#Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.

How melanoma acquire a metastatic phenotype is a key issue. One possible mechanism is that metastasis is driven by microenvironment-induced switching between noninvasive and invasive states. However, whether switching is a reversible or hierarchical process is not known and is difficult to assess by comparison of primary and metastatic tumors. We address this issue in a model of melanoma metastasis using a novel intravital imaging method for melanosomes combined with a reporter construct in which the Brn-2 promoter drives green fluorescent protein (GFP) expression. A subpopulation of cells containing little or no pigment and high levels of Brn2::GFP expression are motile in the primary tumor and enter the vasculature. Significantly, the less differentiated state of motile and intravasated cells is not maintained at secondary sites, implying switching between states as melanoma cells metastasize. We show that melanoma cells can switch in both directions between high-and low-pigment states. However, switching from Brn2::GFP high to low was greatly favored over the reverse direction. Microarray analysis of high-and lowpigment populations revealed that transforming growth factor (TGF)B2 was up-regulated in the poorly pigmented cells. Furthermore, TGFB signaling induced hypopigmentation and increased cell motility. Thus, a subset of less differentiated cells exits the primary tumor but subsequently give rise to metastases that include a range of more differentiated and pigment-producing cells. These data show reversible phenotype switching during melanoma metastasis. [Cancer Res 2009;69(20):7969-77]

In cystic fibrosis (CF) patients, the major alteration in pulmonary function is due to peripheral airway obstruction. In the present study, we investigated the possibility that alterations in the extrathoracic airways, particularly in the trachea that expresses high levels of CFTR (CF transmembrane conductance regulator), may contribute to respiratory dysfunction. We performed morphological analyses of the trachea and airway functional studies in adult Cftr knockout (Cftr −/− ) and F508del-CFTR mice and their controls. Macroscopic and histological examination of the trachea showed the presence of one to seven disrupted or incomplete cartilage rings in Cftr −/− mice (23/25) while only a few Cftr +/+ mice (6/25) had one abnormal ring. Tracheal defects were mainly localized in the proximal trachea. In 14 Cftr −/− mice, frontal disruption of the first three to six rings below the cricoid cartilage were associated with upper tracheal constriction. Similar tracheal abnormalities were detected in adult F508del-CFTR and in newborn Cftr −/− and F508del-CFTR mice. Tracheal and ventilatory function analyses showed in Cftr −/− mice a decreased contractile response of the proximal trachea and a reduced breathing rate due to an increase in the inspiratory and expiratory times. In F508del-CFTR mice, the expiratory time was longer than in controls. Therefore, these structural and functional abnormalities detected in adult and newborn CF mouse models may represent congenital malformations related to CFTR dysfunction. These results raise important questions concerning the mechanisms governing tracheal development within the context of CFTR protein dysfunction and the implication of such abnormalities in the pathogenesis of airway disease in CF.

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

bDeregulation of transcription arising from mutations in key signaling pathways is a hallmark of cancer. In melanoma, the most aggressive and lethal form of skin cancer, the Brn-2 transcription factor (POU3F2) regulates proliferation and invasiveness and lies downstream from mitogen-activated protein kinase (MAPK) and Wnt/␤-catenin, two melanoma-associated signaling pathways. In vivo Brn-2 represses expression of the microphthalmia-associated transcription factor, MITF, to drive cells to a more stem cell-like and invasive phenotype. Given the key role of Brn-2 in regulating melanoma biology, understanding the signaling pathways that drive Brn-2 expression is an important issue. Here, we show that inhibition of phosphatidylinositol 3-kinase (PI3K) signaling reduces invasiveness of melanoma cells in culture and strongly inhibits Brn-2 expression. Pax3, a transcription factor regulating melanocyte lineage-specific genes, directly binds and regulates the Brn-2 promoter, and Pax3 expression is also decreased upon PI3K inhibition. Collectively, our results highlight a crucial role for PI3K in regulating Brn-2 and Pax3 expression, reveal a mechanism by which PI3K can regulate invasiveness, and imply that PI3K signaling is a key determinant of melanoma subpopulation diversity. Together with our previous work, the results presented here now place Brn-2 downstream of three melanoma-associated signaling pathways.

