Protocol for detecting chromatin dynamics and screening chromatin relaxer by FRAP assay.

We describe a fluorescence recovery after photobleaching (FRAP) protocol for assessing the dynamics of heterochromatin/euchromatin and identifying chromatin relaxers for cell fate transition. Here, we developed a system to track heterochromatin foci with HP1α-cherry and performed FRAP assay of H1-GFP to analyze the dynamics of heterochromatin and euchromatin during somatic cell reprogramming. This protocol is used to screen factors that impact chromatin structure, which could also be used to identify chromatin relaxers and repressors in various cell fate transitions. For complete details on the use and execution of this protocol, please refer to Chen et al. (2016) and Chen et al. (2020).

Epigenetic Landscape Changes Due to Acupuncture Treatment: From Clinical to Basic Research.

Acupuncture has been widely used for treating diseases since the ancient days in China, but the mechanism by which acupuncture exerts such powerful roles is unclear. Epigenetics, including DNA methylation, histone modification, and post-transcriptional regulation of miRNAs, is the study of heritable changes in gene expression that do not include DNA sequence alterations. Epigenetics has become a new strategy for the basic and clinical research of acupuncture in the last decade. Some investigators have been trying to illustrate the mechanism of acupuncture from an epigenetics perspective, which has shed new lights on the mechanisms and applications of acupuncture. Moreover, the introduction of epigenetics into the regulatory mechanism in acupuncture treatment has provided more objective and scientific support for acupuncture theories and brought new opportunities for the improvement of acupuncture studies. In this paper, we reviewed the literatures that has demonstrated that acupuncture could directly or indirectly affect epigenetics, in order to highlight the progress of acupuncture studies correlated to epigenetic regulations. We do have to disclose that the current evidence in this review is not enough to cover all the complex interactions between multiple epigenetic modifications and their regulations. However, the up-to-date results can help us to better understand acupuncture's clinical applications and laboratory research.

Epigenetic regulation of agronomical traits in Brassicaceae.

Epigenetic regulation, covalent modification of DNA and changes in histone proteins are closely linked to plant development and stress response through flexibly altering the chromatin structure to regulate gene expression. In this review, we will illustrate the importance of epigenetic influences by discussing three agriculturally important traits of Brassicaceae. (1) Vernalization, an acceleration of flowering by prolonged cold exposure regulated through epigenetic silencing of a central floral repressor, FLOWERING LOCUS C. This is associated with cold-dependent repressive histone mark accumulation, which confers competency of consequence vegetative-to-reproductive phase transition. (2) Hybrid vigor, in which an F1 hybrid shows superior performance to the parental lines. Combination of distinct epigenomes with different DNA methylation states between parental lines is important for increase in growth rate in a hybrid progeny. This is independent of siRNA-directed DNA methylation but dependent on the chromatin remodeler DDM1. (3) Self-incompatibility, a reproductive mating system to prevent self-fertilization. This is controlled by the S-locus consisting of SP11 and SRK which are responsible for self/non-self recognition. Because self-incompatibility in Brassicaceae is sporophytically controlled, there are dominance relationships between S haplotypes in the stigma and pollen. The dominance relationships in the pollen rely on de novo DNA methylation at the promoter region of a recessive allele, which is triggered by siRNA production from a flanking region of a dominant allele.

Human INO80/YY1 chromatin remodeling complex transcriptionally regulates the BRCA2- and CDKN1A-interacting protein (BCCIP) in cells.

