Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins

Fluorescent fusion proteins are widely used to study protein localization and interaction dynamics in living cells. However, to fully characterize proteins and to understand their function it is crucial to determine biochemical characteristics such as enzymatic activity and binding specificity. Here we demonstrate an easy, reliable and versatile medium/high-throughput method to study biochemical and functional characteristics of fluorescent fusion proteins. Using a new system based on 96-well micro plates comprising an immobilized GFP-binding protein (GFP-mulitTrap), we performed fast and efficient one-step purification of different GFP- and YFP-fusion proteins from crude cell lysate. After immobilization we determined highly reproducible binding ratios of cellular expressed GFP-fusion proteins to histone-tail peptides, DNA or selected RFP-fusion proteins. In particular, we found Cbx1 preferentially binding to di-and trimethylated H3K9 that is abolished by phosphorylation of the adjacent serine. DNA binding assays showed, that the MBD domain of MeCP2 discriminates between fully methylated over unmethylated DNA and protein-protein interactions studies demonstrate, that the PBD domain of Dnmt1 is essential for binding to PCNA. Moreover, using an ELISA-based approach, we detected endogenous PCNA and histone H3 bound at GFP-fusions. In addition, we quantified the level of H3K4me2 on nucleosomes containing different histone variants. In summary, we present an innovative medium/high-throughput approach to analyse binding specificities of fluroescently labeled fusion proteins and to detect endogenous interacting factors in a fast and reliable manner in vitro

Structure-guided mutational analysis reveals the functional requirements for product specificity of DOT1 enzymes

DOT1 enzymes are conserved methyltransferases that catalyse the methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, with different enzymatic activities. DOT1A mediates mono-and dimethylation of H3K76, the homologue of H3K79 in other organisms, whereas DOT1B additionally catalyses H3K76 trimethylation. However, it is unclear how these different enzymatic activities are achieved. Here we employ a trypanosomal nucleosome reconstitution system and structure-guided homology modelling to identify critical residues within and outside the catalytic centre that modulate product specificity. Exchange of these residues transfers the product specificity from one enzyme to the other, and reveals the existence of distinct regulatory domains adjacent to the catalytic centre. Our study provides the first evidence that a few crucial residues in DOT1 enzymes are sufficient to catalyse methyl-state-specific reactions. These results might also have far-reaching consequences for the functional understanding of homologous enzymes in higher eukaryotes

Structure-guided mutational analysis reveals the functional requirements for product specificity of DOT1 enzymes

DOT1 enzymes are conserved methyltransferases that catalyse the methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, with different enzymatic activities. DOT1A mediates mono-and dimethylation of H3K76, the homologue of H3K79 in other organisms, whereas DOT1B additionally catalyses H3K76 trimethylation. However, it is unclear how these different enzymatic activities are achieved. Here we employ a trypanosomal nucleosome reconstitution system and structure-guided homology modelling to identify critical residues within and outside the catalytic centre that modulate product specificity. Exchange of these residues transfers the product specificity from one enzyme to the other, and reveals the existence of distinct regulatory domains adjacent to the catalytic centre. Our study provides the first evidence that a few crucial residues in DOT1 enzymes are sufficient to catalyse methyl-state-specific reactions. These results might also have far-reaching consequences for the functional understanding of homologous enzymes in higher eukaryotes

H3K56me3 is a novel, conserved heterochromatic mark that largely but not completely overlaps with H3K9me3 in both regulation and localization.

Histone lysine (K) methylation has been shown to play a fundamental role in modulating chromatin architecture and regulation of gene expression. Here we report on the identification of histone H3K56, located at the pivotal, nucleosome DNA entry/exit point, as a novel methylation site that is evolutionary conserved. We identify trimethylation of H3K56 (H3K56me3) as a modification that is present during all cell cycle phases, with the exception of S-phase, where it is underrepresented on chromatin. H3K56me3 is a novel heterochromatin mark, since it is enriched at pericentromeres but not telomeres and is thereby similar, but not identical, to the localization of H3K9me3 and H4K20me3. Possibly due to H3 sequence similarities, Suv39h enzymes, responsible for trimethylation of H3K9, also affect methylation of H3K56. Similarly, we demonstrate that trimethylation of H3K56 is removed by members of the JMJD2 family of demethylases that also target H3K9me3. Furthermore, we identify and characterize mouse mJmjd2E and its human homolog hKDM4L as novel, functionally active enzymes that catalyze the removal of two methyl groups from trimethylated H3K9 and K56. H3K56me3 is also found in C. elegans, where it co-localizes with H3K9me3 in most, but not all, tissues. Taken together, our findings raise interesting questions regarding how methylation of H3K9 and H3K56 is regulated in different organisms and their functional roles in heterochromatin formation and/or maintenance

Germinal Center B Cells Regulate Their Capability to Present Antigen by Modulation of HLA-DO

Peptide acquisition by MHC class II molecules is catalyzed by HLA-DM (DM). In B cells, HLA-DO (DO) inhibits or modifies the peptide exchange activity of DM. We show here that DO protein levels are modulated during B cell differentiation. Remarkably, germinal center (GC) B cells, which have low levels of DO relative to naive and memory B cells, are shown to have enhanced antigen presentation capabilities. DM protein levels also were somewhat reduced in GC B cells; however, the ratio of DM to DO in GC B cells was substantially increased, resulting in more free DM in GC B cells. We conclude that modulation of DM and DO in distinct stages of B cell differentiation represents a mechanism by which B cells regulate their capacity to function as antigen-presenting cells. Efficient antigen presentation in GC B cells would promote GC B cell–T cell interactions that are essential for B cells to survive positive selection in the GC

The CENP-T C-terminus is exclusively proximal to H3.1 and not to H3.2 or H3.3.

The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere-kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1(C96A) and H3.1(C110A) nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2

Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y

The expression of a new histone variant H3.Y increases during cellular stress to regulate cell cycle progression and gene expression

PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex.

Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial-facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels

Identification of Histone H3 Lysine 36 Acetylation as a Highly Conserved Histone Modification

Histone lysine (K) acetylation is a major mechanism by which cells regulate the structure and function of chromatin, and new sites of acetylation continue to be discovered. Here we identify and characterize histone H3K36 acetylation (H3K36ac). By mass spectrometric analyses of H3 purified from Tetrahymena thermophila and Saccharomyces cerevisiae (yeast), we find that H3K36 can be acetylated or methylated. Using an antibody specific to H3K36ac, we show that this modification is conserved in mammals. In yeast, genome-wide ChIP-chip experiments show that H3K36ac is localized predominantly to the promoters of RNA polymerase II-transcribed genes, a pattern inversely related to that of H3K36 methylation. The pattern of H3K36ac localization is similar to that of other sites of H3 acetylation, including H3K9ac and H3K14ac. Using histone acetyltransferase complexes purified from yeast, we show that the Gcn5-containing SAGA complex that regulates transcription specifically acetylates H3K36 in vitro. Deletion of GCN5 completely abolishes H3K36ac in vivo. These data expand our knowledge of the genomic targets of Gcn5, show H3K36ac is highly conserved, and raise the intriguing possibility that the transition between H3K36ac and H3K36me acts as an “acetyl/methyl switch” governing chromatin function along transcription units

Organismal Differences in Post-translational Modifications in Histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined