The CRISPR/CAS9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional... more The CRISPR/CAS9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens employing CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features, and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent datasets, the model achieved significant results in both positive and negative selection conditions, and outperformed existing models. We also found that the sequence preference for CR...
The predominance of meningiomas in females, their accelerated growth during the luteal phase of t... more The predominance of meningiomas in females, their accelerated growth during the luteal phase of the menstrual cycle and during pregnancy, and the association between meningiomas and breast cancer have led to a number of studies examining the potential role of steroids on the growth of meningiomas. There are numerous discrepancies in the literature about the mitogenic effects of steroids on meningiomas in both in vitro and in vivo models. The aim of this study was to examine the expression of three steroid receptor coactivators, along with progesterone receptor and estrogen receptor in meningiomas. This additional regulatory layer may explain the heterogeneity of hormone responses observed in these tumors. Using Western blot analysis and immunohistochemistry, we demonstrate the expression of the steroid coactivators steroid receptor cofactor (SRC-1), amplified in breast cancer protein (AIB1), and transcriptional intermediary factor 2 (TIF2) in 81, 76, and 76% of meningiomas, respecti...
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, Jan 8, 2015
Obesity and physical activity have been hypothesized to affect breast cancer risk partly via the ... more Obesity and physical activity have been hypothesized to affect breast cancer risk partly via the androgen signaling pathway. We conducted the first study to evaluate these associations by tumor androgen receptor (AR) status. Height, weight, and physical activity were assessed using questionnaires in the Nurses' Health Study. AR, estrogen receptor (ER) and progesterone receptor (PR) status were determined using immunohistochemistry on tumor tissue and medical/pathology reports. 1,701 AR+ and 497 AR- cases were documented during 26 years of follow-up of 103,577 women. After adjusting for ER/PR status and other risk factors, the relative risks (RRs) and 95% confidence intervals (95%CIs) for every 5 kg/m2 increase in body mass index (BMI) were 1.07(1.01-1.13) for AR+ and 1.16(1.05-1.29) for AR- tumors (p-heterogeneity=0.17). The RRs(95%CIs) per 5 hours of brisk walking/week were 0.87(0.73-1.04) for AR+ and 0.67(0.45-0.99) for AR- tumors (p-heterogeneity=0.22). Further, BMI, but not ...
Molecular chaperones encompass a group of unrelated proteins that facilitate the correct assembly... more Molecular chaperones encompass a group of unrelated proteins that facilitate the correct assembly and disassembly of other macromolecular structures, which they themselves do not remain a part of. They associate with a large and diverse set of coregulators termed cochaperones that regulate their function and specificity. Amongst others, chaperones and cochaperones regulate the activity of several signaling molecules including steroid receptors, which upon ligand binding interact with discrete nucleotide sequences within the nucleus to control the expression of diverse physiological and developmental genes. Molecular chaperones and cochaperones are typically known to provide the correct conformation for ligand binding by the steroid receptors. While this contribution is widely accepted, recent studies have reported that they further modulate steroid receptor action outside ligand binding. They are thought to contribute to receptor turnover, transport of the receptor to different subc...
A baculovirus expression system was used to overproduce the human estrogen receptor in insect cel... more A baculovirus expression system was used to overproduce the human estrogen receptor in insect cells. The estrogen receptor made in this system is full-length, binds estrogen specifically, and is recognized by a monoclonal antibody to the human estrogen receptor. The recombinant estrogen receptor binds the estrogen response element (ERE) in both the absence and presence of estrogen if the binding is carried out in the absence of Mg2+. In the presence of Mg2+, the estrogen receptor binds the ERE in a hormone-dependent fashion. This effect is more pronounced at higher temperatures. Tamoxifen, a nonsteroidal anti-estrogen, is able to stimulate ERE binding to the same extent and under the same conditions as estradiol. Estradiol stimulates formation of an estrogen receptor-ERE complex with an increased mobility in native gels as compared with the complex formed without hormone or with tamoxifen. These results demonstrate that specific DNA binding of the estrogen receptor is not absolutely...
