Heterochromatin dynamics during epithelial-to-mesenchymal transition

Although heterochromatin is enriched with repressive traits, it is actively transcribed, giving rise to large amounts of non-coding RNAs. These transcripts are responsible for the formation and maintenance of heterochromatin, but little is known about how their transcription is regulated. In this thesis we show that Snail1 transcription factor represses mouse pericentromeric transcription and regulates heterochromatin organization through the action of the H3K4 deaminase LOXL2. Snail1 has a key role in epithelial-to-mesenchymal transition (EMT). We show that, also during this process, Snail1 is responsible for pericentromeric transcription regulation. At the onset of EMT, one of the major structural heterochromatin proteins, HP1α, is transiently released from heterochromatin foci in a Snail1/LOXL2 dependent manner, concomitantly with a down-regulation of major satellite transcription. Moreover, prevention of major satellite transcripts down-regulation compromises the migratory and invasive behaviour of EMT resulting mesenchymal cells. We propose that Snail1 and LOXL2 regulate heterochromatin during this process, which may be crucial to allow the genome reorganization required to complete EMT.Tot i estar enriquida en marques repressores, l’heterocromatina es transcriu activament i dóna lloc a grans quantitats d’ARNs no codificants. Aquests trànscrits són responsables de la formació i el manteniment de l’heterocromatina, però com es regula la seva transcripció segueix sent quelcom poc clarificat. En aquesta tesi demostrem que el factor de transcripció Snail1 reprimeix la transcripció pericentromèrica en cèl·lules de ratolí i regula l’organització de l’heterocromatina a través de l’acció de la LOXL2, que deamina l’H3K4. Snail1 té un paper clau en la transició epiteli-mesènquima (EMT). Aquí demostrem que, també durant aquest procés, Snail1 és responsable de la regulació de la transcripció pericentromèrica. A l’inici de l’EMT, l’HP1α, una de les principals proteïnes estructurals de l’heterocromatina, es desprèn de forma transitòria de l’heterocromatina. Aquest esdeveniment està regulat per Snail1 i LOXL2 i coincideix amb una disminució de la transcripció pericentromèrica. El bloqueig de la baixada dels trànscrits durant l’EMT compromet les capacitats migratòries i invasives de les cèl·lules mesenchimals que en resulten. Així doncs, proposem que Snail1 i LOXL2 regulen l’heterocromatina durant aquest procés, i així permeten que tingui lloc la reorganització genòmica que deu ser necessària per tal que es completi la EMT

Splicing of a non-coding antisense transcript controls LEF1 gene expression.

In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.Funded by a grant from Association for International Cancer Research (AICR) with additional support from Ministerio de Economía y Competitividad [SAF2010-16089, SAF2013-4889-C2-1R] and the Instituto Carlos III [RD012/0036/0005, part of the Plan Nacional I+D+I and cofounded by the ISCIII-Subdireccion General de Evaluacion and Fondo Europeo de Desarrollo Regional- FEDER]. M.B. was supported by a Predoctoral Fellowship awarded by Instituto Carlos III, an EMBO Long-term Fellowship/nand an ERC Starting Grant to R.G.J., R.M., by a Juan de la Cierva Contract. Funding for open access charge: Ministerio de Economıa y Ciencia [SAF2013-4889-C2-1R]. Conflict of interest statement. None declared

Splicing of a non-coding antisense transcript controls LEF1 gene expression.

In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.Funded by a grant from Association for International Cancer Research (AICR) with additional support from Ministerio de Economía y Competitividad [SAF2010-16089, SAF2013-4889-C2-1R] and the Instituto Carlos III [RD012/0036/0005, part of the Plan Nacional I+D+I and cofounded by the ISCIII-Subdireccion General de Evaluacion and Fondo Europeo de Desarrollo Regional- FEDER]. M.B. was supported by a Predoctoral Fellowship awarded by Instituto Carlos III, an EMBO Long-term Fellowship/nand an ERC Starting Grant to R.G.J., R.M., by a Juan de la Cierva Contract. Funding for open access charge: Ministerio de Economıa y Ciencia [SAF2013-4889-C2-1R]. Conflict of interest statement. None declared

Snail1 transcription factor controls telomere transcription and integrity

Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFβ-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes

Snail1 transcription factor controls telomere transcription and integrity

Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFβ-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes

Regulation of heterochromatin transcription by Snail1/LOXL2 during epithelial-to-mesenchymal transition

Although heterochromatin is enriched with repressive traits, it is also actively transcribed, giving rise to large amounts of noncoding RNAs. Although these RNAs are responsible for the formation and maintenance of heterochromatin, little is known about how their transcription is regulated. Here, we show that the Snail1 transcription factor represses mouse pericentromeric transcription, acting through the H3K4 deaminase LOXL2. Since Snail1 plays a key role in the epithelial-to-mesenchymal transition (EMT), we analyzed the regulation of heterochromatin transcription in this process. At the onset of EMT, one of the major structural heterochromatin proteins, HP1α, is transiently released from heterochromatin foci in a Snail1/LOXL2-dependent manner, concomitantly with a downregulation of major satellite transcription. Moreover, preventing the downregulation of major satellite transcripts compromised the migratory and invasive behavior of mesenchymal cells. We propose that Snail1 regulates heterochromatin transcription through LOXL2, thus creating the favorable transcriptional state necessary for completing EMT

Lysyl oxidase-like 2 deaminates lysine 4 in histone H3

Methylation of lysine 4 (K4) within histone H3 has been linked to active transcription and is removed by LSD1 and the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here, we describe the deamination catalyzed by Lysyl oxidase-like 2 protein (LOXL2) as an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, LOXL2 activity is linked with the transcriptional control of CDH1 gene by regulating H3K4me3 deamination. These results reveal another H3 modification and provide a different mechanism for H3K4 modification