Integrating high-throughput analysis to create an atlas of replication origins in Trypanosoma cruzi in the context of genome structure and variability.

Trypanosoma cruzi is the etiologic agent of the most prevalent human parasitic disease in Latin America, Chagas disease. Its genome is rich in multigenic families that code for virulent antigens and are present in the rapidly evolving genomic compartment named Disruptive. DNA replication is a meticulous biological process in which flaws can generate mutations and changes in chromosomal and gene copy numbers. Here, integrating high-throughput and single-molecule analyses, we were able to identify Predominant, Flexible, and Dormant Orc1Cdc6-dependent origins as well as Orc1Cdc6-independent origins. Orc1Cdc6-dependent origins were found in multigenic family loci, while independent origins were found in the Core compartment that contains conserved and hypothetical protein-coding genes, in addition to multigenic families. In addition, we found that Orc1Cdc6 density is related to the firing of origins and that Orc1Cdc6-binding sites within fired origins are depleted of a specific class of nucleosomes that we previously categorized as dynamic. Together, these data suggest that Orc1Cdc6-dependent origins may contribute to the rapid evolution of the Disruptive compartment and, therefore, to the success of T. cruzi infection and that the local epigenome landscape is also involved in this process.IMPORTANCETrypanosoma cruzi, responsible for Chagas disease, affects millions globally, particularly in Latin America. Lack of vaccine or treatment underscores the need for research. Parasite's genome, with virulent antigen-coding multigenic families, resides in the rapidly evolving Disruptive compartment. Study sheds light on the parasite's dynamic DNA replication, discussing the evolution of the Disruptive compartment. Therefore, the findings represent a significant stride in comprehending T. cruzi's biology and the molecular bases that contribute to the success of infection caused by this parasite.

Integrating high-throughput analysis to create an atlas of replication origins in Trypanosoma cruzi in the context of genome structure and variability

Trypanosoma cruzi is the etiologic agent of the most prevalent human parasitic disease in Latin America, Chagas disease. Its genome is rich in multigenic families that code for virulent antigens and are present in the rapidly evolving genomic compartment named Disruptive. DNA replication is a meticulous biological process in which flaws can generate mutations and changes in chromosomal and gene copy numbers. Here, integrating high-throughput and single-molecule analyses, we were able to identify Predominant, Flexible, and Dormant Orc1Cdc6-dependent origins as well as Orc1Cdc6-independent origins. Orc1Cdc6-dependent origins were found in multigenic family loci, while independent origins were found in the Core compartment that contains conserved and hypothetical protein-coding genes, in addition to multigenic families. In addition, we found that Orc1Cdc6 density is related to the firing of origins and that Orc1Cdc6-binding sites within fired origins are depleted of a specific class of nucleosomes that we previously categorized as dynamic. Together, these data suggest that Orc1Cdc6-dependent origins may contribute to the rapid evolution of the Disruptive compartment and, therefore, to the success of T. cruzi infection and that the local epigenome landscape is also involved in this process.

The antiproliferative effect of FGF2 in K-Ras-driven tumor cells involves modulation of rRNA and the nucleolus.

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

Odontologia hospitalar no ambiente digital: o que se pública sobre esta habilitação? / Hospital dentistry in the digital environment: what is published about this qualification?

Objetivo: O objetivo deste trabalho foi caracterizar as publicações sobre odontologia hospitalar disponíveis em páginas da internet e em páginas do Instagram, além de avaliar a qualidade dos conteúdos disponíveis do ponto de vista da evidência científica e do uso de referências. Métodos: Foi realizado um estudo transversal e exploratório. A partir da pesquisa da chave de busca "odontologia hospitalar" foram localizadas páginas do Instagram e da Internet via Google. Resultados: No Instagram foram localizadas 15 páginas que atendiam aos critérios de inclusão, e as últimas 5 publicações foram analisadas (n=75). Para o Google foram avaliados conteúdos que correspondessem à projeto de lei, entre os primeiros 25 resultados da pesquisa, já que se trata dos mais relevantes a partir da marcação do algoritmo. Percebeu-se que o uso de referências não é frequente nem nas publicações do Instagram, tampouco nos conteúdos da Internet. Tal fato é tão importante, que os resultados deste trabalho demonstram essa urgência na mudança de cenário dos conteúdos publicados na rede mundial de computadores. Conclusão: Nesse sentido, os professores e os próprios conselhos podem atuar como bússolas, na orientação da divulgação e consumo de conteúdo aos estudantes e aos profissionais de saúde.(AU)

Aim: The objective of this study was to characterize the publications on hospital dentistry available on websites and Instagram pages, in addition to evaluating the quality of the available content from the point of view of scientific evidence and the use of references. Methods: A cross-sectional and exploratory study was carried out. From the research of the search key "hospital dentistry", Instagram and Internet pages were located via Google. Results: On Instagram, 15 pages that met the inclusion criteria were located, and the last 5 publications were analyzed (n=75). For Google, content that corresponded to the bill was evaluated, among the first 25 search results, since these are the most relevant based on the algorithm's marking. It was noticed that the use of references is not frequent either in Instagram publications or in Internet content. This fact is so important that the results of this work demonstrate this urgency in changing the scenario of content published on the World Wide Web. Conclusion: In this sense, professors and the councils themselves can act as compasses, guiding the dissemination and consumption of content to students and health professionals. (AU)

TgKDAC4: A Unique Deacetylase of Toxoplasma's Apicoplast.

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world's population and has no effective cure. Epigenetic regulation is one of the mechanisms controlling gene expression and plays an essential role in all organisms. Lysine deacetylases (KDACs) act as epigenetic regulators affecting gene silencing in many eukaryotes. Here, we focus on TgKDAC4, an enzyme unique to apicomplexan parasites, and a class IV KDAC, the least-studied class of deacetylases so far. This enzyme shares only a portion of the specific KDAC domain with other organisms. Phylogenetic analysis from the TgKDAC4 domain shows a putative prokaryotic origin. Surprisingly, TgKDAC4 is located in the apicoplast, making it the only KDAC found in this organelle to date. Transmission electron microscopy assays confirmed the presence of TgKDAC4 in the periphery of the apicoplast. We identified possible targets or/and partners of TgKDAC4 by immunoprecipitation assays followed by mass spectrometry analysis, including TgCPN60 and TgGAPDH2, both located at the apicoplast and containing acetylation sites. Understanding how the protein works could provide new insights into the metabolism of the apicoplast, an essential organelle for parasite survival.

Navigating the boundaries between metabolism and epigenetics in trypanosomes.

Epigenetic marks enable cells to acquire new biological features that favor their adaptation to environmental changes. These marks are chemical modifications on chromatin-associated proteins and nucleic acids that lead to changes in the chromatin landscape and may eventually affect gene expression. The chemical tags of these epigenetic marks are comprised of intermediate cellular metabolites. The number of discovered associations between metabolism and epigenetics has increased, revealing how environment influences gene regulation and phenotype diversity. This connection is relevant to all organisms but underappreciated in digenetic parasites, which must adapt to different environments as they progress through their life cycles. This review speculates and proposes associations between epigenetics and metabolism in trypanosomes, which are protozoan parasites that cause human and livestock diseases.

Do funcionamento autoerótico à desfusão pulsional: um modelo teórico para a etiologia do recurso à droga na esquizofrenia / From autoerotic functioning to instinctual defusion: a theoretical model for the etiology of drug use in schizophrenia / Du fonctionnement autoérotique à la défusion pulsionnelle: un modèle théorique pour l'étiologie de l'usage de drogues dans la schizophrénie / Del funcionamiento autoerótico a la defusión pulsional: un modelo teórico para la etiologia del consumo de drogas en la esquizofenia

O artigo apresenta a construção de um modelo teórico-clínico em psicopatologia psicanalítica, acerca dos determinantes estruturais do recurso à droga na psicose e, em especial, na esquizofrenia. Partimos da tese de Lacan acerca dos efeitos de adesão ao gozo autoerótico decorrente da ruptura com o gozo fálico, que coloca o problema relativo ao estatuto dessa ruptura na toxicomania e, em especial, da função do recurso à droga sobre os efeitos desta ruptura. Nossa hipótese é que o recurso à droga obedece a uma tentativa de refusão pulsional com consequências catastróficas. Conduzimos o levantamento dos textos de Freud que se organizam em torno da economia pulsional, tanto na sua abordagem do recurso à droga, como na sua abordagem da etiologia da psicose e da função reparadora do delírio. Como resultado, obtivemos os índices estruturais para o diagnóstico do recurso à droga na psicose.