Abstract A nucleoprotein complex was isolated from simian virus (SV40)-infected African green mon... more Abstract A nucleoprotein complex was isolated from simian virus (SV40)-infected African green monkey kidney (AGMK) cells by the Sarkosyl extraction procedure [Gariglio, P., and Mousset, S.(1975). FEBS Lett. 56, 149155]. The majority of the viral transcriptional ...
The biological outcome of TMPRSS2:ERG chromosomal translocations in prostate cancer (PC) remains ... more The biological outcome of TMPRSS2:ERG chromosomal translocations in prostate cancer (PC) remains poorly understood. To address this, we compared the transcriptional effects of TMPRSS2:ERG expression in a transgenic mouse model with those of ERG knockdown in a TMPRSS2:ERG-positive PC cell line. This reveals that ERG represses the expression of a previously unreported set of androgen receptor (AR)-independent neuronal genes that are indicative of neuroendocrine (NE) cell differentiation-in addition to previously reported AR-regulated luminal genes. Cell sorting and proliferation assays performed after sustained ERG knockdown indicate that ERG drives proliferation and blocks the differentiation of prostate cells to both NE and luminal cell types. Inhibition of ERG expression in TMPRSS2:ERG-positive PC cells through blockade of AR signaling is tracked with increased NE gene expression. We also provide evidence that these NE cells are resistant to pharmacological AR inhibition and can revert to the phenotype of parental cells upon restoration of AR/ERG signaling. Our findings highlight an ERG-regulated mechanism capable of repopulating the parent tumor through the transient generation of an anti-androgen therapy-resistant cell population, suggesting that ERG may have a direct role in preventing resistance to anti-androgen therapy.Oncogene advance online publication, 29 September 2014; doi:10.1038/onc.2014.308.
BRCA1 promotes homologous recombination-mediated DNA repair (HRR). However, HRR must be tightly r... more BRCA1 promotes homologous recombination-mediated DNA repair (HRR). However, HRR must be tightly regulated to prevent illegitimate recombination. We previously found that BRCA1 HRR function is regulated by the RAP80 complex, but the mechanism was unclear. We have now observed that PARP1 interacts with and poly-ADP-ribosylates (aka PARsylates) BRCA1. PARsylation is directed at the BRCA1 DNA binding domain and downmodulates its function. Moreover, RAP80 contains a poly-ADP-ribose-interacting domain that binds PARsylated BRCA1 and helps to maintain the stability of PARP1-BRCA1-RAP80 complexes. BRCA1 PARsylation is a key step in BRCA1 HRR control. When BRCA1 PARsylation is defective, it gives rise to excessive HRR and manifestations of genome instability. BRCA1 PARsylation and/or RAP80 expression is defective in a subset of sporadic breast cancer cell lines and patient-derived tumor xenograft models. These observations are consistent with the possibility that such defects, when chronic, ...
Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the... more Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the lung parenchyma and is associated with abnormal smooth muscle proliferation affecting airways, lymphatics, and blood vessels. LAM occurs sporadically or in association with the tuberous sclerosis complex (TSC). Recent evidence demonstrates the role of aberrant β-catenin signaling in TSC. To further understand the pathogenesis of LAM and to examine the diagnostic usefulness of β-catenin, we examined protein expression in 28 pulmonary LAM cases and 10 cases of renal angiomyolipoma resected from patients with sporadic LAM. Immunohistochemical analysis was performed for established markers of LAM cells (HMB45, estrogen receptor [ER]-α, and progesterone receptor [PR]) and β-catenin. All LAM cases were positive for β-catenin and demonstrated high specificity with overall immunoreactivity superior to HMB45, ER-α, and PR. Similar expression was demonstrated in renal angiomyolipoma. Our results ...
