Gerald M. Pao

BRCA1 tumour suppression occurs via heterochromatin-mediated silencing

Mutations in the tumour suppressor gene BRCA1 lead to breast and/or ovarian cancer. Here we show that loss of Brca1 in mice results in transcriptional de-repression of the tandemly repeated satellite DNA. Brca1 deficiency is accompanied by a reduction of condensed DNA regions in the genome and loss of ubiquitylation of histone H2A at satellite repeats. BRCA1 binds to satellite DNA regions and ubiquitylates H2A in vivo. Ectopic expression of H2A fused to ubiquitin reverses the effects of BRCA1 loss, indicating that BRCA1 maintains heterochromatin structure via ubiquitylation of histone H2A. Satellite DNA de-repression was also observed in 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 tumour suppressor functions.

Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration.

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.

Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein.

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 Lys48- and Lys63-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys63, 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.

Genic regions of a large salamander genome contain long introns and novel genes

BackgroundThe basis of genome size variation remains an outstanding question because DNA sequence data are lacking for organisms with large genomes. Sixteen BAC clones from the Mexican axolotl (Ambystoma mexicanum: c-value = 32 × 109 bp) were isolated and sequenced to characterize the structure of genic regions.ResultsAnnotation of genes within BACs showed that axolotl introns are on average 10× longer than orthologous vertebrate introns and they are predicted to contain more functional elements, including miRNAs and snoRNAs. Loci were discovered within BACs for two novel EST transcripts that are differentially expressed during spinal cord regeneration and skin metamorphosis. Unexpectedly, a third novel gene was also discovered while manually annotating BACs. Analysis of human-axolotl protein-coding sequences suggests there are 2% more lineage specific genes in the axolotl genome than the human genome, but the great majority (86%) of genes between axolotl and human are predicted to be 1:1 orthologs. Considering that axolotl genes are on average 5× larger than human genes, the genic component of the salamander genome is estimated to be incredibly large, approximately 2.8 gigabases!ConclusionThis study shows that a large salamander genome has a correspondingly large genic component, primarily because genes have incredibly long introns. These intronic sequences may harbor novel coding and non-coding sequences that regulate biological processes that are unique to salamanders.

Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration-wound healing, cellular dedifferentiation and blastemal formation-will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.

Role of BRCA1 in brain development

Significance The developing brain is highly sensitive to ionizing radiation and DNA damage. Here we report that tumor suppressor breast cancer susceptibility gene 1 (BRCA1) plays a novel role in regulating the embryonic brain development and postnatal brain size. We found that loss of BRCA1 induces p53-dependent proapoptotic pathways in the CNS. BRCA1 possibly functions as a centrosomal factor in establishing the cellular polarity of the neural progenitors through the DNA damage sensor kinase ATM. Our data provide new insight in understanding the control of DNA damage sensitivity and brain size during development and evolution. Breast cancer susceptibility gene 1 (BRCA1) is a breast and ovarian cancer tumor suppressor whose loss leads to DNA damage and defective centrosome functions. Despite its tumor suppression functions, BRCA1 is most highly expressed in the embryonic neuroepithelium when the neural progenitors are highly proliferative. To determine its functional significance, we deleted BRCA1 in the developing brain using a neural progenitor–specific driver. The phenotype is characterized by severe agenesis of multiple laminated cerebral structures affecting most notably the neocortex, hippocampus, cerebellum, and olfactory bulbs. Major phenotypes are caused by excess apoptosis, as these could be significantly suppressed by the concomitant deletion of p53. Certain phenotypes attributable to centrosomal and cell polarity functions could not be rescued by p53 deletion. A double KO with the DNA damage sensor kinase ATM was able to rescue BRCA1 loss to a greater extent than p53. Our results suggest distinct apoptotic and centrosomal functions of BRCA1 in neural progenitors, with important implications to understand the sensitivity of the embryonic brain to DNA damage, as well as the developmental regulation of brain size.

Retrotransposon long interspersed nucleotide element-1 (LINE-1) is activated during salamander limb regeneration

Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline‐specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline‐like state. In this work, we focus on one of these genes, the long interspersed nucleotide element‐1 (LINE‐1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE‐1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE‐1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE‐1 retrotransposition is indeed active during regeneration. Active LINE‐1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE‐1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE‐1‐related piRNAs in the limb blastema. Transposable element‐related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline‐like state is established during axolotl limb regeneration, and the re‐activation of LINE‐1 may serve as a marker for cellular dedifferentiation in the early‐stage of limb regeneration.

Glioblastoma Model Using Human Cerebral Organoids

SUMMARY 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 HRasG12V-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.

Abstract LB-330: A novel role of tumor suppressor BRCA1 in regulating integrity of heterochromatin structure.

Introduction: BRCA1, as well as BRCA2, were identified as the hereditary breast and ovarian cancer susceptibility genes that can account for almost all of inherited cases of breast cancers. BRCA1 is a multifunctional nuclear protein with a RING domain at the N-terminus. Heterochromatin is chromatin containing inactive genes. Pericentromeric heterochromatin is composed of highly repetitive tandem DNA repeats whose transcription is constitutively silenced. In mice, the repeats are classified as major and minor satellite repeats. Heterochromatin protein 1 (HP1) plays a critical role in maintaining heterochromatin structure. Methods: To study the biological function of the tumor suppressor BRCA1, we deleted BRCA1 in nestin positive neuronal stem cells in the brain and showed that the animals displayed severe neuro-developmental deficits. In the course of this work we discovered a novel molecular defect in BRCA1 deficient cells, i.e., a failure to maintain integrity of the heterochromatic structure. Results: In the absence of BRCA1, the number of heterochromatic centers is severely reduced in mouse neuronal cells. HP1 proteins are destabilized, and more importantly, the transcription of satellite DNA is derepressed in mouse neural progenitor cells, mouse fibroblasts, and human cancer cells, including a BRCA1 deficient breast cancer cell line. The defect in silencing invariably appears after the loss of the BRCA1 allele in our paradigm prior to exhibiting any neoplastic phenotype. What is the mechanism of BRCA1 in regulating the heterochromatin integrity? ChIP analysis showed that BRCA1 physically associates with both major satellite DNA and minor satellite DNA, whereas the association is diminished in the BRCA1 knockout animals. More intriguingly, the absence of BRCA1 binding to satellite DNA correlates with a drastic reduction of ubiquitinated histone H2A at the constitutive heterochromatin region. Because BRCA1 contains an E3 ubiquitin ligase activity which is enhanced by BARD1 and the complex ubiquitinate histone H2A in vitro, we examined the heterochromatin silencing in cells with different BRCA1 mutants. These experiments showed that the E3 ligase activity of BRCA1 is required for repression of satellite DNA transcription. Conclusions: Our working model is that mutations of BRCA1 may destabilize the protein complex and lead to defects in the heterochromatin integrity, thereby affecting the stability of the genome which is a hallmark of cancer. Given that mutants in the RING domains are the same as in women who have pathological diseases, we hypothesize that deregulation of satellite DNA transcription can be explored as an early detection marker for breast cancer progression in BRCA1 patients.