Mutations in tumor suppressor BRCA1 lead to breast and/or ovarian cancer. Here we show that loss of BRCA1 in mice results in transcriptional derepression of the tandemly repeated satellite DNA. BRCA1 deficiency is accompanied by reduction of condensed DNA regions in the genome and loss of ubiquitylation of histone H2A at satellite repeats. BRCA1 binds to satellite DNA regions in vivo and ubiquitylates H2A in vitro. Ectopic expression of an H2A fused to ubiquitin reverses the effects of BRCA1 loss, suggesting that BRCA1 maintains heterochromatin structure via ubiquitylation of histone H2A. Satellite DNA derepression was also observed mouse and human BRCA1 deficient breast cancers. Ectopic expression of satellite DNA can phenocopy BRCA1 loss in centrosome amplification, cell cycle checkpoint defects, DNA damage and genomic instability. We propose that the role of BRCA1 in maintaining global heterochromatin integrity accounts for many of its tumor suppressor functions.

We have developed a cancer model of gliomas in human cerebral organoids that allows direct observation of tumor initiation as well as continuous microscopic observations. We used CRISPR/Cas9 technology to target an HRas-IRES-tdTomato construct by homologous recombination into the TP53 locus. Results show that transformed cells rapidly become invasive and destroy surrounding organoid structures, overwhelming the entire organoid. Tumor cells in the organoids can be orthotopically xenografted into immunodeficient NOD/SCID IL2RG animals, exhibiting an invasive phenotype. Organoid-generated putative tumor cells show gene expression profiles consistent with mesenchymal subtype human glioblastoma. We further demonstrate that human-organoid-derived tumor cell lines or primary human-patient-derived glioblastoma cell lines can be transplanted into human cerebral organoids to establish invasive tumor-like structures. Our results show potential for the use of organoids as a platform to test human cancer phenotypes that recapitulate key aspects of malignancy.

BackgroundMicroarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration.ResultsConsiderable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences.ConclusionMany new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8 -142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys 48 -and Lys 63 -linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys 63 , which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.

BRCA1 is a breast and ovarian cancer-specific tumor suppressor, with properties of a transcription factor involved in DNA repair. We previously have shown the transactivation of heterologous promoters by the carboxyl terminus of BRCA1. We now describe that BRCA1-mediated transactivation is enhanced by p300͞CBP (CREB binding protein) and that this effect was suppressed by the adenovirus E1A oncoprotein. We show a physical association of BRCA1 with the transcriptional coactivators͞acetyltransferases p300 and CBP. Endogenous as well as overexpressed BRCA1 and p300 were found to associate in a phosphorylation-independent manner. BRCA1 interacts with the cAMP response element binding protein (CREB) domain of p300͞CBP via both its amino and carboxyl termini. Finally, full-length BRCA1 is shown to transcriptionally activate the Rous sarcoma virus-long terminal repeat promoter, which was further stimulated by p300. Immunocolocalization analyses suggest that BRCA1 and p300 associate in a cell cycle-dependent manner. Our results support a role for BRCA1 in transcription. BRCA1 is the first of the two identified familial breast cancer genes (1). The human BRCA1 gene encodes a 1,863-aa-long, predominantly nuclear phosphoprotein (2-5). At its amino terminus BRCA1 possesses a RING finger domain thought to mediate protein-protein interactions. At its extreme carboxyl terminus BRCA1 has two BRCT repeats that are present in a large number of DNA damage-responsive cell cycle checkpoint proteins found from bacteria to humans (6, 7). Interestingly, BARD1, a protein that physically interacts with the RING finger binding domain of BRCA1 also possesses BRCT repeats as well as a RING finger (8). BRCA1 is essential in mouse embryonic development (9-11). BRCA1 Ϫ͞Ϫ embryos do not die at a specific time point but over a period starting from embryonic day (E) 6.5 to E13 because of a gastrulation failure or defects in neural tube closure depending on the mutation (9-11). However, there is one report of a naturally occurring BRCA1 knockout in a breast cancer patient (12). Tissuespecific conditional knockouts of BRCA1 do indeed induce breast epithelial hyperplasias in mice, albeit at a low frequency after 10-13 months (13). If true, this difference might not be unexpected given the surprisingly poor sequence conservation of 58% identity at the amino acid level between the human and mouse BRCA1 orthologues (14). The levels of BRCA1 fluctuate through the cell cycle, reaching a maximum in S phase and a minimum during G 1 (4,15,16). This change parallels the phosphorylation state of BRCA1, which becomes hyperphosphorylated predominantly on serine residues (4,15,17). BRCA1 is known to associate with RAD51, the mammalian homologue of the bacterial RecA protein (5), suggesting that BRCA1 is involved in the maintenance of genomic integrity and͞or in DNA damage-dependent responses. Further evidence pointing in this direction stems from the recent observation that DNA-damaging agents can induce the hyperphosphorylation of BRCA1 on serine residues (17, 1...