Roger B. Deal

Genome-Wide Kinetics of Nucleosome Turnover Determined by Metabolic Labeling of Histones

Everything Changes Nucleosomes package DNA, and their assembly and disassembly regulate access to the genome. The ability to follow these changes is limited to steady-state methods in higher eukaryotes, although direct kinetic analyses are available in yeast. To address this deficiency, Deal et al. (p. 1161) developed a general method for following the genome-wide dynamics of nucleosome assembly and disassembly. High levels of nucleosome turnover were observed across gene bodies and at the sites of epigenetic regulatory elements in fruit fly tissue culture cells. Nucleosomes were replaced multiple times during each 20-hour cell cycle, suggesting that histone modifications themselves are unlikely to transmit epigenetic information. Furthermore, analysis of replication origins indicates that they are determined by chromatin dynamics and not by sequence features. Differences between active and silent chromatin states are critical for maintaining the epigenome. Nucleosome disruption and replacement are crucial activities that maintain epigenomes, but these highly dynamic processes have been difficult to study. Here, we describe a direct method for measuring nucleosome turnover dynamics genome-wide. We found that nucleosome turnover is most rapid over active gene bodies, epigenetic regulatory elements, and replication origins in Drosophila cells. Nucleosomes turn over faster at sites for trithorax-group than polycomb-group protein binding, suggesting that nucleosome turnover differences underlie their opposing activities and challenging models for epigenetic inheritance that rely on stability of histone marks. Our results establish a general strategy for studying nucleosome dynamics and uncover nucleosome turnover differences across the genome that are likely to have functional importance for epigenome maintenance, gene regulation, and control of DNA replication.

A Simple Method for Gene Expression and Chromatin Profiling of Individual Cell Types within a Tissue

Understanding the production and function of specialized cells during development requires the isolation of individual cell types for analysis, but this is currently a major technical challenge. Here we describe a method for cell type-specific RNA and chromatin profiling that circumvents many of the limitations of current methods for cell isolation. We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. We identified hundreds of genes that are preferentially expressed in each cell type and show that genes with the largest expression differences between hair and non-hair cells also show differences between cell types in the trimethylation of histone H3 at lysines 4 and 27. This method should be applicable to any organism that is amenable to transformation.

Repression of Flowering in Arabidopsis Requires Activation of FLOWERING LOCUS C Expression by the Histone Variant H2A.Z

The histone variant H2A.Z has been implicated in numerous chromatin-mediated processes, including transcriptional activation, euchromatin maintenance, and heterochromatin formation. In yeast and humans, H2A.Z is deposited into chromatin by a conserved protein complex known as SWR1 and SRCAP, respectively. Here, we show that mutations in the Arabidopsis thaliana homologs of two components of this complex, ACTIN-RELATED PROTEIN6 (ARP6) and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1), produce similar developmental phenotypes and result in the misregulation of a common set of genes. Using H2A.Z-specific antibodies, we demonstrate that ARP6 and PIE1 are required for the deposition of H2A.Z at multiple loci, including the FLOWERING LOCUS C (FLC) gene, a central repressor of the transition to flowering. Loss of H2A.Z from chromatin in arp6 and pie1 mutants results in reduced FLC expression and premature flowering, indicating that this histone variant is required for high-level expression of FLC. In addition to defining a novel mechanism for the regulation of FLC expression, these results support the existence of a SWR1-like complex in Arabidopsis and show that H2A.Z can potentiate transcriptional activation in plants. The finding that H2A.Z remains associated with chromatin throughout mitosis suggests that it may serve an epigenetic memory function by marking active genes and poising silenced genes for reactivation.

The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana

Genomic studies of cell differentiation and function within a whole organism depend on the ability to isolate specific cell types from a tissue, but this is technically difficult. We developed a method called INTACT (isolation of nuclei tagged in specific cell types) that allows affinity-based isolation of nuclei from individual cell types of a tissue, thereby circumventing the problems associated with mechanical purification techniques. In this method nuclei are affinity-labeled through transgenic expression of a biotinylated nuclear envelope protein in the cell type of interest. Total nuclei are isolated from transgenic plants and biotin-labeled nuclei are then purified using streptavidin-coated magnetic beads, without the need for specialized equipment. INTACT gives high yield and purity of nuclei from the desired cell types, which can be used for genome-wide analysis of gene expression and chromatin features. The entire procedure, from nuclei purification through cDNA preparation or chromatin immunoprecipitation (ChIP), can be completed within 2 d. The protocol we present assumes that transgenic lines are already available, and includes procedural details for amplification of cDNA or ChIP DNA prior to microarray or deep sequencing analysis.

