Publicação
Role of histone H1 in chromatin and gene expression in the african trypanosome : broad skills, specific functions?
| Resumo: | Trypanosoma brucei is a unicellular protozoan parasite that lives in the bloodstream and interstitial spaces of a mammalian host and that is transmitted by the tsetse, an insect vector. In humans, it causes sleeping sickness. T. brucei has a complex life cycle that progresses through different developmental stages. Inside the mammal, the parasite lives as bloodstream forms (BSFs), which differentiates differentiates into procyclic forms (PFs) upon transmission to the insect vector. T. brucei is an evolutionarily early-branching organism that possesses many unusual features for an eukaryote. Most of its transcription is constitutive and driven by RNA polymerase II (Pol II) and RNA polymerase I (Pol I) transcribes not only ribosomal RNA genes, but also protein-coding genes, including genes encoding variant surface glycoproteins (VSGs) and procyclins. These are highly abundant proteins that cover the surface of the BSFs and the PFs, respectively, and whose expression is tightly developmentally regulated. In BSFs, VSGs are transcribed in subtelomeric bloodstream expression sites (BESs). There are ~15 BESs in the genome, however only one is fully transcriptionally active at a time. The remaining BESs need to be kept silent to maintain monoallelic expression of BES. To evade the host immune system, parasites switch the expressed VSG from time to time by a mechanism called antigenic variation. In addition, in BSFs procyclin loci are also partially repressed to prevent illegitimate VSG replacement for procyclins. Some epigenetic factors have been found to regulate Pol I transcriptional activation/silencing at BESs and procyclin loci. Besides, a number of histone variants and histone modifications seem to be associated with Pol II transcription initiation and termination. Inside the eukaryotic nucleus, DNA is wrapped around an octamer of histones that forms the nucleosome, the basic unit of chromatin. Linker histone or histone H1 (H1) binds to the DNA entering and exiting the nucleosome, stabilizing its structure and contributing to the establishment of higher-order chromatin structures. In T. brucei, H1 is encoded by at least five genes and, based on the predicted protein sequence, they can be split in three classes. In my PhD work I aimed at analyzing the roles of H1 in chromatin structure, genome-wide gene expression and transcription in T. brucei. In addition, the importance of H1 for T. brucei mammalian infections in vivo was also addressed. Functional studies were performed by simultaneously depleting all classes of H1 by RNA interference (RNAi). Using chromatin immunoprecipitation (ChIP) of histone H3 and formaldehyde-assisted isolation of regulatory elements (FAIRE), in H1-depleted parasites we detected a global chromatin opening across the genome, even though this happened at silent BESs promoters more prominently and procyclin loci, to a lesser extent. Curiously, RNA sequencing (RNA-Seq) revealed that global chromatin opening due to H1 depletion is not associated with widespread alterations in gene expression but rather with derepression of a specific subset of genes. Out of ~9,000 genes, only 26 were significantly upregulated, mostly from silent BESs (including silent VSGs) and procyclin loci genes. I adapted and used 4-thiouridine (4sU) metabolic labeling of RNA in T. brucei to purify and quantify nascent transcripts emanating from Pol I and Pol II-transcribed loci. 4sU-labeling has the advantage over classical nuclear run-on of measuring transcription de novo in unperturbed cells. Quantification of 4sU-labeled nascent RNA showed that H1 is necessary to repress Pol I transcription at silent BESs and procyclin loci but apparently not at Pol II genes. Altogether our data strongly supports a model in which H1 induces global chromatin compaction, but in what concerns gene expression, H1 plays more specific functions, namely on transcriptional regulation of silent Pol I transcribed genes. This is consistent with the long-stand notion that polycistronic transcription by Pol II is constitutive and most genes are controlled essentially at the post-transcriptional level. On the other hand, Pol I genes are known to be regulated at the transcriptional level, consistent with a role of H1 in silencing Pol I transcription initiation/elongation. An additional phenotype observed in H1-depleted parasites was its increased resistance to methyl methanoesulfonate (MMS)-induced DNA damage. This might indicate that H1 also plays a more general role such as suppressing homologous recombination and perhaps genomic instability. Interestingly, H1-depleted T. brucei parasites revealed an apparent loss-of-fitness during in vivo infections. In mice infected with H1-depleted parasites, progression of parasitaemia (percentage of parasites in the blood) was delayed and mice succumbed later in infection. Curiously, this phenotype was completely abolished in highly immune-compromised mice that lack T, B and NK lymphocytes, demonstrating that fitness of H1-depleted parasites inside the host is impaired because, somehow, these parasites are more susceptible to host immune system. Altogether our results show that H1 of the early divergent T. brucei has ‘broad skills’ because it compacts chromatin throughout the genome, but it plays ‘specific functions’ at the transcription level because it represses transcription of a limited cohort of genes, namely silent Pol I loci. H1-mediated regulation of a specific set of genes was also observed in other higher and lower eukaryotes, which suggests that despite being the most divergent family of histones, its functions were conserved throughout evolution. |
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| Autores principais: | Pena, Ana Catarina Dias, 1985- |
