Spatiotemporal control of estrogen-responsive transcription in ERα-positive breast cancer cells.

Recruitment of transcription machinery to target promoters for aberrant gene expression has been well studied, but underlying control directed by distant-acting enhancers remains unclear in cancer development. Our previous study demonstrated that distant estrogen response elements (DEREs) located on chromosome 20q13 are frequently amplified and translocated to other chromosomes in ERα-positive breast cancer cells. In this study, we used three-dimensional interphase fluorescence in situ hybridization to decipher spatiotemporal gathering of multiple DEREs in the nucleus. Upon estrogen stimulation, scattered 20q13 DEREs were mobilized to form regulatory depots for synchronized gene expression of target loci. A chromosome conformation capture assay coupled with chromatin immunoprecipitation further uncovered that ERα-bound regulatory depots are tethered to heterochromatin protein 1 (HP1) for coordinated chromatin movement and histone modifications of target loci, resulting in transcription repression. Neutralizing HP1 function dysregulated the formation of DERE-involved regulatory depots and transcription inactivation of candidate tumor-suppressor genes. Deletion of amplified DEREs using the CRISPR/Cas9 genomic-editing system profoundly altered transcriptional profiles of proliferation-associated signaling networks, resulting in reduction of cancer cell growth. These findings reveal a formerly uncharacterized feature wherein multiple copies of the amplicon congregate as transcriptional units in the nucleus for synchronous regulation of function-related loci in tumorigenesis. Disruption of their assembly can be a new strategy for treating breast cancers and other malignancies.

Elevated snoRNA biogenesis is essential in breast cancer.

Hyperactive ribosomal biogenesis is widely observed in cancer, which has been partly attributed to the increased rDNA transcription by Pol I in cancer. However, whether small nucleolar RNAs (snoRNAs), a class of non-coding RNAs crucial in ribosomal RNA (rRNA) maturation and functionality, are involved in cancer remains elusive. We report that snoRNAs and fibrillarin (FBL, an enzymatic small nucleolar ribonucleoprotein, snoRNP) are frequently overexpressed in both murine and human breast cancer as well as in prostate cancers, and significantly, that this overexpression is essential for tumorigenicity in vitro and in vivo. We demonstrate that when the elevated snoRNA pathway is suppressed, the tumor suppressor p53 can act as a sentinel of snoRNP perturbation, the activation of which mediates the growth inhibitory effect. On the other hand, high level of FBL interferes with the activation of p53 by stress. We further show that p53 activation by FBL knockdown is not only regulated by the ribosomal protein-MDM2-mediated protein stabilization pathway, but also by enhanced PTB-dependent, cap-independent translation. Together, our data uncover an essential role of deregulated snoRNA biogenesis in tumors and a new mechanism of nucleolar modulation of p53.

DNA methylation and allelic losses on chromosome arm 14q in oligodendroglial tumours.

Cytogenetic and molecular genetic studies have shown frequent losses on the long arm of chromosome 14 in different types of human gliomas. Using differential methylation hybridization as a genome-wide screening approach to determine DNA methylation patterns in gliomas, we recently identified two DNA fragments in 14q23.1 (CGI-clone musical sharp396) and 14q32.12 (CGI-clone musical sharp519) that were differentially methylated between astrocytic gliomas and mixed oligoastrocytomas. To validate this observation, we examined these 14q32.12 locus for methylation in an extended series of 43 astrocytic and oligodendroglial gliomas. All tumours were additionally investigated for loss of heterozygosity (LOH). Microsatellite analysis showed LOH in seven of 28 (25%) oligodendroglial tumours and three of 15 (20%) astrocytic tumours. Seven tumours demonstrated LOH at all informative 14q loci whereas three tumours carried partial deletions defining a commonly deleted region at 14q22.3-q32.1 between the microsatellite markers D14S282 and D14S995. Methylation-specific PCR analysis of the 14q32.12 locus revealed hypermethylation in 12 of 43 gliomas (28%). Hypermethylation was restricted to tumours with oligodendroglial differentiation (12 of 28 tumours, 43%). However, none of the hypermethylated tumours demonstrated LOH on 14q and vice versa. In total, 19 of 28 oligodendroglial tumours (68%) showed either hypermethylation at the 14q32.12 locus or LOH at 14q22.3-q32.2. Taken together, our data lend further support for the location of one or more yet to be identified glioma-associated tumour suppressor gene(s) on 14q. In addition, the restriction of 14q32.12 methylation to oligodendroglial tumours suggests a role for epigenetic DNA modifications in these particular gliomas.

ERTargetDB: an integral information resource of transcription regulation of estrogen receptor target genes.

The estrogen receptor (ER) plays an important role in several physiologic functions of both the reproductive and non-reproductive systems. Malignancies of the ER have been associated with the development of cancers, including those of the prostate and breast. Hence it has become of significant importance to characterize the transcriptional regulation of ER target genes. We have created ERTargetDB in order to integrate the previously published ER target gene information that is available in various publications and databases. This information resource provides researchers with an easy access to ER target genes and the regulatory mechanisms in the corresponding promoters. The current version contains 40 genes with experimentally verified estrogen response elements (EREs), 32 experimentally verified ERE tethering sites, 40 genes identified by the chromatin immunoprecipitation microarray, 381 genes from gene expression microarray and 2948 genes from computational prediction. ERTargetDB provides an integral information resource for direct target genes of ERs for the endocrinology research community. It should prove useful in the investigation of gene regulation and aid the development of computational tools for the prediction of ER target genes.

Uterine responsiveness to estradiol and DNA methylation are altered by fetal exposure to diethylstilbestrol and methoxychlor in CD-1 mice: effects of low versus high doses.

We examined the effects on female CD-1 mice of fetal exposure to low doses of the drug diethylstilbestrol (DES) (0.1 microg/kg/day) and the insecticide methoxychlor (MXC) (10 microg/kg/day) as well as 1000-fold higher doses: 100 microg/kg/day DES and 10,000 microg/kg/day MXC. Pregnant females were administered these chemicals on gestation days 12-18. At 7-8 months of age, female offspring were ovariectomized and implanted for 7 days with a Silastic capsule containing estradiol. Relative to controls, females exposed to the 0.1 microg DES dose showed significantly heavier uteri, while females exposed to the 100 microg DES dose showed significantly lighter uteri. Females exposed prenatally to the 10 microg/kg dose of MXC had significantly heavier uteri relative to females exposed to the 10,000 microg/kg dose of MXC, but neither group differed significantly from controls. Liver weight for females exposed to both doses of DES was significantly greater than controls. Using a microarray approach to analyze DNA methylation, an increase in ribosomal DNA (rDNA) methylation was observed. Sequence data and Southern analysis indicate an increase in 18S rDNA and 45S pre-rDNA methylation in uterine samples exposed prenatally to low and high doses of DES. We thus found opposite effects of fetal exposure to a low and a high dose of DES on the uterine response to estradiol (inverted-U dose-response relationship). In contrast, there was a monotonic dose-response relationship found for prenatal DES exposure on both liver weight and ribosomal DNA hypermethylation.