LOXL2-mediated chromatin compaction is required to maintain the oncogenic properties of triple-negative breast cancer cells.

Oxidation of histone H3 at lysine 4 (H3K4ox) is catalyzed by lysyl oxidase homolog 2 (LOXL2). This histone modification is enriched in heterochromatin in triple-negative breast cancer (TNBC) cells and has been linked to the maintenance of compacted chromatin. However, the molecular mechanism underlying this maintenance is still unknown. Here, we show that LOXL2 interacts with RuvB-Like 1 (RUVBL1), RuvB-Like 2 (RUVBL2), Actin-like protein 6A (ACTL6A), and DNA methyltransferase 1associated protein 1 (DMAP1), a complex involved in the incorporation of the histone variant H2A.Z. Our experiments indicate that this interaction and the active form of RUVBL2 are required to maintain LOXL2-dependent chromatin compaction. Genome-wide experiments showed that H2A.Z, RUVBL2, and H3K4ox colocalize in heterochromatin regions. In the absence of LOXL2 or RUVBL2, global levels of the heterochromatin histone mark H3K9me3 were strongly reduced, and the ATAC-seq signal in the H3K9me3 regions was increased. Finally, we observed that the interplay between these series of events is required to maintain H3K4ox-enriched heterochromatin regions, which in turn is key for maintaining the oncogenic properties of the TNBC cell line tested (MDA-MB-231).

Immune translational control by CPEB4 regulates intestinal inflammation resolution and colorectal cancer development.

Upon tissue injury, cytokine expression reprogramming transiently remodels the inflammatory immune microenvironment to activate repair processes and subsequently return to homeostasis. However, chronic inflammation induces permanent changes in cytokine production which exacerbate tissue damage and may even favor tumor development. Here, we address the contribution of post-transcriptional regulation, by the RNA-binding protein CPEB4, to intestinal immune homeostasis and its role in inflammatory bowel diseases (IBD) and colorectal cancer (CRC) development. We found that intestinal damage induces CPEB4 expression in adaptive and innate immune cells, which is required for the translation of cytokine mRNA(s) such as the one encoding interleukin-22. Accordingly, CPEB4 is required for the development of gut-associated lymphoid tissues and to maintain intestinal immune homeostasis, mediating repair and remodeling after acute inflammatory tissue damage and promoting the resolution of intestinal inflammation. CPEB4 is chronically overexpressed in inflammatory cells in patients with IBD and in CRC, favoring tumor development.

mRNA spindle localization and mitotic translational regulation by CPEB1 and CPEB4.

Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This regulation involves dynamic reprogramming of gene expression at multiple transcriptional and posttranscriptional levels. In transcriptionally silent oocytes, the CPEB-family of RNAbinding proteins coordinates temporal and spatial translation regulation of stored maternal mRNAs to drive meiotic progression. CPEB1 mediates mRNA localization to the meiotic spindle, which is required to ensure proper chromosome segregation. Temporal translational regulation also takes place in mitosis, where a large repertoire of transcripts are activated or repressed in specific cell cycle phases. However, whether control of localized translation at the spindle is required for mitosis is unclear, as mitotic and acentriolar-meiotic spindles are functionally and structurally different. Furthermore, the large differences in scale-ratio between cell volume and spindle size in oocytes compared to somatic mitotic cells may generate distinct requirements for gene expression compartmentalization in meiosis and mitosis. Here we show that mitotic spindles contain CPE-localized mRNAs and translating ribosomes. Moreover, CPEB1 and CPEB4 localize in the spindles and they may function sequentially in promoting mitotic stage transitions and correct chromosome segregation. Thus, CPEB1 and CPEB4 bind to specific spindle-associated transcripts controlling the expression and/or localization of their encoded factors that, respectively, drive metaphase and anaphase/cytokinesis.

The RNA binding protein CPEB2 regulates hormone sensing in mammary gland development and luminal breast cancer.

Organogenesis is directed by coordinated cell proliferation and differentiation programs. The hierarchical networks of transcription factors driving mammary gland development and function have been widely studied. However, the contribution of posttranscriptional gene expression reprogramming remains largely unexplored. The 3' untranslated regions of messenger RNAs (mRNAs) contain combinatorial ensembles of cis-regulatory elements that define transcript-specific regulation of protein synthesis through their cognate RNA binding proteins. We analyze the contribution of the RNA binding cytoplasmic polyadenylation element-binding (CPEB) protein family, which collectively regulate mRNA translation for about 30% of the genome. We find that CPEB2 is required for the integration of hormonal signaling by controlling the protein expression from a subset of ER/PR- regulated transcripts. Furthermore, CPEB2 is critical for the development of ER-positive breast tumors. This work uncovers a previously unknown gene expression regulation level in breast morphogenesis and tumorigenesis, coordinating sequential transcriptional and posttranscriptional layers of gene expression regulation.

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 TGFß-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.

Lysyl oxidase-like 2 (LOXL2) oxidizes trimethylated lysine 4 in histone H3.

Methylation of histone H3 lysine 4 is linked to active transcription and can be removed by LSD1 or the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here we describe that its deamination can be catalyzed by lysyl oxidase-like 2 protein (LOXL2), presenting an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, by regulating H3K4me3 deamination, LOXL2 activity is linked with the transcriptional control of the CDH1 gene. These results reveal the existence of further H3 modification as well as a novel mechanism for H3K4me3 demethylation. DATABASE: The GEO accession number for the data referred to this paper is GSE35600.

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.

The Epithelial-to-Mesenchymal Transition (EMT), a Particular Case.

Constitutive heterochromatin, an essential structure that has been conserved throughout evolution, is required to maintain genome stability. Although heterochromatin is enriched for repressive traits, it can be actively transcribed to generate thousands of noncoding RNAs that are required for correct chromatin assembly. Despite the importance of this structure, how and why heterochromatin transcription is regulated, and the proteins responsible for this regulation, remain poorly understood. Here, we summarize recent findings in heterochromatin transcription regulation during different cellular processes with a focus on the epithelial-mesenchymal transition (EMT), which elicits important changes in cell behavior, has a key role in early development, and is involved in cancer progression.

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.