Structure and dynamics of transcriptionally active chromatin domains of Drosophila melanogaster interphase chromosomes

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Our current data show that the 61C7-8 locus in salivary glands is formed by a ~20 kb open chromatin domain that contains four protein coding genes and one noncoding transcript. By site directed recombination we generated a wild type and partial deleted versions of the locus at the same ectopic position that were initially characterized by cytogenetic methods. In the proposed project we want to study the function of key DNA-elements for the formation of 61C7-8 chromatin at high resolution with the following objectives:1. Determination of the 61C7-8 open domain structure (histone modification and insulator protein binding) in different tissues and stages of development. 2. Determination of the influence of DNA elements on a 61C7-8 domain structure and transcription.3. Determination of the influence of BEAF-binding- and promoter-elements on the 3D folding of the 61C7-8 domain.4. Mapping and initial characterization of BEAF regulated active chromosomal domains in Drosophila.Taking 61C7-8 as a model the experiments will provide understanding on how an open chromatin domain is formed in development. It will further provide information on the stability of basic domain structure and the extent of its modifications in different tissues in correlation to gene expression. The experiments will show the importance of the insulator proteins BEAF, CP190 and Chriz for the formation of chromatin modifications and folding of an open chromatin domain. Moreover, they will address how Chriz and CP190 may contribute to both aspects independently of BEAF binding and will address the mutual dependency of factor binding and transcriptional activity. The elimination of single BEAF binding sites within the domain will allow us to understand the mutual interaction between factors bound at individual sites and possibly the hierarchy of interactions within the domain.Finally, the experiments are intended to extend our 61C7-8 related studies to further open chromatin domains with a similar BEAF connected architecture. Mapping of more open domains will influence our current concepts on polytene and diploid chromatin domain structure, in particular our concepts on interbands as open chromatin domains. In the future the methods established for the 61C7-8 region may be used and extended for the study of open chromatin domains that depend on other insulator proteins.