Heinrich Leonhardt

A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei

Tissue-specific patterns of methylated deoxycytidine residues in the mammalian genome are preserved by postreplicative methylation of newly synthesized DNA. DNA methyltransferase (MTase) is here shown to associate with replication foci during S phase but to display a diffuse nucleoplasmic distribution in non-S phase cells. Analysis of DNA MTase-beta-galactosidase fusion proteins has shown that association with replication foci is mediated by a novel targeting sequence located near the N-terminus of DNA MTase. This sequence has the properties expected of a targeting sequence in that it is not required for enzymatic activity, prevents proper targeting when deleted, and, when fused to beta-galactosidase, causes the fusion protein to associate with replication foci in a cell cycle-dependent manner.

Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy

Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.

A guide to super-resolution fluorescence microscopy

For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

Dynamic Readers for 5-(Hydroxy)Methylcytosine and Its Oxidized Derivatives

Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins

Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein.

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

Targeting and tracing antigens in live cells with fluorescent nanobodies.

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

Reversal of terminal differentiation and control of DNA replication: Cyclin A and cdk2 specifically localize at subnuclear sites of DNA replication

DNA replication in mammalian cells occurs in discrete nuclear foci. Here we show that terminally differentiated myotubes can be induced to reenter S phase and show the same pattern of replication foci as cycling cells. We used this cellular system to analyze the interaction of cell cycle proteins with these foci in vivo. Cyclin A and cdk2, but not cyclin B1 and cdc2, were specifically localized at nuclear replication foci, just like the replication protein proliferating cell nuclear antigen. A potential target of cyclin A and cdk2 is the 34 kd subunit of replication protein A (RPA34). In contrast with the 70 kd subunit, which localizes to the foci, RPA34 was not detected at these replication sites, which may reflect a transient interaction. The specific localization of cyclin A and cdk2 at nuclear replication foci provides a direct link between cell cycle regulation and DNA replication.

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.

LBR and Lamin A/C Sequentially Tether Peripheral Heterochromatin and Inversely Regulate Differentiation

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.