Distinct binding of BRCA2 BRC repeats to RAD51 generates differential DNA damage sensitivity.

BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5-8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1-4. Furthermore, BRC5-8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5-8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage.

Mice carrying a hypomorphic Evi1 allele are embryonic viable but exhibit severe congenital heart defects.

The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1's critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1(fl3)) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1(δex3/δex3) mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1(fl3/fl3) mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1(δex3/δex3) knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1(δex3/δex3) mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.

PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function.

The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). ß-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function.

Regulation of podosome formation in macrophages by a splice variant of the sodium channel SCN8A.

Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells.

Epithelial V-like antigen regulates permeability of the blood-CSF barrier.

Epithelial V-like antigen (EVA), a CD3-binding immunoglobulin-like protein, regulates embryonic thymic development. Here we demonstrate that EVA is expressed in choroid plexus from mature immune competent and lymphocyte-deficient (RAG-/-) mice. Choroid plexus epithelial cells from RAG-/- mice demonstrated reduced junctional integrity and enhanced permeability that was associated with decreased expression of E-cadherin and EVA mRNA as compared to wild-type mice. Following iv infusion of an anti-CD3 antibody (145-2C11) that also binds EVA, expression of E-cadherin and EVA mRNA approached levels seen in wild-type mice. Immuno-fluorescent staining for cadherin also revealed decreased expression in untreated RAG-/- mice that could be increased by 145-2C11 treatment. Expression of mouse EVA in HEK-293 cells followed by challenge with 145-2C11 resulted in increased cytosolic calcium that was not seen in control cells. These results suggest that EVA expressed in choroid plexus cells may regulate the permeability of the blood-CSF barrier.

The mouse Ifi200 gene cluster: genomic sequence, analysis, and comparison with the human HIN-200 gene cluster.

The interferon-activatable Ifi200 gene cluster is located on mouse Chromosome 1q21-q23. We report here our analysis of two genomic regions encoding at least 10 closely related 200 family genes (Ifi201, Ifi202a, Ifi202b, Ifi202c, Ifi203a, Ifi203b, Ifi203c, Ifi203', Ifi204, and Ifi204') in 129/SvJ mice. Through a BAC-based sequencing approach, the exact structure and organization of these highly similar Ifi200 genes were obtained. A high degree of conservation (99% identity) was observed between Ifi202a and b and between Ifi203a and b. The presence of an additional transcribed region in intron 4 of Ifi203a and b suggests the possibility of alternative splicing, and a spliced variant of the Ifi204' mRNA exhibits 91% sequence identity with a related but unmapped D3 mRNA. Comparative analysis of the mouse and human clusters indicates an absence of significant sequence conservation in noncoding sequences, suggesting that the 200 family emerged prior to human-mouse speciation and subsequently diverged after gene duplication.

Acceleration of mouse mammary tumor virus-induced murine mammary tumorigenesis by a p53 172H transgene: influence of FVB background on tumor latency and identification of novel sites of proviral insertion.

We previously showed that a mammary-specific dominant-negative p53 transgene (WAP-p53(172H)) could accelerate ErbB2-induced mammary tumorigenesis in mice, but was not tumorigenic on its own. To identify other genes that cooperate with WAP-p53(172H) in tumorigenesis, we performed mouse mammary tumor virus (MMTV) proviral mutagenesis. We derived F1, N2, and N4/N5 mice from p53(172H) transgenic FVB mice backcrossed onto MMTV+ C3H/He mice. Results show the latency of MMTV tumorigenesis is correlated with FVB contribution. F1 tumors had the shortest latency (217 days), had a higher rate of metastasis, and were less differentiated than the N2 and N4/N5 tumors. The latency was 269 days in N2 mice, and lengthened to 346 days in N4/N5 mice. p53(172H) significantly accelerated MMTV tumorigenesis only in N2 mice, indicating cooperativity between p53(172H) and MMTV in this cohort. To identify genes that may be causally involved in MMTV-induced mammary tumorigenesis, we identified 60 sites of proviral insertion in the N2 tumors. Among the insertions in p53(172H) transgenic tumors were 10 genes not previously found as sites of MMTV insertion including genes involved in signaling (Pdgfra, Pde1b, Cnk1), cell adhesion (Cd44), angiogenesis (Galgt1), and transcriptional regulation (Olig1, Olig2, and Uncx4.1). These may represent cellular functions that are likely not deregulated by mutation in p53.