Caracterização da região MHM em aves: padrões diferenciais de metilação em machos e fêmeas
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2016-06-21
Autores
Jeronimo, Bruna Cristina [UNESP]
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Universidade Estadual Paulista (Unesp)
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Em contraste ao padrão de cromossomos sexuais de mamíferos (XX/XY), as aves apresentam um sistema de determinação sexual em que os machos representam o sexo homogamético (ZZ) e as fêmeas constituem o sexo heterogamético (ZW). Adicionalmente, embora mamíferos apresentem um mecanismo de compensação de dose, a inativação completa de um dos cromossomos Z não é observada em machos de aves e, portanto, estes possuem um maior nível de expressão de vários genes presentes nesse cromossomo. A despeito disso, um mecanismo ainda não completamente esclarecido de compensação de dose parcial em aves resulta em expressão equivalente entre os sexos para alguns genes do cromossomo Z. A região MHM (Male Hypermethylated), localizada no cromossomo Z de Galliformes, está associada a um padrão de hipermetilação em machos e hipometilação em fêmeas, levando à síntese de um RNA não-codificante longo (lncRNA) somente em fêmeas. A presença deste lncRNA é associada ao aumento da expressão de genes próximos à região MHM em fêmeas, o que parece resultar em uma compensação de dose local entre os sexos. Dado que, até o momento, segmentos MHM foram somente identificados em Galiformes e Anseriformes, o presente estudo visou isolar e caracterizar esta região em Galliformes (galinha doméstica, codorna européia, peru) e também em Struthioniformes (avestruz), Strigiformes (coruja-orelhuda, corujinha-do-mato, coruja-da-igreja, coruja-buraqueira), Piciformes (tucanuçu), Psittaciformes (arara-azul-grande) e Apodiformes (beija-flor-da-banda-branca, beija-flor-tesoura, beija-flor-preto). Indivíduos adultos e embriões com seis dias de desenvolvimento foram sexados com base em caracteres morfológicos e moleculares - por meio de PCR (Polymerase Chain Reaction) para amplificação de uma região intrônica dos genes CHD1-Z e CHD1-W, seguida de eletroforese em gel de agarose e poliacrilamida, análise SSCP e análise automatizada de fragmentos de DNA. Métodos de sexagem molecular mostraram-se adequados para identificação de machos e fêmeas de galinha doméstica, codorna européia, peru, tucanuçu, arara-azul-grande, coruja-orelhuda, corujinha-do-mato, coruja-buraqueira, beija-flor-da-banda-branca, beija-flor-tesoura e beija-flor-preto. Entretanto, as técnicas moleculares utilizadas não permitiram identificar diferenças entre machos e fêmeas de avestruz. Análises in silico da região MHM de galinha doméstica mostraram que esta se encontra localizada no braço curto do cromossomo Z, sendo constituída por 260Kb (chrZ:27,000,000-27,260,000) e alto conteúdo de CG. Esta região é delimitada por duas LINES (Long Interspersed Nuclear Elements) e possui múltiplos elementos repetitivos da classe LTR (Long Terminal Repeat), especialmente os pertencentes à família EVRL (Endogenous Retrovirus), denominados GGLTR5A. Com base em sua composição genômica, a região MHM de galinha doméstica foi subdividida em sub-regiões - denominadas de 1 (chrZ:27,176,712-27,260,282), 2 (chrZ:27,132,044-27,174,901), 3a (chrZ:27,094,512-27,132,043), 4 (chrZ:27,036,950-27,094,511) e 3b (chrZ:27,000,000-27,036,949) - que apresentam-se compostas por três unidades de repetições diferentes (denominadas de repeats 1, 2 e 3) flanqueadas por LTRs específicas. PCR multiplex em amostras de galinha doméstica levou à amplificação de dois fragmentos de DNA de aproximadamente 240 e 750 pares de bases, sendo o fragmento maior correspondente à repeat 1 da região MHM. Assim como observado para galinha doméstica, também foram gerados, via PCR, dois fragmentos de DNA de diferentes tamanhos associados à região MHM para as outras espécies de aves estudadas. Um maior nível de identidade (80-97%) foi observado entre a região MHM de galinha doméstica e as sequências nucleotídicas obtidas de codorna européia, peru e avestruz, o que demonstra que a presença de segmentos MHM não se restringe ao genoma de Galliformes e Anseriformes. Ensaios de digestão enzimática em DNA genômico de galinha doméstica, codorna européia e peru, por meio do uso de endonucleases de restrição sensíveis à metilação e dependentes de metilação (MspI, HpaII e McrBC), seguidos de amplificação de um fragmento de DNA associado à sub-região 1 MHM, evidenciaram padrões diferenciais de metilação dessa região entre os sexos, sendo hipometilada em fêmeas e hipermetilada em machos. Tais padrões diferenciais mostram-se potencialmente adequados para aplicação em testes de sexagem molecular em espécies de aves.
