Pax6 is a highly conserved developmental regulator with a complex temporal, spatial and quantitative expression pattern, that is crucial for correct development of the central nervous system, the eye, and pancreas. Accordingly, the Pax6 gene resides in a complex genomic locus containing a large array of long-range tissue-specific cis-regulatory elements primarily identified through multispecies sequence conservation and reporter studies. I have set out to understand how the chromatin architecture of the locus contributes to the mechanism and specificity of cis-regulatory interactions. As well as addressing whether the DNA looping model for regulatory interactions applies to the mouse Pax6 locus, I will identify which elements facilitate such interactions and if they vary between cell types. Utilising ChIP-array technology the distribution and variability of key regulatory histone modifications and factors were assessed in a set of Pax6 expressing and non-expressing mouse cell lines, acting as models for different regulatory states of the locus. Work in other loci suggests a key role for CTCF and cohesin (subunit Rad21) in chromatin organisation and long distance regulatory interactions. ChIP-chip for CTCF/Rad21 across the Pax6 locus identified numerous sites within the gene and at distal regulatory locations. The majority of these sites are cell type invariable. The active enhancer modification H3K27ac identified both known and several novel putative enhancer elements distributed through the locus that are highly cell type specific. A subset of CTCF/Rad21 sites also acquire the active enhancer modification H3K27ac in a cell type dependent manor, suggesting that CTCF/Rad21 may facilitate looping to the target gene from these sites. Using reporter based assays, putative regulatory elements marked by the looping factors and active histone modifications showed a diverse range of functional activities. Unexpectedly only 3 of the 7 CTCF sites tested showed classical insulator activity in an enhancer blocking reporter assay. Surprisingly the strongest insulator tested resided within intron 7 the Pax6 gene. Other CTCF/Rad21 sites were neutral or enhancers in the insulator assay. This reveals the disparity between predicting regulatory properties using ChIP binding profiles alone and the actual outcome of functional reporter experiments. A novel element, CTCF6 showed a ChIP signature of CTCF/Rad21/H3K27ac in all Pax6 expressing tissues, and functioned as a strong enhancer in transient transfection and stable LacZ reporter assays. CTCF6 recapitulated a broad range of Pax6 expression patterns, at multiple embryonic stages, including the brain, neural tube and pancreas. A second novel element, E-120 identified in the pancreatic derived cell line, drove stable embryonic reporter expression in the embryonic pancreas and sub set of brain regions. Together this has expanded the repertoire and size of Pax6’s regulatory landscape particular in the upstream region. Chromatin conformation capture (3C) was used to characterise the dynamic chromatin architecture of the locus and identify the interaction profiles from three CTCF/Rad21 binding regulatory locations within the Pax6 locus. This revealed a core set of regulatory interactions with the Pax6 gene, while individual elements showed a more variable set of cell type specific interactions. The CTCF6 enhancer showed highly cell type specific promoter interactions throughout the Pax6 gene, indicative of enhancer-promoter looping not detected in the non-expressing cells. While the downstream site CTCF5 at the edge of a cluster of regulatory elements known as the DRR (differentially regulated region), interacted with both the gene and an upstream element CTCF7 300 kb away only in the Pax6 expressing locus. Together these results reveal Pax6 has a chromatin hub structure with regulatory loops from upstream and downstream bringing distant yet variable active elements in to the vicinity of the Pax6 promoters where they can act. This work has revealed new roles for CTCF/cohesin sites in transcriptional regulation of Pax6 and how the cis-regulatory activity and structure of the locus varies across different cell types.

