[Show abstract][Hide abstract] ABSTRACT: Background:
Heterochromatin has been reported to be a major silencing compartment during development and differentiation. Prominent heterochromatin compartments are located at the nuclear periphery and inside the nucleus (e.g., pericentric heterochromatin). Whether the position of a gene in relation to some or all heterochromatin compartments matters remains a matter of debate, which we have addressed in this study. Answering this question demanded solving the technical challenges of 3D measurements and the large-scale morphological changes accompanying cellular differentiation.
Here, we investigated the proximity effects of the nuclear periphery and pericentric heterochromatin on gene expression and additionally considered the effect of neighboring genomic features on a gene's nuclear position. Using a well-established myogenic in vitro differentiation system and a differentiation-independent heterochromatin remodeling system dependent on ectopic MeCP2 expression, we first identified genes with statistically significant expression changes by transcriptional profiling. We identified nuclear gene positions by 3D fluorescence in situ hybridization followed by 3D distance measurements toward constitutive and facultative heterochromatin domains. Single-cell-based normalization enabled us to acquire morphologically unbiased data and we finally correlated changes in gene positioning to changes in transcriptional profiles. We found no significant correlation of gene silencing and proximity to constitutive heterochromatin and a rather unexpected inverse correlation of gene activity and position relative to facultative heterochromatin at the nuclear periphery.
In summary, our data question the hypothesis of heterochromatin as a general silencing compartment. Nonetheless, compared to a simulated random distribution, we found that genes are not randomly located within the nucleus. An analysis of neighboring genomic context revealed that gene location within the nucleus is rather dependent on CpG islands, GC content, gene density, and short and long interspersed nuclear elements, collectively known as RIDGE (regions of increased gene expression) properties. Although genes do not move away/to the heterochromatin upon up-/down-regulation, genomic regions with RIDGE properties are generally excluded from peripheral heterochromatin. Hence, we suggest that individual gene activity does not influence gene positioning, but rather chromosomal context matters for sub-nuclear location.
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome is a neurological, X chromosomal-linked disorder associated with mutations in the MECP2 gene. MeCP2 protein has been proposed to play a role in transcriptional regulation as well as in chromatin architecture. Since MeCP2 mutant cells exhibit surprisingly mild changes in gene expression, we have now explored the possibility that Rett mutations may affect the ability of MeCP2 to bind and organize chromatin. We found that all but one of the 21 missense MeCP2 mutants analyzed accumulated at heterochromatin and about half of them were significantly affected. Furthermore, two-thirds of all mutants showed a significantly decreased ability to cluster heterochromatin. Three mutants containing different proline substitutions (P101H, P101R and P152R) were severely affected only in heterochromatin clustering and located far away from the DNA interface in the MeCP2 methyl-binding domain structure. MeCP2 mutants affected in heterochromatin accumulation further exhibited the shortest residence time on heterochromatin, followed by intermediate binding kinetics for clustering impaired mutants. We propose that different interactions of MeCP2 with methyl cytosines, DNA and likely other heterochromatin proteins are required for MeCP2 function and their dysfunction lead to Rett syndrome.
Human Molecular Genetics 08/2011; 20(21):4187-95. DOI:10.1093/hmg/ddr346 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The HUPO Brain Proteome Project (HUPO BPP) held its 11th workshop in Kolymbari on March 3, 2009. The principal aim of this project is to obtain a better understanding of neurodiseases and ageing, with the ultimate objective of discovering prognostic and diagnostic biomarkers, in addition to the development of novel diagnostic techniques and new medications. The attendees came together to discuss sub-project progress in the clinical neuroproteomics of human or mouse models of Alzheimer's and Parkinson's disease, and to define the needs and guidelines required for more advanced proteomics approaches. With the election of new steering committees, the members of the HUPO BPP elaborated an actual plan promoting activities, outcomes, and future directions of the HUPO BPP to acquire new funding and new participants.
[Show abstract][Hide abstract] ABSTRACT: In proteomics, rapid developments in instrumentation led to the acquisition of increasingly large data sets. Correspondingly, ProDaC was founded in 2006 as a Coordination Action project within the 6th European Union Framework Programme to support data sharing and community-wide data collection. The objectives of ProDaC were the development of documentation and storage standards, setup of a standardized data submission pipeline and collection of data. Ending in March 2009, ProDaC has delivered a comprehensive toolbox of standards and computer programs to achieve these goals.
