The BRMS1 metastasis suppressor interacts with the protein AT-rich interactive domain 4A (ARID4A, RBBP1) as part of SIN3⅐histone deacetylase chromatin remodeling complexes. These transcriptional co-repressors regulate diverse cell phenotypes depending upon complex composition. To define BRMS1 complexes and their roles in metastasis suppression, we generated BRMS1 mutants (BRMS1 mut ) and mapped ARID4A interactions. BRMS1L174D disrupted direct interaction with ARID4A in yeast two-hybrid genetic screens but retained an indirect association with ARID4A in MDA-MB-231 and -435 human breast cancer cell lines by co-immunoprecipitation. Deletion of the first coiled-coil domain (BRMS1 ⌬CC1 ) did not disrupt direct interaction in yeast two-hybrid screens but did prevent association by co-immunoprecipitation. These results suggest altered complex composition with BRMS1 mut . Although basal transcription repression was impaired and the pro-metastatic protein osteopontin was differentially down-regulated by BRMS1 L174D and BRMS1 ⌬CC1 , both down-regulated the epidermal growth factor receptor and suppressed metastasis in MDA-MB-231 and -435 breast cancer xenograft models. We conclude that BRMS1 mut , which modifies the composition of a SIN3⅐histone deacetylase chromatin remodeling complex, leads to altered gene expression profiles. Because metastasis requires the coordinate expression of multiple genes, down-regulation of at least one important gene, such as the epidermal growth factor receptor, had the ability to suppress metastasis. Understanding which interactions are necessary for particular biochemical/ cellular functions may prove important for future strategies targeting metastasis.The ability of a cancer cell to complete all steps of the metastatic cascade requires diverse tumor-host interactions that are dependent on the coordinate expression of specific genes both intrinsically and extrinsically (1-3). The metastasis suppressor BRMS1 3 has been shown to regulate the expression of multiple genes leading to the suppression of metastasis in multiple model systems, including human breast carcinoma (4, 5), melanoma (6), and ovarian carcinoma (7), without preventing orthotopic tumor growth. Specifically, down-regulation of the pro-metastatic genes osteopontin (OPN) and urokinase-type plasminogen activator has been linked to BRMS1 expression (8, 9). Gap junctional intercellular communication is restored by BRMS1 through a change in connexin expression (10). Microarray and proteomic analyses have also been performed showing multiple changes in gene and protein expression when BRMS1 was introduced (11-13). Clinically, loss of BRMS1 protein has been correlated with progesterone receptor expression and inversely correlated with HER2 expression in breast cancer patients (14).BRMS1 has been proposed to regulate transcription of genes by interaction with a large SIN3⅐HDAC chromatin remodeling complex through interaction with the protein AT-rich interactive domain 4A (ARID4A) that suppresses basal transcription in vivo using a Gal...

Vitamin D has been shown to have anti-angiogenic properties and to play a protective role in several types of cancer, including breast, prostate and cutaneous melanoma. Similarly, vitamin D levels have been shown to be protective for risk of a number of conditions, including cardiovascular disease and chronic kidney disease, as well as numerous autoimmune disorders such as multiple sclerosis, inflammatory bowel diseases and type 1 diabetes mellitus. A study performed by Parekh et al. was the first to suggest a role for vitamin D in age-related macular degeneration (AMD) and showed a correlation between reduced serum vitamin D levels and risk for early AMD. Based on this study and the protective role of vitamin D in diseases with similar pathophysiology to AMD, we examined the role of vitamin D in a family-based cohort of 481 sibling pairs. Using extremely phenotypically discordant sibling pairs, initially we evaluated the association of neovascular AMD and vitamin D/sunlight-related epidemiological factors. After controlling for established AMD risk factors, including polymorphisms of the genes encoding complement factor H (CFH) and age-related maculopathy susceptibility 2/HtrA serine peptidase (ARMS2/HTRA1), and smoking history, we found that ultraviolet irradiance was protective for the development of neovascular AMD (p = 0.001). Although evaluation of serum vitamin D levels (25-hydroxyvitamin D [25(OH)D]) was higher in unaffected individuals than in their affected siblings, this finding did not reach statistical significance.Based on the relationship between ultraviolet irradiance and vitamin D production, we employed a candidate gene approach for evaluating common variation in key vitamin D pathway genes (the genes encoding the vitamin D receptor [VDR]; cytochrome P450, family 27, subfamily B, polypeptide 1 [CYP27B1]; cytochrome P450, family 24, subfamily A, polypeptide 1 [CYP24A1]; and CYP27A1) in this same family-based cohort. Initial findings were then validated and replicated in the extended family cohort, an unrelated case-control cohort from central Greece and a prospective nested case-control population from the Nurse's Health Study and Health Professionals Follow-Up Studies, which included patients with all subtypes of AMD for a total of 2,528 individuals. Single point variants in CYP24A1 (the gene encoding the catabolising enzyme of the vitamin D pathway) were demonstrated to influence AMD risk after controlling for smoking history, sex and age in all populations, both separately and, more importantly, in a meta-analysis. This is the first report demonstrating a genetic association between vitamin D metabolism and AMD risk. These findings were also supplemented with expression data from human donor eyes and human retinal cell lines. These data not only extend previous biological studies in the AMD field, but further emphasise common antecedents between several disorders with an inflammatory/immunogenic component such as cardiovascular disease, cancer and AMD.

