TET2-mediated epigenetic reprogramming of breast cancer cells impairs lysosome biogenesis.

Methylation and demethylation of cytosines in DNA are believed to act as keystones of cell-specific gene expression by controlling the chromatin structure and accessibility to transcription factors. Cancer cells have their own transcriptional programs, and we sought to alter such a cancer-specific program by enforcing expression of the catalytic domain (CD) of the methylcytosine dioxygenase TET2 in breast cancer cells. The TET2 CD decreased the tumorigenic potential of cancer cells through both activation and repression of a repertoire of genes that, interestingly, differed in part from the one observed upon treatment with the hypomethylating agent decitabine. In addition to promoting the establishment of an antiviral state, TET2 activated 5mC turnover at thousands of MYC-binding motifs and down-regulated a panel of known MYC-repressed genes involved in lysosome biogenesis and function. Thus, an extensive cross-talk between TET2 and the oncogenic transcription factor MYC establishes a lysosomal storage disease-like state that contributes to an exacerbated sensitivity to autophagy inducers.

Changes in Gene Expression and Estrogen Receptor Cistrome in Mouse Liver Upon Acute E2 Treatment.

Transcriptional regulation by the estrogen receptor-α (ER) has been investigated mainly in breast cancer cell lines, but estrogens such as 17ß-estradiol (E2) exert numerous extrareproductive effects, particularly in the liver, where E2 exhibits both protective metabolic and deleterious thrombotic actions. To analyze the direct and early transcriptional effects of estrogens in the liver, we determined the E2-sensitive transcriptome and ER cistrome in mice after acute administration of E2 or placebo. These analyses revealed the early induction of genes involved in lipid metabolism, which fits with the crucial role of ER in the prevention of liver steatosis. Characterization of the chromatin state of ER binding sites (BSs) in mice expressing or not ER demonstrated that ER is not required per se for the establishment and/or maintenance of chromatin modifications at the majority of its BSs. This is presumably a consequence of a strong overlap between ER and hepatocyte nuclear factor 4α BSs. In contrast, 40% of the BSs of the pioneer factor forkhead box protein a (Foxa2) were dependent upon ER expression, and ER expression also affected the distribution of nucleosomes harboring dimethylated lysine 4 of Histone H3 around Foxa2 BSs. We finally show that, in addition to a network of liver-specific transcription factors including CCAAT/enhancer-binding protein and hepatocyte nuclear factor 4α, ER might be required for proper Foxa2 function in this tissue.

Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities.

Estradiol signaling is ideally suited for analyzing the molecular and functional linkages between the different layers of information directing transcriptional regulations: the DNA sequence, chromatin modifications, and the spatial organization of the genome. Hence, the estrogen receptor (ER) can bind at a distance from its target genes and engages timely and spatially coordinated processes to regulate their expression. In the context of the coordinated regulation of colinear genes, identifying which ER binding sites (ERBSs) regulate a given gene still remains a challenge. Here, we investigated the coordination of such regulatory events at a 2-Mb genomic locus containing the estrogen-sensitive trefoil factor (TFF) cluster of genes in breast cancer cells. We demonstrate that this locus exhibits a hormone- and cohesin-dependent reduction in the plasticity of its three-dimensional organization that allows multiple ERBSs to be dynamically brought to the vicinity of estrogen-sensitive genes. Additionally, by using triplex-forming oligonucleotides, we could precisely document the functional links between ER engagement at given ERBSs and the regulation of particular genes. Hence, our data provide evidence of a formerly suggested cooperation of enhancers toward gene regulation and also show that redundancy between ERBSs can occur.

Cyclical DNA methylation of a transcriptionally active promoter.

Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.

COUP-TFI modulates estrogen signaling and influences proliferation, survival and migration of breast cancer cells.

We previously showed that COUP-TFI interacts with the Estrogen Receptor alpha (ER alpha) to recruit Extracellular signal Regulated Kinases (ERKs) in an Estradiol (E2)-independent manner, resulting in an enhancement of ER alpha transcriptional activity. However, the involvement of COUP-TFI in physiologically relevant functions of ER alpha, such as the mitogenic activity that E2 has on breast cancer cells, remains poorly understood. Here, we first showed that the amounts of COUP-TFI protein are higher in dedifferentiated mammary cell lines (MDA-MB-231) and tumor breast cells as compared to the differentiated MCF-7 cell line and normal breast cells. To evaluate the functional relevance of the COUP-TFI/ER alpha interplay in mammary cells, we generated MCF-7 cells that stably over-express COUP-TFI. We found that the over-expression of COUP-TFI enhances motility and invasiveness of MCF-7 cells. COUP-TFI also promotes the proliferation of MCF-7 cells through ER alpha-dependent mechanisms that target cell cycle progression and cell survival. To further investigate the mechanisms underlying these effects of COUP-TFI, we evaluated the expression of known E2-target genes in breast cancer, and found that COUP-TFI differentially regulated genes involved in cell proliferation, apoptosis, and migration/invasion. Notably, Cathepsin D (CTSD) transcript and protein levels were significantly higher in presence and absence of E2 in MCF-7 over-expressing COUP-TFI. Chromatin Immunoprecipitation assays showed that ER alpha, phospho-RNA Polymerase II, as well as p68 RNA Helicase, a phospho-Serine 118 dependent co-activator of ER alpha, were preferentially recruited onto the CTSD gene proximal promoter in COUP-TFI over-expressing cells. These results suggest that COUP-TFI selectively regulates the expression of endogenous E2-target genes and consequently modifies ER alpha positive mammary cells response to E2.

