RESUMEN
UTX (also known as KDM6A) encodes a histone H3K27 demethylase and is an important tumour suppressor that is frequently mutated in human cancers1. However, as the demethylase activity of UTX is often dispensable for mediating tumour suppression and developmental regulation2-8, the underlying molecular activity of UTX remains unknown. Here we show that phase separation of UTX underlies its chromatin-regulatory activity in tumour suppression. A core intrinsically disordered region (cIDR) of UTX forms phase-separated liquid condensates, and cIDR loss caused by the most frequent cancer mutation of UTX is mainly responsible for abolishing tumour suppression. Deletion, mutagenesis and replacement assays of the intrinsically disordered region demonstrate a critical role of UTX condensation in tumour suppression and embryonic stem cell differentiation. As shown by reconstitution in vitro and engineered systems in cells, UTX recruits the histone methyltransferase MLL4 (also known as KMT2D) to the same condensates and enriches the H3K4 methylation activity of MLL4. Moreover, UTX regulates genome-wide histone modifications and high-order chromatin interactions in a condensation-dependent manner. We also found that UTY, the Y chromosome homologue of UTX with weaker tumour-suppressive activity, forms condensates with reduced molecular dynamics. These studies demonstrate a crucial biological function of liquid condensates with proper material states in enabling the tumour-suppressive activity of a chromatin regulator.
Asunto(s)
Diferenciación Celular , Cromatina , Genes Supresores de Tumor , Histona Demetilasas/genética , Animales , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células HEK293 , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Ratones , Proteínas de Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Células THP-1RESUMEN
Neighboring genes transcribing in the same direction can form chimeric RNAs via cis-splicing (cis-SAGe). Previously, we reported 16 novel cis-SAGe chimeras in prostate cancer cell lines, and performed in silico validation on 14 pairs of normal and tumor samples from Chinese patients. However, whether these fusions exist in different populations, as well as their clinical implications, remains unclear. To investigate, we developed a bioinformatics pipeline using modified Spliced Transcripts Alignment to a Reference (STAR) to quantify these fusion RNAs simultaneously in silico. From RNA-Seq data of 100 paired normal and prostate cancer samples from TCGA, we find that most fusions are not specific to cancer. However, D2HGDH-GAL3ST2 is more frequently seen in cancer samples, and seems to be enriched in the African American group. Further validation with our own collection as well as from commercial sources did not detect this fusion RNA in 29 normal prostate samples, but in 19 of 93 prostate cancer samples. It is more frequently detected in late stage cancer, suggesting a role in cancer progression. Consistently, silencing this fusion resulted in dramatic reduction of cell proliferation rate and cell motility.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Biomarcadores de Tumor/genética , Fusión Génica , Neoplasias de la Próstata/genética , Empalme del ARN , ARN Neoplásico/genética , Sulfurtransferasas/genética , Negro o Afroamericano/genética , Oxidorreductasas de Alcohol/metabolismo , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Línea Celular , Movimiento Celular , Proliferación Celular , Biología Computacional , Bases de Datos Genéticas , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Humanos , Masculino , Neoplasias de la Próstata/etnología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Interferencia de ARN , ARN Neoplásico/metabolismo , Análisis de Secuencia de ARN , Sulfotransferasas , Sulfurtransferasas/metabolismo , Transfección , Población Blanca/genéticaRESUMEN
The chimeric RNA, SLC45A3-ELK4, was found to be a product of cis-splicing between the two adjacent genes (cis-SAGe). Despite the biological and clinical significance of SLC45A3-ELK4, its generating mechanism has not been elucidated. It was shown in one cell line that the binding of transcription factor CTCF to the insulators located at or near the gene boundaries, inversely correlates with the level of the chimera. To investigate the mechanism of such cis-SAGe events, we sequenced potential regions that may play a role in such transcriptional read-through. We could not detect mutations at the transcription termination site, insulator sites, splicing sites, or within CTCF itself in LNCaP cells, thus suggesting a "soft-wired" mechanism in regulating the cis-SAGe event. To investigate the role CTCF plays in regulating the chimeric RNA expression, we compared the levels of CTCF binding to the insulators in different cell lines, as well as clinical samples. Surprisingly, we did not find an inverse correlation between CTCF level, or its bindings to the insulators and SLC45A3-ELK4 expression among different samples. However, in three prostate cancer cell lines, different environmental factors can cause the expression levels of the chimeric RNA to change, and these changes do inversely correlate with CTCF level, and/or its bindings to the insulators. We thus conclude that CTCF and its bindings to the insulators are not the primary reasons for differential SLC45A3-ELK4 expression in different cell lines, or clinical cases. However, they are the likely mechanism for the same cells to respond to different environmental cues, in order to regulate the expression of SLC45A3-ELK4 chimeric RNA. This response to different environmental cues is not general to other cis-SAGe events, as we only found one out of 16 newly identified chimeric RNAs showing a pattern similar to SLC45A3-ELK4.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , ARN/genética , Proteínas Represoras/metabolismo , Proteína Elk-4 del Dominio ets/metabolismo , Secuencia de Bases , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Células HEK293 , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos , Mutación , Mutación Puntual , Neoplasias de la Próstata/metabolismo , Proteínas Represoras/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Genes or their encoded products are not expected to mingle with each other unless in some disease situations. In cancer, a frequent mechanism that can produce gene fusions is chromosomal rearrangement. However, recent discoveries of RNA trans-splicing and cis-splicing between adjacent genes (cis-SAGe) support for other mechanisms in generating fusion RNAs. In our transcriptome analyses of 28 prostate normal and cancer samples, 30% fusion RNAs on average are the transcripts that contain exons belonging to same-strand neighboring genes. These fusion RNAs may be the products of cis-SAGe, which was previously thought to be rare. To validate this finding and to better understand the phenomenon, we used LNCaP, a prostate cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. About half of the fusions are expressed at a significant level compared to their parental genes. Silencing one of the in-frame fusions resulted in reduced cell motility. Most out-of-frame fusions are likely to function as non-coding RNAs. The majority of the 16 fusions are also detected in other prostate cell lines, as well as in the 14 clinical prostate normal and cancer pairs. By studying the features associated with these fusions, we developed a set of rules: 1) the parental genes are same-strand-neighboring genes; 2) the distance between the genes is within 30kb; 3) the 5' genes are actively transcribing; and 4) the chimeras tend to have the second-to-last exon in the 5' genes joined to the second exon in the 3' genes. We then randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells. These results suggest that splicing between neighboring gene transcripts is a rather frequent phenomenon, and it is not a feature unique to cancer cells.