Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase

We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.     

Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology

Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.     

Detection of in vivo protein-DNA interactions using DamID in mammalian cells

Understanding gene regulatory networks in mammalian cells requires detailed knowledge of protein-DNA interactions. Commonly used methods for genome-wide mapping of these interactions are based on chromatin immunoprecipitation. However, these methods have some drawbacks, such as the use of crosslinking reagents, the need for highly specific antibodies and relatively large amounts of starting material. We present DamID, an alternative technique to map genome-wide occupancy of interaction sites in vivo, that bypasses these limitations. DamID is based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays. Using DamID, genome-wide maps of the binding of DNA-interacting proteins in mammalian cells can be constructed efficiently. Depending on the strategy used for expression of the Dam-fusion proteins, genome-wide binding maps can be obtained in as little as 2 weeks.     

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.     

DNA-binding properties of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS.

The MADS domain proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) specify the identity of Arabidopsis floral organs. AP1 and AG homocomplexes and AP3-PI heterocomplexes bind to CArG-box sequences. The DNA-binding properties of these complexes were investigated. We find that AP1, AG and AP3-PI are all capable of recognizing the same DNA-binding sites, although with somewhat different affinities. In addition, the three complexes induce similar conformational changes on a CArG-box sequence. Phasing analysis reveals that the induced distortion is DNA bending, oriented toward the minor groove. The molecular dissection of AP1, AP3, PI and AG indicates that the boundaries of the dimerization domains of these proteins vary. The regions required to form a DNA-binding complex include, in addition to the MADS box, the entire L region (which follows the MADS box) and the first putative amphipathic helix of the K box in the case of AP3-PI, while for AP1 and AG only a part of the L region is needed. The similarity of the DNA-binding properties of AP1, AP3-PI and AG is discussed with regard to the biological specificity that these proteins exhibit.     

Molecular characterization of the tobacco SET domain protein NtSET1 unravels its role in histone methylation, chromatin binding, and segregation

Plants contain a great number of genes encoding a distinctive class of SET domain proteins which harbor a plant-specific N-terminal part together with a C-terminal part showing highest sequence similarity to the catalytic domain of the yeast CLR4, the human SUV39H1 and G9a histone-methyltransferases (HMTases). Here we show that NtSET1, a representative member of this class from tobacco, methylated both K9 and K27 of histone H3 in vitro. Ectopic expression of NtSET1, by an inducible promoter, increased the amount of dimethylated H3K9 and induced chromosome-segregation defects in tobacco BY2 cells. Deletion analyses show that the HMTase activity, the association with specific chromatin regions and with condensed chromosomes, and the cellular effects largely depended on the C-terminal region including the SET domain of the protein. Nevertheless, the N-terminal part of NtSET1 was capable of targeting the green fluorescent protein to interphase chromatin. Finally, we show that NtSET1 bound LHP1, the Arabidopsis homolog of animal heterochromatin protein 1 (HP1), and that LHP1 co-localized with heterochromatin containing high amounts of dimethylated H3K9, suggesting a role for NtSET1 in heterochromatic function. Taken together, our results provide new insights into the molecular and global chromatin-binding activities of this particular class member of plant SET domain proteins.     

Different domains control the localization and mobility of LIKE HETEROCHROMATIN PROTEIN1 in Arabidopsis nuclei

Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type-dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localization signal was identified within the hinge region. Fluorescence recovery after photobleaching showed that Sl LHP1 is a highly mobile protein whose localization and retention are controlled by distinct domains; retention at the nucleolus and chromocenters is conferred by the CSD. Our results imply that LHP1 recruitment to chromatin is mediated, at least in part, through interaction with methyl K9 and that LHP1 controls different nuclear processes via transient binding to its nuclear sites.     

Structural basis for the recognition of methylated histone H3 by the Arabidopsis LHP1 chromodomain

Arabidopsis LHP1 (LIKE HETEROCHROMATIN PROTEIN 1), a unique homolog of HP1 in Drosophila, plays important roles in plant development, growth, and architecture. In contrast to specific binding of the HP1 chromodomain to methylated H3K9 histone tails, the chromodomain of LHP1 has been shown to bind to both methylated H3K9 and H3K27 histone tails, and LHP1 carries out its function mainly via its interaction with these two epigenetic marks. However, the molecular mechanism for the recognition of methylated histone H3K9/27 by the LHP1 chromodomain is still unknown. In this study, we characterized the binding ability of LHP1 to histone H3K9 and H3K27 peptides and found that the chromodomain of LHP1 binds to histone H3K9me2/3 and H3K27me2/3 peptides with comparable affinities, although it exhibited no binding or weak binding to unmodified or monomethylated H3K9/K27 peptides. Our crystal structures of the LHP1 chromodomain in peptide-free and peptide-bound forms coupled with mutagenesis studies reveal that the chromodomain of LHP1 bears a slightly different chromodomain architecture and recognizes methylated H3K9 and H3K27 peptides via a hydrophobic clasp, similar to the chromodomains of human Polycomb proteins, which could not be explained only based on primary structure analysis. Our binding and structural studies of the LHP1 chromodomain illuminate a conserved ligand interaction mode between chromodomains of both animals and plants, and shed light on further functional study of the LHP1 protein.     