The human estrogen receptor-alpha isoform hERalpha46 antagonizes the proliferative influence of hERalpha66 in MCF7 breast cancer cells.

The expression of two human estrogen receptor-alpha (hERalpha) isoforms has been characterized within estrogen receptor-alpha-positive breast cancer cell lines such as MCF7: the full-length hERalpha66 and the N terminally deleted hERalpha46, which is devoid of activation function (AF)-1. Although hERalpha66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of hERalpha46 in these cells remains undefined. Here we show that, during MCF7 cell growth, hERalpha46 is mainly expressed in the nucleus at relatively low levels, whereas hERalpha66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with hERalpha46 accumulating within the nucleus. Although hERalpha46 expression remains rather stable during an estrogen-induced cell cycle, its overexpression in proliferating MCF7 cells provokes a cell-cycle arrest in G(0)/G(1) phases. To gain further details on the influence of hERalpha46 on cell growth, we used PC12 estrogen receptor-alpha-negative cell line, in which stable transfection of hERalpha66 but not hERalpha46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, overexpression of hERalpha46 inhibits the hERalpha66-mediated estrogenic induction of all AF-1-sensitive reporters: c-fos and cyclin D1 as well as estrogen-responsive element-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, hERalpha46 antagonizes the proliferative action of hERalpha66 in MCF7 cells in part by inhibiting hERalpha66 AF-1 activity.

Intracellular trafficking of heat shock factor 2.

HSF2 is an enigmatic member of the heat shock factor family, identified in the homeotherm classes of birds and mammals. We report the characterization of HSF2 from an evolutionary ancient vertebrate, the fish rainbow trout (rtHSF2). rtHSF2 appears closely related to its mammalian counterparts at structural and functional levels. The conservation of the distinctive features of HSF2 from fish to human suggests that it should ensure important biological functions, not redundant with those of HSF1. Proteasome inhibition, reported as a potent stimulator of HSF2, leads to the stabilization and to a striking nuclear trafficking of rtHSF2-GFP fusion protein. Upon treatment with the proteasome inhibitor MG132, rtHSF2-GFP accumulates into PML nuclear bodies (NBs) independently of its sumoylation and, if expressed at moderate level, moves to nucleoli. The translocation of rtHSF2-GFP from NBs to nucleoli is greatly favored by overexpression of the heat shock protein Hsp70. The mammalian counterpart mouse HSF2 (mHSF2) also exhibited changes in intracellular distribution upon MG132 treatment. mHSF2 partitioned between a juxtanuclear area that we characterized as an aggresome and the nucleoli. These relocalizations are likely to reflect common structural changes of mouse and trout HSF2 upon activation.

Multiple phosphorylation events control chicken ovalbumin upstream promoter transcription factor I orphan nuclear receptor activity.

Chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) is an orphan member of the nuclear hormone receptor superfamily that comprises key regulators of many biological functions, such as embryonic development, metabolism, homeostasis, and reproduction. Although COUP-TFI can both actively silence gene transcription and antagonize the functions of various other nuclear receptors, the COUP-TFI orphan receptor also acts as a transcriptional activator in certain contexts. Moreover, COUP-TFI has recently been shown to serve as an accessory factor for some ligand-bound nuclear receptors, suggesting that it may modulate, both negatively and positively, a wide range of hormonal responses. In the absence of any identified cognate ligand, the mechanisms involved in the regulation of COUP-TFI activity remain unclear. The elucidation of several putative phosphorylation sites for MAPKs, PKC, and casein kinase II within the sequence of this orphan receptor led us to investigate phosphorylation events regulating the various COUP-TFI functions. After showing that COUP-TFI is phosphorylated in vivo, we provide evidence that in vivo inhibition of either MAPK or PKC signaling pathway leads to a specific and pronounced decrease in COUP-TFI-dependent transcriptional activation of the vitronectin gene promoter. Focusing on the molecular mechanisms underlying the MAPK- and PKC-mediated regulation of COUP-TFI activity, we show that COUP-TFI can be directly targeted by PKC and MAPK. These phosphorylation events differentially modulate COUP-TFI functions: PKC-mediated phosphorylation enhances COUP-TFI affinity for DNA and MAPK-mediated phosphorylation positively regulates the transactivation function of COUP-TFI, possibly through enhancing specific coactivator recruitment. These data provide evidence that COUP-TFI is likely to integrate distinct signaling pathways and raise the possibility that multiple extracellular signals influence biological processes controlled by COUP-TFI.