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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - SFSASP (Structural and Functional Studies of ATRX- Syndrome Protein)

Teaser

Summary

The project aimed at understanding the molecular basis of function of ATRX, a protein that when mutated causes ATRX syndrome. Using structure-function approaches, our investigations addressed different aspects of ATRX function in cells.

ATRX syndrome is characterized by severe mental retardation, thalassemia, facial deformities and disability. ATRX is a serious genetic disorder, which results in a considerable increase in both acute and chronic morbidity, and mortality. Although ATRX is a rare disease, treatment is intensive and the lifetime cost of treating an ATRX patient is estimated to be ~Â£800,000. There is potential for commercialisation and exploitation of the scientific knowledge originated from this research as understanding how ATRX interacts with its target sequences may provide new druggable targets. Moreover, ATRX has also been implicated in many cancers, particularly in sarcomas, and the knowledge gained from our work will also improve the tools available to diagnose and treat cancers that display ATRX deregulation.

The overall aim of the project was to define at a molecular level how ATRX remodels chromatin.

Objective 1: To elucidate the structure of ATRX Snf2 domain by X-ray crystallography at various steps in ATP hydrolysis cycle
Objective 2: To investigate the structural preference of ATRX for various DNA substrates that are likely to be formed at interstitial repeat regions where ATRX is known to bind in vivo.
Objective 3: To investigate the interaction of ATRX with DAXX and Histone 3.3

Our main result from this project was the dissection of the ATRX - MeCP2 interaction in the context of the ATRX/RETT syndrome mutations. We were able to show direct interaction between ATRX and MeCP2. We have observed that this interaction is abrogated by most of the RETT-causing mutations in MeCP2. We are at present investigating if ATRX syndrome-causing mutations in ATRX also affect this interaction.

Work performed

The project took a multi-tongued approach in investigating ATRX function.

One approach was to understand how ATRX localizes on heterochromatin and how it interacts with the complex DNA structure. In this approach we made a battery of different ATRX constructs and attempted to express them in bacteria. We were able to generate many constructs of different regions of ATRX and we managed to express and purify the HELICc construct of ATRX Snf2 domain.

The second approach was to investigate the interactions of ATRX with other chromatin proteins. Two of the many partner proteins of ATRX are DAXX and Histone H3.3. DAXX is a histone chaperon protein, particularly Histone H3.3. The interaction of DAXX is crucial in successful deposition of histone H3.3 onto promyelocytic leukemia bodies. In this aim, we made a series of constructs covering different regions of DAXX in addition to the constructs of ATRX. The proteins were expressed in E. coli and purification protocol was set-up. We were also able to successfully generate the Histone H3.3 construct as well as the ADD domain construct of ATRX.

Finally our third approach was to investigate the interactions of ATRX with a methyl-DNA binding protein, MeCP2, which also localizes on heterochromatin. Our results showed that the two proteins interact directly with each other. We attempted crystallizing this complex and performed SAXS analysis to gain low resolution information on the structural features of the ternary complex. We also performed MicroScale thermophoresis (MST) to estimate the affinity of interaction of MeCP2 and ATRX. In addition, we generated RETT syndrome causing mutations in MeCP2 AxID construct and performed a pull-down assay with the RETT mutants against ATRX HELICc protein. Our results showed that out of the twelve mutants we considered, eight mutations completely abrogated the binding while other three reduced the binding significantly. This strongly suggests that the interaction between ATRX and MeCP2 is clinically important. Moreover, RETT mutations found in the AxID region have a direct effect in disrupting this interaction. We are at present performing a similar complementary experiment with the ATRX syndrome mutants in HELICc domain and wild type MeCP2 AxID. We are very close to finishing these studies and we will soon publish the data in order to disseminate our findings.

The molecular basis of ATRX function in cells is currently not clear. Our work here has highlighted one of the crucial previously unexplored function of ATRX, its interaction with MeCP2. We have characterized the interaction at molecular level. An X-ray structure of the complex, which is on-going work at present, will provide even more detailed characterization of this interaction. The results of our studies will provide key insights into the molecular mechanism of how ATRX interacts with MeCP2. Our investigation will further provide answers as to how the disease causing mutations affect the interaction between these two proteins.

Final results

This project has advanced the current knowledge in the area of ATRX research, in that we have discovered a crucial link between the RETT and the ATRX syndromes, by virtue of the interaction between ATRX and MeCP2 proteins. This finding is novel and of likely impact, in the sense that it has the potential to progress the understanding of two mental retardation syndromes simultaneously. Just like mutations in ATRX lead to the ATRX syndrome, mutations in MeCP2 gene cause RETT syndrome, accounting for more than 90 % of cases. RETT is one of the leading causes of mental retardation in females but despite extensive research not enough is known about the molecular basis of its function, signalling and interaction with partner proteins.

The main impact of this research will be an academic one. The likely beneficiaries will be cell and molecular biologists who want to understand the mechanism of chromatin remodelling and medical scientists who want to study the aetiology of mental retardation syndromes. Our research will provide fellow academics and researchers important information in better understanding the mechanisms of both ATRX and MeCP2 function and regulation. The structural data of complexes produced from our results will form the basis of further drug discovery projects in academia and industry. This aspect of our work will benefit researchers from both RETT as well as ATRX syndrome fields. Our interaction studies will not only increase our knowledge in MeCP2 function but will also unravel previously unknown regulation pathways of ATRX in the cells.

Because of the potential broad range of interest arising from the generated data, the results will be widely disseminated in scientific peer-reviewed periodicals, in presentation at scientific meetings and through the early deposition in public access databases. The crystallographic and EM details will be deposited with the Protein Data Bank.

Our studies might lead to better diagnosis and treatment of RETT and ATRX syndromes, which in turn would benefit healthcare institutions around the world. At present the treatments for these syndromes are symptomatic and better treatments would dramatically reduce the cost of care and support for such patients. The outcomes in terms of better drugs and treatment of RETT and ATRX syndromes, might generate revenue as well as save financial resources that could be redirected from this area into other healthcare areas.