It is presently unknown whether any member of the IGFBP (insulin-like growth factor binding protein) family directly participates in the control of cell proliferation. We have previously documented that induction of IGFBP-2 was associated with inhibition of DNA synthesis in lung alveolar epithelial cells. In the present study, we investigated the relationship between IGFBP-2 and the cell cycle inhibitor p21CIP1/WAF1 further. We used serum deprivation to inhibit the proliferation of MLE (mouse lung epithelial)-12 cells, and characterized the spatial localization of IGFBP-2. We found that growth inhibition, which was supported by the strong induction of p21CIP1/WAF1, was correlated with increased secretion of IGFBP-2 and, unexpectedly, with its increased localization in the nucleus and particularly in the cytoplasm. By coimmunoprecipitation, we discovered that IGFBP-2 is capable of binding to p21CIP1/WAF1. Interaction between these two proteins was further supported by colocalization of the proteins within growth-arrested cells, as visualized by confocal microscopy. Furthermore, this interaction increased with the duration of the stress, but was suppressed when proliferation was restimulated by the addition of serum. The recombinant expression of GFP (green fluorescent protein)-tagged IGFBP-2 in transfected MLE-12 cells demonstrated its ability to bind specifically to p21CIP1/WAF1. Taken together, these results provide a link between IGFBP-2 and p21CIP1/WAF1 in the regulation of alveolar lung cell proliferation.

f MITF-M and PAX3 are proteins central to the establishment and transformation of the melanocyte lineage. They control various cellular mechanisms, including migration and proliferation. BRN2 is a POU domain transcription factor expressed in melanoma cell lines and is involved in proliferation and invasion, at least in part by regulating the expression of MITF-M and PAX3. The T361 and S362 residues of BRN2, both in the POU domain, are conserved throughout the POU protein family and are targets for phosphorylation, but their roles in vivo remain unknown. To examine the role of this phosphorylation, we generated mutant BRN2 in which these two residues were replaced with alanines (BRN2TS¡BRN2AA). When expressed in melanocytes in vitro or in the melanocyte lineage in transgenic mice, BRN2TS induced proliferation and repressed migration, whereas BRN2AA repressed both proliferation and migration. BRN2TS and BRN2AA bound and repressed the MITF-M promoter, whereas PAX3 transcription was induced by BRN2TS but repressed by BRN2AA. Expression of the BRN2AA transgene in a Mitf heterozygous background and in a Pax3 mutant background enhanced the coat color phenotype. Our findings show that melanocyte migration and proliferation are controlled both through the regulation of PAX3 by nonphosphorylated BRN2 and through the regulation of MITF-M by the overall BRN2 level. POU family transcription factors are expressed in a wide variety of cell types. They are involved in diverse functions, such as cell type determination, proliferation, renewal, invasion, and migration. The members of the POU domain family of transcription factors share the POU DNA-binding domain (DBD) called the POU domain. The POU domain consists of two DNA-binding units (POUs for POU specific and POUh for POU homeodomain) connected by a flexible linker (3). This molecular structure allows POU proteins to recognize a large set of DNA targets and also to bind different transactivator proteins, depending on the spacing and the positioning adopted by the two subdomains of the POU DBD (50).POU transcription factor function can be modulated by posttranslational modifications, including sumoylation, oxidation, ubiquitinylation, glycosylation, and particularly phosphorylation (5,20,32). Two residues in the DBD domain, a threonine and a serine, are conserved in all mammalian POU domains (see Table S1 at http://umr3347.curie.fr/fr/quipes-de -recherche/d-veloppement-normal-et-pathologie-des-m-lanocytes /differential-function-non-pho). These serine and/or threonine residues in Oct-1, Pit-1, and BRN2 are phosphorylated by protein kinase A (PKA) (31,46,55).Several lines of evidence suggest that PKA is involved in melanocyte lineage proliferation. Forskolin stimulates the proliferation of human melanocytes (54). Proliferation of human melanocytes is induced in a dose-dependent manner by alphamelanocyte-stimulating hormone (1,57,58). Dibutyryl adenosine cyclic AMP induces the proliferation of epidermal melanocytes in culture (29). PKA phosphorylates claudin-1 and allows its tr...

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