Resumos The article presents the construction of a theoretical-clinical model in psychoanalytic psychopathology about the structural determinants of drug use in psychosis and in schizophrenia. We start from Lacan's thesis about the effects of adherence to autoerotic jouissance resulting from the rupture with phallic jouissance, which poses the problem concerning the status of this rupture in drug addiction and the role of drug use on the effects of this rupture. Our hypothesis is that the use of drugs follows an attempt at instinctual refusion with catastrophic consequences. We conducted a survey of Freud's texts that are organized around the drive economy, both in his approach to the use of drugs and in his approach to the etiology of psychosis and the reparative function of delusion. As a result, we obtained the structural indices for the diagnosis of drug use in psychosis..

L'article présente la construction d'un modèle théorico-clinique, en psychopathologie psychanalytique à propos des déterminants structurels de l'usage de drogues dans la psychose et, en particulier, dans la schizophrénie. Nous partons de la thèse lacanienne sur les effets d'adhésion à la jouissance autoérotique résultant de la rupture avec la jouissance phallique, ce qui pose le problème du statut de cette rupture dans la toxicomanie et, en particulier, du rôle de l'usage de drogues sur les effets de cette rupture. Notre hypothèse est que le recours aux drogues obéit à une tentative de refusion pulsionnelle aux conséquences catastrophiques. Nous avons mené l'enquête sur les textes de Freud qui s'organisent autour de l'économie pulsionnelle, tant dans son approche de la ressource médicamenteuse que dans son approche de l'étiologie de la psychose et de la fonction réparatrice du délire. Nous avons ainsi obtenu les indices structurels pour le diagnostic de l'usage de drogues dans la psychose.

Este artículo presenta la construcción de un modelo teórico-clínico en psicopatología psicoanalítica sobre los determinantes estructurales del consumo de drogas en la psicosis y, en particular, en la esquizofrenia. Partimos de la tesis de Lacan sobre los efectos de la adhesión al goce autoerótico resultante de la ruptura con el goce fálico, que plantea el problema del estatuto de esta ruptura en la drogadicción y, en particular, el papel del consumo de drogas en los efectos de esta ruptura. Nuestra hipótesis es que el uso de drogas sigue un intento de rechazo instintivo con consecuencias catastróficas. Realizamos un recorrido por los textos de Freud que se organizan en torno a la economía pulsional, tanto en su abordaje del uso de drogas como en su abordaje de la etiología de la psicosis y de la función reparadora del delirio. Como resultado obtuvimos los índices estructurales para el diagnóstico del consumo de drogas en psicosis.

The antiproliferative effect of FGF2 in K-Ras-driven tumor cells involves modulation of rRNA and the nucleolus

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

The antiproliferative effect of FGF2 in K-Ras-driven tumor cells involves modulation of rRNA and the nucleolus

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus-, and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression, and chromatin remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression may be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

Navigating the boundaries between metabolism and epigenetics in trypanosomes

Epigenetic marks enable cells to acquire new biological features that favor their adaptation to environmental changes. These marks are chemical modifications on chromatin-associated proteins and nucleic acids that lead to changes in the chromatin landscape and may eventually affect gene expression. The chemical tags of these epigenetic marks are comprised of intermediate cellular metabolites. The number of discovered associations between metabolism and epigenetics has increased, revealing how environment influences gene regulation and phenotype diversity. This connection is relevant to all organisms but underappreciated in digenetic parasites, which must adapt to different environments as they progress through their life cycles. This review speculates and proposes associations between epigenetics and metabolism in trypanosomes, which are protozoan parasites that cause human and livestock diseases.

TgKDAC4: a unique deacetylase of toxoplasma's apicoplast

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world’s population and has no effective cure. Epigenetic regulation is one of the mechanisms controlling gene expression and plays an essential role in all organisms. Lysine deacetylases (KDACs) act as epigenetic regulators affecting gene silencing in many eukaryotes. Here, we focus on TgKDAC4, an enzyme unique to apicomplexan parasites, and a class IV KDAC, the least-studied class of deacetylases so far. This enzyme shares only a portion of the specific KDAC domain with other organisms. Phylogenetic analysis from the TgKDAC4 domain shows a putative prokaryotic origin. Surprisingly, TgKDAC4 is located in the apicoplast, making it the only KDAC found in this organelle to date. Transmission electron microscopy assays confirmed the presence of TgKDAC4 in the periphery of the apicoplast. We identified possible targets or/and partners of TgKDAC4 by immunoprecipitation assays followed by mass spectrometry analysis, including TgCPN60 and TgGAPDH2, both located at the apicoplast and containing acetylation sites. Understanding how the protein works could provide new insights into the metabolism of the apicoplast, an essential organelle for parasite survival.

