Characterization of mammary tumors from Brg1 heterozygous mice.

Mammalian SWI/SNF-related complexes have been implicated in cancer based on some of the subunits physically interacting with retinoblastoma (RB) and other proteins involved in carcinogenesis. Additionally, several subunits are mutated or not expressed in tumor-derived cell lines. Strong evidence for a role in tumorigenesis in vivo, however, has been limited to SNF5 mutations that result primarily in malignant rhabdoid tumors (MRTs) in humans and MRTs as well as other sarcomas in mice. We previously generated a null mutation of the Brg1 catalytic subunit in the mouse and reported that homozygotes die during embryogenesis. Here, we demonstrate that Brg1 heterozygotes are susceptible to mammary tumors that are fundamentally different than Snf5 tumors. First, mammary tumors are carcinomas not sarcomas. Second, Brg1+/- tumors arise because of haploinsufficiency rather than loss of heterozygosity. Third, Brg1+/- tumors exhibit genomic instability but not polyploidy based on array comparative genomic hybridization results. We monitored Brg1+/-, Brm-/- double-mutant mice but did not observe any tumors resembling those from Snf5 mutants, indicating that the Brg1+/- and Snf5+/- tumor phenotypes do not differ simply because Brg1 has a closely related paralog whereas Snf5 does not. These findings demonstrate that BRG1 and SNF5 are not functionally equivalent but protect against cancer in different ways. We also demonstrate that Brg1+/- mammary tumors have relatively heterogeneous gene expression profiles with similarities and differences compared to other mouse models of breast cancer. The Brg1+/- expression profiles are not particularly similar to mammary tumors from Wap-T121 transgenic line where RB is perturbed. We were also unable to detect a genetic interaction between the Brg1+/- and Rb+/- tumor phenotypes. These latter findings do not support a BRG1-RB interaction in vivo.

Pc-G/trx-G and the SWI/SNF connection: developmental gene regulation through chromatin remodeling.

Pc-G and trx-G genes are responsible for maintenance of transcriptional regulation and provide a cellular memory mechanism throughout development. Studies in fly, yeast, mouse, and human have implicated modulation of higher-order chromatin structure as an important component in this process. Specifically, connections between SWI/SNF complexes and trx-G genes have provided a mechanistic link between chromatin remodeling and transcriptional regulation. Here we discuss recent genetic and biochemical data that has shed light on the molecular mechanisms and pathways associated with Pc-G and trx-G function in developmental processes such as cell cycle control and hematopoiesis. genesis 26:189-197, 2000.

A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes.

Mammalian SWI/SNF complexes utilize either brahma (Brm) or brahma-related gene 1 (Brg1) catalytic subunits to remodel nucleosomes in an ATP-dependent manner. Brm was previously shown to be dispensable, suggesting that Brm and Brg1 are functionally redundant. To test this hypothesis, we have generated a Brg1 null mutation by gene targeting, and, surprisingly, homozygotes die during the periimplantation stage. Furthermore, blastocyst outgrowth studies indicate that neither the inner cell mass nor trophectoderm survives. However, experiments with other cell types demonstrate that Brg1 is not a general cell survival factor. In addition, Brg1 heterozygotes are predisposed to exencephaly and tumors. These results provide evidence that biochemically similar chromatin-remodeling complexes have dramatically different functions during mammalian development.

Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes.

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle-regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for chi2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

A model system to study genomic imprinting of human genes.

Somatic-cell hybrids have been shown to maintain the correct epigenetic chromatin states to study developmental globin gene expression as well as gene expression on the active and inactive X chromosomes. This suggests the potential use of somatic-cell hybrids containing either a maternal or a paternal human chromosome as a model system to study known imprinted genes and to identify as-yet-unknown imprinted genes. Testing gene expression by using reverse transcription followed by PCR, we show that functional imprints are maintained at four previously characterized 15q11-q13 loci in hybrids containing a single human chromosome 15 and at two chromosome 11p15 loci in hybrids containing a single chromosome 11. In contrast, three gamma-aminobutyric acid type A receptor subunit genes in 15q12-q13 are nonimprinted. Furthermore, we have found that differential DNA methylation imprints at the SNRPN promoter and at a CpG island in 11p15 are also maintained in somatic-cell hybrids. Somatic-cell hybrids therefore are a valid and powerful system for studying known imprinted genes as well as for rapidly identifying new imprinted genes.

Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome.

Ulnar-mammary syndrome is a rare pleiotropic disorder affecting limb, apocrine gland, tooth and genital development. We demonstrate that mutations in human TBX3, a member of the T-box gene family, cause ulnar-mammary syndrome in two families. Each mutation (a single nucleotide deletion and a splice-site mutation) is predicted to cause haploinsufficiency of TBX3, implying that critical levels of this transcription factor are required for morphogenesis of several organs. Limb abnormalities of ulnar-mammary syndrome involve posterior elements. Mutations in TBX5, a related and linked gene, cause anterior limb abnormalities in Holt-Oram syndrome. We suggest that during the evolution of TBX3 and TBX5 from a common ancestral gene, each has acquired specific yet complementary roles in patterning the mammalian upper limb.

Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.

Holt-Oram syndrome is a developmental disorder affecting the heart and upper limb, the gene for which was mapped to chromosome 12 two years ago. We have now identified a gene for this disorder (HOS1). The gene (TBX5) is a member of the Brachyury (T) family corresponding to the mouse Tbx5 gene. We have identified six mutations, three in HOS families and three in sporadic HOS cases. Each of the mutations introduces a premature stop codon in the TBX5 gene product. Tissue in situ hybridization studies on human embryos from days 26 to 52 of gestation reveal expression of TBX5 in heart and limb, consistent with a role in human embryonic development.

Identification, characterization, and localization to chromosome 17q21-22 of the human TBX2 homolog, member of a conserved developmental gene family.

The T-box motif is present in a family of genes whose structural features and expression patterns support their involvement in developmental gene regulation. Previously, sequence comparisons among the T-box domains of ten vertebrate and invertebrate T-box (Tbx) genes established a phylogenetic tree with three major branches. The Tbx2-related branch includes mouse Mm-Tbx2 and Mm-Tbx3, Drosophila optomotor-blind (Dm-Omb), and Caenorhabditis elegans Ce-Tbx2 and Ce-Tbx2 and Ce-Tbx7 genes. From the localization of Mm-Tbx2 to Chromosome (Chr) 11, we focused our search for the human homolog, Hs-TBX2, within a region of synteny conservation on Chr 17q. We used Dm-Omb polymerase chain reaction (PCR) primers to amplify a 137-basepair(bp)_ product from human genomic, Chr 17 monochromosome hybrid, and fetal kidney cDNA templates. The human PCR product showed 89% DNA sequence identity and 100% peptide sequence identity to the corresponding T-box segment of Mm-Tbx2. The putative Hs-TBX2 locus was isolated within a YAC contig that included three anonymous markers, D17S792, and D17S948, located at Chr 17q21-22. Hybridization-and PCR-based screening of a 15-week fetal kidney cDNA library yielded several TBX2 clones. Sequence analysis of clone lambda omicron TBX2-1 confirmed homology to Mm-TBx2-90% DNA sequence identity over 283 nt, and 96% peptide sequence identity over 94 amino acids. Similar analysis of Hs-TBX2 cosmid 154F11 confirmed the cDNA coding sequence and also identified a 1.7-kb intron located at the same relative position as in Mm-Tbx2. Phylogenetic analyses of the T-box domain sequences found in several vertebrate and invertebrate species further suggested that the putative human TBX2 and mouse Tbx2 are true homologs. Northern blot analysis identified two major TBX2 expression fetal kidney and lung; and in adult kidney, lung, ovary, prostate, spleen, and testis. Reduced expression levels were seen in heart, white blood cells, small intestine, and thymus. These results suggest that Hs-TBX2 could play important roles in both developmental and postnatal gene regulation.