Die UVB-abhängige Induktion des Vaskulären Endothelialen Wachstumsfaktors (VEGF) wird in epidermalen Zellen autokrin durch den Transformierenden Wachstumsfaktor alpha (TGF@) reguliert: Bedeutung für die UVB-induzierte

Ein Zusammenhang zwischen chronischer Sonnenexposition der Haut und vermehrter Bildung von Gefäßen bei der Photoalterung und Photokarzinogenese der Haut mit vorzeitiger Alterung und Ausbildung nicht-melanozytärer und melanozytärer Tumoren der Haut kann als wissenschaftlich gesichert angesehen werden. Teleangiektasien, die durch eine Zunahme und Vergrößerung kutaner Blutgefäße gekennzeichnet sind, und die Entwicklung stark vaskularisierter Hauttumoren sind charakteristische Endpunkte einer chronisch sonnenexponierten und sonnengeschädigten Haut. Die zugrundeliegenden molekularen Mechanismen der UV-induzierten pathophysiologischen Angiogenese sind weitgehend unbekannt. In der Tumorprogression sind Angiogenesefaktoren wie der vaskuläre endotheliale Wachstumsfaktor VEGF, ein multifunktionales Zytokin mit mitogener Aktivität für Endothelzellen, von zentraler Bedeutung, da Tumorwachstum und -metastasierung von der Ausbildung eines tumorversorgenden Blutgefäßsystems abhängig sind. In der vorliegenden Arbeit sollte mit molekularbiologischen und zellbiologischen Methoden untersucht werden, ob und in welcher Form solare UVB-Strahlung als schädigende Komponente des Sonnenlichts auf der Erdoberfläche die Regulation von VEGF beeinflußt. Es konnte gezeigt werden, daß UVB-Bestrahlung den Angiogenesefaktor VEGF in epidermalen Zellen auf Proteinebene mit biphasischer Expression nach 4 bzw. 24 h maximal induziert. In Untersuchungen der UVB-induzierten VEGF-Promotoraktivität wurde durch transiente Transfektionen von HaCaT-Zellen mit Deletionskonstrukten des humanen VEGF-Promotors und in Experimenten unter Verwendung neutralisierender Antikörper der transformierende Wachstumsfaktor alpha (TGF) als Mediator der sekundären VEGF-Expression nach UVBBestrahlung identifiziert. Die primäre Induktion von VEGF und die Synthese von TGF sind abhängig von der UVB-Aktivierung des EGF-Rezeptors (EGF-R) und konnten durch den EGF-R-spezifischen Tyrosinkinase-Inhibitor PD 153035 signifikant inhibiert werden. Die UVB-abhängige VEGF-Expression wurde in vivo in menschlicher Haut immunohistologisch bestätigt. Durch die signifikante Verminderung der UVB-abhängig gesteigerten Angiogenese in der Epidermis repetitiv bestrahlter haarloser Mäuse konnte nach intraperitonealer Injektion neutralisierender Antikörper gegen VEGF die kausale Bedeutung von VEGF in vivo im Mausmodell belegt werden. Zusammengefaßt konnten in dieser Arbeit in vitro und in vivo die Bedeutung von VEGF für angiogenetische Prozesse nach UVB-Exposition herausgearbeitet und wesentliche Wege seiner Signaltransduktion identifiziert werden

Subunits of the Histone Chaperone CAF1 Also Mediate Assembly of Protamine-Based Chromatin

One of the most dramatic forms of chromatin reorganization occurs during spermatogenesis, when the paternal genome is repackaged from a nucleosomal to a protamine-based structure. We assessed the role of the canonical histone chaperone CAF1 in Drosophila spermatogenesis. In this process, CAF1 does not behave as a complex, but its subunits display distinct chromatin dynamics. During histone-to-protamine replacement, CAF1-p180 dissociates from the DNA while CAF1-p75 binds and stays on as a component of sperm chromatin. Association of CAF1-p75 with the paternal genome depends on CAF1-p180 and protamines. Conversely, CAF1-p75 binds protamines and is required for their incorporation into sperm chromatin. Histone removal, however, occurs independently of CAF1 or protamines. Thus, CAF1-p180 and CAF1-p75 function in a temporal hierarchy during sperm chromatin assembly, with CAF1-p75 acting as a protamine-loading factor. These results show that CAF1 subunits mediate the assembly of two fundamentally different forms of chromatin

Stress-Induced PARP Activation Mediates Recruitment of Drosophila Mi-2 to Promote Heat Shock Gene Expression

Eukaryotic cells respond to genomic and environmental stresses, such as DNA damage and heat shock (HS), with the synthesis of poly-[ADP-ribose] (PAR) at specific chromatin regions, such as DNA breaks or HS genes, by PAR polymerases (PARP). Little is known about the role of this modification during cellular stress responses. We show here that the nucleosome remodeler dMi-2 is recruited to active HS genes in a PARP–dependent manner. dMi-2 binds PAR suggesting that this physical interaction is important for recruitment. Indeed, a dMi-2 mutant unable to bind PAR does not localise to active HS loci in vivo. We have identified several dMi-2 regions which bind PAR independently in vitro, including the chromodomains and regions near the N-terminus containing motifs rich in K and R residues. Moreover, upon HS gene activation, dMi-2 associates with nascent HS gene transcripts, and its catalytic activity is required for efficient transcription and co-transcriptional RNA processing. RNA and PAR compete for dMi-2 binding in vitro, suggesting a two step process for dMi-2 association with active HS genes: initial recruitment to the locus via PAR interaction, followed by binding to nascent RNA transcripts. We suggest that stress-induced chromatin PARylation serves to rapidly attract factors that are required for an efficient and timely transcriptional response

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location

Chromatin dynamics during spermiogenesis

The function of sperm is to safely transport the haploid paternal genome to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made during the post-meiotic stages of spermatogenesis. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodeling complexes, as well as transient DNA strand breaks. The consequences of mutations have revealed that a protamine-based chromatin is essential for fertility in mice but not in Drosophila. Nevertheless, loss of protamines in Drosophila increases the sensitivity to X-rays and thus supports the hypothesis that protamines are necessary to protect the paternal genome. Pharmaceutical approaches have provided the first mechanistic insights and have shown that hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. We conclude with a model that illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved

Chromatin dynamics during spermiogenesis

The function of sperm is to safely transport the haploid paternal genome to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made during the post-meiotic stages of spermatogenesis. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodeling complexes, as well as transient DNA strand breaks. The consequences of mutations have revealed that a protamine-based chromatin is essential for fertility in mice but not in Drosophila. Nevertheless, loss of protamines in Drosophila increases the sensitivity to X-rays and thus supports the hypothesis that protamines are necessary to protect the paternal genome. Pharmaceutical approaches have provided the first mechanistic insights and have shown that hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. We conclude with a model that illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved