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Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus

2014-03-25 12:55:36

Cell Research; 2014 March 25; DOI: 10.1038/cr.2014.35



Jian-Feng Xiang, Qing-Fei Yin, Tian Chen, Yang Zhang, Xiao-Ou Zhang, Zheng Wu, Shaofeng Zhang, Hai-Bin Wang, Junhui Ge, Xuhua Lu, Li Yang, Ling-Ling Chen



Abstract



The human 8q24 gene desert contains multiple enhancers that form tissue-specific long-range chromatin loops with the MYC oncogene, but how chromatin looping at the MYC locus is regulated remains poorly understood. Here we demonstrate that a long noncoding RNA (lncRNA), CCAT1-L, is transcribed specifically in human colorectal cancers from a locus 515 kb upstream of MYC. This lncRNA plays a role in MYC transcriptional regulation and promotes long-range chromatin looping. Importantly, the CCAT1-L locus is located within a strong super-enhancer and is spatially close to MYC. Knockdown of CCAT1-L reduced long-range interactions between the MYC promoter and its enhancers. In addition, CCAT1-L interacts with CTCF and modulates chromatin conformation at these loop regions. These results reveal an important role of a previously unannotated lncRNA in gene regulation at the MYC locus.



INTRODUCTION



The increased sensitivity of experimental assays has revealed that long noncoding RNAs (lncRNAs) impact a variety of important biological processes. Aberrant expression of lncRNAs has been linked to cancers with distinct modes of action. For example, HOTAIR is highly expressed in breast tumors and has been reported to promote cancer metastasis by targeting chromatin repressor Polycomb proteins to specific genomic loci. LincRNA-p21 in association with hnRNP-K serves as a repressor in p53-dependent transcriptional responses or suppresses target mRNA translation in coordination with the RNA-binding protein HuR6. MALAT1 has been implicated in the regulation of cell growth and tumor metastasis. These findings suggest that lncRNAs may serve as important regulators in tumorigenesis although the expression regulation of lncRNAs in specific human tumors and their mechanisms involved in tumorigenesis remain to be explored.



Enhancers are a class of DNA regulatory sequences that can activate gene expression independent of their proximity or orientation to their target genes. Enhancers often form long-range chromatin loops with their target genes to control temporal- and tissue-specific gene expression during development and their mis-regulation contributes to human diseases. A large portion of enhancers can be transcribed into enhancer RNAs (eRNAs), which have been proposed to contribute to gene activation. In addition, super-enhancers were recently identified and shown to consist of large clusters of transcriptional enhancers formed by binding of master transcription factors/mediators and to be associated with genes that control and define cell identity. Thus, it would be interesting to know whether super-enhancers are transcribed and whether they are regulated by RNA transcripts.



The expression of the human MYC oncogene is complex and is regulated at multiple levels, including enhancers, promoters, transcription factors and chromatin state. The human 8q24 region includes a gene desert containing enhancers forming chromatin loops with the MYC promoter located several hundred kilobases telomerically. These chromatin interactions are tissue-specific in prostate, breast and colorectal cancers. In colorectal cancer (CRC), one well-characterized loop is between a locus 335 kb upstream of MYC (MYC-335) and the MYC promoter. MYC-335, the site harboring an important CRC risk SNP (rs6983267), is a transcriptional enhancer that promotes the binding of transcription factor 4 (TCF4) specifically in CRC. Importantly, mice lacking MYC-335 were resistant to intestinal tumors, although MYC transcripts were only modestly reduced. Very recently, the region upstream of MYC has been reported to contain an exceptionally large super-enhancer and such a super-enhancer is tumor type specific in cancer cells, but not in its healthy counterparts. However, how these chromatin loops at the 8q24 MYC locus are regulated remains unknown.



Human 8q24 has recently been reported to express several lncRNAs in different human tumors. PRNCR1 (8q24) binds to the androgen receptor (AR) and is involved in the AR-mediated gene activation in prostate cancers26. However, it is not expressed in CRC. Instead, two other CRC-specific lncRNAs transcribed from 8q24 were recently reported. CCAT1 (Colorectal Cancer Associated Transcript 1) is 2 600 nt in length and is a highly specific marker for CRC27, and its upregulation is evident in both pre-malignant conditions and through all disease stages in CRC28. CCAT2, a 340 nt ncRNA transcribed from the MYC-335 region, appeared to enhance invasion and metastasis through MYC-regulated miRNAs miR-17-5p and miR-20a29. However, we have been unable to detect CCAT2 in any human CRC tissue samples or CRC-derived cell lines examined.



Here we report that a novel 5 200 nt CRC-specific lncRNA, CCAT1-L (CCAT1, the Long isoform), is transcribed from a locus 515 kb upstream of MYC (MYC-515), a super-enhancer region of MYC and plays a role in MYC transcriptional regulation. We demonstrate that CCAT1-L localizes to its site of transcription and functions in the maintenance of chromatin looping between the MYC promoter and its enhancers in coordination with CTCF. Together, these results reveal a novel connection between the chromatin organization regulated by a lncRNA and MYC expression in a specific human cancer.




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