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Biomedical subjects

Myles Brown

Publications and source records attributed to Myles Brown.

At least 19 recordsLinked to original sources

scATAnno: Automated Cell Type Annotation for Single-cell ATAC-seq Data.

Recent advances in single-cell epigenomic techniques have increased the demand for single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) analysis. One key analytical task is to determine cell type identity based on epigenetic data. Here, we introduce scATAnno, a Python package designed to automatically annotate scATAC-seq data using large-scale scATAC-seq reference atlases. This workflow generates reference atlases from publicly available datasets, enabling accurate cell type annotation by integrating query data with reference atlases without the use of single-cell RNA sequencing (scRNA-seq) data. To enhance annotation accuracy, we incorporated k-nearest neighbors (KNN)-based and weighted distance-based uncertainty scores to effectively detect cell populations within the query data that are distinct from all cell types in the reference data. We compared and benchmarked scATAnno against five other published cell annotation approaches, demonstrating its superior performance across multiple datasets and metrics. We further showcased the utility of scATAnno across multiple datasets, including peripheral blood mononuclear cells (PBMCs), triple-negative breast cancer (TNBC), and basal cell carcinoma (BCC), and demonstrated that scATAnno accurately annotates cell types across diverse biological conditions. Overall, scATAnno is a useful tool for scATAC-seq reference atlas construction and cell type annotation and can facilitate the interpretation of new scATAC-seq datasets in complex biological systems. scATAnno is publicly available at https://scatanno-main.readthedocs.io/.

Single-Cell Analysis↗

Targeting the AIB1 oncogene through mammalian target of rapamycin inhibition in the mammary gland.

Amplified in breast cancer 1 (AIB1), an estrogen receptor (ER) coactivator, is frequently amplified or overexpressed in human breast cancer. We previously developed a transgenic mouse model in which AIB1 can act as an oncogene, giving rise to a premalignant hyperplastic mammary phenotype as well as to a high incidence of mammary tumors that are primarily ER(+). In this model, the AIB1 transgene is responsible for continued activation of the insulin-like growth factor-I receptor, suggesting a role for the activation of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway in the premalignant phenotype and tumor development. Here we show that treatment of AIB1 transgenic mice with the mTOR inhibitor RAD001 reverts the premalignant phenotype. Furthermore, treatment of cell lines derived from AIB1-dependent mammary tumors with RAD001 in culture leads to a G(1) cell cycle arrest. Lastly, tumor growth after injection of ER(+) AIB1 tumor cell lines into wild-type animals is inhibited by RAD001 treatment. In this ER(+) model, inhibition of tumor growth by RAD001 was significantly better than inhibition by the antiestrogen 4-hydroxytamoxifen alone, whereas a combination of both RAD001 and 4-hydroxytamoxifen was most effective. Based on these results, we propose that the combination of mTOR inhibition and ER-targeted endocrine therapy may improve the outcome of the subset of ER(+) breast cancers overexpressing AIB1. These studies provide preclinical support for the clinical development of RAD001 and suggest that AIB1 may be a predictive factor of RAD001 response.

Animals↗

Genome-wide analysis of estrogen receptor binding sites.

The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.

Adaptor Proteins, Signal Transducing↗

A cell-type-specific transcriptional network required for estrogen regulation of cyclin D1 and cell cycle progression in breast cancer.

Estrogen stimulates the proliferation of the most common type of human breast cancer that expresses estrogen receptor alpha (ERalpha) through the activation of the cyclin D1 (CCND1) oncogene. However, our knowledge of ERalpha transcriptional mechanisms remains limited. Hence, it is still elusive why ERalpha ectopically expressed in ER-negative breast cancer cells (BCC) is functional on ectopic reporter constructs but lacks activity on many endogenous target genes, including CCND1. Here, we show that estradiol (E2) stimulation of CCND1 expression in BCC depends on a novel cell-type-specific enhancer downstream from the CCND1 coding region, which is the primary ERalpha recruitment site in estrogen-responsive cells. The pioneer factor FoxA1 is specifically required for the active chromatin state of this enhancer and as such is crucial for both CCND1 expression and subsequent cell cycle progression. Interestingly, even in BCC, CCND1 levels and proliferation are tightly controlled by E2 through the establishment of a negative feedforward loop involving the induction of NFIC, a putative tumor suppressor capable of directly repressing CCND1 transcription. Taken together, our results reveal an estrogen-regulated combinatorial network including cell-specific cis- and trans-regulators of CCND1 expression where ERalpha collaborates with other transcription factors associated with the ER-positive breast cancer phenotype, including FoxA1 and NFIC.

Adaptor Proteins, Signal Transducing↗

Model-based analysis of tiling-arrays for ChIP-chip.

We propose a fast and powerful analysis algorithm, titled Model-based Analysis of Tiling-arrays (MAT), to reliably detect regions enriched by transcription factor chromatin immunoprecipitation (ChIP) on Affymetrix tiling arrays (ChIP-chip). MAT models the baseline probe behavior by considering probe sequence and copy number on each array. It standardizes the probe value through the probe model, eliminating the need for sample normalization. MAT uses an innovative function to score regions for ChIP enrichment, which allows robust P value and false discovery rate calculations. MAT can detect ChIP regions from a single ChIP sample, multiple ChIP samples, or multiple ChIP samples with controls with increasing accuracy. The single-array ChIP region detection feature minimizes the time and monetary costs for laboratories newly adopting ChIP-chip to test their protocols and antibodies and allows established ChIP-chip laboratories to identify samples with questionable quality that might contaminate their data. MAT is developed in open-source Python and is available at http://chip.dfci.harvard.edu/ approximately wli/MAT. The general framework presented here can be extended to other oligonucleotide microarrays and tiling array platforms.

Algorithms↗

Primate-specific evolution of an LDLR enhancer.

BACKGROUND: Sequence changes in regulatory regions have often been invoked to explain phenotypic divergence among species, but molecular examples of this have been difficult to obtain. RESULTS: In this study we identified an anthropoid primate-specific sequence element that contributed to the regulatory evolution of the low-density lipoprotein receptor. Using a combination of close and distant species genomic sequence comparisons coupled with in vivo and in vitro studies, we found that a functional cholesterol-sensing sequence motif arose and was fixed within a pre-existing enhancer in the common ancestor of anthropoid primates. CONCLUSION: Our study demonstrates one molecular mechanism by which ancestral mammalian regulatory elements can evolve to perform new functions in the primate lineage leading to human.

Amino Acid Motifs↗

p63 regulates an adhesion programme and cell survival in epithelial cells.

p63 is critical for epithelial development yet little is known about the transcriptional programmes it regulates. By characterising transcriptional changes and cellular effects following modulation of p63 expression, we have defined a vital role for p63 in cellular adhesion. Knockdown of p63 expression caused downregulation of cell adhesion-associated genes, cell detachment and anoikis in mammary epithelial cells and keratinocytes. Conversely, overexpression of the TAp63gamma or deltaNp63alpha isoforms of p63 upregulated cell adhesion molecules, increased cellular adhesion and conferred resistance to anoikis. Apoptosis induced by loss of p63 was rescued by signalling downstream of beta4 integrin. Our results implicate p63 as a key regulator of cellular adhesion and survival in basal cells of the mammary gland and other stratified epithelial tissues.

Anoikis↗

Estrogen receptor target gene: an evolving concept.

Estrogen receptor (ER) functions as a transcription factor to induce gene expression events sufficient for cell division and breast cancer progression. A significant body of work exists on the identification of ER gene targets and the cofactors that contribute to these transcription events, yet surprisingly little is known of the cis-regulatory elements involved. In this review, we investigate the advances in technology that contribute to a comprehensive understanding of ER target genes and explore recent work identifying cis-regulatory domains that augment transcription of these targets. Specifically, we find that ER association with gene targets results from an association with the pioneer factor FoxA1, responsible for recruitment of ER to the genome. Recruitment of ER to the genome does not occur at promoter proximal regions, but instead involves distal enhancer elements that function to tether the ER complex to the target gene promoters. These advances in technology permit a more detailed investigation of ER activity and may aid in the development of superior drug interventions.

Animals↗

X chromosomal abnormalities in basal-like human breast cancer.

Sporadic basal-like cancers (BLC) are a distinct class of human breast cancers that are phenotypically similar to BRCA1-associated cancers. Like BRCA1-deficient tumors, most BLC lack markers of a normal inactive X chromosome (Xi). Duplication of the active X chromosome and loss of Xi characterized almost half of BLC cases tested. Others contained biparental but nonheterochromatinized X chromosomes or gains of X chromosomal DNA. These abnormalities did not lead to a global increase in X chromosome transcription but were associated with overexpression of a small subset of X chromosomal genes. Other, equally aneuploid, but non-BLC rarely displayed these X chromosome abnormalities. These results suggest that X chromosome abnormalities contribute to the pathogenesis of BLC, both inherited and sporadic.

Alleles↗

The joint effect of smoking and AIB1 on breast cancer risk in BRCA1 mutation carriers.

