Metilação de histonas em oócitos e embriões mamíferos / Methylation of histones in mammalian oocytes and embryos

A epigenética consiste no estudo de mudanças na função gênica que não dependem de mudanças na estrutura primária do DNA e que podem ser hereditárias. As mudanças epigenéticas desempenham um importante papel no processo de diferenciação celular, permitindo que as células mantenham características estáveis diferentes, apesar de conterem o mesmo material genético. As histonas possuem uma importante função na estrutura do DNA, e alterações bioquímicas em sua cauda aminoterminal, alteram a estrutura da cromatina e suas funções. Dessa forma são fundamentais para o controle da expressão gênica, ativação do genoma embrionário, metilação do DNA e inativação do cromossomo X. O desenvolvimento oocitário e embrionário envolvem uma série de modificações epigenéticas importantes, incluindo as modificações pós-traducionais das histonas, que são imprescindíveis para o desenvolvimento normal do embrião. O melhor entendimento a respeito do epigenoma e sua influência sobre a oogênese e embriogênese podem permitir a otimização das biotecnologias da reprodução.(AU)

Epigenetic is the study of changes in genetic function which do not depend on change in the primary structure of DNA, and can be heritable. Epigenetic changes play an important role in cellular differentiation process, allowing the cells to keep different stable characteristics, though they contain the same genomic material. Histones are not only structural proteins, but are also critical to the control of gene expression, embryonic genome activation, DNA methylation and inactivation of the X chromosome. The histones have an important function in the structure of the DNA, consequently histone methylation can alter its structure and its functions. The oocyte and embryo development involve several important epigenetic modifications, including post-translation modifications of histones, which are essential for normal development of the embryo. The best understanding of the epigenome and its influence on oogenesis and embryogenesis may allow the optimization of reproduction biotechnologies.(AU)

Metilação de histonas em oócitos e embriões mamíferos / Methylation of histones in mammalian oocytes and embryos

A epigenética consiste no estudo de mudanças na função gênica que não dependem de mudanças na estrutura primária do DNA e que podem ser hereditárias. As mudanças epigenéticas desempenham um importante papel no processo de diferenciação celular, permitindo que as células mantenham características estáveis diferentes, apesar de conterem o mesmo material genético. As histonas possuem uma importante função na estrutura do DNA, e alterações bioquímicas em sua cauda aminoterminal, alteram a estrutura da cromatina e suas funções. Dessa forma são fundamentais para o controle da expressão gênica, ativação do genoma embrionário, metilação do DNA e inativação do cromossomo X. O desenvolvimento oocitário e embrionário envolvem uma série de modificações epigenéticas importantes, incluindo as modificações pós-traducionais das histonas, que são imprescindíveis para o desenvolvimento normal do embrião. O melhor entendimento a respeito do epigenoma e sua influência sobre a oogênese e embriogênese podem permitir a otimização das biotecnologias da reprodução.

Epigenetic is the study of changes in genetic function which do not depend on change in the primary structure of DNA, and can be heritable. Epigenetic changes play an important role in cellular differentiation process, allowing the cells to keep different stable characteristics, though they contain the same genomic material. Histones are not only structural proteins, but are also critical to the control of gene expression, embryonic genome activation, DNA methylation and inactivation of the X chromosome. The histones have an important function in the structure of the DNA, consequently histone methylation can alter its structure and its functions. The oocyte and embryo development involve several important epigenetic modifications, including post-translation modifications of histones, which are essential for normal development of the embryo. The best understanding of the epigenome and its influence on oogenesis and embryogenesis may allow the optimization of reproduction biotechnologies.