The BCCIP (BRCA2- and CDKN1A-interacting protein) is an important cofactor for BRCA2 in tumor suppression. Although the low expression of BCCIP is observed in multiple clinically diagnosed primary tumor tissues such as ovarian cancer, renal cell carcinoma and colorectal carcinoma, the mechanism of how BCCIP is regulated in cells is still unclear. The human INO80/YY1 chromatin remodeling complex composed of 15 subunits catalyzes ATP-dependent sliding of nucleosomes along DNA. Here, we first report that BCCIP is a novel target gene of the INO80/YY1 complex by presenting a series of experimental evidence. Gene expression studies combined with siRNA knockdown data locked candidate genes including BCCIP of the INO80/YY1 complex. Silencing or over-expressing the subunits of the INO80/YY1 complex regulates the expression level of BCCIP both in mRNA and proteins in cells. Also, the functions of INO80/YY1 complex in regulating the transactivation of BCCIP were confirmed by luciferase reporter assays. Chromatin immunoprecipitation (ChIP) experiments clarify the enrichment of INO80 and YY1 at +0.17 kb downstream of the BCCIP transcriptional start site. However, this enrichment is significantly inhibited by either knocking down INO80 or YY1, suggesting the existence of both INO80 and YY1 is required for recruiting the INO80/YY1 complex to BCCIP promoter region. Our findings strongly indicate that BCCIP is a potential target gene of the INO80/YY1 complex.

Differential histone modification and protein expression associated with cell wall removal and regeneration in rice (Oryza sativa).

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

Identification of inhibitors of chromatin modifying enzymes using the yeast phenotypic screens.

A multitude of enzymes that modify histones and remodel nucleosomes are required for the formation, maintenance, and propagation of the transcriptionally repressed chromatin state in eukaryotes. Robust phenotypic screens in yeast S. cerevisiae have proved instrumental in identifying these activities and for providing mechanistic insights into epigenetic regulation. These phenotypic assays, amenable for high throughput small molecule screening, enable identification and characterization of inhibitors of chromatin modifying enzymes largely bypassing traditional biochemical approaches.

The linker-protein network: control of nucleosomal DNA accessibility.

Numerous studies have recently addressed the accessibility of nucleosomal DNA to protein factors. Two popular concepts - the histone code and chromatin remodeling - consider the nucleosome as a passive entity that 'waits' to be marked by histone modifications and is 'mobilized' by ATP-dependent remodelers. Here, we propose a holistic view of the nucleosome as an active, dynamic entity, the accessibility of which is controlled by binding of different linker proteins to the DNA entry/exit site. The linker proteins might directly compete for this binding site; alternatively, protein chaperones and/or chromatin remodelers might exchange one linker protein for another. Finally, according to our proposed model, the exchange factors are themselves controlled by post-translational modifications or binding of protein partners, to respond to the ever-changing intra- and extra-cellular environment.

Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus.

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. Jurkat lymphoma cells were cultured in media containing 0.025, 0.25, or 10 nmol/l biotin. The nuclear translocation of HCS correlated with biotin concentrations in media; the relative enrichment of both HCS and K12Bio H4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/l biotin compared with 0.25 nmol/l biotin. This increase of K12Bio H4 at the SMVT promoter decreased SMVT expression by up to 86%. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12Bio H4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.

Single-molecule and population probing of chromatin structure using DNA methyltransferases.

Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein-DNA interactions. Here, we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary single-molecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here, we describe the application of MAP in living Saccharomyces cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule.

Developmental regulation of Eed complex composition governs a switch in global histone modification in brain.

Originally discovered as epigenetic regulators of developmental gene expression, the Polycomb (PcG) and trithorax (trxG) group of proteins form distinct nuclear complexes governing post-translational modification of histone tails. This study identified a novel, developmentally regulated interface between Eed and Mll, pivotal constituents of PcG and trxG pathways, respectively, in mouse brain. Although the PcG proteins Eed and EzH2 (Enhancer of Zeste protein-2) engaged in a common complex during neurodevelopment, Eed associated with the trxG protein Mll upon brain maturation. Comprehensive analysis of multiple histone modifications revealed differential substrate specificity of the novel Eed-Mll complex in adult brain compared with the developmental Eed-EzH2 complex. Newborn brain from eed heterozygotes and eed;Mll double heterozygotes exhibited decreased trimethylation at lysine 27 of histone H3, as well as hyperacetylation of histone H4. In contrast, adult hippocampus from Mll heterozygotes was remarkable for decreased acetylation of histone H4, which restored to wild-type levels in eed;Mll double heterozygotes. A physiological role for the Eed-Mll complex in adult brain was evident from complementary defects in synaptic plasticity in eed and Mll mutant hippocampi. These results support the notion that developmental regulation of complex composition bestows the predominant Eed complex with the chromatin remodeling activity conducive for gene regulation during neurodevelopment and adult brain function. Thus, this study suggests dynamic regulation of chromatin complex composition as a molecular mechanism to co-opt constituents of developmental pathways into the regulation of neuronal memory formation in adult brain.