Women with BRCA1 mutations are at an elevated risk for breast cancer. AIB1 (NCOA3/SRC3) genotype and smoking may alter this risk. We examined the differences in breast cancer risk by AIB1 polyglutamine repeat polymorphism and pre-diagnosis smoking habits for BRCA1 mutation carriers to determine if there was an interaction between smoking and AIB1 genotype. Multivariate Cox proportional hazards regression was used with 316 female BRCA1 mutation carriers to model breast cancer risk. Ever having smoked was associated with a decreased breast cancer risk [Hazard Ratio (HR) = 0.63, 95% CI, 0.47-0.87]. A dose-response relationship between number of pack-years smoked and breast cancer risk was also found for women who smoked <20 pack years of cigarettes (HR = 0.72, 95% CI, 0.52-1.00) and for women who smoked >/=20 pack years (HR = 0.41, 95% CI, 0.23-0.71; P for trend = 0.0007). Women with a 28 repeat allele for AIB1 had a significantly reduced risk of breast cancer (HR = 0.72, 95% CI, 0.51-1.00). Women who smoked >/=20 pack-years with a 28 repeat allele had an even greater reduced risk of breast cancer (HR = 0.19, 95% CI, 0.07-0.54) compared to women who were never smokers with no 28 allele. Since AIB1 appears to modulate the effect of endogenous hormones via the estrogen receptor, and smoking affects circulating hormone levels, these results support evidence that steroid hormones play an important role in breast carcinogenesis in BRCA1 mutation carriers, and suggest mechanisms for developing novel cancer prevention strategies for BRCA1 mutation carriers.

Acetyltransferases↗

Spatial and temporal recruitment of androgen receptor and its coactivators involves chromosomal looping and polymerase tracking.

Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer, where it is a key therapeutic target. Here we report that, in contrast to estrogen receptor transcription complexes which form within minutes and recycle hourly, the levels of regulatory regions bound by AR complexes rise over a 16 hr period and then slowly decline. AR regulation of the prostate specific antigen (PSA) gene involves both a promoter-proximal sequence as well as an enhancer approximately 4 kb upstream. Recruitment of AR and its essential coactivators at both sites creates a chromosomal loop that allows RNA polymerase II (pol II) to track from the enhancer to the promoter. Phosphorylation of the pol II C-terminal domain is required for pol II tracking but not chromosomal looping. Development of improved hormonal therapies for prostate cancer must take in account the specific spatial and temporal modes of AR-mediated gene regulation.

Androgens↗

Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1.

Estrogen plays an essential physiologic role in reproduction and a pathologic one in breast cancer. The completion of the human genome has allowed the identification of the expressed regions of protein-coding genes; however, little is known concerning the organization of their cis-regulatory elements. We have mapped the association of the estrogen receptor (ER) with the complete nonrepetitive sequence of human chromosomes 21 and 22 by combining chromatin immunoprecipitation (ChIP) with tiled microarrays. ER binds selectively to a limited number of sites, the majority of which are distant from the transcription start sites of regulated genes. The unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct ER binding requires the presence of Forkhead factor binding in close proximity. Furthermore, knockdown of FoxA1 expression blocks the association of ER with chromatin and estrogen-induced gene expression demonstrating the necessity of FoxA1 in mediating an estrogen response in breast cancer cells.

Animals↗

Modulator recognition factor 1, an AT-rich interaction domain family member, is a novel corepressor for estrogen receptor alpha.

Cardiovascular tissues are important targets of estrogen action. Vascular cells express the two known estrogen receptors (ERs), ERalpha and ERbeta, ligand-activated transcription factors that regulate gene transcription through interactions with both coactivator and corepressor molecules. To isolate ERalpha coregulators in vascular cells, we performed a yeast two-hybrid screen for ERalpha-interacting proteins using a human aorta library. Here we report the identification of modulator recognition factor 1 (MRF1) as an ERalpha-interacting corepressor protein. Full-length MRF1 binds to both the N terminus and the C terminus of ERalpha. ERalpha and MRF1 coimmunoprecipitate in an estradiol-independent manner, and recombinant ERalpha binds to both full-length and COOH-terminal MRF1 in the absence of estradiol. MRF1 also interacts in a ligand-dependent manner with thyroid receptor alpha, retinoid X receptor alpha, and androgen receptor, and in a ligand-independent manner with ERbeta and the retinoic acid receptor. MRF1 RNA is highly expressed in aorta, heart, skeletal muscle, and liver. MRF1 has intrinsic repressor activity in an in vitro GAL reporter assay. Transient transfection studies show that MRF1 represses transcription by ERalpha activated by estradiol in a dose-dependent manner, as well as by the selective ER modulators 4-hydroxy-tamoxifen and raloxifene. MRF1 repression is not influenced by pharmacological inhibition of histone deacetylase. These data identify MRF1 as a repressor of ERalpha-mediated transcriptional activation and support a role for MRF1 in regulating ER-dependent gene expression in cardiovascular and other cells.

Aorta↗

Cell cycle progression stimulated by tamoxifen-bound estrogen receptor-alpha and promoter-specific effects in breast cancer cells deficient in N-CoR and SMRT.