In contrast to the sexual chromosomes pattern found in mammals (XX/XY), birds present a sex determination system in which males represent the homogametic sex (ZZ) and females correspond to the heterogametic sex (ZW). Furthermore, although mammals present a dosage compensation mechanism, the complete inactivation of one Z chromosome is not observed in male birds and, therefore, they have a higher expression level of several genes that are found in this chromosome. Despite this, a mechanism of partial dosage compensation that was not clearly explained so far for birds results on an equivalent expression between sexes for some of the genes found at the Z chromosome. The MHM region (Male Hypermethylated), localized at the Z chromosome of Galliformes, is associated to a hypermethylation pattern in males and hypomethylation in females, which leads to the synthesis of a long non-coding RNA (lncRNA) only in females. The presence of this lncRNA is associated with a higher expression of genes that are located near to the MHM region in females, which seems to result in a local dosage compensation between sexes. As MHM segments were so far identified only in Galliformes and Anseriformes, the present study aimed to isolate and characterize this region on Galliformes (chicken, European quail, turkey) and also on Struthioniformes (ostrich), Strigiformes (striped owl, tropical screech-owl, barn owl, burrowing owl), Piciformes (toco toucan), Psittaciformes (hyacinth macaw), and Apodiformes (versicolored emerald, swallow-tailed hummingbird, black jacobin). Adult individuals and six-day embryos were sexed based on morphological and molecular characters - throughout PCR (Polymerase Chain Reaction) to amplify an intronic region of the CHD1-Z e CHD1-W genes, followed by agarose and polyacrylamide electrophoresis, SSCP analysis and automated fragment DNA analysis. Molecular sexing methodologies were useful for the identification of males and females of chicken, European quail, turkey, toco toucan, hyacinth macaw, striped owl, tropical screech-owl, burrowing owl, versicolored emerald, swallow-tailed hummingbird, and black jacobin. However, the applied techniques were not effective to identify differences between male and female ostriches. In silico analyses of the chicken MHM region showed that it is localized at the short arm of the Z chromosome and is constituted by 260Kb (chrZ:27,000,000-27,260,000) and a high CG content. This region is delimited by two LINES (Long Interspersed Nuclear Elements) and presents multiple repetitive elements of the LTR (Long Terminal Repeat) class, especially those of the EVRL (Endogenous Retrovirus) family, denominated GGLTR5A. Based on its genomic composition, the MHM region was subdivided into sub regions – denominated as 1 (chrZ:27,176,712-27,260,282), 2 (chrZ:27,132,044-27,174,901), 3a (chrZ:27,094,512-27,132,043), 4 (chrZ:27,036,950-27,094,511), and 3b (chrZ:27,000,000-27,036,949) - that are composed by three different repeat units (denominated as repeats 1, 2 e 3) flanked by specific LTRs. Multiplex PCR on chicken samples resulted in the amplification of two different size DNA fragments of around 240 and 750 base pairs, and the larger fragment corresponds to the repeat 1 of the MHM region. As observed for chicken, two different DNA fragments associated to the MHM region were also generated, by PCR, for the other studied species. A higher identity index (80-97%) was recognized between the chicken MHM region and the obtained nucleotide sequences of European quail, turkey and ostrich, which evidences that the presence of MHM segments is not restricted to the Galliformes and Anseriformes genomes. Enzymatic digestion assays in genomic DNA samples of chicken, European quail and turkey, through the use of methylation sensitive and methylation dependent restriction endonucleases (MspI, HpaII e McrBC), followed by the amplification of a DNA fragment associated to the sub region 1 MHM, showed differential methylation patterns between sexes - hypomethylated in females and hypermethylated in males. These differential patterns are potentially applicable for molecular sexing tests in bird species.