[Show abstract][Hide abstract] ABSTRACT: The Proteomics Data Collection (ProDaC) consortium, a "Coordination Action" funded by the 6th EU Framework Programme, started in October 2006. Its aim was to facilitate the collection and distribution of proteomics data and the public availability of data sets from proteomics experiments. Within the consortium standard formats are created and tools are developed to allow extensive data collection within the proteomics community. An important part of ProDaC is the organization of workshops twice a year to inform about the consortium's progress and to stimulate communication between the ProDaC partners and between partners and interested members of the proteomics community. ProDaC ends on March 31, 2009. The most recent (and final) workshop was the 5th ProDaC workshop held on March 4, 2009 in Kolympari, Crete, Greece. The progress since the last meeting and an overall summary was presented by the work package coordinators and partners. Four external speakers presented talks about their work in relation to ProDaC.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms by which cells accurately distinguish between DNA double-strand break (DSB) ends and telomeric DNA ends remain poorly defined. Recent investigations have revealed intriguing interactions between DNA repair and telomeres. We were the first to report a requirement for the nonhomologous end-joining (NHEJ) protein DNA-dependent protein kinase (DNA-PK) in the effective end-capping of mammalian telomeres. Here, we report our continued characterization of uncapped (as opposed to shortened) dysfunctional telomeres in cells deficient for the catalytic subunit of DNA-PK (DNA-PKcs) and shed light on their consequence. We present evidence in support of our model that uncapped telomeres in this repair-deficient background are inappropriately detected and processed as DSBs and thus participate not only in spontaneous telomere-telomere fusion but, importantly, also in ionizing radiation-induced telomere-DSB fusion events. We show that phosphorylation of DNA-PKcs itself (Thr-2609 cluster) is a critical event for proper telomere end-processing and that ligase IV (NHEJ) is required for uncapped telomere fusion. We also find uncapped telomeres in cells from the BALB/c mouse, which harbors two single-nucleotide polymorphisms that result in reduced DNA-PKcs abundance and activity, most markedly in mammary tissue, and are both radiosensitive and susceptible to radiogenic mammary cancer. Our results suggest mechanistic links between uncapped/dysfunctional telomeres in DNA-PKcs-deficient backgrounds, radiation-induced instability, and breast cancer. These studies provide the first direct evidence of genetic susceptibility and environmental insult interactions leading to a unique and ongoing form of genomic instability capable of driving carcinogenesis.
Cancer Research 03/2009; 69(5):2100-7. DOI:10.1158/0008-5472.CAN-08-2854 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ProDaC (Proteomics Data Collection), a "Coordination Action" within the 6(th) EU framework programme, was created to support the collection, distribution and public availability of data from proteomics experiments. Within the consortium standards are created and maintained enabling an extensive data collection within the proteomics community. Important elements of ProDaC are workshops held twice a year to allow communication between the ProDaC partners and to report the ongoing progress. The most recent assembly was the 4(th) ProDaC workshop on August 15(th), 2008, in Amsterdam, The Netherlands. It took place directly before the 7(th) HUPO Annual World Congress (Human Proteome Organisation). Work package coordinators and partners presented the progress achieved since the last meeting. Additionally, an EU official presented funding opportunities for proteomics in the next EU framework programme and five external speakers presented talks about their work in relation to ProDaC.
[Show abstract][Hide abstract] ABSTRACT: The “Coordination Action” ProDaC (Proteomics Data Collection) – funded by the EU within the 6th framework programme – was created to support the dissemination, utilization and publication of proteomics data. Within this international consortium, standards are developed and maintained to support extensive data collection by the proteomics community. An important part of ProDaC are workshops organized on a regular basis (two per year) to allow discussions and communication between the ProDaC partners and to report on the progress of the project. The kick-off meeting took place in October 2006 in Long Beach, CA, USA. The 1st ProDaC workshop was held in Lyon, France (April 2007) and the 2nd in Seoul, Korea in October 2007. ProDaC organized the 3rd ProDaC workshop at the Beatriz Hotel, Toledo, on 22nd April, 2008, directly before the HUPO - PSI spring meeting (Human Proteome Organisation - Proteomics Standards Initiative). The work package coordinators presented talks about the progress achieved during the past six months. Additionally four external speakers presented their work on data conversion and data repositories. The concluding discussion session was chaired by the Journal's representative.
[Show abstract][Hide abstract] ABSTRACT: The Human Brain Proteome Project (HUPO BPP) aims at advancing knowledge and the understanding of neurodiseases and aging with the purpose of identifying prognostic and diagnostic biomarkers, as well as to push new diagnostic approaches and medications. The participating groups meet in semi-annual workshops to discuss the progress, as well as the needs, within the field of proteomics. The 9(th) HUPO BPP workshop took place in Barbados from 9-10 January, 2008. Discussing the future HUPO BPP Roadmap, the attendees drafted the so called HUPO BPP wish list containing timelines, suggestions and missions. This wish list will be updated regularly and will serve as a guideline for the next phase.
[Show abstract][Hide abstract] ABSTRACT: The Human Proteome Organisation was launched in February 2001 as a result of the need of an international proteomic forum to improve the understanding of human diseases. The initiative dealing with the brain is the Human Proteome Organisation Brain Proteome Project chaired in Germany and Korea. In order to estimate the existing approaches in brain proteomics, as well as to establish a standardized data reprocessing pipeline, pilot studies were initiated including both mouse and human samples. Data had to be submitted to a Data Collection Center for central re-processing and are now publicly accessible at the PRIDE database serving as reference data for future analysis. It became clear that heterogeneity, for example, different analysis strategies and data formats, are a real challenge when comparing results and when working in a consortium. Therefore, standardization, the organisation of data management and the synergistic effects of a consortium of collaborators are of outstanding importance to any big proteome analysis. The following manuscript will highlight these activities and aims of the Human Proteome Organisation Brain Proteome Project, summarizing its historical timeline and its two pilot studies.
[Show abstract][Hide abstract] ABSTRACT: What are the current approaches in brain proteomics? Can we combine different, but complementary study designs to obtain better results concerning brain diseases? What are the neuro-hotspots, especially in Korea? These were some of the questions the participants of the 8(th) HUPO Brain Proteome Project Workshop tried to answer prior to the 6(th) HUPO World Congress in Seoul, Korea. Around 100 scientists came together during the afternoon of 7 October, 2007, to discuss and to catch up on the latest results and strategies concerning Huntington's disease, glioblastoma and standardization.
[Show abstract][Hide abstract] ABSTRACT: Proteomics Data Collection (ProDaC) is an EU-funded "Coordination Action" within the 6th framework programme. It aims to simplify the publication, dissemination and utilization of proteomics data by establishing standards that will support broad data collection from the research community. As a part of ProDaC, regular workshops are organized on a half-yearly basis to enable communication and discussion of the involved partners and to report on project progress. After the kick-off meeting (October 2006) in Long Beach, CA, USA and the 1st workshop in Lyon, France (April 2007), the 2nd ProDaC workshop took place at the COEX InterContinental Hotel in Seoul, Korea, on 5th October 2007, shortly before the HUPO World Congress. The progress achieved within the first year was presented by the leaders of the work packages. Additionally, a Journal's representative talked about his experiences and future plans concerning Proteomics standards; and two further external speakers presented their research related to data handling and Proteomics repositories.
[Show abstract][Hide abstract] ABSTRACT: In 2001, the German Federal Ministry of Education and Research (BMBF) initiated the National Genome Research Network (NGFN; www.ngfn.de) as a nation-wide multidisciplinary networking platform aiming at the analysis of common human diseases and aging. Within the NGFN the Human Brain Proteome Project (HBPP; www.smp-proteomics.de) focuses on the analysis of the human brain in health and disease. The concept is based on two consecutive steps: (i) Elaborating and establishing the necessary technology platforms. (ii) Application of the established technologies for research in Alzheimer's disease and Parkinson's disease. In the first funding period, HBPP1, running from 2001 to 2004, necessary technologies were established and optimized. In HBPP2, which started 2004 and will end in May 2008, the developed technologies are used for large-scale experiments, offering new links for disease related research and therapies. The following overview describes structure, aims and outcome of this unique German Brain Proteome Project.
[Show abstract][Hide abstract] ABSTRACT: The symposium High Performance Proteomics was held in Dortmund on May 14-16, 2007, to celebrate the opening of the Zentrum für Angewandte Proteomik as well as the 6(th) anniversary of the German Human Brain Proteome Project. It offered an outstanding opportunity to obtain a broad overview about all fields of proteomics and related fields, combining the expertise of biochemists, physicians, bioinformatics, mathematicians and other researchers in Life Sciences. The main topics were the presentation of state-of-the-art proteomics technologies as well as possible transfer models for industrial applications. An accompanying industrial exhibition, as well as a discussion panel, offered the possibility to get in contact with colleagues and potential industrial partners. A visit to the former colliery Zeche Zollern and the social event at the Harenberg City-Center with an excellent view around Dortmund also left time for further communication between the more than 200 attendees.