Molecular mechanisms of transcriptional and chromatin remodeling around embryonic genome activation

The early stages of embryonic development in all metazoans are characterized by profound remodeling of chromatin organization and transcriptomics. This remodeling involves modifying two differentiated cells (oocyte and sperm) into a totipotent embryonic configuration capable of forming all the specialized cells that make up the adult animal. The early cleavage stages of preimplantation animal embryos occur in the absence of active transcription and relies on maternal factors stored in the cytoplasm of the oocyte during oogenesis. Chromatin and transcriptomic remodeling during preimplantation development are key for the initiation of embryonic transcriptional activity at species-specific stage, yet the exact mechanisms that trigger embryonic genome activation (EGA) are still unknown. Evidence of key transcriptional regulators and permissive chromatin configuration accompanied by specific epigenetic marks has been revealed as some of the mechanisms needed for EGA in the past few years. In this review, we will revisit the latest advances in the understanding of the mechanisms involved in the activation of the embryonic genome across several species, focusing on data generated by next generation sequencing technologies.

Molecular mechanisms of transcriptional and chromatin remodeling around embryonic genome activation

The early stages of embryonic development in all metazoans are characterized by profound remodeling of chromatin organization and transcriptomics. This remodeling involves modifying two differentiated cells (oocyte and sperm) into a totipotent embryonic configuration capable of forming all the specialized cells that make up the adult animal. The early cleavage stages of preimplantation animal embryos occur in the absence of active transcription and relies on maternal factors stored in the cytoplasm of the oocyte during oogenesis. Chromatin and transcriptomic remodeling during preimplantation development are key for the initiation of embryonic transcriptional activity at species-specific stage, yet the exact mechanisms that trigger embryonic genome activation (EGA) are still unknown. Evidence of key transcriptional regulators and permissive chromatin configuration accompanied by specific epigenetic marks has been revealed as some of the mechanisms needed for EGA in the past few years. In this review, we will revisit the latest advances in the understanding of the mechanisms involved in the activation of the embryonic genome across several species, focusing on data generated by next generation sequencing technologies.(AU)

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos / Suplementação com soro fetal bovino altera a modificação de histona H3R26me2 durante o período pré-implantacional em embriões bovinos produzidos in vitro

In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.(AU)

A produção in vitro (PIV) de embriões de bovinos não é apenas de grande importância econômica para a pecuária, mas é também um importante modelo para estudar o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a modificação de histona, H3R26me2 durante o desenvolvimento pré-implantacional em embriões bovinos produzidos in vitro, cultivados com ou sem suplementação de soro fetal bovino (SFB), bem como comparar essa modificação específica entre mórulas produzidas in vitro e in vivo. Após a maturação in vitro e fertilização, embriões foram cultivados com suplementação de 0 ou 2,5% SFB. O desenvolvimento embrionário foi avaliado e embriões foram coletados e fixados em diferentes fases durante o desenvolvimento (2, 4, 8 e 16 células, mórula e blastocisto). Os embriões fixados foram avaliados por imunofluorescência utilizando um anticorpo para H3R26me2. Imagens de embriões corados foram analisadas baseadas na porcentagem do DNA total. Embriões cultivados com 2,5% SFB tiveram uma taxa de desenvolvimento ao estágio de blastocisto maior que o grupo que não recebeu suplementação com SFB (34.85±5,43% vs 23.38±,93%; P<0,05). Níveis de H3R26me2 variaram para ambos os grupos ao longo do desenvolvimento. No grupo 0% SFB, a marcação para H3R26me2 foi mais intensa nos estágios de 4 células (P<0,05), 16 células (P<0,05) e mórula (P<0.05). No grupo 2.5% SFB, apenas os embriões de 4 células tiveram marcação significativamente maior que todas as outras fases (P<0,01). Mórulas produzidas in vivo apresentaram níveis de H3R26me2 semelhantes ao grupo 0% SFB, e ambos foram significativamente maiores que o grupo 2.5% SFB. Estes resultados sugerem que a modificação de histona H3R26me2 é regulada durante o desenvolvimento pré-implantacional de embriões bovinos, e que as condições de cultura alteram de maneira importante esta regulação.(AU)

Efeitos da tricostatina A sobre a acetilação de histonas, proliferação celular e diferenciação de células tronco embrionárias murinas / Effects of Trichostatin A on Histone Acetylation, Cell Proliferation and Differentiation of Murine Embryonic Stem Cells

