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).
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.
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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