Age-related macular degeneration (AMD) is a progressive degenerative disease which leads to blindness, affecting the quality of life of millions of Americans. More than 1.75 million individuals in the United States are affected by the advanced form of AMD. The etiological pathway of AMD is not yet fully understood, but there is a clear genetic influence on disease risk. To date, the 1q32 (CFH) and 10q26 (PLEKHA1/ARMS2/HTRA1) loci are the most strongly associated with disease; however, the variation in these genomic regions alone is unable to predict disease development with high accuracy. Therefore, current genetic studies are aimed at identifying new genes associated with AMD and their modifiers, with the goal of discovering diagnostic or prognostic biomarkers. Moreover, these studies provide the foundation for further investigation into the pathophysiology of AMD by utilizing a systems-biology-based approach to elucidate underlying mechanistic pathways.

To identify novel genes and pathways associated with AMD, we performed microarray gene expression and linkage analysis which implicated the candidate gene, retinoic acid receptor-related orphan receptor alpha (RORA, 15q). Subsequent genotyping of 159 RORA single nucleotide polymorphisms (SNPs) in a family-based cohort, followed by replication in an unrelated case-control cohort, demonstrated that SNPs and haplotypes located in intron 1 were significantly associated with neovascular AMD risk in both cohorts. This is the first report demonstrating a possible role for RORA, a receptor for cholesterol, in the pathophysiology of AMD. Moreover, we found a significant interaction between RORA and the ARMS2/HTRA1 locus suggesting a novel pathway underlying AMD pathophysiology.

Tartrate-resistant acid phosphatase (TRAP) plays an important role in bone resorption. TRAP expression in osteoclasts is regulated by receptor activator of NF-B (RANKL), a potent activator of osteoclast differentiation. However, the molecular mechanism underlying the RANKL-induced TRAP expression remains unknown. Here we show that two regions in the mouse TRAP promoter (one at ؊1858 to ؊1239 and the other at ؊1239 to ؊1039, relative to the translation start site) are implicated in RANKL-induced TRAP transcription in RAW264.7 cells. A detailed characterization of the region at ؊1239 to ؊1039 identifies a 12-bp sequence, AGC-CACGTGGTG, that specifically binds nuclear proteins from RAW264.7 cells and primary bone marrow macrophages (BMMs) in an electrophoretic mobility shift assay (EMSA). Moreover, the binding is significantly enhanced in EMSA with nuclear extracts from RANKLtreated RAW264.7 cells and BMMs, suggesting that the 12-bp sequence may be involved in RANKL-induced TRAP transcription. Various assays reveal that nuclear proteins binding to the 12-bp sequence are upstream stimulatory factors (USF) 1 and 2. Importantly, mutation of the USF-binding site partially blocks RANKLinduced TRAP transcription in RAW264.7 cells, confirming that USF1 and USF2 are functionally involved in RANKL-induced TRAP transcription. In summary, our data show that USF1 and USF2 play a functional role in RANKL-dependent TRAP expression during osteoclast differentiation.

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2 -nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases.

BRMS1 and SUDS3 are related members of SIN3-HDAC chromatin remodeling complexes. We hypothesized that they might have overlapping functions and that SUDS3 over-expression could compensate for BRMS1 deficiency. SUDS3 expression was ubiquitous in seven breast cell lines, regardless of metastatic potential. SUDS3 over-expression in BRMS1-non-expressing metastatic cells did not suppress metastasis, motility, osteopontin secretion nor EGF receptor expression, phenotypes associated with BRMS1-mediated metastasis suppression. This study demonstrates functional differences for BRMS1 family members and highlights how the composition of SIN3-HDAC (BRMS1/SUDS3) complexes uniquely affects protein expression and biological behaviors.

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