Mechanisms of histone H3 lysine 27 trimethylation remodeling during early mammalian development

During fertilization, two of the most differentiated cells in the mammalian organism, a sperm and oocyte, are combined to form a pluripotent embryo. Dynamic changes in chromatin structure allow the transition of the chromatin on these specialized cells into an embryonic configuration capable of generating every cell type. Initially, this reprogramming activity is supported by oocyte-derived factors accumulated during oogenesis as proteins and mRNAs; however, the underlying molecular mechanisms that govern it remain poorly characterized. Trimethylation of histone H3 at lysine 27 (H3K27me3) is a repressive epigenetic mark that changes dynamically during pre-implantation development in mice, bovine and pig embryos. Here we present data and hypotheses related to the potential mechanisms behind H3K27me3 remodeling during early development. We postulate that the repressive H3K27me3 mark is globally erased from the parental genomes in order to remove the gametic epigenetic program and to establish a pluripotent embryonic epigenome. We discuss information gathered in mice, pigs, and bovine, with the intent of providing a comparative analysis of the reprogramming of this epigenetic mark during early mammalian development.     

Mechanisms of histone H3 lysine 27 trimethylation remodeling during early mammalian development

During fertilization, two of the most differentiated cells in the mammalian organism, a sperm and oocyte, are combined to form a pluripotent embryo. Dynamic changes in chromatin structure allow the transition of the chromatin on these specialized cells into an embryonic configuration capable of generating every cell type. Initially, this reprogramming activity is supported by oocyte-derived factors accumulated during oogenesis as proteins and mRNAs; however, the underlying molecular mechanisms that govern it remain poorly characterized. Trimethylation of histone H3 at lysine 27 (H3K27me3) is a repressive epigenetic mark that changes dynamically during pre-implantation development in mice, bovine and pig embryos. Here we present data and hypotheses related to the potential mechanisms behind H3K27me3 remodeling during early development. We postulate that the repressive H3K27me3 mark is globally erased from the parental genomes in order to remove the gametic epigenetic program and to establish a pluripotent embryonic epigenome. We discuss information gathered in mice, pigs, and bovine, with the intent of providing a comparative analysis of the reprogramming of this epigenetic mark during early mammalian development.     

Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis

Trimethylation of histone H3 lysine 27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. We identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways, such as DNA methylation or RNA interference. Unlike in animals, where H3K27m3 occupies large genomic regions, in Arabidopsis, we found that H3K27m3 domains were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low–nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.     

Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27

The plant Polycomb-group (Pc-G) protein CURLY LEAF (CLF) is required to repress targets such as AGAMOUS (AG) and SHOOTMERISTEMLESS (STM). Using chromatin immunoprecipitation, we identify AG and STM as direct targets for CLF and show that they carry a characteristic epigenetic signature of dispersed histone H3 lysine 27 trimethylation (H3K27me3) and localised H3K27me2 methylation. H3K27 methylation is present throughout leaf development and consistent with this, CLF is required persistently to silence AG. However, CLF is not itself an epigenetic mark as it is lost during mitosis. We suggest a model in which Pc-G proteins are recruited to localised regions of targets and then mediate dispersed H3K27me3. Analysis of transgenes carrying AG regulatory sequences confirms that H3K27me3 can spread to novel sequences in a CLF-dependent manner and further shows that H3K27me3 methylation is not sufficient for silencing of targets. We suggest that the spread of H3K27me3 contributes to the mitotic heritability of Pc-G silencing, and that the loss of silencing caused by transposon insertions at plant Pc-G targets reflects impaired spreading.     

The CHD3 remodeler PICKLE promotes trimethylation of histone H3 lysine 27

CHD3 proteins are ATP-dependent chromatin remodelers that contribute to repression of developmentally regulated genes in both animal and plant systems. In animals, this repression has been linked to a multiple subunit complex, Mi-2/NuRD, whose constituents include a CHD3 protein, a histone deacetylase, and a methyl-CpG-binding domain protein. In Arabidopsis, PICKLE (PKL) codes for a CHD3 protein that acts during germination to repress expression of seed-associated genes. Repression of seed-associated traits is promoted in pkl seedlings by the plant growth regulator gibberellin (GA). We undertook a microarray analysis to determine how PKL and GA act to promote the transition from seed to seedling. We found that PKL and GA act in separate pathways to repress expression of seed-specific genes. Comparison of genomic datasets revealed that PKL-dependent genes are enriched for trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation studies demonstrate that PKL promotes H3K27me3 in both germinating seedlings and in adult plants but do not identify a connection between PKL-dependent expression and acetylation levels. Taken together, our analyses illuminate a new pathway by which CHD3 remodelers contribute to repression in eukaryotes.     

