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Abstract
Introduction
Long non-coding RNAs are considered as transcripts that do not code for protein and are longer than
200 nucleotides. LncRNAs are not well studied yet and it is a new emerging field. Once it was
discovered that these sequences are well conserved lncRNAs were considered as functional RNAs
because conservation means that they are constrained due to structural or sequence-specific
functions (Ponting, Oliver, & Reik, 2009).
Gene expression is regulated by a large diversity of protein complexes and RNA molecules and is
highly dependent on the chromatin structure. RNA is a component of chromatin and is also involved
in chromatin modification. LncRNAs are shown to have influence on the gene expression by
remodelling the epigenetic state of the cells. LncRNAs have been shown to act as cis and trans
regulatory molecules. Where cis acting molecules influence the expression on the same chromosome
they are transcribed from and trans acting molecules act on a different chromosome than the
chromosome coding for the lncRNA.
HOTAIR and HOTTIP are our lncRNAs of interest, HOTAIR stands for HOX antisense intergenic RNA.
The gene coding for this lncRNA is located on the HOXC locus on chromosome 12 but the molecule
acts on the HOXD locus on chromosome 2, therefore it acts in trans. It binds to PRC2 complex and
has been proposed to target PRC2 complex to the HOXD locus. PRC2 stands for polycomb repressive
complex 2. The recruitment of PRC2 by HOTAIR leads to trimethylation of H3K27 and silencing of
metastasis suppressor genes (Wan & Chang, 2010 and Tsai, et al., 2010). HOTAIR is showing an
increased expression in primary breast tumours which eventually go into metastasis and is therefore
a tool to predict patient survival (Wan & Chang, 2010). HOTTIP, the other lncRNA of interest, is short
for Hox transcript at the distal tip. It has been shown that this lncRNA is involved in the upregulation
of 5’ end genes on the HOXA locus. The sequence coding for HOTTIP is also on the HOXA locus thus it
is a cis acting molecule. Together with WDR5 and MLL, functioning as methyltransferase, it leads to
trimethylation of H3K4 (Wang, et al., 2011).
Goal
The goal of this project is to create a system to recruit lncRNAs to specific places in the genome. This
will allow analyzing the effects of lncRNA recruitment on the local chromatin structure and the
expression of the genes located in that area of the genome.
Strategy
A set of mES cell lines is available which have GFP reporter genes integrated in different parts of the
genome. These reporter genes have 14 Gal4 binding sites upstream of the promoter. These sites will
be used to recruit HOTAIR to the reporter genes. This can be done by utilizing the ability of MS2 coat
protein to bind the MS2 RNA repeats. Constructs will be made where the Gal4 DNA binding domain
(Gal4-DBD) is fused with the MS2 coat protein. The HOTAIR coding sequence will then be fused with
the MS2 RNA repeats. Co-expression of these constructs will produce a system with the RNA-protein
complex that will bind HOTAIR to the reporter gene and the effect of this binding on the expression
of the reporter gene will be studied.
For the preparation of Gal4-DBD-MS2, MS2-Gal4-DBD and MS2repeats-HOTAIR constructs, the DNA
coding for these different parts will be PCR amplified and cloned into a vector containing mPGK
promotor. After cloning all the constructs will be validated by sequencing.
The plasmids that are constructed will be transfected into mES cell lines with the GFP reporter genes.
When this is done the gene expression will be analyzed by qPCR. The expression of HOTAIR will be
monitored by RT-PCR, the effect of these proteins on the expression of the reporter gene will be
assessed by looking at the GFP expression from the transgene.