The CRISPR/CAS9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional... more The CRISPR/CAS9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens employing CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features, and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent datasets, the model achieved significant results in both positive and negative selection conditions, and outperformed existing models. We also found that the sequence preference for CR...
The predominance of meningiomas in females, their accelerated growth during the luteal phase of t... more The predominance of meningiomas in females, their accelerated growth during the luteal phase of the menstrual cycle and during pregnancy, and the association between meningiomas and breast cancer have led to a number of studies examining the potential role of steroids on the growth of meningiomas. There are numerous discrepancies in the literature about the mitogenic effects of steroids on meningiomas in both in vitro and in vivo models. The aim of this study was to examine the expression of three steroid receptor coactivators, along with progesterone receptor and estrogen receptor in meningiomas. This additional regulatory layer may explain the heterogeneity of hormone responses observed in these tumors. Using Western blot analysis and immunohistochemistry, we demonstrate the expression of the steroid coactivators steroid receptor cofactor (SRC-1), amplified in breast cancer protein (AIB1), and transcriptional intermediary factor 2 (TIF2) in 81, 76, and 76% of meningiomas, respecti...
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, Jan 8, 2015
Obesity and physical activity have been hypothesized to affect breast cancer risk partly via the ... more Obesity and physical activity have been hypothesized to affect breast cancer risk partly via the androgen signaling pathway. We conducted the first study to evaluate these associations by tumor androgen receptor (AR) status. Height, weight, and physical activity were assessed using questionnaires in the Nurses' Health Study. AR, estrogen receptor (ER) and progesterone receptor (PR) status were determined using immunohistochemistry on tumor tissue and medical/pathology reports. 1,701 AR+ and 497 AR- cases were documented during 26 years of follow-up of 103,577 women. After adjusting for ER/PR status and other risk factors, the relative risks (RRs) and 95% confidence intervals (95%CIs) for every 5 kg/m2 increase in body mass index (BMI) were 1.07(1.01-1.13) for AR+ and 1.16(1.05-1.29) for AR- tumors (p-heterogeneity=0.17). The RRs(95%CIs) per 5 hours of brisk walking/week were 0.87(0.73-1.04) for AR+ and 0.67(0.45-0.99) for AR- tumors (p-heterogeneity=0.22). Further, BMI, but not ...
Molecular chaperones encompass a group of unrelated proteins that facilitate the correct assembly... more Molecular chaperones encompass a group of unrelated proteins that facilitate the correct assembly and disassembly of other macromolecular structures, which they themselves do not remain a part of. They associate with a large and diverse set of coregulators termed cochaperones that regulate their function and specificity. Amongst others, chaperones and cochaperones regulate the activity of several signaling molecules including steroid receptors, which upon ligand binding interact with discrete nucleotide sequences within the nucleus to control the expression of diverse physiological and developmental genes. Molecular chaperones and cochaperones are typically known to provide the correct conformation for ligand binding by the steroid receptors. While this contribution is widely accepted, recent studies have reported that they further modulate steroid receptor action outside ligand binding. They are thought to contribute to receptor turnover, transport of the receptor to different subc...
A baculovirus expression system was used to overproduce the human estrogen receptor in insect cel... more A baculovirus expression system was used to overproduce the human estrogen receptor in insect cells. The estrogen receptor made in this system is full-length, binds estrogen specifically, and is recognized by a monoclonal antibody to the human estrogen receptor. The recombinant estrogen receptor binds the estrogen response element (ERE) in both the absence and presence of estrogen if the binding is carried out in the absence of Mg2+. In the presence of Mg2+, the estrogen receptor binds the ERE in a hormone-dependent fashion. This effect is more pronounced at higher temperatures. Tamoxifen, a nonsteroidal anti-estrogen, is able to stimulate ERE binding to the same extent and under the same conditions as estradiol. Estradiol stimulates formation of an estrogen receptor-ERE complex with an increased mobility in native gels as compared with the complex formed without hormone or with tamoxifen. These results demonstrate that specific DNA binding of the estrogen receptor is not absolutely...
