ABSTRACT
The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/metabolism , Drosophila melanogaster/embryology , Mass Spectrometry/methods , Proteomics/methods , Animals , Chromatin/genetics , DNA/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/metabolism , Histones/metabolism , In Vitro Techniques , KineticsABSTRACT
Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.
Subject(s)
Histone Demethylases/antagonists & inhibitors , Histones/chemistry , Peptides/chemistry , Animals , Antibodies/chemistry , Biotinylation , Drug Discovery , Epigenomics , Fluorescence , Histone Demethylases/chemistry , Humans , Immunoassay , Insecta , Kinetics , Lysine/chemistry , Mass Spectrometry , Protein Processing, Post-Translational , Reproducibility of Results , Spectrometry, FluorescenceABSTRACT
Recent progress in the synthesis of nucleotides from prebiotically plausible precursors has opened up new ways to explain the origin of genetic matter. Mechanisms for the polymerization of nucleotides without the help of catalysts are, however, rare. Complementary to the experiments done by Costanzo et al., we found that drying 3',5'-cyclic GMP leads to poly-G RNA strands with lengths of up to 40 nucleotides. We also show that the polymerization to long RNA strands is considerably more efficient under dry conditions than for cGMP polymerization in water. The length depends on the incubation time of dry nucleotides at temperatures of 40-80 °C. No enzymes or other catalysts are needed for successful polymerization.
Subject(s)
Cyclic GMP/metabolism , RNA/metabolism , Polymerization , RNA/chemistry , Ribonuclease T1/metabolism , Temperature , Water/chemistryABSTRACT
Much progress has been made concerning histone function in the nucleus; however, following their synthesis, how their marking and subcellular trafficking are regulated remains to be explored. To gain an insight into these issues, we focused on soluble histones and analyzed endogenous and tagged H3 histones in parallel. We distinguished six complexes that we could place to account for maturation events occurring on histones H3 and H4 from their synthesis onward. In each complex, a different set of chaperones is involved, and we found specific post-translational modifications. Interestingly, we revealed that histones H3 and H4 are transiently poly(ADP-ribosylated). The impact of these marks in histone metabolism proved to be important as we found that acetylation of lysines 5 and 12 on histone H4 stimulated its nuclear translocation. Furthermore, we showed that, depending on particular histone H3 modifications, the balance in the presence of the different translocation complexes changes. Therefore, our results enabled us to propose a regulatory means of these marks for controlling cytoplasmic/nuclear shuttling and the establishment of early modification patterns.
