[show abstract][hide abstract] ABSTRACT: 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.
Genome Research 01/2012; 22(4):766-77. · 14.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: The dynamic interplay between DNA-binding proteins and nucleosomes underlies essential nuclear processes such as transcription, replication, and DNA repair. Manifestations of this interplay include the assembly, eviction, and replacement of nucleosomes. Hence, measurements of nucleosome turnover kinetics can lead to insights into the regulation of dynamic chromatin processes. In this chapter, we describe a genome-wide method for measuring nucleosome turnover that uses metabolic labeling followed by capture of newly synthesized histones, which we have termed Covalent Attachment of Tagged Histones to Capture and Identify Turnover (CATCH-IT). Although CATCH-IT can be used with any genome-wide mapping procedure, high-resolution profiling is attainable using paired-end sequencing of native chromatin. Our protocol also includes an efficient Solexa DNA sequencing library preparation protocol that can be used for single base-pair resolution mapping of both nucleosome and subnucleosomal particles. We not only describe the use of these protocols in the context of a Drosophila cell line but also provide the necessary changes for adaptation to other model systems.
Methods in enzymology 01/2012; 513:169-84. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Current opinion in plant biology 04/2011; 14(2):116-22. · 10.33 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: When environmental temperatures rise, plants seek help from their core molecular mechanisms to adapt. The chromatin protein H2A.Z, which regulates gene expression, is one such rescue molecule.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Actin-related protein 5 (ARP5) is a conserved subunit of the INO80 chromatin-remodeling complex in yeast and mammals. We have characterized the expression and subcellular distribution of Arabidopsis thaliana ARP5 and explored its role in the epigenetic control of multicellular development and DNA repair. ARP5-specific monoclonal antibodies localized ARP5 protein to the nucleoplasm of interphase cells in Arabidopsis and Nicotiana tabacum. ARP5 promoter-reporter fusions and the ARP5 protein are ubiquitously expressed. A null mutant and a severe knockdown allele produced moderately dwarfed plants with all organs smaller than the wild type. The small and slightly deformed organs such as leaves and hypocotyls were composed of small-sized cells. The ratio of leaf stomata to epidermal cells was high in the mutant, which also exhibited a delayed stomatal development compared with the wild type. Mutant plants were hypersensitive to DNA-damaging reagents including hydroxyurea, methylmethane sulfonate, and bleocin, demonstrating a role for ARP5 in DNA repair. Interestingly, the hypersensitivity phenotype of ARP5 null allele arp5-1 is stronger than the severe knockdown allele arp5-2. Moreover, a wild-type transgene fully complemented all developmental and DNA repair mutant phenotypes. Despite the common participation of both ARP4 and ARP5 in the INO80 complex, ARP4- and ARP5-deficient plants displayed only a small subset of common phenotypes and each displayed novel phenotypes, suggesting that in Arabidopsis they have both shared and unique functions.
[show abstract][hide abstract] ABSTRACT: Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igalpha and Igbeta of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igalpha expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).
Journal of immunological methods 02/2009; 343(1):28-41. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Actin depolymerizing factors (ADF/cofilin) modulate the rate of actin filament turnover, networking cellular signals into cytoskeletal-dependent developmental pathways. Plant and animal genomes encode families of diverse ancient ADF isovariants. One weakly but ubiquitously expressed member of the Arabidopsis ADF gene family, ADF9, is moderately expressed in the shoot apical meristem (SAM). Mutant alleles adf9-1 and adf9-2 showed a 95% and 50% reduction in transcript levels, respectively. Compared to wild-type, mutant seedlings and plants were significantly smaller and adult mutant plants had decreased numbers of lateral branches and a reduced ability to form callus. The mutants flowered very early during long-day light cycles, but not during short days. adf9-1showed a several-fold lower expression of FLOWERING LOCUS C (FLC), a master repressor of the transition to flowering, and increased expression of CONSTANS, an activator of flowering. Transgenic ADF9 expression complemented both developmental and gene expression phenotypes. FLC chromatin from adf9-1 plants contained reduced levels of histone H3 lysine 4 trimethylation and lysine 9 and 14 acetylation, as well as increased nucleosome occupancy consistent with a less active chromatin state. We propose that ADF9 networks both cytoplasmic and nuclear processes within the SAM to control multicellular development.
[show abstract][hide abstract] ABSTRACT: Regulating developmental transitions, cell proliferation and cell death through differential gene expression is essential to the ontogeny of all multicellular organisms. Chromatin remodeling is an active process that is necessary for managing the genome-wide suppression of gene activities resulting from DNA compaction. Recent data in plants suggest a general theme, whereby chromatin remodeling complexes containing nuclear actin-related proteins (ARPs) potentiate the activities of crucial regulatory genes involved in plant growth and development, in addition to their basal activities on a much larger set of genes.
Trends in cell biology 08/2007; 17(7):325-32. · 12.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 Plant Cell 02/2007; 19(1):74-83. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Expressing stem-loop RNAs in plants, fungi, and animals efficiently silences homologous target gene expression. We devised a novel PCR strategy, called inverted repeat PCR (IR-PCR), which allows rapid assembly and cloning of stem-loop-containing constructs in any vector. IR-PCR relies on differentially tagging antisense and sense copies of the target in one round of PCR and assembling them in a second. We used IR-PCR to assemble constructs targeting profilin, actin, and actin-related protein (ARP) transcripts from Arabidopsis. Immunoblotting of lines expressing a profilin PRF1 3' untranslated region (UTR)-specific construct demonstrated a 77 to 97% reduction in PRF1 protein, but not other profilin isovariants.
Plant and Cell Physiology 12/2005; 46(11):1872-8. · 4.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
The Plant Cell 11/2005; 17(10):2633-46. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.