In contrast to the sexual chromosomes pattern found in mammals (XX/XY), birds present a sex determination system in which males represent the homogametic sex (ZZ) and females correspond to the heterogametic sex (ZW). Furthermore, although mammals present a dosage compensation mechanism, the complete inactivation of one Z chromosome is not observed in male birds and, therefore, they have a higher expression level of several genes that are found in this chromosome. Despite this, a mechanism of partial dosage compensation that was not clearly explained so far for birds results on an equivalent expression between sexes for some of the genes found at the Z chromosome. The MHM region (Male Hypermethylated), localized at the Z chromosome of Galliformes, is associated to a hypermethylation pattern in males and hypomethylation in females, which leads to the synthesis of a long non-coding RNA (lncRNA) only in females. The presence of this lncRNA is associated with a higher expression of genes that are located near to the MHM region in females, which seems to result in a local dosage compensation between sexes. As MHM segments were so far identified only in Galliformes and Anseriformes, the present study aimed to isolate and characterize this region on Galliformes (chicken, European quail, turkey) and also on Struthioniformes (ostrich), Strigiformes (striped owl, tropical screech-owl, barn owl, burrowing owl), Piciformes (toco toucan), Psittaciformes (hyacinth macaw), and Apodiformes (versicolored emerald, swallow-tailed hummingbird, black jacobin). Adult individuals and six-day embryos were sexed based on morphological and molecular characters - throughout PCR (Polymerase Chain Reaction) to amplify an intronic region of the CHD1-Z e CHD1-W genes, followed by agarose and polyacrylamide electrophoresis, SSCP analysis and automated fragment DNA analysis. Molecular sexing methodologies were useful for the identification of males and females of chicken, European quail, turkey, toco toucan, hyacinth macaw, striped owl, tropical screech-owl, burrowing owl, versicolored emerald, swallow-tailed hummingbird, and black jacobin. However, the applied techniques were not effective to identify differences between male and female ostriches. In silico analyses of the chicken MHM region showed that it is localized at the short arm of the Z chromosome and is constituted by 260Kb (chrZ:27,000,000-27,260,000) and a high CG content. This region is delimited by two LINES (Long Interspersed Nuclear Elements) and presents multiple repetitive elements of the LTR (Long Terminal Repeat) class, especially those of the EVRL (Endogenous Retrovirus) family, denominated GGLTR5A. Based on its genomic composition, the MHM region was subdivided into sub regions – denominated as 1 (chrZ:27,176,712-27,260,282), 2 (chrZ:27,132,044-27,174,901), 3a (chrZ:27,094,512-27,132,043), 4 (chrZ:27,036,950-27,094,511), and 3b (chrZ:27,000,000-27,036,949) - that are composed by three different repeat units (denominated as repeats 1, 2 e 3) flanked by specific LTRs. Multiplex PCR on chicken samples resulted in the amplification of two different size DNA fragments of around 240 and 750 base pairs, and the larger fragment corresponds to the repeat 1 of the MHM region. As observed for chicken, two different DNA fragments associated to the MHM region were also generated, by PCR, for the other studied species. A higher identity index (80-97%) was recognized between the chicken MHM region and the obtained nucleotide sequences of European quail, turkey and ostrich, which evidences that the presence of MHM segments is not restricted to the Galliformes and Anseriformes genomes. Enzymatic digestion assays in genomic DNA samples of chicken, European quail and turkey, through the use of methylation sensitive and methylation dependent restriction endonucleases (MspI, HpaII e McrBC), followed by the amplification of a DNA fragment associated to the sub region 1 MHM, showed differential methylation patterns between sexes - hypomethylated in females and hypermethylated in males. These differential patterns are potentially applicable for molecular sexing tests in bird species.
Descrição
Palavras-chave
MHM, Aves, Metilação, Sexagem molecular, Birds, Methylation, Molecular sexing