The CHD3 remodeler PICKLE associates with genes enriched for trimethylation of histone H3 lysine 27

In Arabidopsis (Arabidopsis thaliana), the ATP-dependent chromatin remodeler PICKLE (PKL) determines expression of genes associated with developmental identity. PKL promotes the epigenetic mark trimethylation of histone H3 lysine 27 (H3K27me3) that facilitates repression of tissue-specific genes in plants. It has previously been proposed that PKL acts indirectly to promote H3K27me3 by promoting expression of the POLYCOMB REPRESSIVE COMPLEX2 complex that generates H3K27me3. We undertook expression and chromatin immunoprecipitation analyses to further characterize the contribution of PKL to gene expression and developmental identity. Our expression data support a critical and specific role for PKL in expression of H3K27me3-enriched loci but do not support a role for PKL in expression of POLYCOMB REPRESSIVE COMPLEX2. Moreover, our chromatin immunoprecipitation data reveal that PKL protein is present at the promoter region of multiple H3K27me3-enriched loci, indicating that PKL directly acts on these loci. In particular, we find that PKL is present at LEAFY COTYLEDON1 and LEAFY COTYLEDON2 during germination, which is when PKL acts to repress these master regulators of embryonic identity. Surprisingly, we also find that PKL is present at the promoters of actively transcribed genes that are ubiquitously expressed such as ACTIN7 and POLYUBIQUITIN10 that do not exhibit PKL-dependent expression. Taken together, our data contravene the previous model of PKL action and instead support a direct role for PKL in determining levels of H3K27me3 at repressed loci. Our data also raise the possibility that PKL facilitates a common chromatin remodeling process that is not restricted to H3K27me3-enriched regions.     

Vernalization-induced trimethylation of histone H3 lysine 27 at FLC is not maintained in mitotically quiescent cells

Vernalization promotes flowering in Arabidopsis through epigenetic repression of the floral repressor, FLOWERING LOCUS C (FLC). Vernalization, like other polycomb-mediated repression events, occurs in two stages; FLC repression is established at low temperatures, then maintained during subsequent growth at 22 degrees C. Low temperatures induce VIN3 activity, which is required for changes in histone modifications and the associated FLC repression. Plant polycomb proteins FIE, VRN2, CLF, and SWN, together with VIN3, form a complex that adds histone H3 lysine 27 methylation at FLC in vernalized plants. VRN1 and LHP1 are required for maintenance of FLC repression. Tissue must be undergoing cell division during low-temperature treatments for acceleration of flowering to occur. We show that low-temperature treatments repress FLC in cells that are not mitotically active, but this repression is not fully maintained. Trimethyl-lysine 27 (K27me3), is enriched at the start of the FLC gene during the cold, before spreading across the locus after vernalization. In the absence of DNA replication, K27me3 is added to chromatin at the start of FLC but is removed on return to 22 degrees C. This suggests that DNA replication is essential for maintenance of vernalization-induced repression of FLC.     

The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation

Polycomb proteins are required for maintenance of silent chromatin states via histone H3 Lys27 trimethylation (H3K27me3) in animals, but homologs are not found in plant genomes. Using a DamID-chip method, we found that the Arabidopsis thaliana chromodomain-containing protein LHP1 colocalizes with H3K27me3 genome-wide. The LHP1 chromodomain also binds H3K27me3 with high affinity, suggesting that LHP1 has functions similar to those of Polycomb.     

Dynamic changes of histone H3 lysine 9 following trimethylation in bovine oocytes and pre-implantation embryos

Objectives

We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos.

Results

There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes (P < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels.

Conclusion

Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

     

Long noncoding RNA ATB promotes ovarian cancer tumorigenesis by mediating histone H3 lysine 27 trimethylation through binding to EZH2

Ovarian cancer (OC) remains one of the most lethal gynecological malignancies. The unfavourable prognosis is mainly due to the lack of early-stage diagnosis, drug resistance and recurrence. Therefore, it needs to investigate the mechanism of OC tumorigenesis and identify effective biomarkers for the clinical diagnosis. It is reported that long noncoding RNAs (lncRNAs) play important roles during the tumorigenesis of OC. Therefore, the present study aimed to study the role and clinical significance of LncRNAs ATB (lnc-ATB) in the development and progression of OC. In our research, lnc-ATB expression in OC tissues was elevated compared with adjacent normal tissues and high expression of lnc-ATB was associated with poor outcomes of OC patients. The silencing of lnc-ATB blocked cell proliferation, invasion and migration in SKOV3 and A2780 cells. RNA immunoprecipitation and RNA pull-down results showed that lnc-ATB positively regulated the expression of EZH2 via directly interacting with EZH2. Besides, the overexpression of EZH2 partly rescued lnc-ATB silencing-inducing inhibition of cell proliferation, invasion and migration. Chromatin immunoprecipitation assay results demonstrated that the silencing of lnc-ATB reduced the occupancy of caudal-related homeobox protein 1, Forkhead box C1, Large tumour suppressor kinase 2, cadherin-1 and disabled homolog 2 interacting protein promoters on EZH2 and H3K27me3. These data revealed the oncogenic of lnc-ATB and provided a novel biomarker for OC diagnosis. Furthermore, these findings indicated the mechanism of lnc-ATB functioning in the progression of OC, which provided a new target for OC therapy.     

Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo

The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.     

Dynamics of H3K27me3 methylation and demethylation in plant development

Epigenetic regulation controls multiple aspects of the plant development. The N-terminal tail of histone can be differently modified to regulate various chromatin activities. One of them, the trimethylation of histone H3 lysine 27 (H3K27me3) confers a repressive chromatin state with gene silencing. H3K27me3 is dynamically deposited and removed throughout development. While components of the H3K27me3 writer, Polycomb repressive complex 2 (PRC2), have been reported for almost 2 decades, it is only recently that JUMONJI (JMJ) proteins are reported as H3K27me3 demethylases, affirming the dynamic nature of histone modifications. This review highlights recent progress in plant epigenetic research, focusing on the H3K27me3 demethylases.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 marks. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.