Bioinformatic analysis of proteomics data

Most biochemical reactions in a cell are regulated by highly specialized proteins, which are the prime mediators of the cellular phenotype. Therefore the identification, quantitation and characterization of all proteins in a cell are of utmost importance to understand the molecular processes that mediate cellular physiology. With the advent of robust and reliable mass spectrometers that are able to analyze complex protein mixtures within a reasonable timeframe, the systematic analysis of all proteins in a cell becomes feasible. Besides the ongoing improvements of analytical hardware, standardized methods to analyze and study all proteins have to be developed that allow the generation of testable new hypothesis based on the enormous pre-existing amount of biological information. Here we discuss current strategies on how to gather, filter and analyze proteomic data sates using available software packages

Epigenetics and Metabolism in Health and Disease

In the next 10 years, one billion people are estimated to suffer from disabling consequences of metabolic disorders, making them the number one noncommunicable disease on a global scale by 2030. Lots of risk factors such as dietary intake, lack of exercise and other life style behaviors are considered to play a role in the development of metabolic disorders. Despite the efforts that have been undertaken to unravel their potential causes, the underlying molecular mechanisms remain elusive. Evidence suggests that the pathogenesis involves changes on chromatin and chromatin-modifying enzymes, which can contribute to a persistent dysregulated metabolic phenotype. Indeed, a rising number of studies links epigenetic alterations with the diagnosis and prognosis of metabolic disorders. A prerequisite for exploiting these findings for pharmacological intervention is a detailed understanding of how differential epigenetic modifications control cell metabolism. In this mini review, we summarize the recent advances in uncovering the interplay between epigenetics and metabolic pathways on a cellular level and highlight potential new avenues for alternative treatment strategies

Phosphorylation of SU(VAR)3-9 by the chromosomal kinase JIL-1

The histone methyltransferase SU(VAR)3-9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR)3-9's activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR)3-9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR)3-9 N-terminus. Among several other proteins known to bind Su(VAR)3-9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR)3-9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR)3-9 and JIL-1

Global and specific responses of the histone acetylene to systematic perturbation

Regulation of histone acetylation is fundamental to the utilization of eukaryotic genomes in chromatin. Aberrant acetylation contributes to disease and can be clinically combated by inhibiting the responsible enzymes. Our knowledge of the histone acetylation system is patchy because we so far lacked themethodology to describe acetylation patterns and their genesis by integrated enzyme activities. We devised a generally applicable, mass spectrometry-based strategy to precisely and accurately quantify combinatorial modification motifs. This was applied to generate a comprehensive inventory of acetylation motifs on histones H3 and H4 in Drosophila cells. Systematic depletion of known or suspected acetyltransferases and deacetylases revealed specific alterations of histone acetylation signatures, established enzyme-substrate relationships, and unveiled an extensive crosstalk between neighboring modifications. Unexpectedly, overall histone acetylation levels remained remarkably constant upon depletion of individual acetyltransferases. Conceivably, the acetylation level is adjusted to maintain the global charge neutralization of chromatin and the stability of nuclei

Drosophila RISC Component VIG and Its Homolog Vig2 Impact Heterochromatin Formation

Heterochromatin formation plays an important role in gene regulation and the maintenance of genome integrity. Here we present results from a study of the D. melanogaster gene vig, encoding an RNAi complex component and its homolog vig2 (CG11844) that support their involvement in heterochromatin formation and/or maintenance. Protein null mutations vigEP812 and vig2PL470 act as modifiers of Position Effect Variegation (PEV). VIG and Vig2 are present in polytene chromosomes and partially overlap with HP1. Quantitative immunoblots show depletion of HP1 and HP2 (large isoform) in isolated nuclei from the vigEP812 mutant. The vig2PL470 mutant strain demonstrates a decreased level of H3K9me2. Pull-down experiments using antibodies specific to HP1 recovered both VIG and Vig2. The association between HP1 and both VIG and Vig2 proteins depends on an RNA component. The above data and the developmental profiles of the two genes suggest that Vig2 may be involved in heterochromatin targeting and establishment early in development, while VIG may have a role in stabilizing HP1/HP2 chromatin binding during later stages

A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

In human Estrogen Receptor alpha (ERalpha)-positive breast cancers, 5' end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ERalpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation.We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ERalpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 5' end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ERalpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ERalpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ERalpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated.MBD2 and ERalpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ERalpha and MBD2

Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs) for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development

Drosophila HP1c Is Regulated by an Auto-Regulatory Feedback Loop through Its Binding Partner Woc

HP1 is a major component of chromatin and regulates gene expression through its binding to methylated histone H3. Most eukaryotes express at least three isoforms of HP1 with similar domain architecture. However, despite the common specificity for methylated histone H3, the three HP1 isoforms bind to different regions of the genome. Most of the studies so far focused on the HP1a isoform and its role in transcriptional regulation. As HP1a requires additional factors to bind methylated chromatin in vitro, we wondered whether another isoform might also require additional targeting factors. Indeed, we found that HP1c interacts with the DNA binding factors Woc and Row and requires Woc to become targeted to chromatin in vivo. Moreover, we show that the interaction between HP1c and Woc constitutes a transcriptional feedback loop that operates to balance the concentration of HP1c within the cell. This regulation may prevent HP1c from binding to methylated heterochromatin