Prokaryotic chromosomes code for toxin–antitoxin (TA) loci, often in multiple copies. In E.coli, experimental evidence indicates that TA loci are stress-response elements that help cells survive unfavorable growth conditions. The first gene in a TA operon codes for an antitoxin that combines with and neutralizes a regulatory ‘toxin’, encoded by the second gene. RelE and MazF toxins are regulators of translation that cleave mRNA and function, in interplay with tmRNA, in quality control of gene expression. Here, we present the results from an exhaustive search for TA loci in 126 completely sequenced prokaryotic genomes (16 archaea and 110 bacteria). We identified 671 TA loci belonging to the seven known TA gene families. Surprisingly, obligate intracellular organisms were devoid of TA loci, whereas free-living slowly growing prokaryotes had particularly many (38 in Mycobacterium tuberculosis and 43 in Nitrosomonas europaea). In many cases, TA loci were clustered and closely linked to mobile genetic elements. In the most extreme of these cases, all 13 TA loci of Vibrio cholerae were bona fide integron elements located in the V.cholerae mega-integron. These observations strongly suggest that TA loci are mobile cassettes that move frequently within and between chromosomes and also lend support to the hypothesis that TA loci function as stress-response elements beneficial to free-living prokaryotes.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.

The steroid hormone oestrogen can signal through several receptors and pathways. Although the transcriptional responses mediated by the nuclear oestrogen receptors (ER) have been extensively characterized, the changes in gene expression elicited by signalling through the membraneassociated ER GPR30 have not been studied. We show here for ER-negative human breast cancer cells that the activation of GPR30 signalling by oestrogen or by hydroxytamoxifen (OHT), an ER antagonist but GPR30 agonist, induces a transcription factor network, which resembles that induced by serum in fibroblasts. The most strongly induced gene, CTGF, appears to be a target of these transcription factors. We found that the secreted factor connective tissue growth factor (CTGF) not only contributes to promote proliferation but also mediates the GPR30-induced stimulation of cell migration. These results provide a framework for understanding the physiological and pathological functions of GPR30. As the activation of GPR30 by OHT also induces CTGF in fibroblasts from breast tumour biopsies, these pathways may be involved in promoting aggressive behaviour of breast tumours in response to endogenous oestrogens or to OHT being used for endocrine therapy.

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Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.

Estrogen receptor ␣ (ER␣) is a ligand-regulated transcription factor with a broad range of physiological functions and one of the most important classifiers in breast cancer. MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as important regulators of gene expression in a plethora of physiological and pathological processes. Upon binding the 3 untranslated region (UTR) of target mRNAs, miRNAs typically reduce their stability and/or translation. The ER␣ mRNA has a long 3 UTR of about 4.3 kb which has been reported to reduce mRNA stability and which bears evolutionarily conserved miRNA target sites, suggesting that it might be regulated by miRNAs. We have performed a comprehensive and systematic assessment of the regulatory role of all miRNAs that are predicted to target the 3 UTR of the ER␣ mRNA. We found that miR-22 represses ER␣ expression most strongly and by directly targeting the ER␣ mRNA 3 UTR. Of the three predicted miR-22 target sites in the 3 UTR, the evolutionarily conserved one is the primary target. miR-22 overexpression leads to a reduction of ER␣ levels, at least in part by inducing mRNA degradation, and compromises estrogen signaling, as exemplified by its inhibitory impact on the ER␣-dependent proliferation of breast cancer cells.The steroid hormone 17␤-estradiol (E2) regulates a number of developmental and physiological processes, such as growth and differentiation, in a range of tissues, including the male and female reproductive tracts, breast epithelium, and a plethora of other organs (9, 23). These physiological effects are mediated, at least in part, by two nuclear receptors, estrogen receptor ␣ (ER␣) and ER␤. Upon binding E2, they are activated as transcription factors and regulate target genes by binding directly to specific regulatory sequences or indirectly to other DNA-bound transcription factors (23). In addition, rapid nongenomic effects of E2 can be elicited by the same ERs as membrane-associated receptors (22). ER␣ is the more widely expressed of the two ER isoforms, and its levels are regulated by multiple mechanisms in a development-and tissue-specific manner (23). ER␣ levels are regulated at all levels of gene expression, beginning with transcriptional control by several transcription factors (48) and continuing with regulation at the posttranscriptional (26, 27) and posttranslational levels (2), including the control of protein turnover (44).Breast cancer is the most frequent form of cancer in women, and ER␣ is still the most important classifier of breast tumors. ER␣-positive tumors, which represent nearly 70% of all breast tumors, respond to E2 for growth and survival, and consequently, ER␣-positive tumors can be inhibited with antiestrogens such as tamoxifen. Why and how most tumors eventually become resistant to antiestrogen therapy and why some tumors do not express ER␣ from the outset remain hotly debated questions. Multiple mechanisms have been invoked to explain

After publication, we became aware of some inaccurate and incomplete statements. The conclusions are in no way affected by the additional and corrected information listed below. Page 3784, column 1, Materials and Methods, paragraph 2, line 10: "the full-length ER␣ 3Ј UTR" should read "the ER␣ 3Ј UTR (which we will refer to as full length even though it lacked the 5Ј-most 348 nucleotides out of 4.3 kb)." Page 3785, column 1, lines 1 to 4: The sentence beginning "Although. . ." should be replaced with the following. "We had initially excluded it from our list of miRNAs because its target sites are not conserved and because TargetScan, even with relaxed search criteria, fails to identify the primary target site located at the very 5Ј end of the 3Ј UTR reported in that publication. Hence, the latter is not shown in Fig. 1, which does show two other nonconserved miR-206 sites, and it is not included in our ER␣ 3Ј UTR constructs. Nevertheless, we decided to investigate miR-206 in parallel in some of the key experiments." Page 3788, column 2, line 22: The following sentence should be added after "in MCF7-SH cells." "Note that high levels of both miR-22 and miR-206 were reached in transient-transfection experiments with 293T cells (103-fold and 40,258-fold, respectively, compared to a control transfection) and in stable MCF7-SH transformants (levels comparable to those of the endogenous miRNAs let-7i and let-7g) (data not shown)." Page 3788, column 2, line 24: "protein levels" should read "mRNA levels." Page 3788, column 2, lines 25 to 27: "since MCF7 cells. .. express miR-206 (36)" should read "since MCF7-SH cells, derived from MCF7 cells, do not express miR-206 (data not shown) and since another study could not find miR-206 expression in MCF7 cells (36).

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma. Cancer Res; 73(20); 6323-33. Ó2013 AACR.