Final Activity Report Summary - CHROMOSOMAL DOMAINS (A novel ex vivo approach to study the role of chromatin in the regulation of transcription)
In the eukaryotic cell the genetic information encoded in the sequence of the DNA is accommodated by the structure of chromatin. Chromatin composition and structure change dramatically depending on the transcriptional state of a genomic locus. Consequently, research of the past two decades has indicated that the cell employs chromatin for coordinated expression of its genome. The molecular mechanisms underlying this regulation are still poorly understood.
We aim to understand how chromatin related processes influence gene expression. For this purpose we have developed a technique, allowing us to purify native chromatin of a chromosomal locus of interest in the yeast S. cerevisiae (Griesenbeck et al., 2003). We want to obtain a full molecular description of chromatin composition and structure associated with distinct transcriptional states of a gene. At the same time we try to establish experimental systems to test the in vivo relevance of the results obtained by analysis of the purified biological material.
We have initially focused on the RNA Polymerase II transcribed single copy gene, PHO5, encoding for a secreted acidic phosphatase. This gene has served as a paradigm for a defined chromatin transition controlling transcription of the locus. However, isolation of sufficient material for the analyses intended proofed to be difficult. This was one reason directing our interest to the RNA Polymerase I transcribed 35S ribosomal RNA (rRNA) genes, which are present in 150 copies in average in a wild type yeast cell. In the last two years we have successfully established the basis to explore this locus: We are currently performing large scale purifications from which we expect detailed information about the chromatin composition of active and inactive genes. Furthermore we developed new methods for analysis of ribosomal DNA chromatin in vivo. Thus, we can unambiguously show if a known factor associates with either one of the two distinct trancriptional states of rRNA genes present in every living cell. Data obtained by the latter analyses allowed us to directly test hypotheses about regulation of rRNA gene transcription.
The combination of a straight-forward biochemical approach to purify and analyse natural chromatin, together with cell biological methods investigating chromatin in vivo will eventually lead us to understand important aspects of the relationship between local chromatin structure and transcriptional regulation.
We aim to understand how chromatin related processes influence gene expression. For this purpose we have developed a technique, allowing us to purify native chromatin of a chromosomal locus of interest in the yeast S. cerevisiae (Griesenbeck et al., 2003). We want to obtain a full molecular description of chromatin composition and structure associated with distinct transcriptional states of a gene. At the same time we try to establish experimental systems to test the in vivo relevance of the results obtained by analysis of the purified biological material.
We have initially focused on the RNA Polymerase II transcribed single copy gene, PHO5, encoding for a secreted acidic phosphatase. This gene has served as a paradigm for a defined chromatin transition controlling transcription of the locus. However, isolation of sufficient material for the analyses intended proofed to be difficult. This was one reason directing our interest to the RNA Polymerase I transcribed 35S ribosomal RNA (rRNA) genes, which are present in 150 copies in average in a wild type yeast cell. In the last two years we have successfully established the basis to explore this locus: We are currently performing large scale purifications from which we expect detailed information about the chromatin composition of active and inactive genes. Furthermore we developed new methods for analysis of ribosomal DNA chromatin in vivo. Thus, we can unambiguously show if a known factor associates with either one of the two distinct trancriptional states of rRNA genes present in every living cell. Data obtained by the latter analyses allowed us to directly test hypotheses about regulation of rRNA gene transcription.
The combination of a straight-forward biochemical approach to purify and analyse natural chromatin, together with cell biological methods investigating chromatin in vivo will eventually lead us to understand important aspects of the relationship between local chromatin structure and transcriptional regulation.