Struktur und Dynamik transkriptionell aktiver Chromatindomänen von Drosophila melanogaster Interphasechromosomen


The interphase chromatin is folded into spatial domains that compact the DNA and organize the regulation of gene activity, replication, repair and recombination. High throughput methods give valuable information on the chromatin organization on a genomic scale, however, relatively few domains have been studied in detail so far. Therefore, we engineered a condensed chromatin cassette with an internal attB sequence for site specific recombination of attP tagged DNA elements triggered by C31 recombinase. Insertion of this condensed chromatin cassette into the Drosophila genome formed an additional band on polytene chromosomes that can be used to screen for DNA elements with the capacity to form open chromatin. We demonstrated that site specific recombination of DNA forming decondensed chromatin at their endogenous sites (3C6-7 or 61C7-8) resulted in splitting of the condensed chromatin domain cassette by less condensed chromatin. The 61C7-8 interband (~20 kb) contains four protein coding genes and one noncoding transcript that are moderately transcribed in salivary glands. It also contains several overlapping binding sites for the insulator proteins BEAF and CP190 and the insulator associated chromatin protein Chriz that bind both within the endogenous and the ectopic 61C7-8 domain. The Chriz protein together with the zinc finger protein Z4 and the H3S10 kinase Jil-1 targets H3S10 phosphorylation (Gan et al., 2011) that is responsible for to open chromatin formation (Deng et al., 2008). By site specific recombination of several 61C7-8 deletions we mapped the DNA essential for open chromatin domain formation of the whole 61C7-8 locus to a proximal 490 bp fragment. (Zielke and Saumweber, 2014) that overlaps strong binding sites for Chriz, BEAF and CP190. Elimination of this fragment abrogates the binding of all three proteins. According to available data the chromatin configuration of the 61C7-8 open domain is remarkable stable in different cell lines and tissues. Our project is aimed at the understanding of the structure and 3D folding of this open chromatin domain in relation to insulator protein binding and gene activity by comparing the endogenous locus with modified versions at the ectopic position. In addition to the deletions already available we will remove selected BEAF binding sites or promoter elements in 61C7-8 and test their requirement for the decondensed state by our cassette system. Our project then has the following subjects: First, to explore the dynamics of the chromatin domain, we will test 61C6-7 chromatin protein binding, histone modifications and gene expression at different stages of development (early embryos, larval and adult tissues) using a home made tiling array of the region. Second, by ChIP-PCR and RT-PCR experiments with chromatin from 3rd instar salivary glands we will compare protein binding, histone modifications and transcription at the ectopic domain with that at the endogenous locus. The sequence of the ectopic and the endogenous 61C7-8 DNA varies at a number positions, which will be used to distinguish between them by PCR. This will allow us to assay the requirement of DNA elements for protein binding, histone modifications and transcription at both sites simultaneously. Third, we will perform a domain wide 3D interaction analysis by 4C comparing ectopic domains with different deletions and point mutations with the endogenous locus. This will explain how rather restricted elements can influence the chromatin structure of the whole domain. Fourth, investigation of interband chromatin will allow generalizations on mechanisms responsible for the formation of open chromatin domains. Using our condensed chromatin domain cassette we obtained 43 insertions into interbands at different genomic positions. Their genomic coordinates will be used as starting point for fine mapping of open domains by a combination of data mining for protein binding and histone modifications from modENCODE and high resolution in situ mapping.


Projektleitung
Saumweber, Harald Prof. i.R. Dr. rer. nat. (Details) (Zytogenetik)

Laufzeit
Projektstart: 09/2014
Projektende: 10/2016

Publikationen
J Cell Sci (2014) 127, 2365-75

Zuletzt aktualisiert 2020-11-11 um 10:34