Estrogen receptor alpha (ERalpha) mediates the effects of estrogens in breast cancer development and growth via transcriptional regulation of target genes. Tamoxifen can antagonize ERalpha activity and has been used in breast cancer therapy. Tamoxifen-bound ERalpha associates with nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) at certain target genes. Here we show the effects of reducing N-CoR and SMRT levels on the actions of estrogen and tamoxifen in breast cancer cells. Silencing both corepressors led to tamoxifen-stimulated cell cycle progression without activation of the ERalpha target genes c-myc, cyclin D1, or stromal cell-derived factor 1, which play a role in estrogen-induced proliferation. By contrast, expression of X-box binding protein 1 was markedly elevated in tamoxifen-treated cells in which N-CoR and SMRT had been silenced. The gain in cell cycle entry seen with tamoxifen when N-CoR and SMRT were silenced was dependent on ERalpha and not observed upon treatment with estradiol or epidermal growth factor. These results suggest that N-CoR and SMRT play an active role in preventing tamoxifen from stimulating proliferation in breast cancer cells through repression of a subset of target genes involved in ERalpha function and cell proliferation.

Antineoplastic Agents, Hormonal↗

Androgen mediated regulation and functional implications of fkbp51 expression in prostate cancer.

PURPOSE: Androgen ablation continues to be the most effective therapy for metastatic prostate cancer, although the biologically active androgen receptor (AR) target genes remain largely unknown. Because AR signaling continues in hormone refractory disease, effector AR target genes may have therapeutic import. MATERIALS AND METHODS: We used oligonucleotide microarrays to identify genes with expression induced by androgen and associated with androgen independent growth. The androgen induced expression of FKBP51, a steroid receptor chaperone, was further investigated in LNCaP cells by Northern and Western analysis, and in primary prostate specimens using immunohistochemistry. We used stable clones over expressing FKBP51 to test the functional effects of FKBP51. RESULTS: Many genes had expression that correlates with androgen stimulation in LNCaP cells but relatively few had reproducible, androgen mediated changes in expression across multiple prostate cancer cell lines. FKBP51 had androgen induced RNA and protein expression in LNCaP cells and decreased expression in normal prostate epithelial cells following castration. Further study demonstrated that FKBP51 induction was not a generalized response to cell proliferation, FKBP51 protein physically interacts with AR and LNCaP cells constitutively over expressing FKBP51 have increased ligand mediated AR activation of an exogenous AR reporter construct and endogenous prostate specific antigen. CONCLUSIONS: Taken together these results confirm FKBP51 as an androgen induced gene, demonstrate a physical interaction between FKBP51 and AR and suggest that FKBP51 over expression increases AR transcriptional activity in prostate cancer.

HSP90 Heat-Shock Proteins↗

Coactivator AIB1 links estrogen receptor transcriptional activity and stability.

Agonist-mediated degradation of estrogen receptor alpha (ERalpha) has been associated with its transcriptional activity. However, the mechanism by which ERalpha is targeted for degradation and whether there is a direct functional link between ERalpha stability and ERalpha-mediated transactivation have not been elucidated. Here we provide evidence that the p160 coactivator, AIB1, uniquely mediates agonist-induced, but not antagonist-induced, ERalpha degradation. We show that AIB1 recruitment by ERalpha is not only necessary but also sufficient to promote degradation. Suppression of AIB1 levels leads to ERalpha stabilization in the presence of 17beta-estradiol and, despite increased ERalpha levels, reduced recruitment of ERalpha to endogenous target gene promoters. In addition, association of RNA polymerase II with ERalpha target promoters is lost when AIB1 is suppressed, leading to inhibition of target gene transcription. AIB1 thus plays a dual role in regulating ERalpha activity, one in recruiting transcription factors including other coactivators involved in gene activation and the other in regulating ERalpha protein degradation mediated by the ubiquitin-proteosome machinery.

Cell Line, Tumor↗

High tumor incidence and activation of the PI3K/AKT pathway in transgenic mice define AIB1 as an oncogene.

The gene encoding AIB1, an estrogen receptor coactivator, is amplified in a subset of human breast cancers. Here we show that overexpression of AIB1 in transgenic mice (AIB1-tg) leads to mammary hypertrophy, hyperplasia, abnormal postweaning involution, and the development of malignant mammary tumors. Tumors are also increased in other organs, including the pituitary and uterus. AIB1 overexpression increases mammary IGF-I mRNA and serum IGF-I protein levels. In addition, IGF-I receptor and downstream signaling molecules are activated in primary mammary epithelial cells and mammary tumor cells derived from AIB1-tg mice. Knockdown of AIB1 expression in cultured AIB1-tg mammary tumor cells leads to reduced IGF-I mRNA levels and increased apoptosis, suggesting that an autocrine IGF-I loop underlies the mechanism of AIB1-induced oncogenesis.

Acetyltransferases↗