Se transcribe el Resumen del Proyecto FONDECYT obtenido por el Dra. Soraya Gutiérrez Gallegos, luego que 9 grupos de investigación de la Facultad de Ciencias Biológicas se adjudicaron proyectos presentados al Concurso de Proyectos Regular Fondecyt 2013.
Título del Proyecto
Identification and characterization of a novel antisense RNA encoded in intron5 of the RUNX1 gene (Identificación y caracterización de un nuevo ARN antisentido codificado en el intron5 del gen RUNX1).
Describe the main issues you plan to address, including goals, methodology and expected outcomes. A good summary facilitates an understanding of what you intend to achieve and the proposal review process. The abstract of funded proposals may be published on CONICYT website. The maximum length for this section is 1 page (Use Verdana font size 10). Acute myelogenous leukemis (AML) is a genetically heterogeneous disease in which somatic mutations that disturb cellular growth, proliferation and differentiation accumulate in hematopoietic progenitor cells. Chromosomal translocations are present in about half of the patients at the time of diagnosis with RUNX1 gene being the most frequent target of chromosomal translocations in cells of myeloid lineage. Interestingly, the RUNX1 gene covers 260kb of chromosome 21 but surprisingly, all genomic breakpoints for the leukemia causing translocation (8;21) and (16;21) are found in intron5 of the gene. Its partner in the (8;21) translocation, the ETO gene spans 150kb, is located on chromosome 8 and like RUNX1, all the breakpoints map to intron1 of the gene. These breakpoint regions lack any sequence homology that may explain the high recombination frequency. Interestingly, topoisomerase II and DNAase I hypersensitive sites were found to correlate with genomic breakpoints. Genome wide analysis of chromatin architecture has demonstrated that the presence of DNase I hypersensitive regions correlates with the presence of regulatory modules such as promoters, insulators, enhancers, etc. Moreover, we have previously shown that the intronic region of the RUNX1 gene involved in t(8;21) formation exhibit histone H3 acetylation and is depleted of histone H1. Interestingly, H3 enrichment is no homogeneous throughout the whole intron5, but rather specific areas exhibit a high association with hyperacetylated histone H3; suggesting that there are functionally active regions in this intron. Based on this experimental evidence, we performed a bioinformatic analysis of intron5 of the RUNX1 gene using ENCODE database. Surprisingly, the computational analysis predicts two putative promoter regions located at the 3’end of the intron that we named IN5P1 and IN5P2. These putative promoters are located in the antisense DNA strand of the RUNX1 gene. Moreover, analysis of the ENCODE database identify two forms of a natural antisense transcript, (i.e. a RNA that transcribe opposite to RUNX1 and that we have designated as NAT-RUNX1) that apparently can be generated by alternative use of the two promoters identified in intron5 (IN5P1 and IN5P2).
In this proposal we will test the hypothesis that RUNX1 locus gives rise to a natural antisense transcript originated from intronic promoters located in intron5 of the gene. Moreover, based on our EST results an in the fact that the most common pair of sense-antisense RNAs are protein coding-non coding RNA, we postulate that this novel gene encodes a non-coding RNA that is involved in RUNX1 gene regulation. To test our hypothesis, in specific aim 1 we propose to clone the full length NAT-RUNX1 RNA. In specific aims 2 and 3 we propose to determine the tissue specific expression pattern and sub-cellular localization of NAT-RUNX1 gene, respectively. In specific aim 4 we propose to evaluate the effect that NAT-RUNX1 has on RUNX1 expression both at the protein and mRNA level. Finally, in specific aim 5 we proposed to study the cis-regulatory functions of the promoter regions controlling NAT-RUNX1 expression, named IN5P1 and IN5P2, as well as their epigenetics marks in different cell lines.
We expect that results obtained from this project would be relevant for the antisense and transcriptional regulatory field as well as for the AML research where still in approximately 40 to 50% of the patients aged 16-60 years diagnosed with AML the genetic or molecular alteration causing the disease are unknown.