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Epigenetic Modifications of Histones
Published in Cristina Camprubí, Joan Blanco, Epigenetics and Assisted Reproduction, 2018
George Rasti, Alejandro Vaquero
Immediately after fertilization and zygote formation takes place, a rapid demethylation of DNA and histones, which is a critical process for dedifferentiated embryo formation. Both are established during gametogenesis, where they regulate gene promoters involved in gene imprinting in the embryo. However, unlike DNA methylation, histone PTMs are stable during the proliferative and meiotic phases of spermatogenesis. Interestingly, the histone marks H3K9me3 and H3K20me2 are predominately associated with methylated DNA in both embryo and placenta. These parent-of-origin-specific epigenetic changes cause differential expression of specific genes in the embryo, mainly encoding transcription and growth factors. Gene imprinting in sperm depends on the PTMs of the remaining histones, like H3K9me3 and H4K20me3 (38). The female pronucleus carries a number of active marks like H3K4me2/3, H4K8/12 ac, all of which are not altered globally during fertilization. In contrast, sperm chromatin undergoes major changes such as replacement of protamines with H3K4me1, H3K9me1, and H3K27me1 and subsequent appearance of H3K4me3, H3K9me2, H3K27me3, and acetylated form of H4K5/8/12/16 (39) (Figure 2.1 and Table 2.1). It was shown that Suv4-20h1/h2, the H4K20me2,3 HMTs, are mainly absent in preimplantation mouse embryos, leading to a rapid decrease of H4K20me3 from the 2-cell stage onward until the peri-implantation period (40). Other histone marks are also important in regulation of gene expression during early stages of development. After fertilization, H3K27ac progressively decreases from the early pronuclear stage to 8-cell stage, corresponding to major embryonic genome activation, followed by H3K27 re-acetylation from the morula stage onward (41). In contrast, the non-canonical H3K4me3 is subjected to massive reprogramming. This involves a massive loss from the early to the late 2-cell stage embryo, followed by a re-establishment of H3K4me3 on promoter regions, which is maintained till blastocyst stage (Figure 2.2). Meanwhile, H3K27me3 levels decrease massively from the late 2-cell stage embryo to blastocyst. The distinct features of H3K4me3 and H3K27me3 are essential for zygotic genome activation and the further development (42). H3K9me2,me3 levels increase from the 4-cell stage and peak at morula stage, but their levels decrease again during the blastocyst stage (Figure 2.2) (43,44). HDAC1 regulates gene expression in mouse preimplantation embryos by deacetylating histone H4, and especially H4K5Ac. Sirtuins are also important in gametogenesis and early embryo development in the context of the response to metabolic and stress conditions. Thus, during post-fertilization events, maternally derived SIRT3 is essential for protecting early-stage embryos from stress conditions by inhibiting of the ROS-p53 pathway. Moreover, SIRT3 plays a protective role in mouse preimplantation embryos under in vitro culture conditions. Although Sirt3-/- mice are fertile, embryos derived from in vitro-cultured Sirt3-/- are susceptible to developmental defects (45).
Effect of titanium dioxide nanoparticles on histone modifications and histone modifying enzymes expression in human cell lines
Published in Nanotoxicology, 2022
Marta Pogribna, Beverly Word, Beverly Lyn-Cook, George Hammons
The effects of TiO2 nanoparticles exposure on the level of global histone modifications in each of the cell lines were assessed after 72 h of treatment by using histone H3 or histone H4 multiplex assay kits (Epigentek), which detect and quantify specific modified histone H3 patterns (15 different lysine methylations, 4 lysine acetylations, 2 serine phosphorylations and 1 citrullination) or modified H4 patterns (5 methylations, 4 acetylations, and 1 serine phosphorylation) (Figures 1 and 2). Among the histone modifications in the array analysis, when compared to control cells increased levels were observed in Caco-2 cells for H3cit, H3K4me2, H3K9me3, H3K27me2, H3K27me3, H3K36me3, H3K18ac, H3K56ac, H4K8ac, and H4K20m1; H3K9ac was slightly decreased. In NL20 cells, levels of H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K36me1, H3K36me3, H3K14ac, H3K18ac, and H4K5ac were higher compared to controls; levels of H3K9ac and H4ser1P were lower. Subsequent Western blot analysis confirmed the global upregulation of several of the histone modifications identified in the array analysis. In Caco-2 cells levels of H3cit, H3K9me3, H3K27me3, H3K36me3, and H4K8ac (Figure 3) and in NL20 cells H3K4me3, H3K27me3, and H3K18ac in NL20 cells were significantly increased (Figure 4). Although H3K9ac levels were observed to be decreased in both cell lines by array analysis, a significant increase in the level of this histone modification was shown using Western blotting.
Application of NanoString technologies in companion diagnostic development
Published in Expert Review of Molecular Diagnostics, 2019
Jennifer Mary Eastel, Ka Wai Lam, Nga Lam Lee, Wing Yan Lok, Andy Hin Fung Tsang, Xiao Meng Pei, Amanda Kit Ching Chan, William Chi Shing Cho, Sze Chuen Cesar Wong
ChIP-string was reported to be applied in systematic analysis to reveal any epigenomic changes in melanoma progression. A strategy using a ChIP-string assay with 96 designed test probes was used to evaluate the key epigenetic features observed in different cell lines. Six selected histone regions were selected for investigation. They were H2BK5ac, H4K5ac, H3K27ac, H3K4me1, H3K4me3, and H3K27me3, which represented promoters, enhancers or polycomb-repressed regions. Results revealed that the loss of histone acetylations and H3K4me3 on regulatory regions were associated with melanoma development. Upon treatment with histone deacetylase inhibitors, restoration of acetylation on deacetylated loci suppressed the aggressive proliferation of tumorigenic cells [29]. There was also a good correlation between the signal generated from ChIP-string and ChIP-sequencing.
Proteomic approaches for cancer epigenetics research
Published in Expert Review of Proteomics, 2019
Dylan M. Marchione, Benjamin A. Garcia, John Wojcik
In fact, H4K5 methylation is no longer believed to be the physiologically important substrate of SMYD3 [74]. In 2014, a follow-up study to that of Van Aller reported that SMYD3 is predominantly cytoplasmic in the cancers in which it is most highly expressed, suggesting that it might have non-histone substrates. To identify these substrates, investigators conducted a broader in vitro SMYD3 methylation screen utilizing a library of nearly 10,000 proteins. This and follow-up experiments revealed that SMYD3 methylates the MAP-kinase MAPK3K2 with a catalytic efficiency nearly two orders of magnitude greater than for H4K5. A series of mechanistic experiments then showed that the ability of SMYD3 to potentiate Ras signaling was largely dependent on its ability to methylate MAP3K2 at K260. These findings fit neatly with the observation that SMYD3 expression is highest in Ras-driven cancers [74].