Integration of prolactin and glucocorticoid signaling at the beta-casein promoter and enhancer by ordered recruitment of specific transcription factors and chromatin modifiers.

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent system in which to perform kinetic studies of chromatin remodeling and transcriptional activation. Using HC11 cells as a model, we have investigated the effects of prolactin (Prl) and glucocorticoids both singly and in combination at different time points after hormone treatment. Using chromatin immunoprecipitation analysis, we have determined the dynamics of assembly and disassembly of signal transducer and activator of transcription 5, glucocorticoid receptor, CCAAT enhancer binding protein beta, and Ying Yang-1 at the hormonally activated beta-casein proximal promoter as well as the distal mouse beta-casein enhancer located approximately -6 kb upstream of the transcription start site. Prl alone resulted in a rapid recruitment of both signal transducer and activator of transcription 5 and histone deacetylase 1 to the beta-casein promoter and enhancer, and reciprocally the dissociation of Ying Yang-1 from the proximal promoter. In addition, we have examined the recruitment of coactivator p300 and determined chromatin acetylation status as a function of hormonal treatment. Finally, we have established the time course of RNA polymerase II and phospho-RNA polymerase II accumulation at the beta-casein promoter and enhancer after stimulation with hydrocortisone and Prl. Although glucocorticoids alone led to a rapid increase in histone H3 acetylation, treatment with both hormones was required for stable association of p300 and phospho-RNA polymerase II at both the promoter and enhancer. Collectively, these data suggest a model for the assembly of a multiprotein complex that helps to define how the signaling pathways controlled by these lactogenic hormones are integrated to regulate beta-casein gene expression.

Histone modifications in status epilepticus induced by kainate.

Animal models of epilepsy have allowed the determination of the basic molecular and cellular mechanisms of epileptogenesis. Generalized limbic seizures and subsequent status epilepticus can be induced by either pilocarpine, the muscarinic acetylcholine receptor agonist or kainate, the glutamate receptor agonist. There has been increasing interest that chromatin remodeling might play a critical role in gene regulation even in non-dividing cells such as neurons. One form of chromatin remodeling is histone amino-terminal modification that can generate synergistic or antagonistic affinities for the interactions of transcriptional factors, in turn causing changes in gene activity. Two widely studied histone modification processes are histone acetylation and phosphorylation. While histone hyperacetylation indicates an increase in gene activity, its hypoacetylation marks gene repression. Both states are controlled by a dynamic interplay of histone acetyltransferase (HAT) and histone deacetylase (HDAC). We have found the upregulation of acetylation and phosphorylation of histones, coupled with status epilepticus after kainate administration. c-fos and c-jun mRNA have been sequentially induced in response to kainate, in different hippocampal subpopulations starting from the dentate gyrus and spreading to the cornus ammonis regions well correlated with the spatio-temporal distribution of histone H4 hyperacetylation. Both histone modifications are associated with the c-fos gene promoter after kainate stimulation, while only histone acetylation with the c-jun gene. Pretreatment with curcumin, which has a HAT inhibitory activity specific for CBP/p300, attenuates histone modifications, IEGs expression and also the severity of status epilepticus after kainate treatment. Histone modifications may have a crucial role in the development of epilepsy induced by kainate.