Trypanosoma cruzi infection changes the chromatin proteome profile of infected human cells

Chagas disease is endemic in 22 Latin American countries, with approximately 8 million individuals infected worldwide and 10,000 deaths yearly. Trypanosoma cruzi presents an intracellular life cycle in mammalian hosts to sustain infection. Parasite infection activates host cell responses, promoting an unbalance in reactive oxygen species (ROS) in the intracellular environment inducing genomic DNA lesions in the host cell during infection. To further understand changes in host cell chromatin induced by parasite infection, we investigated alterations in chromatin caused by infection by performing quantitative proteomic analysis. DNA Damage Repair proteins, such as Poly-ADP-ribose Polymerase 1 (PARP-1) and X-Ray Repair Cross Complementing 6 (XRRC6), were recruited to the chromatin during infection. Also, changes in chromatin remodeling enzymes suggest that parasite infection may shape the epigenome of the host cells. Interestingly, the abundance of oxidative phosphorylation mitochondrial and vesicle-mediated transport proteins increased in the host chromatin at the final stages of infection. In addition, Apoptosis-inducing Factor (AIF) is translocated to the host cell nucleus upon infection, suggesting that cells enter parthanatos type of death. Altogether, this study reveals how parasites interfere with the host cells' responses at the chromatin level leading to significant crosstalk that support and disseminate infection.

Previously Unidentified Histone H1-Like Protein Is Involved in Cell Division and Ribosome Biosynthesis in Toxoplasma gondii.

Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTMs). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes four canonical histones and five histone variants. In contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii ("TgH1-like") that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of the tgh1-like gene does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutation of posttranslationally modified amino acids results in defects in cell division like those in the Δtgh1-like mutant, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii. IMPORTANCE Histone H1, or linker histone, is an important protein that binds to the nucleosome, aiding chromatin compaction. Here, we characterize for the first time a linker histone in T. gondii, named TgH1-like. It is a small and basic protein that corresponds only to the C-terminal portion of the human H1 but is similar to histone H1 from trypanosomatids and bacteria. TgH1-like is located in the nucleus, interacts with nucleosome histones, and acts in chromatin structure and cell division. Our findings show for the first time the presence of a histone H1 protein in an apicomplexan parasite and will provide new insights into cell division and chromatin dynamics in T. gondii and related parasites.

Estratificação de risco para diabetes tipo 2 com base no Findrisc e fatores associados / Risk stratification for type 2 diabetes according to the Findrisc and associated factors / Estratificación del riesgo para diabetes tipo 2 según el Findrisc y factores asociados

Os objetivos deste estudo são estratificar os usuários de um centro de atenção primária segundo o risco de desenvolver diabetes mellitus tipo 2 (DM2), utilizando o Escore Finlandês de Risco de Diabetes (Findrisc), e avaliar fatores associados ao risco elevado de desenvolver DM2. Trata-se de um estudo transversal, com amostra aleatória de duzentos adultos, não diabéticos, de um centro de saúde escola. Utilizou-se regressão logística para investigar fatores associados ao escore elevado (≥ 15 pontos) no Findrisc. Observou-se que 33,5% apresentavam risco discretamente aumentado, 17% risco moderado e 34,5% risco alto/muito alto para desenvolver DM2. Aqueles com menor escolaridade (OR: 3,21; IC: 1,52-6,77) e com histórico de hipercolesterolemia (OR: 2,47; IC: 1,27-4,81) exibiram maior chance de apresentar escore elevado. Em conclusão, a frequência de indivíduos com risco alto/muito alto de desenvolver DM2 foi elevada na população estudada, e o menor nível de escolaridade e o histórico de hipercolesterolemia estavam associados ao escore elevado no Findrisc.

The aim of this study was to stratify users of a primary care center according to the risk of developing type II diabetes mellitus (T2DM) using the Finnish Diabetes Risk Score (FINDRISC) questionnaire and to assess factors associated with elevated risk for T2DM. We conducted a cross-sectional study, with a random sample of 200 non-diabetic adults attending a school primary care center. Logistic regression was used to investigate factors associated with elevated FINDRISC scores (≥ 15 points). We observed that 33.5% of subjects had a slightly elevated risk, 17.0% moderate risk, and 34.5% high/very high risk of developing T2DM. Those with a low level of education (OR: 3.21; 95%CI: 1.52-6.77) and with a history of hypercholesterolemia (OR: 2.47; 95%CI: 1.27-4.81) were more likely to have an elevated score. In conclusion, the frequency of individuals at high/very high risk of developing T2DM was high in the population studied, and the lower level of education and history of hypercholesterolemia were associated with elevated FINDRISC score.

