Histonas H2B.V e H4.V de Trypanosoma cruzi: investigação sobre a abundância, localização celular e genômica e interação com a cromatina
Data
2023-10-11
Autores
Rosón, Juliana Nunes 
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Trypanosoma cruzi é um parasita flagelado causador da doença de Chagas que possui particularidades na expressão gênica, como a transcrição policistrônica. Alterações na conformação da cromatina podem ocorrem pelas modificações pós-traducionais de histonas (MPTs) e deposição de histonas variantes, podendo alterar a expressão gênica. Dados proteômicos quantitativos indicaram que a histona variante H2B.V de T. cruzi está aumentada nas formas tripomastigotas celulares (TCTs) quando comparado às formas epimastigotas. No presente trabalho objetivou-se compreender o possível papel regulatório de duas histonas variantes, H2B.V e H4.V, na estrutura da cromatina e na expressão gênica, avaliando suas abundâncias, níveis de afinidade com a cromatina e localizações genômicas e nucleares nos ciclos celular e de vida. Primeiramente foi identificada uma putativa H4.V de T. cruzi (TcCLB.511681.20) por meio análises in silico. Foram geradas linhagens de parasitas H2B.V-myc e ty1-H4.V por CRISPR-Cas9. Por meio da localização genômica, a putativa H4.V de T. cruzi foi confirmada como H4.V por possuir localização específica principalmente nas cSSRs, em alguns loci de tDNAs, em regiões teloméricas e em genes RHS e trans-sialidases localizados principalmente nas extremidades dos contigs. Quanto a localização genômica da H2B.V, foi detectada enriquecimento nas dSSRs, em alguns loci de tDNAs e em regiões entre os compartimentos genômicos denominados conservado e disruptivo. Nos dados de ChIP-seq da H2B.V, foi observado que as formas TCTs apresentaram pouco enriquecimento em relação às formas epimastigotas, tal resultado mostra-se contraditório em relação aos dados proteômicos já publicados. Assim, foi observado os níveis de interação das histonas variantes com a cromatina, e avaliado que ambas as variantes se apresentam mais ligadas à cromatina nas formas epimastigotas e metacíclicas, enquanto nas formas TCTs está fracamente ligada a cromatina, justificando o possível desligamento da histona H2B.V durante o ensaio de ChIP-seq das formas TCTs. Em adição, foi verificado por ensaios de western blot que a H2B.V está mais abundante nas formas metacíclicas seguida das formas amastigotas, tripomastigotas e epimastigotas. A H4.V está mais abundante nas formas metacíclicas, seguida das formas epimastigotas, enquanto nas formas amastigotas e TCTs encontra-se pouquissimamente abundante. No ciclo celular verificou-se que ambas as variantes possuem marcação nos ensaios de IFA crescente das fases G1 para G2, enquanto a maior abundância de H2B.V está em G1 e S, e da H4.V está em G2 e G2/M. A integração dos resultados desta tese indica que cada variante participa de um cenário diferente do genoma e da cromatina, podendo influenciar atividades da maquinaria de RNA polimerase II, dado que se localizam em regiões associadas ao início e final de transcrição e aos telômeros. Por fim, o fato da abundância e interação com a cromatina das variantes serem distintas entre as formas de vida e fases do ciclo celular do parasita indicam que esta regulação possa ser dinâmica.
Trypanosoma cruzi is a flagellated parasite that causes Chagas disease and has particularities in gene expression, such as polycistronic transcription. Changes in chromatin conformation can occur through histone post-translational modifications (MPTs) and deposition of variant histones, which can alter gene expression. Quantitative proteomic data indicated that the histone variant H2B.V of T. cruzi is increased in cellular trypomastigotes (TCTs) compared to epimastigotes. The present work aimed to understand the possible regulatory role of two variant histones, H2B.V and H4.V, in chromatin structure and gene expression, evaluating their abundances, levels of affinity with chromatin and genomic and nuclear locations in life and cell cycles. First, a putative H4.V from T. cruzi (TcCLB.511681.20) was identified through in silico analyses. H2B.V-myc and ty1-H4.V parasite lines were generated by CRISPR-Cas9. Through genomic localization, the putative H4.V of T. cruzi was confirmed as H4.V because it has a specific location mainly in cSSRs, in some tDNA loci, in telomeric regions and RHS and trans-sialidases genes located mainly at the ends of the contigs. Regarding the genomic location of H2B.V, enrichment was detected in dSSRs, in some tDNA loci and in regions between the genomic compartments called conserved and disruptive. In the H2B.V ChIP-seq data, it was observed that the TCTs forms showed little enrichment in relation to the epimastigotes forms. This result is contradictory to the proteomic data already published. Thus, the interaction levels of the variant histones with chromatin were observed, and it was assessed that both variants are more linked to chromatin in the epimastigote and metacyclic forms, while in the TCT forms, it is weakly linked to chromatin, justifying the possible disconnection of histone H2B.V during ChIP-seq assay of TCTs forms. In addition, it was verified by western blot assays that H2B.V is most abundant in metacyclic forms, followed by amastigotes, trypomastigotes and epimastigotes. H4.V is most abundant in metacyclic forms, followed by epimastigote forms, while it is very little abundant in amastigote and TCT forms. In the cell cycle, it was found that both variants have increasing marking in the IFA assays from the G1 to G2 phases, while the most incredible abundance of H2B.V is in G1 and S, and of H4.V is in G2 and G2/M. The integration of the results of this thesis indicates that each variant participates in a different scenario of the genome and chromatin and can influence the activities of the RNA polymerase II machinery, given that they are located in regions associated with the beginning and end of transcription and telomeres. Finally, the fact that the abundance and interaction with chromatin of the variants are different between life forms and phases of the parasite's cell cycle indicates that this regulation may be dynamic.
Trypanosoma cruzi is a flagellated parasite that causes Chagas disease and has particularities in gene expression, such as polycistronic transcription. Changes in chromatin conformation can occur through histone post-translational modifications (MPTs) and deposition of variant histones, which can alter gene expression. Quantitative proteomic data indicated that the histone variant H2B.V of T. cruzi is increased in cellular trypomastigotes (TCTs) compared to epimastigotes. The present work aimed to understand the possible regulatory role of two variant histones, H2B.V and H4.V, in chromatin structure and gene expression, evaluating their abundances, levels of affinity with chromatin and genomic and nuclear locations in life and cell cycles. First, a putative H4.V from T. cruzi (TcCLB.511681.20) was identified through in silico analyses. H2B.V-myc and ty1-H4.V parasite lines were generated by CRISPR-Cas9. Through genomic localization, the putative H4.V of T. cruzi was confirmed as H4.V because it has a specific location mainly in cSSRs, in some tDNA loci, in telomeric regions and RHS and trans-sialidases genes located mainly at the ends of the contigs. Regarding the genomic location of H2B.V, enrichment was detected in dSSRs, in some tDNA loci and in regions between the genomic compartments called conserved and disruptive. In the H2B.V ChIP-seq data, it was observed that the TCTs forms showed little enrichment in relation to the epimastigotes forms. This result is contradictory to the proteomic data already published. Thus, the interaction levels of the variant histones with chromatin were observed, and it was assessed that both variants are more linked to chromatin in the epimastigote and metacyclic forms, while in the TCT forms, it is weakly linked to chromatin, justifying the possible disconnection of histone H2B.V during ChIP-seq assay of TCTs forms. In addition, it was verified by western blot assays that H2B.V is most abundant in metacyclic forms, followed by amastigotes, trypomastigotes and epimastigotes. H4.V is most abundant in metacyclic forms, followed by epimastigote forms, while it is very little abundant in amastigote and TCT forms. In the cell cycle, it was found that both variants have increasing marking in the IFA assays from the G1 to G2 phases, while the most incredible abundance of H2B.V is in G1 and S, and of H4.V is in G2 and G2/M. The integration of the results of this thesis indicates that each variant participates in a different scenario of the genome and chromatin and can influence the activities of the RNA polymerase II machinery, given that they are located in regions associated with the beginning and end of transcription and telomeres. Finally, the fact that the abundance and interaction with chromatin of the variants are different between life forms and phases of the parasite's cell cycle indicates that this regulation may be dynamic.
Descrição
Citação
ROSON, Juliana Nunes. Histonas H2B.V e H4.V de Trypanosoma cruzi: investigação sobre a abundância, localização celular e genômica e interação com a cromatina. 2023. 188 f. Tese (Doutorado em Microbiologia e Imunologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (são Paulo), São Paulo, 2023.