5(S)-hydroxyeicosatetraenoic acid stimulates DNA synthesis in human microvascular endothelial cells via activation of Jak/STAT and phosphatidylinositol 3-kinase/Akt signaling,leading to induction of expression of basic fibroblast growth factor 2

To understand the role of eicosanoids in angiogenesis, we have studied the effect of lipoxygenase metabolites of arachidonic acid on human microvascular endothelial cell (HMVEC) DNA synthesis. Among the various lipoxygenase metabolites of arachidonic acid tested, 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE) induced DNA synthesis in HMVEC. 5(S)-HETE also stimulated Jak-2, STAT-1, and STAT-3 tyrosine phosphorylation and STAT-3-DNA binding activity. Tyrphostin AG490, a specific inhibitor of Jak-2, significantly reduced tyrosine phosphorylation and DNA binding activity of STAT-3 and DNA synthesis induced by 5(S)-HETE. In addition, 5(S)-HETE stimulated phosphatidylinositol 3-kinase (PI3-kinase) activity and phosphorylation of its downstream targets Akt, p70S6K, and 4E-BP1 and their effector molecules ribosomal protein S6 and eIF4E. LY294002 and rapamycin, potent inhibitors of PI3-kinase and mTOR, respectively, also blocked the DNA synthesis induced by 5(S)-HETE. Interestingly, AG490 attenuated 5(S)-HETE-induced PI3-kinase activity and phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. 5(S)-HETE induced the expression of basic fibroblast growth factor 2 (bFGF-2) in a Jak-2- and PI3-kinase-dependent manner. In addition, a neutralizing anti-bFGF-2 antibody completely blocked 5(S)-HETE-induced DNA synthesis in HMVEC. Together these results suggest that 5(S)-HETE stimulates HMVEC growth via Jak-2- and PI3-kinase-dependent induction of expression of bFGF-2. These findings also reveal a cross-talk between Jak-2 and PI3-kinase in response to 5(S)-HETE in HMVEC.     

An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation

Cytosine methylation is critical for correct development and genome stability in mammals and plants. In order to elucidate the factors that control genomic DNA methylation patterning, a genetic screen for mutations that disrupt methylation-correlated silencing of the endogenous gene PAI2 was conducted in Arabidopsis: This screen yielded seven loss-of-function alleles in a SET domain protein with histone H3 Lys9 methyltransferase activity, SUVH4. The mutations conferred reduced cytosine methylation on PAI2, especially in non-CG sequence contexts, but did not affect methylation on another PAI locus carrying two genes arranged as an inverted repeat. Moreover, an unmethylated PAI2 gene could be methylated de novo in the suvh4 mutant background. These results suggest that SUVH4 is involved in maintenance but not establishment of methylation at particular genomic regions. In contrast, a heterochromatin protein 1 homolog, LHP1, had no effect on PAI methylation.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> LHP1 protein is a component of euchromatin

The HP1 family proteins are involved in several aspects of chromatin function and regulation in Drosophila, mammals and the fission yeast. Here we investigate the localization of LHP1, the unique Arabidopsis thaliana HP1 homolog known at present time, to approach its function. A functional LHP1–GFP fusion protein, able to restore the wild-type phenotype in the lhp1 mutant, was used to analyze the subnuclear distribution of LHP1 in both A. thaliana and Nicotiana tabacum. In A. thaliana interphase nuclei, LHP1 was predominantly located outside the heterochromatic chromocenters. No major aberrations were observed in heterochromatin content or chromocenter organization in lhp1 plants. These data indicate that LHP1 is mainly involved in euchromatin organization in A. thaliana. In tobacco BY-2 cells, the LHP1 distribution, although in foci, slightly differed suggesting that LHP1 localization is determined by the underlying genome organization of plant species. Truncated LHP1 proteins expressed in vivo allowed us to determine the function of the different segments in the localization. The in foci distribution is dependent on the presence of the two chromo domains, whereas the hinge region has some nucleolus-targeting properties. Furthermore, like the animal HP1β and HP1γ subtypes, LHP1 dissociates from chromosomes during mitosis. In transgenic plants expressing the LHP1–GFP fusion protein, two major localization patterns were observed according to cell types suggesting that localization evolves with age or differentiation states. Our results show conversed characteristics of the A. thaliana HP1 homolog with the mammal HP1γ isoform, besides specific plant properties.