Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

Lane, Timothy F.; Lin, Chenwei; Brown, Melissa A.; Solomon, Ellen; Leder, Philip

doi:10.1038/sj.onc.1203760

Cited by 25 publications

(20 citation statements)

References 30 publications

(50 reference statements)

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“…Since mouse and human BRCA1 proteins are interchangeable in a number of assays (11,12,48), we hypothesized that chimeric fusions of mouse and human BRCA1-CTD sequences should have a modest effect on activities that are important for gene function. Using conserved restriction sites within the mouse and human CTD, we constructed chimeric proteins in which mouse sequences (referred to as regions A, B, and C in Fig.…”

Section: Domain Swapping Between the Human And Mouse Ctd Leads To Chimentioning

confidence: 99%

“…BRCA1 complexes contain E3 ubiquitin-ligase activity, but the precise target(s) remains an avenue of active study (9,10). Although BRCA1 proteins show surprisingly low sequence identity, human BRCA1 can replace the mouse gene in genetically engineered animals (11,12), a result that implies general conservation of important functional motifs. Comparative functional studies are thus likely to play an important role in identifying critical targets of BRCA1 in human cells.…”

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confidence: 99%

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BRCA1 Associates with Processive RNA Polymerase II

Krum¹,

Miranda²,

Lin³

et al. 2003

Journal of Biological Chemistry

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The human BRCA1 tumor suppressor interacts with transcriptional machinery, including RNA polymerase II (RNA pol II). We demonstrated that interaction with RNA pol II is a conserved feature of BRCA1 proteins from several species. We found that full-length BRCA1 proteins universally fail to activate transcription in classic GAL4-UAS one-hybrid assays and that the activity associated with the human BRCA1 C terminus was poorly conserved in closely related homologs of the gene. Fractionation studies demonstrated that BRCA1 proteins from all species tested interacted specifically with hyperphosphorylated pol II (IIO), in preference to hypophosphorylated RNA pol II (IIA) expected at promoters. BRCA1-RNA pol II complexes showed evidence of a multiply phosphorylated heptad repeat domain in the catalytic subunit (p220) of RNA pol II, and the complex was highly functional in transcriptional run-off assays. Interestingly, endogenous BRCA1 associated with a large fraction of the processive RNA pol II activity present in undamaged cells, and the interaction was disrupted by DNA-damaging agents. Preferential interaction with processive RNA pol II in undamaged cells places BRCA1 in position to link late events in transcription with repair processes in eukaryotic cells.Mutations in the BRCA1 tumor suppressor gene are associated with an increased risk of breast and ovarian cancer and an elevated incidence of certain other cancers (1-3). Genetic and biochemical data place BRCA1 as a downstream target of ATM/ ATR in cellular responses to genotoxic stress, and the BRCA1 protein has been implicated in chromatin remodeling and homologous recombination functions in mitotic and meiotic cells (4 -8). BRCA1 complexes contain E3 ubiquitin-ligase activity, but the precise target(s) remains an avenue of active study (9, 10). Although BRCA1 proteins show surprisingly low sequence identity, human BRCA1 can replace the mouse gene in genetically engineered animals (11, 12), a result that implies general conservation of important functional motifs. Comparative functional studies are thus likely to play an important role in identifying critical targets of BRCA1 in human cells.A distinct literature has evolved linking BRCA1 to roles in transcription. BRCA1 protein has been shown to associate with RNA polymerase II (RNA pol II) 1 and various transcriptional regulators (13). Early on, several groups demonstrated that the C-terminal domain (CTD; amino acids 1380 -1863) of human BRCA1 scored positively in transcriptional activator trap experiments using various forms of the so-called "one-hybrid" assay (14 -18). While not elucidating a specific function, ectopic expression of full-length human BRCA1 was then shown to increase expression of stress-responsive genes including p21 (19), GADD45 (20), and p27 (21) and decreased expression of other genes, including c-Myc-regulated genes (22) and certain estrogen-regulated genes (23). However, it remains an open question whether endogenous BRCA1 directly mediates recruitment of RNA pol II to promoters of these...

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Section: Domain Swapping Between the Human And Mouse Ctd Leads To Chimentioning

confidence: 99%

mentioning

confidence: 99%

BRCA1 Associates with Processive RNA Polymerase II

Krum¹,

Miranda²,

Lin³

et al. 2003

Journal of Biological Chemistry

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“…A 140-kb P1 artificial chromosome containing human BRCA1 plus 60 kb of flanking sequence can rescue the embryonic lethal phenotype of Brca1 null mice (18), suggesting that all of the sequences required for correct temporal and spatial expression are contained within this sequence. The identity of these elements, how they associate with one another, and whether they contribute to breast tumorigenesis is unknown.…”

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confidence: 99%

Dynamic interactions between the promoter and terminator regions of the mammalian BRCA1 gene

