Characterization of Alternative Splicing of LSD1 in Neuroblastoma Cell Lines

Weichun Li


DNA in the nucleus of the cell is wrapped around octamers of histone protein, called nucleosomes that allow for compaction of DNA (Luger, K et al. 1997). Besides the structural role in nucleosome, histone proteins are also key players in the regulation of gene expression. The known regulations depend partially on their post-translational modification including phosphorylation, acetylation, methylation, etc primarily on the N-terminal tail of histones. These epigenetic markers endowed by various modifications are under tight regulation of specific chromatin-modifying enzymes, which usually function as part of large multiprotein complexes (Kouzaride, T 2007).
LSD-1 is the gene that encodes a histone demethylase, catalyzing demethylation of mono- and di-methylated histone at H3K4 or H3K9 (Qingjun Zhu et al. 2008). This protein contains a SWIRM domain, which functions as a putative protein-protein interaction motif, an amine oxidase domain which harbors the demethylase activity and requires FAD as the cofactor (Eric Metzger, et al 2005). Besides, LSD1 usually functions as a subunit in protein complexes such as Co-REST, NRD and BRAF35HDAC (Lee, M.G. et al. 2005). The crystal structure and biochemical studies of LSD1 reveal that this enzyme requires H3 peptides at least 16 residues in length for substrate recognition (Ronen Marmorstein, Raymond C. Trievel 2009) and the distinction between methylated H3K4, H3K9, both of which are substrates of this enzyme and other methylated histone sites depends on chemical selection instead of steric control (Pete Stavropoulos, et al. 2006).
As a histone demethylase, LSD1 can either activate or repress certain gene transcription. There is report that it silences genes by targeting at H3K4me1/2, a methylation site usually associated with transcriptional active genes (Federico Forneris, et al. 2008). However, there are reports that LSD1 could demethylate repressive histone markers to promote transcription. Like in normal human prostate and prostate tumor, people find LSD1 co-localizes with the androgen receptor and stimulates androgen-receptor-dependent transcription by demethylating H3K9 (Eric Metzger, et al. 2005). Such regulatory effect on gene expression predicts a potential link between cancer and LSD1 (Wang, G.G et al. 2007). Moreover, people have found in prostate cancer, the over-expression of LSD1 is associated with increasing grade and may be used as a prognostic predictor (Kahl et al. 2006). Although major substrate of LSD1 is histone, it also demethylates and stabilizes Dnmt1 (DNA methyltrasferase 1), thereby influencing DNA methylation system (Jing Wang, et al 2009).
Although the structure and function of the protein encoded by LSD 1 is under intensive investigation, which is proportional to its importance, the alternative splicing of this gene is not fully characterized. According to Entrez Gene, this gene maps on chromosome 1, at 1p36.12. It covers 64.25 kb (NCBI 36, March 2006). There are 19 exons, 27 different gt-ag introns. Transcription produces 12 alternatively spliced variants. The mRNAs appear to differ by truncation of the 5 end, truncation of the 3 end, presence or absence of 7 cassette exons, overlapping exons with different boundaries (Genebank). However, up to now, little is known about the expression of this gene in neuroblastoma cells. Therefore, the goal of this project is to characterize alternative splicing of LSD1 in neuroblastoma cells and see if there is any differential expression among the variants observed.


Figure 1-The location of each pair of primer and the size of possible products when different alternative splicing events happen.

Figure 2-Two bands are amplified by primer A and B in each cell lines. According to the size, the large band include the 60-base insertion while the small band does not.

Figure 3-The sequence of acceptor site of the insertion is the same as consensus acceptor site sequence (the blue color). The sequence of donor site of insertion is quite similar to the sequence of the consus donor site (the pink color) except for the last nucleotide, which explains why this insertion is sometimes included in mature RNA but not guranteed.

Figure 4-Six cell lines produce the same product amplified from primer C. The size indicates that exon 7 is preserved and sequence result confirms it.

LSD1 is a gene that encodes a chromatin modifying protein which functions as a histone demethylase. Although numerous researches have been done to investigate how this protein regulates gene transcription and interacts with DNA methylation, only a few is known about the alternative splicing of this gene. In this project, I detect three possible alternative spliced forms in neuroblastoma cell lines, the skipping of exon 2, exon 7 and a novel insertion between exon 2 and 3. Also, RT-PCR is conducted to see if there is any differential expression between transcript variants among six cell lines. According to the RT-PCR and sequencing results, exon 2 and 7 are preserved in the RNA of six neuroblastoma cell lines. There is a novel insertion of 60 bases between exon 2 and 3 which encodes 20 amino acids in one transcript variant observed. The sequences before and after this insertion are in great similarity to the sequences of consensus donor and acceptor site with only one nucleotide difference. This may explain why in some cases, this insertion is cut out while in other cases, it is preserved. Meanwhile, the location of this insertion is right in front of the RNA that encodes SWIRM domain which is a highly conserved domain present in many modifying and remolding complexes. According to recently published papers, this domain functions by improving the stability of LSD1 protein and interaction with amine oxidase domain to form catalytic cavity. Therefore, this 20 amino-acid peptide encoded by the 60-base insertion may exert structural and functional influence on this domain and the protein but details remain to be identified.

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I would like to thank Bo Liu, Leleesha Samaraweera for their help throughout th ecourse.I would like to thank Leleesha Samaraweera for providing neuroblastoma cell lines. I would like to thank Bo Liu for really helpful discussion and Dorris for her suggestions. I would also like to acknowledge Dr. Berish Rubin without whose guidance this project would be impossible.

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