SUMMARY Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.

Genome instability is a characteristic of malignant cells; however, evidence for its contribution to tumorigenesis has been enigmatic. In this study, we demonstrate that the retinoblastoma protein, E2F1, and Condensin II localize to discrete genomic locations including major satellite repeats at pericentromeres. In the absence of this complex, aberrant replication ensues followed by defective chromosome segregation in mitosis. Surprisingly, loss of even one copy of the retinoblastoma gene reduced recruitment of Condensin II to pericentromeres and caused this phenotype. Using cancer genome data and gene-targeted mice, we demonstrate that mutation of one copy of RB1 is associated with chromosome copy-number variation in cancer. Our study connects DNA replication and chromosome structure defects with aneuploidy through a dosage-sensitive complex at pericentromeric repeats. SIGNIFICANCE:Genome instability is inherent to most cancers and is the basis for selective killing of cancer cells by genotoxic therapeutics. In this report, we demonstrate that instability can be caused by loss of a single allele of the retinoblastoma gene that prevents proper replication and condensation of pericentromeric chromosomal regions, leading to elevated levels of aneuploidy in cancer. Cancer Discov; 4(7); 840-53.

Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit. FIG 1 CRISPR/Cas9-induced mutations in RB1 cause DNA damage. (A) Ethidium bromide-stained agarose gel showing examples of wild-type, heterozygous, and homozygous mutant RB1 genotypes that are detected by PCR amplification of exon 22 sequences. MW, molecular weight. (B, top) Representative Western blot showing RB expression in control, heterozygous, and homozygous mutant cells. (Bottom) Sp1 loading control.(C) Immunofluorescence microscopy was used to detect RB expression (green) in cultures of control, heterozygous, or homozygous mutants. Cells were counterstained with DAPI to visualize nuclei (blue). (D) Representative confocal microscopy images of ␥H2AX foci (red) in control, heterozygous, and homozygous RB1 mutant cells. Cells were counterstained with DAPI to visualize nuclei (blue). (E) Counts of ␥H2AX foci for each of the U2OS RB1 genotypes. The average proportions of cells with discrete numbers of foci are shown as histograms, while the cumulative frequency of foci for each genotype is shown in the inset. The average distributions of foci for RB1 wild-type (4 different clones), heterozygous (3 different clones), and knockout (4 different clones) cells were compared using the Kolmogorov-Smirnov test (*, P Ͻ 0.05). (F) U2OS cells were transfected with CRISPR/Cas9 constructs targeting either a safe-harbor site in the genome or exon 2 of the RB1 gene. Three clones were selected under both control and knockout conditions, and ␥H2AX foci were quantified by fluorescence microscopy. The average proportions of ␥H2AX foci for both RB1 wild-type and knockout genotypes are shown as histograms, while the cumulative relative frequency of foci is shown in the inset. Focus distributions were again compared by a Kolmogorov-Smirnov test. (G) H460 lung cancer cells were stained for ␥H2AX foci and analyzed as described above for panel F. (H) H1792 non-small cell lung cancer cells were analyzed as described above for panel F. All error bars are ϩ1 standard error of the mean (SEM). *, P Ͻ 0.05. RB1Deletion Causes ...

SHQ1 is essential for biogenesis of H/ACA ribonucleoproteins, a class of molecules important for processing ribosomal RNAs, modifying spliceosomal small nuclear RNAs, and stabilizing telomerase. Components of the H/ACA ribonucleoprotein complex have been linked to neurological developmental defects. Here we report two sibling pairs from unrelated families with compound heterozygous variants in SHQ1. Exome sequencing was used to detect disease causing variants which were submitted to ‘matching’ platforms linked to MatchMaker Exchange. Phenotype comparisons supported these matches. The affected individuals present with early-onset dystonia, with individuals from one family displaying additional neurological phenotypes, including neurodegeneration. As a result of CSF studies suggesting possible abnormal dopamine metabolism, a trial of levodopa replacement therapy was started but no clear response was noted. We show that fibroblasts from affected individuals have dramatic loss of SHQ1 protein. Variants from both families were expressed in S. cerevisiae, resulting in a strong reduction in H/ACA snoRNA production and remarkable defects in rRNA processing and ribosome formation. Our study identifies SHQ1 as associated with neurological disease, including early-onset dystonia, and begins to delineate the molecular etiology of this novel condition.

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Interphase chromosomes are organized into topologically associated domains in order to establish and maintain integrity of transcriptional programs that remain poorly understood. Here, we show that condensin II and TFIIIC are recruited to bidirectionally transcribed promoters by a mechanism that is dependent on the retinoblastoma (RB) protein. Long-range chromosome contacts are disrupted by loss of condensin II loading, which leads to altered expression at bidirectional gene pairs. This study demonstrates that mammalian condensin II functions to organize long-range chromosome contacts and regulate transcription at specific genes. In addition, RB dependence of condensin II suggests that widespread misregulation of chromosome contacts and transcriptional alterations are a consequence of RB mutation.

ObjectiveHuman genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683).MethodsIndividuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed.ResultsA total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414‐2_414‐1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574‐6_574‐3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574‐6_574‐3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism.InterpretationThe ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1‐related neurological disease, (2) highlights the importance of genotype‐driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304–321

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