Background: Embryonic stem cells are cells derived from early-stage embryos that are characterized by pluripotency and self- renewal capacity. The in vitro cultured murine embryonic stem cells can indefinitely propagate in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). However, when stimulated, these cells can differentiate into cell lines derived from all three embryonic germ layers. The trichostatin A (TSA) is an epigenetic modifier agent and several studies have used the TSA to stimulate cellular differentiation. However, most of these studies only assessed one TSA concentration. Therefore, this study aimed to evaluate the effects of different TSA concentrations on histone hyperacetylation during in vitro cell differentiation of murine pluripotent embryonic stem cells, cultured with or without LIF, in the quest of to standardize their application on early cultures of embryonic stem cells. Materials, Methods & Results: Undifferentiated murine embryonic stem cells were plated in the presence of different TSA concentrations (0 nM, 15 nm, 50 nM and 100 nM) in the presence or absence of LIF. Thus, the treatments were evaluated in undifferentiated embryonic stem cells cultured in the presence of LIF (Control group: 0 nM LIF + ; Group 15 nM LIF + ; Group 50 nM LIF + and Group 100 nM LIF + ), and in embryonic stem cells cultured in the absence of LIF (Control group: 0 nM LIF - ; Group 15 nM LIF - ; Group 50 nM LIF - and Group 100 nM LIF - ). Treatment with TSA was performed for 24 h. After that the medium was replaced with fresh medium without TSA. Samples were collected at 0, 12, 24, 36 and 48 h after the beginning of the experiment. Three replicates were performed in each experimental group. The relative amount of Histone H3 lysine 9 acetylation was analyzed in all groups, as well as the cell proliferation in the embryonic stem cells cultured in the presence of LIF.

Efeitos da tricostatina A sobre a acetilação de histonas, proliferação celular e diferenciação de células tronco embrionárias murinas / Effects of Trichostatin A on Histone Acetylation, Cell Proliferation and Differentiation of Murine Embryonic Stem Cells

Background: Embryonic stem cells are cells derived from early-stage embryos that are characterized by pluripotency and self- renewal capacity. The in vitro cultured murine embryonic stem cells can indefinitely propagate in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). However, when stimulated, these cells can differentiate into cell lines derived from all three embryonic germ layers. The trichostatin A (TSA) is an epigenetic modifier agent and several studies have used the TSA to stimulate cellular differentiation. However, most of these studies only assessed one TSA concentration. Therefore, this study aimed to evaluate the effects of different TSA concentrations on histone hyperacetylation during in vitro cell differentiation of murine pluripotent embryonic stem cells, cultured with or without LIF, in the quest of to standardize their application on early cultures of embryonic stem cells. Materials, Methods & Results: Undifferentiated murine embryonic stem cells were plated in the presence of different TSA concentrations (0 nM, 15 nm, 50 nM and 100 nM) in the presence or absence of LIF. Thus, the treatments were evaluated in undifferentiated embryonic stem cells cultured in the presence of LIF (Control group: 0 nM LIF + ; Group 15 nM LIF + ; Group 50 nM LIF + and Group 100 nM LIF + ), and in embryonic stem cells cultured in the absence of LIF (Control group: 0 nM LIF - ; Group 15 nM LIF - ; Group 50 nM LIF - and Group 100 nM LIF - ). Treatment with TSA was performed for 24 h. After that the medium was replaced with fresh medium without TSA. Samples were collected at 0, 12, 24, 36 and 48 h after the beginning of the experiment. Three replicates were performed in each experimental group. The relative amount of Histone H3 lysine 9 acetylation was analyzed in all groups, as well as the cell proliferation in the embryonic stem cells cultured in the presence of LIF.(AU)