Abstract A nucleoprotein complex was isolated from simian virus (SV40)-infected African green mon... more Abstract A nucleoprotein complex was isolated from simian virus (SV40)-infected African green monkey kidney (AGMK) cells by the Sarkosyl extraction procedure [Gariglio, P., and Mousset, S.(1975). FEBS Lett. 56, 149155]. The majority of the viral transcriptional ...
The biological outcome of TMPRSS2:ERG chromosomal translocations in prostate cancer (PC) remains ... more The biological outcome of TMPRSS2:ERG chromosomal translocations in prostate cancer (PC) remains poorly understood. To address this, we compared the transcriptional effects of TMPRSS2:ERG expression in a transgenic mouse model with those of ERG knockdown in a TMPRSS2:ERG-positive PC cell line. This reveals that ERG represses the expression of a previously unreported set of androgen receptor (AR)-independent neuronal genes that are indicative of neuroendocrine (NE) cell differentiation-in addition to previously reported AR-regulated luminal genes. Cell sorting and proliferation assays performed after sustained ERG knockdown indicate that ERG drives proliferation and blocks the differentiation of prostate cells to both NE and luminal cell types. Inhibition of ERG expression in TMPRSS2:ERG-positive PC cells through blockade of AR signaling is tracked with increased NE gene expression. We also provide evidence that these NE cells are resistant to pharmacological AR inhibition and can revert to the phenotype of parental cells upon restoration of AR/ERG signaling. Our findings highlight an ERG-regulated mechanism capable of repopulating the parent tumor through the transient generation of an anti-androgen therapy-resistant cell population, suggesting that ERG may have a direct role in preventing resistance to anti-androgen therapy.Oncogene advance online publication, 29 September 2014; doi:10.1038/onc.2014.308.
BRCA1 promotes homologous recombination-mediated DNA repair (HRR). However, HRR must be tightly r... more BRCA1 promotes homologous recombination-mediated DNA repair (HRR). However, HRR must be tightly regulated to prevent illegitimate recombination. We previously found that BRCA1 HRR function is regulated by the RAP80 complex, but the mechanism was unclear. We have now observed that PARP1 interacts with and poly-ADP-ribosylates (aka PARsylates) BRCA1. PARsylation is directed at the BRCA1 DNA binding domain and downmodulates its function. Moreover, RAP80 contains a poly-ADP-ribose-interacting domain that binds PARsylated BRCA1 and helps to maintain the stability of PARP1-BRCA1-RAP80 complexes. BRCA1 PARsylation is a key step in BRCA1 HRR control. When BRCA1 PARsylation is defective, it gives rise to excessive HRR and manifestations of genome instability. BRCA1 PARsylation and/or RAP80 expression is defective in a subset of sporadic breast cancer cell lines and patient-derived tumor xenograft models. These observations are consistent with the possibility that such defects, when chronic, ...
Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the... more Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the lung parenchyma and is associated with abnormal smooth muscle proliferation affecting airways, lymphatics, and blood vessels. LAM occurs sporadically or in association with the tuberous sclerosis complex (TSC). Recent evidence demonstrates the role of aberrant β-catenin signaling in TSC. To further understand the pathogenesis of LAM and to examine the diagnostic usefulness of β-catenin, we examined protein expression in 28 pulmonary LAM cases and 10 cases of renal angiomyolipoma resected from patients with sporadic LAM. Immunohistochemical analysis was performed for established markers of LAM cells (HMB45, estrogen receptor [ER]-α, and progesterone receptor [PR]) and β-catenin. All LAM cases were positive for β-catenin and demonstrated high specificity with overall immunoreactivity superior to HMB45, ER-α, and PR. Similar expression was demonstrated in renal angiomyolipoma. Our results ...
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