[Show abstract][Hide abstract] ABSTRACT: Proteomics Data Collection (ProDaC) is an EU funded "Coordination Action" within the 6(th) framework programme. It aims to simplify the publication, dissemination and utilization of proteomics data by establishing standards that will support broad data collection from the research community. The 1(st) ProDaC workshop 2007 (succeeding the kick-off meeting last year at the HUPO World Congress 2006) took place at the Ecole Normale Supérieur in Lyon, France. These workshops take place as regular meetings on a half-year basis. On Thursday April 26(th) 2007 the progress of the first six months of the project was presented by the leaders of each of the seven work packages.
[Show abstract][Hide abstract] ABSTRACT: There is increasing evidence of crosstalk between epigenetic modifications such as histone and DNA methylation, recognized by HP1 and methyl CpG-binding proteins, respectively. We have previously shown that the level of methyl CpG-binding proteins increased dramatically during myogenesis leading to large-scale heterochromatin reorganization. In this work, we show that the level of HP1 isoforms did not change significantly throughout myogenic differentiation but their localization did. In particular, HP1gamma relocalization to heterochromatin correlated with MeCP2 presence. Using co-immunoprecipitation assays, we found that these heterochromatic factors interact in vivo via the chromo shadow domain of HP1 and the first 55 amino acids of MeCP2. We propose that this dynamic interaction of HP1 and MeCP2 increases their concentration at heterochromatin linking two major gene silencing pathways to stabilize transcriptional repression during differentiation.
Nucleic Acids Research 02/2007; 35(16):5402-8. DOI:10.1093/nar/gkm599 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spectral karyotyping (SKY) is a widely used methodology to identify genetic aberrations. Multicolor fluorescence in situ hybridization using chromosome painting probes in individual colors for all metaphase chromosomes at once is combined with a unique spectral measurement and analysis system to automatically classify normal and aberrant chromosomes. Based on countless studies and investigations in many laboratories worldwide, numerous new chromosome translocations and other aberrations have been identified in clinical and tumor cytogenetics. Thus, gene identification studies have been facilitated resulting in the dissection of tumor development and progression. For example, different translocation partners of the TEL/ETV6 transcription factor that is specially required for hematopoiesis within the bone marrow were identified. Also, the correct classification of complex karyotypes of solid tumors supports the prognostication of cancer patients. Important accomplishments for patients with genetic diseases, leukemias and lymphomas, mesenchymal tumors and solid cancers are summarized and exemplified. Furthermore, studies of disease mechanisms such as centromeric DNA breakage, DNA double strand break repair, telomere shortening and radiation-induced neoplastic transformation have been accompanied by SKY analyses. Besides the hybridization of human chromosomes, mouse karyotyping has also contributed to the comprehensive characterization of mouse models of human disease and for gene therapy studies.
Cytogenetic and Genome Research 02/2006; 114(3-4):199-221. DOI:10.1159/000094203 · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reaction of 5,5,5-trifluoro-4-(trifluoromethyl)penta-3-en-2-one (1) with p-toluene-sulfonylhydrazines under basic conditions provided anti-Michael addition products. Acid-catalyzed reactions yielded the expected hydrazones. In the absence of solvent the reaction of (CH3)3SiCl with 1 gave the corresponding silylenolether (3), which did not yield cross condensation products with ketones. In the presence of TiCl4 silylenolether (3) reacted at −78°C to give the corresponding nonadienone (11). Analogously at 20°C condensation with water elimination gave the pyran system (12). The yields of 11 and 12 were increased by adding ZnCl2. It is interesting to note that trimethylsiloxycyclohexene reacted with 1 in a Mukaiyama-aldol-condensation to the corresponding cross condensation product 13. Starting from (CF3)2CC(CN)2 and a mixture of (E/Z)-1,3-pentadiene the expected cyclohexene was formed. One of the two CN-groups was removed with NaOH in C2H5OH/H2O. A second double bond was introduced in an allylic position of the hexene ring by bromination and elimination with (C2H5)3N to yield the diene. Reduction with DIBAlH provided the aldehyde 7,7,7,8,8,8-hexafluorosafranal. Hydrogenation of the C3–C4 double bond in the presence of palladium on a charcoal catalyst led to 7,7,7,8,8,8-hexafluoro-β-cyclocitral. X-ray structures of selected compounds are provided.