Tan-Wong

French

Proudfoot

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

134

121

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The 85-kb breast cancer-associated gene BRCA1 is an established tumor suppressor gene, but its regulation is poorly understood. We demonstrate by gene conformation analysis in both human cell lines and mouse mammary tissue that gene loops are imposed on BRCA1 between the promoter, introns, and terminator region. Significantly, association between the BRCA1 promoter and terminator regions change upon estrogen stimulation and during lactational development. Loop formation is transcription-dependent, suggesting that transcriptional elongation plays an active role in BRCA1 loop formation. We show that the BRCA1 terminator region can suppress estrogen-induced transcription and so may regulate BRCA1 expression. Significantly, BRCA1 promoter and terminator interactions vary in different breast cancer cell lines, indicating that defects in BRCA1 chromatin structure may contribute to dysregulated expression of BRCA1 seen in breast tumors.transcriptional regulation ͉ chromatin conformation ͉ gene repression ͉ mammary gland ͉ breast cancer E xpression of the tumor suppressor gene BRCA1 is reduced in a significant proportion of human breast tumors (1-3). Although up to one-third of these cases can be explained by promoter hypermethylation (4, 5) for most cases the cause is unknown. Understanding the underlying mechanisms of BRCA1 gene repression is critical for generating effective strategies for re-establishing BRCA1 expression and thus restoring its tumor suppressor function.Transcriptional initiation of protein-coding genes depends on a coordinated interplay of protein-DNA and protein-protein interactions (6). In addition to the assembly of RNA polymerase II (Pol II) with basal transcription machinery on the gene promoter, numerous transcription factors are recruited to either activate or repress transcription. As many of these factors associate with DNA sequences distant to the promoter, transcriptional regulation often involves long-range DNA associations, possibly mediated by the formation of chromatin loops (7). Chromatin loops can be detected by the chromosome conformation capture (3C) technique (8), which involves formaldehyde cross-linking of chromatin in live cells, digesting DNA with restriction enzymes, and then religating DNA in dilute solution to favor intramolecular ligation. PCR is then used to detect the presence of such ligation products. 3C has been used to study the normal regulation of genes in multiple eukaryotic species and supports a looping model for gene activation and repression. For example, transcriptional activation of the ␤-globin gene in mouse is associated with interactions between multiple hypersensitive sites spanning Ͼ50 kb of DNA (9), whereas repression of the maternal IGF2 gene is linked to a long-range association between IGF2 and H19 loci, restricting access to an IGF2 enhancer (10).Several human diseases are associated with mutations in long-range control elements (11). Examples include Campomelic dysplasia, which can be caused by deletion of critical regulatory elements Ϸ50 kb upst...

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“…Translation of this sequence predicts a protein of 1849 amino acids (Figure 1a) with an estimated mass of 206 kDa. In addition, several potential splice forms (accession numbers AF479648 and AF479649) were identified from spleen, a tissue shown in other species to express high levels of BRCA1 mRNA (Lane et al, 2000). From the analysis of multiple PCR products, major alternative splice forms in spleen include one variant (D11) lacking all sequences homologous to human and mouse exon 11, and a second form (D11b) in which a cryptic splice site is used within this exon (Figure 1b).…”

Section: Cloning Of Bovine Brca1mentioning

confidence: 99%

“…Within mammals, BRCA1 genes show high degrees of divergence (Madsen et al, 2001). However, it is likely that important structural and functional elements have been conserved since human BRCA1 (hBRCA1) can genetically complement loss-of-function alleles in mice, in which BRCA1 has less than 60% amino-acid identity (Lane et al, 2000;Chandler et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Bovine BRCA1 shows classic responses to genotoxic stress but low in vitro transcriptional activation activity

Krum¹,

Womack

Lane

2003

Oncogene

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Human BRCA1 has a genetically demonstrated role in DNA repair, and has been proposed to act as a transcriptional activator in a limited number of specialized settings. To gain insight into biologically conserved functional motifs, we isolated an ortholog of BRCA1 from cattle (Bos taurus). The predicted protein product shows 72.5% sequence identity with the human protein and conservation of amino acids involved in BRCA1 structure and function. Although the bovine C-terminus is truncated by seven amino acids as compared to human, bovine BRCA1 protein exhibited a similar cell cycleregulated nuclear expression pattern. Expression was characteristically low and diffuse in the nucleus of G1/G0 cells, followed by increasing BRCA1-positive nuclear speckles in late S phase and G2/M phase cells. Bovine BRCA1 was phosphorylated and nuclear speckling was enhanced in response to DNA-damaging agents. Consistent with evidence from studies of human BRCA1, bovine BRCA1 was shown to interact with RNA polymerase II in vivo, an activity that was mapped to the C-terminal domain (CTD) (bBRCA ). Interestingly, when tested in the GAL4 transcriptional activation assay, fulllength bovine and human BRCA1 lacked any ability to act as transcriptional activators and the CTD of bovine BRCA1 had five-fold lower activity when compared to the more acidic human C-terminus. These results provide evidence that phosphorylation and nuclear relocalization are highly conserved features of the BRCA1 response to genotoxic stress. In addition, bovine BRCA1 binds the RNA polymerase II holoenzyme, but this interaction lacks significant ability to correctly orient or recruit RNA polymerase II for transcription in the classic GAL4 transcriptional activation system.

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Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

Cited by 25 publications

References 30 publications

BRCA1 Associates with Processive RNA Polymerase II

BRCA1 Associates with Processive RNA Polymerase II

Dynamic interactions between the promoter and terminator regions of the mammalian BRCA1 gene

Bovine BRCA1 shows classic responses to genotoxic stress but low in vitro transcriptional activation activity

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