Orientadores: Maricilda Palandi de Mello, Monica Barbosa de Melo, Fernanda Caroline Soardi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T10:51:43Z (GMT). No. of bitstreams: 1 Franca_EmersonSalvadordeSouza_M.pdf: 3804995 bytes, checksum: 694d80ade56340a0a1125338c03238ad (MD5) Previous issue date: 2009 / Resumo: O gene PAX6 é o principal gene para o controle da organização do sistema ocular durante a embriogênese. Este gene pertence a uma família de reguladores de transcrição denominada PAX, sendo que seus membros compartilham um domínio funcional de 128 aminoácidos chamado de paired domain. O PAX6 é o mais bem estudado dessa família. O gene PAX6 está localizado na banda 13 do braço curto do cormossomo11 em humanos, e apresenta 14 exons sendo que os três primeiros e parte do quarto exon não são traduzidos. A proteína do PAX6 possui dois domínios funcionais: o paired domain e o homeo domain, que são separados por um segmento de ligação denominado LNK e seguidos por uma região com importante função na ativação transcricional denominada PST. A proteína do gene PAX6 é um fator de regulação transcricional altamente conservado com funções importantes para o desenvolvimento normal dos olhos e do sistema nervoso. Alterações no gene PAX6 em humanos foram associadas ao fenótipo de aniridia, da síndrome de Morning Glory (MGS) e também de doenças associadas ao desenvolvimento ocular. A aniridia é um defeito congênito raro, a qual provoca uma formação incompleta ou a ausência da íris. Embora seus efeitos variem entre os indivíduos, pode causar perda de visão. A doença pode ser de herança autossômica dominante ou de manifestação esporádica. MGS é uma anomalia congênita do nervo óptico, comumente unilateral, podendo encontrar-se associada estrabismo, ambliopia e nistagmo. Freqüentemente essa síndrome pode encontrar-se associada a anomalias endócrinas, renais e do sistema nervoso central. Sendo assim, o projeto teve por objetivos a análise molecular do gene PAX6 através de seqüenciamento e da técnica de MLPA em pacientes com aniridia e pacientes portadores da síndrome de MGS, a fim de se detectar mutações e/ou polimorfismos que pudessem estar ligados ao quadro clínico dos indivíduos. A casuística do projeto foi de três famílias com segregação aniridia, um indivíduo esporádico com aniridia e quatro indivíduos portadores da síndrome de Morning Glory. O grupo controle consistiu de 50 indivíduos triados como não tendo alterações oftalmológicas. Foi encontrada uma mutação p.R240X no gene PAX6, que causa uma troca de uma arginina por um stop codon, segregando nos indivíduos afetados da Família 1 e assim explicando o fenótipo dos indivíduos. Essa mutação é a mais freqüente associada à aniridia, porém é a primeira vez que ela foi descrita na população brasileira. Também foram encontradas diversas alterações descritas e não descritas que necessitam de mais estudos para que possam ser associadas à manifestação do fenótipo dos indivíduos afetados. Além disso, foi observada pela técnica de MLPA, uma possível micro-deleção ou alteração nucleotídica no exon 1 do gene RCN1 encontrada nos filhos das Famílias 1 e 3 podendo estar envolvida na regulação 5' do gene PAX6. Outra possível micro-deleção ou alteração nucleotídica foi também observada no exon 9 do gene ELP4, que pode estar associada a regulação 3' do gene PAX6. Esse estudo demonstrou que para a etiologia de aniridia e síndrome Morning Glory devem existir outros genes cuja expressão possa estar alterada durante o desenvolvimento. / Abstract: PAX6 gene is the major gene in the control of eye organization during development. It belongs to a family of transcription regulators called PAX, formed by several members which share a 128 aminoacid functional domain called paired domain. PAX6 is best studied within this family. In humans, it is located on chromosome 11p13 and is formed by 14 exons; the first three and part of the fourth are not translated. PAX6 protein comprises two functional domains: the paired domain and the homeo domain which are separated by a linker called LNK and followed by an important region with trancriptional activity called PST. The PAX6 protein is a highly conserved transcriptional regulator factor that is important for normal ocular and neural development. Mutations on human PAX6 gene were associated to aniridia, Morning Glory Syndrome and other ocular diseases. Aniridia is rare birth defect which leads to an incomplete formation or the absence of the iris. Although their effects vary between individual, aniridia can cause loss of vision. The disease may be autosomal dominant or sporadic event. The Morning Glory Syndrome (MGS) is a congenital optic disk dysplasia, generally unilateral, which can be associated with strabismus, amblyopia and nystagmus. This syndrome may be often associated with endocrine, renal and central nervous system abnormalities. Thus the aim of this investigation was to evaluate the molecular composition of PAX6 gene using direct sequencing and MLPA technique in patients with Aniridia and Morning Glory Syndrome, to detect mutations and/or polymorphisms associated with the patient's phenotypes. Were included in the study 1 family with segregation of aniridia, 1 family with Axenfeld-Rieger Syndrome, 1 sporadic individual with aniridia and 4 individuals carrying MGS. The control group comprised 50 individuals considered ophthalmologically normal. The nonsense mutation p.R240X was found in the PAX6 gene, segregating with the affected members in family 1, what explains their phenotypes. This mutation is one of the most frequent nonsense mutations associated with the aniridia, however this is the first report on a PAX6 gene mutation familial case of aniridia in Brazil. Several described and non-described nucleotide variations were found, but additional studies are required to correlate them to the phenotype of affected individuals. Furthermore, the MLPA technique showed possible micro-deletions or mutations in exon 1 of RCN1 gene, located 5' to PAX6. This result was observed in both male children of families 1 and 3. Other possible micro-deletion or mutation was observed in exon 9 of ELP4 gene, which can be associated to 3' regulation of PAX6 gene. This study demonstrated that the involvement of other gene whose expressions may be altered during the development cannot be excluded for the etiology of aniridia and Morning Glory Syndrome. / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular