method is universal and can be used to detect all diseases related to chromosome variation, such as solid tumors, liquid tumors and birth defects.

Monosomies, trisomies and segmental aneuploidies differentially affect chromosomal stability

Aneuploidy and chromosomal instability are both commonly found in cancer. Chromosomal instability leads to karyotype heterogeneity in tumors and is associated with therapy resistance, metastasis and poor prognosis. It has been hypothesized that aneuploidy per se is sufficient to drive CIN, however due to limited models and heterogenous results, it has remained controversial which aspects of aneuploidy can drive CIN. In this study we systematically tested the impact of different types of aneuploidies on the induction of CIN. We generated a plethora of isogenic aneuploid clones harboring whole chromosome or segmental aneuploidies in human p53-deficient RPE-1 cells. We observed increased segregation errors in cells harboring trisomies that strongly correlated to the number of gained genes. Strikingly, we found that clones harboring only monosomies do not induce a CIN phenotype. Finally, we found that an initial chromosome breakage event and subsequent fusion can instigate breakage-fusion-bridge cycles in segmental aneuploidies. This suggests that monosomies, trisomies and segmental aneuploidies have fundamentally different effects on chromosomal instability and these results help us to decipher the complex relationship between aneuploidy and CIN.

Extracellular and Intracellular Factors in Brain Cancer

The external and internal factors of the cell are critical to glioma initiation. Several factors and molecules have been reported to be implicated in the initiation and progression of brain cancer. However, the exact sequence of events responsible for glioma initiation is still unknown. Existing reports indicate that glioma stem cells are the cell of glioma origin. During cell division, chromosome breakage, DNA alteration increases the chance of cell genome modifications and oncogene overexpression. Although there is a high risk of gene alteration and oncogene overexpression, not everyone develops cancer. During embryogenesis, the same oncogenes that promote cancers have also been reported to be highly expressed, but this high expression which does not lead to carcinogenesis raises questions about the role of oncogenes in carcinogenesis. The resistance of cancer cells to drugs, apoptosis, and immune cells does not rely solely on oncogene overexpression but also on the defect in cell organelle machinery (mitochondria, endoplasmic reticulum, and cytoskeleton). This review discusses factors contributing to cancer; we report the dysfunction of the cell organelles and their contribution to carcinogenesis, while oncogene overexpression promotes tumorigenesis, maintenance, and progression through cell adhesion. All these factors together represent a fundamental requirement for cancer and its development.

IL-17-Mediated Inflammation Promotes Cigarette Smoke-Induced Genomic Instability

(1) Background: Chronic inflammation has been regarded as a risk factor for the onset and progression of human cancer, but the critical molecular mechanisms underlying this pathological process have yet to be elucidated. (2) Methods: In this study, we investigated whether interleukin (IL)-17-mediated inflammation was involved in cigarette smoke-induced genomic instability. (3) Results: Higher levels of both IL-17 and the DNA damage response (DDR) were found in the lung tissues of smokers than in those of non-smokers. Similarly, elevated levels of IL-17 and the DDR were observed in mice after cigarette smoke exposure, and a positive correlation was observed between IL-17 expression and the DDR. In line with these observations, the DDR in the mouse lung was diminished in IL-17 KO when exposed to cigarette smoke. Besides this, the treatment of human bronchial epithelium cells with IL-17 led to increased levels of the DDR and chromosome breakage. (4) Conclusions: These results suggest that cigarette smoke induces genomic instability at least partially through IL-17-mediated inflammation, implying that IL-17 could play an important role in the development of lung cancer.

New insights in phenotype and treatment of lung disease immuno-deficiency and chromosome breakage syndrome (LICS)

We report five patients with lung disease immuno-deficiency and chromosome breakage syndrome (LICS) but without recurrent infections and severe immunodeficiency. One patient had extended survival to 6.5 years. Hematopoietic stem-cell transplantation failed to cure another patient. Our findings suggest that the immunological abnormalities can be limited and do not fully explain the LICS phenotype.

Palindromes in DNA—A Risk for Genome Stability and Implications in Cancer

A palindrome in DNA consists of two closely spaced or adjacent inverted repeats. Certain palindromes have important biological functions as parts of various cis-acting elements and protein binding sites. However, many palindromes are known as fragile sites in the genome, sites prone to chromosome breakage which can lead to various genetic rearrangements or even cell death. The ability of certain palindromes to initiate genetic recombination lies in their ability to form secondary structures in DNA which can cause replication stalling and double-strand breaks. Given their recombinogenic nature, it is not surprising that palindromes in the human genome are involved in genetic rearrangements in cancer cells as well as other known recurrent translocations and deletions associated with certain syndromes in humans. Here, we bring an overview of current understanding and knowledge on molecular mechanisms of palindrome recombinogenicity and discuss possible implications of DNA palindromes in carcinogenesis. Furthermore, we overview the data on known palindromic sequences in the human genome and efforts to estimate their number and distribution, as well as underlying mechanisms of genetic rearrangements specific palindromic sequences cause.

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.