The Nuclear Actin-Related Protein ARP6 Is a Pleiotropic Developmental Regulator Required for the Maintenance of FLOWERING LOCUS C Expression and Repression of Flowering in Arabidopsis

Actin-related proteins (ARPs) are found in the nuclei of all eukaryotic cells, but their functions are generally understood only in the context of their presence in various yeast and animal chromatin-modifying complexes. Arabidopsis thaliana ARP6 is a clear homolog of other eukaryotic ARP6s, including Saccharomyces cerevisiae ARP6, which was identified as a component of the SWR1 chromatin remodeling complex. We examined the subcellular localization, expression patterns, and loss-of-function phenotypes for this protein and found that Arabidopsis ARP6 is localized to the nucleus during interphase but dispersed away from the chromosomes during cell division. ARP6 expression was observed in all vegetative tissues as well as in a subset of reproductive tissues. Null mutations in ARP6 caused numerous defects, including altered development of the leaf, inflorescence, and flower as well as reduced female fertility and early flowering in both long- and short-day photoperiods. The early flowering of arp6 mutants was associated with reduced expression of the central floral repressor gene FLOWERING LOCUS C (FLC) as well as MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. In addition, arp6 mutations suppress the FLC-mediated late flowering of a FRIGIDA-expressing line, indicating that ARP6 is required for the activation of FLC expression to levels that inhibit flowering. These results indicate that ARP6 acts in the nucleus to regulate plant development, and we propose that it does so through modulation of chromatin structure and the control of gene expression.

Histone H2A.Z regulates the expression of several classes of phosphate starvation response genes but not as a transcriptional activator.

Phosphate (Pi) availability is a major constraint to plant growth. Consequently, plants have evolved complex adaptations to tolerate low Pi conditions. Numerous genes implicated in these adaptations have been identified, but their chromatin-level regulation has not been investigated. The nuclear actin-related protein ARP6 is conserved among all eukaryotes and is an essential component of the SWR1 chromatin remodeling complex, which regulates transcription via deposition of the H2A.Z histone variant into chromatin. Here, we demonstrate that ARP6 is required for proper H2A.Z deposition at a number of Pi starvation response (PSR) genes in Arabidopsis (Arabidopsis thaliana). The loss of H2A.Z at these target loci results in their derepression in arp6 mutants and correlates with the presence of multiple Pi-starvation-related phenotypes, including shortened primary roots and increases in the number and length of root hairs, as well as increased starch accumulation and phosphatase activity in shoots. Our data suggest a model for chromatin-level control of Pi starvation responses in which ARP6-dependent H2A.Z deposition modulates the transcription of a suite of PSR genes.

Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling

An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue- or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotin-labeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types.

Histone variants and modifications in plant gene regulation

Genomes are packaged by complexing DNA with histone proteins, which provides an opportunity to regulate gene expression by dynamically impeding access of transcriptional regulatory proteins and RNA polymerases to DNA. The incorporation of histone variants into nucleosomes and addition of post-translational modifications to histones can alter the physical properties of nucleosomes and thereby serve as a mechanism for regulating DNA exposure. Chromatin-based gene regulation has profound effects on developmental processes including regulation of the vegetative to reproductive transition, as well as responses to pathogens and abiotic factors. Incorporation of the histone variant H2A.Z and methylation of histone H3 lysine residues 4 and 27 have emerged as key elements in the regulation of genes involved in each of these processes.

Arabidopsis ARP7 is an essential actin-related protein required for normal embryogenesis, plant architecture, and floral organ abscission

The actin-related proteins (ARPs) that are localized to the nucleus are present as components of various chromatin-modifying complexes involved in chromatin dynamics and transcriptional regulation. Arabidopsis (Arabidopsis thaliana) ARP7 is a constitutively expressed nuclear protein belonging to a novel plant-specific ARP class. In this study, we demonstrate a vital role for ARP7 protein in embryogenesis and plant development. Knocking out the expression of ARP7 in an arp7-1 T-DNA mutant produced morphologically aberrant, homozygous embryos that were arrested at or before the torpedo stage of development. Hence, the arp7-1 null mutation is homozygous lethal. Knocking down the expression levels of ARP7 protein with RNA interference produced viable Arabidopsis lines affected in multiple developmental pathways and induced dosage-dependent, heritable defects in plant architecture. The transgenic plants containing greatly reduced levels of ARP7 in the nucleus were severely dwarfed with small rosette leaves that are defective in cell expansion and trichome morphology. Moreover, the ARP7-deficient RNA interference plants exhibited retarded root growth, altered flower development, delayed perianth abscission, and reduced fertility. These pleiotropic phenotypic changes suggest a critical role for the Arabidopsis ARP7 protein in the regulation of various phases of plant development through chromatin-mediated, global regulation of gene expression.

Capturing the dynamic epigenome

Traditional methods for epigenomic analysis provide a static picture of chromatin, which is actually a highly dynamic assemblage. Recent approaches have allowed direct measurements of chromatin dynamics, providing deeper insights into processes such as transcription, DNA replication and epigenetic inheritance.