Autophagy transcriptional crosstalk: the LMX1A/LMX1B paradigm

Autophagy is an important quality control process carried out by all cells to assist with their normal biological functions and to protect them from the stresses they inevitably encounter as part of multicellular life. It involves the packaging of damaged and potentially harmful cellular material into membrane-bound sacs (called autophagosomes) for delivery to the waste disposal centre of the cell. For this reason, autophagy is a key facet of normal tissue homeostasis across the lifespan, and is a critical protector against important human diseases including neurodegeneration, heart disease, diabetes and cancer. Over recent years we have learned much about how the autophagy process is regulated within cells - culminating in the award of a Nobel Prize in 2016 to Prof. Yoshinori Ohsumi, one of the pioneers in the field - but we still know relatively little about how the genes that oversee the autophagy process are controlled in response to stress. The process of gene expression (switching genes on) involves the binding of proteins called transcription factors to activating sites on the cell's DNA. We have found that an important pair of transcription factors - called LMX1A and LMX1B - switch on critical autophagy genes to help with the formation and function of cells in important organs such as the kidney and (parts of) the brain. Having confirmed roles for these transcription factors during autophagy gene expression, the challenge has been to better understand how they are themselves regulated. Surprisingly, we have discovered that a key protein needed for the formation of autophagosomes - called LC3 - binds to LMX1A/LMX1B to increase their ability to stimulate autophagy gene expression, meaning that the autophagy machinery can potentially control itself by switching on its own genes. In this proposal we aim to provide a clear picture of the molecular mechanisms central to this exciting finding, and by doing so we will determine whether this novel scenario extends to other autophagy and related transcription factors as part of concerted efforts to better understand how cells cope with stresses and challenges across the lifespan.

This is a frontier bioscience project that seeks to understand fundamental processes in cell biology. That said, autophagy is essential for normal health in humans, animals and plants, being required during various stages of development and helping to keep tissues functioning normally during ageing. For this reason, autophagy is being vigorously researched as a potent pharmaceutical target in various diseases associated with ageing, notably neurodegeneration and cancer, and as a route to improve crop productivity and in bioengineering. Our efforts to better understand the potential crosstalk within the autophagy gene expression pathways in humans has the potential to be exploited in disease therapy, and for our part we will therefore examine how the novel pathway we have discovered impacts on the health of important cell types in brain (neurons) and kidney (podocytes) cell models.

Autophagy is a major cellular stress response that plays key roles during differentiation, development and homeostasis. Relatively little is known about how autophagy is controlled transcriptionally in specific tissues, and large gaps remain in our understanding of how this is in turn regulated during cellular stress adaption. Our investigation into the molecular regulation of two related homeodomain transcription factors (TFs), LMX1A and LMX1B, has revealed new roles for these proteins in the regulation of basal and inducible autophagy in vitro. This proposal is based on further key observations, including: our identification of a conserved LIR (LC3-interacting) motif in LMX1B that enables binding to multiple ATG8 proteins (LC3s; GABARAPs) in vitro and in human cells; our demonstration that LMX1B-LC3B binding is nutrient status and localisation-dependent, being restricted to the nucleus in full nutrient conditions and emerging in the cytoplasm during starvation; and crucially, our finding that nuclear ATG8 binding is needed for LMX1B-mediated transcription and protection against cell stress in human midbrain dopaminergic neurons (mDANs). Thus, we provide the first evidence for ATG8-mediated transcriptional activation to influence cellular stress protection responses. In this proposal we will: (i) further characterise the LIR-dependency of LX1A/LMX1B-ATG8 transcriptional control; (ii) establish how ATG8 binding modulates LMX1A/LMX1B activity to control cellular differentiation and survival in kidney and mDAN models; and (iii) determine how widely our model of ATG8-mediated transcriptional control extends amongst TF classes. Together, this work will establish a novel, non-canonical autophagy role for ATG8 proteins as modulators of the transcriptional landscape across the life-course. This new regulatory hub presents new opportunities for strategies that target the autophagy cell stress response in health and disease.

There is great interest in the possibility to target existing cellular pathways for therapeutic benefit. Particularly, those pathways that are identified as being altered in disease are prominent targets for correction and/or manipulation. Autophagy dysfunction is a common feature of numerous human diseases (including neurodegenerative disease, diabetes, heart disease, cancer), highlighting the importance of a full understanding of autophagy control to guide possible future clinical intervention. Advancing our basic understanding of a critical cellular process such as autophagy, it is most likely our work will inform long term projects in other fields including biotechnological and pharmaceutical industries.

Who might benefit and how?

Clinicians - the challenge of identifying strategies to up-or down-regulate autophagy in given disease contexts depends on a solid base of fundamental knowledge. We understand much about how autophagy is controlled at the protein machinery/post-translational level, but know much less about the control of autophagy gene transcription. This provides an important route to autophagy control, highlighting the value in focussed research in this area. Our work will address, for the first time, how autophagy proteins are able to reciprocally influence their own transcription via direct binding to prominent autophagy transcription factors. Clinicians will benefit along the line from knowledge of autophagy transcriptional control, and how this changes and potentially can be changed in disease settings.

Industry - Our approach to identify and characterise novel interactions between important transcription factors and the autophagy machinery is likely to pinpoint a range of possible pathways for intervention. As ATG8 binding to transcription factors to modify their functions represents an integrated pathway of cofactor control, the ability to stabilise or interfere with this interaction through e.g. small molecules represents an interesting path to novel autophagy modulating compounds.

The public - In addition to the broad benefits that understanding fundamental bioscience brings in the longer term, this work addresses directly key areas of health that have the potential to impact the long-term health of the general (ageing) population. Autophagy is needed for homeostasis of diverse tissues, and is necessary for healthy lifespan benefits in model systems in vivo. Key research into the control of autophagy therefore has the potential to benefit many areas of health.

Bioscience researchers - This project includes considerable opportunity to train the PDRA in areas that go
beyond the day-to-day research methodology. Examples include public communication and outreach opportunities and the extensive network of University schemes to benefit the training and development of research staff (Bristol is at the forefront of research staff development). I have a good track record in facilitating the placement of staff in areas outside our core research activity including computer programming, teaching, patent law. This demonstrates that the environment provided by our own labs a well as our institutions more widely are highly conducive to career development of our staff beyond academic, basic science research, thereby contributing to the economic development of the nation. Our projects are also very data intensive, and the management and analysis of such large (terabyte) datasets is applicable to many areas of professional life.