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| Author(s): |
Rafaela Rosa Ribeiro
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2016-08-25 |
| Examining board members: |
Hernandes Faustino de Carvalho;
Sérgio Luis Felisbino;
Eduardo Moraes Rego Reis;
Murilo Vieira Geraldo;
Leticia Fröhlich Archangelo
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| Advisor: | Hernandes Faustino de Carvalho |
| Abstract | |
In this work we have defined the expression and localization of transcription factors related to control of differentially expressed genes by rat ventral prostate under androgen deprivation. In the first part, using DNA microarray, bioinformatics we have identified genes with annotated function in transcription regulation: EVI1 (Mecom), NFY, ELK1, GATA2, MYBL1, MYBL2, and NF?B member family wich were validated through immunofluorescence and qRT-PCR (Rosa-Ribeiro, Nishan, et al., 2014). In the second part, we characterize Bmyb in the rat ventral prostate after castration and BMYB in prostate human cell lines. We observed that expression of Bmyb has had a five-fold increase in mRNA content and the presence of its active form (phospho-T487-BMYB) in the epithelial cell nuclei in the rat ventral prostate three days after castration. We identified that LNCaP cells (isolated from human prostate cancer epithelial cell) express BMYB and chose theses cells to test the effects of androgen stimulation on BMYB activation and interaction with the genome/chromatin. LNCaP cells were incubated with either 1 nM R1881 (a synthetic androgen) or the vehicle (DMSO) for 6, 12 or 24 hours. Treatment with R1881 for 12 hours resulted in decreased expression of BMYB (qRT-PCR), reduced content of phospho-T487-BMYB (Western Blotting), although we have shown that the content of this phosphorylated form increases along of cellular cycle and accumulates in mitotic cells (immunofluorescence). Double immunofluorescence revealed that AR activation for R1881, accumulates AR in the nucleus by R1881 and excludes phospho-T487-BMYB. To investigate aspects of BMYB interaction with the chromatin in LNCaP cells, submitted the 12h control and treated with R1881 groups to ChIP-seq. The binding regions of BMYB (peaks) identified by ChIP-seq showed distinct and similar aspects between the two experimental groups. How expected, the BMYB binding motif was found enriched in both groups, however, in the control group was also localized the non-canonical sequence to BMYB. Besides BMYB motifs, it was found enrichment motif to SMAD2, STAT5, STAT6 and ZNFX in both groups, while NKX2.5 and ZNF11 were found in control and R1881 groups, respectively. The presence of BMYB motif through the peaks, has a distribution that respect the "power law" showing that peaks with high BMYB motif concentration are huge regions near to TSS suggesting a formation of "activity fields". Analyzing regions near TSS, there is a high frequency of peaks in R1881 group. Despite of BMYB chromosome distribution, its has similar distribution in both groups and preferably centromeric occupation. The genes with closest TSS of BMYB peaks, are mostly "protein coding" in both groups. While in gene regions there is a preferential binding to "intergenic" region with exclusive regions to R1881 in "5¿UTR", "3¿UTR" and "Exon" of genes. The ontologies (10 vs 55, for the control and R1881, respectively) are distinct for each experimental condition and refer to different functions considering to transcriptomes from literature, was found that 5% of genes are the same; and gene like: KLK3, KLK2, TMPRSS22 and NKX2.5, strongly related to prostate biology, were found in R1881 group. In conclusion, the results are novel and demonstrate that BMYB patterns of genome occupation are compatible with its function in determining different chromatin structures in different scales in the androgenic and hypoandrogenic environments (AU) | |
| FAPESP's process: | 12/07512-4 - Sequential gene expression in response to androgen deprivation: orchestrating secretion activity, proliferation and cell death in the rat ventral prostate |
| Grantee: | Rafaela da Rosa Ribeiro |
| Support Opportunities: | Scholarships in Brazil - Doctorate |