El objetivo de este estudio fue estratificar a los usuarios de un centro de atención primaria según el riesgo de desarrollar diabetes mellitus tipo 2 (DM2) mediante el cuestionario Finnish Diabetes Risk Score (FINDRISC), así como evaluar los factores asociados con un mayor riesgo de desarrollar DM2. Se trata de un estudio transversal con una muestra aleatoria de 200 adultos no diabéticos de un centro clínico de salud. Se utilizó regresión logística para investigar los elevados factores asociados con FINDRISC (≥ 15 puntos). Se observó que el 33,5% de los sujetos presentaron un riesgo ligeramente aumentado, el 17,0% riesgo moderado y el 34,5% riesgo alto/muy alto de desarrollar DM2. Aquellos con menos nivel educativo (OR: 3,21; IC: 1,52-6,77) y con antecedentes de hipercolesterolemia (OR: 2,47; IC: 1,27-4,81) tenían mayor probabilidad de tener un puntaje elevado. Se concluye que la frecuencia de individuos con riesgo alto/muy alto de desarrollar DM2 fue alta en la población estudiada, y que el menor nivel educativo y antecedentes de hipercolesterolemia se asociaron con un puntaje elevado en el FINDRISC.

Open chromatin analysis in Trypanosoma cruzi life forms highlights critical differences in genomic compartments and developmental regulation at tDNA loci.

BACKGROUND: Genomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. RESULTS: Here, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using FAIRE-seq approach supported by transmission electron microscopy. We identified differences in T. cruzi genome compartments, putative transcriptional start regions, and virulence factors. In addition, we also detected a developmental chromatin regulation at tRNA loci (tDNA), which could be linked to the intense chromatin remodeling and/or the translation regulatory mechanism required for parasite differentiation. We further integrated the open chromatin profile with public transcriptomic and MNase-seq datasets. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. CONCLUSION: Taken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

H2B.V demarcates divergent strand-switch regions, some tDNA loci, and genome compartments in Trypanosoma cruzi and affects parasite differentiation and host cell invasion.

Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in tissue-derived trypomastigote (TCT) life forms, a nonreplicative stage of Trypanosoma cruzi, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using epimastigotes and TCT life forms, and we found that H2B.V was preferentially located at the edges of divergent transcriptional strand switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of TCT forms was depleted of H2B.V-enriched peaks in comparison to epimastigote forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle.

Previously unidentified histone H1-like protein is involved in cell division and ribosome biosynthesis in toxoplasma gondii

Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTMs). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes four canonical histones and five histone variants. In contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii (“TgH1-like”) that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of the tgh1-like gene does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutation of posttranslationally modified amino acids results in defects in cell division like those in the Δtgh1-like mutant, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii. Histone H1, or linker histone, is an important protein that binds to the nucleosome, aiding chromatin compaction. Here, we characterize for the first time a linker histone in T. gondii, named TgH1-like. It is a small and basic protein that corresponds only to the C-terminal portion of the human H1 but is similar to histone H1 from trypanosomatids and bacteria. TgH1-like is located in the nucleus, interacts with nucleosome histones, and acts in chromatin structure and cell division. Our findings show for the first time the presence of a histone H1 protein in an apicomplexan parasite and will provide new insights into cell division and chromatin dynamics in T. gondii and related parasites.

Open chromatin analysis in Trypanosoma cruzi life forms highlights critical differences in genomic compartments and developmental regulation at tDNA loci

Background Genomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. Results Here, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using FAIRE-seq approach supported by transmission electron microscopy. We identified differences in T. cruzi genome compartments, putative transcriptional start regions, and virulence factors. In addition, we also detected a developmental chromatin regulation at tRNA loci (tDNA), which could be linked to the intense chromatin remodeling and/or the translation regulatory mechanism required for parasite differentiation. We further integrated the open chromatin profile with public transcriptomic and MNase-seq datasets. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. Conclusion Taken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

H2B.V demarcates divergent strand-switch regions, some tDNA loci, and genome compartments in Trypanosoma cruzi and affects parasite differentiation and host cell invasion

Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in tissue-derived trypomastigote (TCT) life forms, a nonreplicative stage of Trypanosoma cruzi, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using epimastigotes and TCT life forms, and we found that H2B.V was preferentially located at the edges of divergent transcriptional strand switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of TCT forms was depleted of H2B.V-enriched peaks in comparison to epimastigote forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle.