Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell death. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the nondividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the ؊701 to ؊921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.

The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628 bp fragment (−613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site of SLUG. SLUG specifically inhibited VDR gene promoter activity. Chromatin-immunoprecipitation (ChIP) assays revealed that SLUG is recruited on the native VDR gene promoter along with the co-repressor protein CtBP1 and the effector protein HDAC1. These data suggests that SLUG binds to the E2-box sequences of the VDR gene promoter and recruits CtBP1 and HDAC1, which results in the inhibition of VDR gene expression by chromatin remodeling. SLUG is a member of the SNAI family of C 2 H 2 -zinc finger family of transcriptional repressors [4][5][6]. It is involved in the epithelial-mesenchymal transition during development [5], acts as an inhibitor of apoptosis [7], and causes tubulogenesis during breast and kidney developments [4,5]. The genes inhibited by SLUG include E-cadherin [8], claudins [9], BRCA2 [10], and cytokeratins [11]. Our ChIP-DSL analysis of 20,000 human gene promoter array revealed that more than 150 promoters bind to SLUG at their promoters (Mittal, M.K. and Chaudhuri, G., unpublished data). VDR gene is one of the candidate SLUG-regulated genes. Here, we report that SLUG indeed binds in vivo to the VDR gene promoter in human breast cell nucleus and inhibits VDR gene expression by chromatin remodeling.

BackgroundBRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. The expression of BRCA2 gene is silenced at the G0/G1 phase of cell growth and is de-silenced at the S/G2 phase. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. Standard techniques like cell synchronization by serum starvation, flow cytometry, N-terminal or C-terminal FLAG epitope-tagged protein expression, immunofluorescence confocal microscopy, dual luciferase assay for promoter evaluation, and chromatin immunoprecipitation assay were employed during this study.ResultsHuman BRCA2 gene promoter is active in both the forward and the reverse orientations. This promoter is 8-20 fold more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1). When the cells are in the dividing state (S/G2), the forward activity of the promoter is 5-8 folds higher than the reverse activity. The reverse activity transcribes the ZAR2 mRNA with 966 nt coding sequence which codes for a 321 amino acid protein. ZAR2 has two C4 type zinc fingers at the carboxyl terminus. In the G0/G1 growth phase ZAR2 is predominantly located inside the nucleus of the breast cells, binds to the BRCA2 promoter and inhibits the expression of BRCA2. In the dividing cells, ZAR2 is trapped in the cytoplasm.ConclusionsBRCA2 gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger containing transcription factor ZAR2. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 binds to BRCA2/ZAR2 bi-directional promoter in vivo and is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.

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The ubiquitin-proteasome system regulates protein degradation in mammalian and other eukaryotic cells to affect a wide range of biological processes, including cellular proliferation and differentiation (1-3). The covalent modification of protein substrates with ubiquitin in the form of polymeric chains, with ubiquitin moieties connected through isopeptide linkage between the ⑀-amide group of lysine and the carboxyl end of glycine, is the critical prerequisite of proteasomal degradation of proteins (4).Proteasomal degradation of proteins is initiated by the concerted actions of three enzymes, E1, E2, and E3, which ubiquitinate the target protein (4 -9). The specificity of ubiquitination is largely determined by E3 ligases, which recognize the protein substrate and facilitate ubiquitin transfer from the E2 ubiquitin-conjugating enzyme onto substrate (4 -9). A single E2 enzyme can usually interact with several E3 ubiquitin ligases and thereby affect multiple targets (10). Recently, the E2 enzymes of the UbcH5 family is drawing the attention of biologists due to their perceived roles in the degradation of key regulatory molecules like IkB (11), cyclin D1 (12, 13), TP53, and MDM2 (14).The human UbcH5 family consists of three homologs: UbcH5a, UbcH5b (also known as Ubc4), and UbcH5c (also known as UbE2D3) (15-18). The detailed mechanism of the polyubiquitination of IkB␣ is shown to begin with the action of the UbcH5c E2 ubiquitin-conjugating enzyme that transfers a single ubiquitin to IkB␣ (11). Subsequently, the Cdc34 E2 functions in the formation of polyubiquitin chains (11). Whether similar modalities involving Cdc34 are operative in the degradation of cyclin D1 or TP53 is not known. Little is also known about the potential mechanisms that may regulate the levels of UbcH5c in the breast cancer cells.Cyclins modulate the cyclin-dependent kinases, which are the key cell cycle regulators (19 -24). These molecules are often regulated by ubiquitination through different mechanisms (20 -24). Binding of cyclin D1 to Cdk4 and Cdk6 leads to the phosphorylation of the retinoblastoma protein (25). Phosphorylation of retinoblastoma protein prevents it from repressing the E2F family of transcription factors and leads to the transcription of several genes required for the G 1 to S phase transition, thereby promoting cellular proliferation (25). Cyclin D1 is overexpressed in ϳ50% of breast cancers and is implicated as the cause of their increased rate of proliferation (26).One of the causes for breast cancer cell proliferation and metastasis is the overexpression of the metastasis modulator transcriptional repressor protein SLUG (27)(28)(29)(30)(31). This protein binds to the E2-box sequence of its target gene promoters and down-regulates their expression by chromatin remodeling (28,32). SLUG induces metastasis through the repression of several genes in breast and other cancer cells (28,29). We report here that SLUG indirectly elevates the levels of cyclin D1

Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process. KeywordsInvasiveness; Breast tumor; SLUG; Cytokeratin 8; Cytokeratin 19; Metallothionein 1G; Metallothionein 1L; siRNA; Microarray A serious gap in our understanding of cancer concerns malignancy and metastasis. We have yet to clearly identify gene defects that specifically permit cells to invade surrounding tissues, spread through the body, and form metastases. Local invasiveness requires a break down of the mechanisms that normally hold epithelial cells together. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. One of the known genotypic differences between these two cells is that BT-549 expresses the zinc finger repressor protein SLUG but the MDA-MB-468 cells do not [1]. Hence, we tested whether any gene overexpressed in the MDA-MB-468 cells is regulated by SLUG.We paid particular attention to SLUG because it is implicated as one of the determinants of tumor cell invasiveness [2][3][4][5][6]. This protein is a member of a superfamily of transcriptional regulators that are implicated in the formation of mesoderm and neural crest, as well as in We then overexpressed SLUG in MDA-MB-468 cells from a doxycycline-inducible promoter and compared the gene expression profile in the presence or absence of the inducer. We identified that cytokeratins 8 and 19 genes are repressed when SLUG expression is induced. We have verified the role of SLUG in regulating these cytokeratins by siRNA-mediated knock down of SLUG in BT-549 cells. We also report here our preliminary characterization of the promoters of these cytokeratin genes as it is regulated by SLUG exp...

Background: High motility of aggressive breast cancer cells is associated with high SLUG and low plakoglobin levels. Results: SLUG binds to plakoglobin gene promoter and represses its expression. Conclusion: SLUG-induced increase in breast cancer cell motility is due to repression of plakoglobin by SLUG. Significance: Management of SLUG level should diminish the motility and thus aggressiveness in breast cancer cells.