Epigenetic regulation of gene transcription via histone modifications

Our laboratory is interested in post-translational modifications of histone proteins, with studies ranging from identification of novel modifications to functional characterization of these marks. Ultimately, we seek to provide a greater understanding of how histone modifications work together to form a ‘histone code’. This code is thought to regulate the recruitment of effector proteins that regulate the diverse functions associated with DNA, including gene transcription and DNA repair. Our recent studies show that RNA polymerase II recruits a variety of chromatin modifying enzymes that contribute to the disruption, reassembly and maintenance of chromatin structure during the transcription elongation process. One enzyme we have focused on is Set2, which associates with the transcribing polymerase and methylates nucleosomal H3 on lysine 36. H3K36 methylation results in the recruitment of a histone deacetylase complex which functions to prevent inappropriate transcription initiation from occurring within the transcribed regions of genes. I will discuss our recent progress toward understanding how Set2 contributes to the organization and function of chromatin. In addition, I will highlight our progress on a proteomics project that is providing new insights into how “readers” of the histone code bind their cognate modifications using high-density histone peptide arrays.

Epigenetic regulation of gene transcription via histone modifications

Our laboratory is interested in post-translational modifications of histone proteins, with studies ranging from identification of novel modifications to functional characterization of these marks. Ultimately, we seek to provide a greater understanding of how histone modifications work together to form a ‘histone code. This code is thought to regulate the recruitment of effector proteins that regulate the diverse functions associated with DNA, including gene transcription and DNA repair. Our recent studies show that RNA polymerase II recruits a variety of chromatin modifying enzymes that contribute to the disruption, reassembly and maintenance of chromatin structure during the transcription elongation process. One enzyme we have focused on is Set2, which associates with the transcribing polymerase and methylates nucleosomal H3 on lysine 36. H3K36 methylation results in the recruitment of a histone deacetylase complex which functions to prevent inappropriate transcription initiation from occurring within the transcribed regions of genes. I will discuss our recent progress toward understanding how Set2 contributes to the organization and function of chromatin. In addition, I will highlight our progress on a proteomics project that is providing new insights into how “readers” of the histone code bind their cognate modifications using high-density histone peptide arrays.(AU)

Epigenetic control of folliculogenesis and luteinization

Folliculogenesis and luteinization are characterized by irreversible and profound physiological and morphological transformation processes, which eventually culminate in the provision of fertilizable eggs and the conversion of the estrogen producing follicle into a progesterone producing corpus luteum. All these processes are preceded by complex alterations of the gene expression profiles in the somatic cell layers granulosa and theca. It has been well established that epigenetic mechanisms, such as DNA methylation, histone modification and local changes of the chromatin structure, are essentially involved in cell type-specific gene activation and silencing. This short review will mainly focus on epigenetic processes that are induced by the gonadotropins FSH and LH during late folliculogenesis and luteinization. Data will be presented demonstrating that histone modification and chromatin modulation, but also DNA methylation are involved in the changing gene expression profiles during folliculogenesis and luteinization. Hence, these epigenetic mechanisms have to be considered to understand the control of the female reproductive cycle and pregnancy as well as pathological aberrations.

Epigenetic control of folliculogenesis and luteinization

Folliculogenesis and luteinization are characterized by irreversible and profound physiological and morphological transformation processes, which eventually culminate in the provision of fertilizable eggs and the conversion of the estrogen producing follicle into a progesterone producing corpus luteum. All these processes are preceded by complex alterations of the gene expression profiles in the somatic cell layers granulosa and theca. It has been well established that epigenetic mechanisms, such as DNA methylation, histone modification and local changes of the chromatin structure, are essentially involved in cell type-specific gene activation and silencing. This short review will mainly focus on epigenetic processes that are induced by the gonadotropins FSH and LH during late folliculogenesis and luteinization. Data will be presented demonstrating that histone modification and chromatin modulation, but also DNA methylation are involved in the changing gene expression profiles during folliculogenesis and luteinization. Hence, these epigenetic mechanisms have to be considered to understand the control of the female reproductive cycle and pregnancy as well as pathological aberrations.(AU)