Final Report Summary - ACCOMPLI (Assembly and maintenance of a co-regulated chromosomal compartment)
Eukaryotic nuclei integrate form (structural organisation) and function in distinct compartments that are enriched in certain molecules, chemical modifications or biochemical activities and therefore specify localised functional outputs. On such compartment is the X chromosome in male cells of Drosophila, which is subject to global transcription regulation. A dedicated transcription regulator, the Male-specific-lethal Dosage Compensation Complex (MSL-DCC) distinguished the X from the autosomes and doubles the transcription output of all genes to match the expression with that of the two X’s in females, a process termed ‘dosage compensation’. The X chromosome forms a chromosomal territory within nuclei, which qualifies as a nuclear compartment, as autosomal genes will be subject to dosage compensation, if integrated into the X.
How is the X chromosome recognised? How is the chromosomal environment formed that imposed activation on any gene that resides in it? ACCOMPLI research explored these questions by studying the structure and functions of the non-coding RNA and protein subunits that form the DCC and by monitoring DCC interactions with the X chromosome in time and space.
The structure of the DCC, which is composed of five proteins and a long, non-coding roX RNA, is key to its function. We determined the folding of the roX RNA and found that the prevailing secondary structure corresponds to an inactive conformation that needs to be altered by one of the DCC subunits, the RNA helicase MLE. The remodeling of roX exposes RNA sequences that serves as binding sites for the MSL proteins, thus reconstituting a switch initiating DCC assembly.
Key to the formation and function of the MSL-DCC is the subunit MSL2, which is only expressed in male cells. We explored diverse functions of MSL2. We found that ML2 bears E3 ligase activity that assures assembly of appropriate levels of the complex. This is critical for its function since excessive amounts of the MSL2 and of the DCC leads to false regulation of autosomes as well. Some ubiquitylation marks on MSL proteins are stable and do not lead to degradation. They may constitute signals that we do not yet understand.
MSL2 is also the DNA binding subunit of the DCC. We identified and studied its ‘CXC’ domain as a DNA binding module. Applying genome-wide DNA binding assays in vitro (DNA immunoprecipitation combined with deep sequencing) we found that the CXC domains can recognise an X chromosome-specific DNA element, which we termed ‘PionX’. PionX sites are early X chromosome-specific determinants involved in faithful dosage compensation. They are preferentially bound by an early intermediate of DCC assembly. They are the first to be occupied during de novo establishment of dosage compensation. Remarkably, the work of others on X chromosome evolution in Drosophila miranda suggests that PionX sites originate early during X chromosome evolution by neoX-specific deletion of a transposon-derived precursor sequence.
We characterised the spatial interactions of PionX sites and other DCC binding sites on the X chromosome in the context of X chromosome folding by HiC and 4C analyses. The DCC profits from long-range interaction of chromosomal segments in the active compartment of the X chromosomal territory. Our ability to induce DCC binding to PionX sites allowed monitoring the reach of its transcription activation activity in space. We found that PionX-bound DCC may activate target genes of megabase distances if the corresponding target genes are brought into proximity by chromosome folding.
How is the X chromosome recognised? How is the chromosomal environment formed that imposed activation on any gene that resides in it? ACCOMPLI research explored these questions by studying the structure and functions of the non-coding RNA and protein subunits that form the DCC and by monitoring DCC interactions with the X chromosome in time and space.
The structure of the DCC, which is composed of five proteins and a long, non-coding roX RNA, is key to its function. We determined the folding of the roX RNA and found that the prevailing secondary structure corresponds to an inactive conformation that needs to be altered by one of the DCC subunits, the RNA helicase MLE. The remodeling of roX exposes RNA sequences that serves as binding sites for the MSL proteins, thus reconstituting a switch initiating DCC assembly.
Key to the formation and function of the MSL-DCC is the subunit MSL2, which is only expressed in male cells. We explored diverse functions of MSL2. We found that ML2 bears E3 ligase activity that assures assembly of appropriate levels of the complex. This is critical for its function since excessive amounts of the MSL2 and of the DCC leads to false regulation of autosomes as well. Some ubiquitylation marks on MSL proteins are stable and do not lead to degradation. They may constitute signals that we do not yet understand.
MSL2 is also the DNA binding subunit of the DCC. We identified and studied its ‘CXC’ domain as a DNA binding module. Applying genome-wide DNA binding assays in vitro (DNA immunoprecipitation combined with deep sequencing) we found that the CXC domains can recognise an X chromosome-specific DNA element, which we termed ‘PionX’. PionX sites are early X chromosome-specific determinants involved in faithful dosage compensation. They are preferentially bound by an early intermediate of DCC assembly. They are the first to be occupied during de novo establishment of dosage compensation. Remarkably, the work of others on X chromosome evolution in Drosophila miranda suggests that PionX sites originate early during X chromosome evolution by neoX-specific deletion of a transposon-derived precursor sequence.
We characterised the spatial interactions of PionX sites and other DCC binding sites on the X chromosome in the context of X chromosome folding by HiC and 4C analyses. The DCC profits from long-range interaction of chromosomal segments in the active compartment of the X chromosomal territory. Our ability to induce DCC binding to PionX sites allowed monitoring the reach of its transcription activation activity in space. We found that PionX-bound DCC may activate target genes of megabase distances if the corresponding target genes are brought into proximity by chromosome folding.