| Assunto: | Histonas Trypanosoma brucei Cromatina Expressão génica Teses de doutoramento - 2015 |
| Ano: | 2015 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Trypanosoma brucei is a unicellular protozoan parasite that lives in the bloodstream and interstitial spaces of a mammalian host and that is transmitted by the tsetse, an insect vector. In humans, it causes sleeping sickness. T. brucei has a complex life cycle that progresses through different developmental stages. Inside the mammal, the parasite lives as bloodstream forms (BSFs), which differentiates differentiates into procyclic forms (PFs) upon transmission to the insect vector. T. brucei is an evolutionarily early-branching organism that possesses many unusual features for an eukaryote. Most of its transcription is constitutive and driven by RNA polymerase II (Pol II) and RNA polymerase I (Pol I) transcribes not only ribosomal RNA genes, but also protein-coding genes, including genes encoding variant surface glycoproteins (VSGs) and procyclins. These are highly abundant proteins that cover the surface of the BSFs and the PFs, respectively, and whose expression is tightly developmentally regulated. In BSFs, VSGs are transcribed in subtelomeric bloodstream expression sites (BESs). There are ~15 BESs in the genome, however only one is fully transcriptionally active at a time. The remaining BESs need to be kept silent to maintain monoallelic expression of BES. To evade the host immune system, parasites switch the expressed VSG from time to time by a mechanism called antigenic variation. In addition, in BSFs procyclin loci are also partially repressed to prevent illegitimate VSG replacement for procyclins. Some epigenetic factors have been found to regulate Pol I transcriptional activation/silencing at BESs and procyclin loci. Besides, a number of histone variants and histone modifications seem to be associated with Pol II transcription initiation and termination. Inside the eukaryotic nucleus, DNA is wrapped around an octamer of histones that forms the nucleosome, the basic unit of chromatin. Linker histone or histone H1 (H1) binds to the DNA entering and exiting the nucleosome, stabilizing its structure and contributing to the establishment of higher-order chromatin structures. In T. brucei, H1 is encoded by at least five genes and, based on the predicted protein sequence, they can be split in three classes. In my PhD work I aimed at analyzing the roles of H1 in chromatin structure, genome-wide gene expression and transcription in T. brucei. In addition, the importance of H1 for T. brucei mammalian infections in vivo was also addressed. Functional studies were performed by simultaneously depleting all classes of H1 by RNA interference (RNAi). Using chromatin immunoprecipitation (ChIP) of histone H3 and formaldehyde-assisted isolation of regulatory elements (FAIRE), in H1-depleted parasites we detected a global chromatin opening across the genome, even though this happened at silent BESs promoters more prominently and procyclin loci, to a lesser extent. Curiously, RNA sequencing (RNA-Seq) revealed that global chromatin opening due to H1 depletion is not associated with widespread alterations in gene expression but rather with derepression of a specific subset of genes. Out of ~9,000 genes, only 26 were significantly upregulated, mostly from silent BESs (including silent VSGs) and procyclin loci genes. I adapted and used 4-thiouridine (4sU) metabolic labeling of RNA in T. brucei to purify and quantify nascent transcripts emanating from Pol I and Pol II-transcribed loci. 4sU-labeling has the advantage over classical nuclear run-on of measuring transcription de novo in unperturbed cells. Quantification of 4sU-labeled nascent RNA showed that H1 is necessary to repress Pol I transcription at silent BESs and procyclin loci but apparently not at Pol II genes. Altogether our data strongly supports a model in which H1 induces global chromatin compaction, but in what concerns gene expression, H1 plays more specific functions, namely on transcriptional regulation of silent Pol I transcribed genes. This is consistent with the long-stand notion that polycistronic transcription by Pol II is constitutive and most genes are controlled essentially at the post-transcriptional level. On the other hand, Pol I genes are known to be regulated at the transcriptional level, consistent with a role of H1 in silencing Pol I transcription initiation/elongation. An additional phenotype observed in H1-depleted parasites was its increased resistance to methyl methanoesulfonate (MMS)-induced DNA damage. This might indicate that H1 also plays a more general role such as suppressing homologous recombination and perhaps genomic instability. Interestingly, H1-depleted T. brucei parasites revealed an apparent loss-of-fitness during in vivo infections. In mice infected with H1-depleted parasites, progression of parasitaemia (percentage of parasites in the blood) was delayed and mice succumbed later in infection. Curiously, this phenotype was completely abolished in highly immune-compromised mice that lack T, B and NK lymphocytes, demonstrating that fitness of H1-depleted parasites inside the host is impaired because, somehow, these parasites are more susceptible to host immune system. Altogether our results show that H1 of the early divergent T. brucei has ‘broad skills’ because it compacts chromatin throughout the genome, but it plays ‘specific functions’ at the transcription level because it represses transcription of a limited cohort of genes, namely silent Pol I loci. H1-mediated regulation of a specific set of genes was also observed in other higher and lower eukaryotes, which suggests that despite being the most divergent family of histones, its functions were conserved throughout evolution. |
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