How do H2A.Z-nucleosomes control the temperature transcriptome?

All living things are sensitive to their temperature. Mammals like ourselves have evolved ways to keep their body temperature fairly constant, and this ensures that our cells can work at their full efficiency. During fever though, our body temperature rises, and during hibernation, animals allow their temperature to drop to very low levels, so even mammals have significant variation in their temperature. Although we have known how important temperature is for life for hundreds of years, how temperature is sensed is not clearly understood. Plants often cannot prevent large fluctuations in their temperature, and they have evolved very sophisticated ways to measure temperature, and adjust the behaviour of their cells to adapt to these changes. Plants are therefore a perfect system to study how higher (non-bacterial) organisms sense temperature. We have found that plants actually change the way their DNA is wrapped when the temperature changes. When temperature increases, plant DNA becomes less tightly packed. This allows genes to be switched on in response to temperature. What we do not know is whether this is a direct effect of temperature on DNA, or is more indirect. In this study, we will determine the mechanism of how this change is controlled, and see if other higher organisms, such as yeast and mammals respond to temperature in the same way. Understanding how temperature is sensed is important because it will help us to create crops that are resilient to climate change. Higher temperatures are particularly damaging to crop yields since they cause the plant to make less grain which is of a poorer quality. For example during the hot summer of 2003, wheat yields in France decreased by about 20 %. The aim of our research is to understand how temperature is sensed well enough that we can breed plants with improved temperature sensing characteristics. In this way it may be possible to create crops that are better able to cope with climate change. Crops have been bred for thousands of years and selected to have a very sensitive response to temperature. While this is normally a good thing, sometimes, for example in the developing grain, it has bad effects on crop yields. If we fully understand the molecules that cause a plant to sense temperature we will be able to alter how different parts of the plant sense and respond to temperature. This would be very valuable, since variation in temperature is a major cause of lost yield in agriculture. Moreover climate change is increasing the severity of temperature shocks, as well as their frequency. Temperature can affect crops in many different ways, for example interfering with crop scheduling by changing flowering time. Some crops are also induced to flower inappropriately (for example lettuces) by temperature changes. Wheat and rice are particularly sensitive to high temperatures during grain filling. Being able to breed crops with optimal temperature responses would enable farmers to be able to grow crops reliably in the face of higher and more variable temperatures. This will be important for sustainability (fewer crops will be lost and yields will be saved) and food security, which will be particularly important in a world of 9 billion people.

How eukaryotic cells sense temperature is a key, but poorly understood area of biology. Plants offer a tractable system to address this problem, having excellent genetics and multiple transcriptional and developmental responses to small changes in temperature. We have previously found that chromatin structure mediates all the developmental responses to warmer temperature that we have assayed, and at the transcriptome level accounts for roughly half of all the transcriptome changes in response to temperature. We have been able to show that these differences are mediated by the alternative histone H2A.Z, and in the absence of H2A.Z incorporation, the warm temperature transcriptome is constitutively expressed. Our results indicate that H2A.Z occupancy changes in response to temperature. This effect could be due to a either a direct effect of temperature on the interaction between DNA and the nucleosome or more indirectly through posttranslational modification of the H2A.Z-nucleosome in response to temperature that changes the behaviour of the nucleosome. We shall test these two possibilities in a series of in vitro and in planta experiments. To test if nucleosomes respond directly to temperature, we shall reconstitute nucleosomes in vitro and measure their dynamics as a function of temperature. We shall complement these experiments with in planta analyses to determine if histones are post-translationally modified in response to temperature. These studies will help us understand how H2A.Z-nucleosomes control the transcriptome in response to temperature. We shall analyse the behaviour of S. pombe and S. cerevisiae H2A.Z mutants to determine the degree of conservation of the pathway. Chimeras between H2A.Z and H2A informed by structural analysis will be expressed in plants. Finally, we shall also use Illumina sequencing to identify further alleles that modify the H2A.Z signalling pathway to determine if these are involved in modulating a temperature signal to chromatin.

Our work has a number of pathways to impact at both the academic and economic level. These are outlined below: This work is of academic interest, and knowledge of how temperature is perceived will be of international academic impact and is likely to be published in high impact journals. Work in the areas of nucleosome structure and temperature from the Rhodes and Wigge lab has been published in Nature and Cell, and received international interest. Our work is innovative and cross-disciplinary, combining structural biology and cutting edge methods for the in vivo synthesis of modified histone proteins with in planta biological assays. Our study will further the academic discipline through contributing to the training of researchers skilled in chromatin analysis in plants from a structural perspective, which is a critical, yet underskilled area in UK science. Apart from the direct advances in plant biology described in this project, discoveries we make are likely to have a broad interest to biologists. Specifically, H2A.Z-nucleosomes are essential for animal development, and they have been shown to play a direct role in stem cell differentiation and cancer progression. The very high degree of H2A.Z sequence and structural conservation across the eukaryotic kingdom and their conserved genomic distribution indicate that advances obtained from understanding how H2A.Z-nucleosomes signal in plants will have impact across the biological systems. This has been reflected in the widespread interest from non-plant scientists in our recent discovery that H2A.Z-nucleosomes control the temperature transcriptome. Our work has considerable potential for societal and economic impact. Our initial discovery that chromatin structure mediates temperature signals has lead to a patent, and we have entered negotiations with Monsanto, Dow Agrosciences, BASF and Pioneer-Dupont to apply the technology to crop plants. A common feature of these negotiations has been a request for a closer molecular understanding underpinning to the role of H2A.Z-nucleosomes that we have described. The chief scientists of these companies said that this greater understanding would greatly facilitate investment in this technology by the industrial sector. We have a clear route to commercialisation and exploitation, and have already filed IP and issued technical description sheets on aspects of the technology with Plant Biosciences Limited (PBL). Climate change is a major concern that directly threatens food security by increasing the frequency and severity of hot summers. From analysing crop data over the last 100 years, it has been shown that for every 1 degree Celsius increase in mean temperature, there is a decrease in yield of between 2.5 and 16 % from high temperature effects alone, excluding the effects of drought or changes in land use etc. The UK chief scientific advisor, John Beddington, has identified a 'perfect storm' scenario during this century when the need for increased global agricultural yields will directly conflict with decreases in yields caused by climate change. Our work directly addresses how tissues sense temperature, and we are exploring mechanisms to selectively alter temperature perception pathways for example in the developing grain to protect it against heat stress in collaboration with industrial partners. Understanding the molecular basis of temperature signaling through H2A.Z-nucleosome offers the prospect of being able to engineer specific versions of H2A.Z-nucleosomes to control temperature responses in a tissue specific manner. The work in this proposal will therefore directly inform our collaborations with the agricultural biotech industry. Improving the yields of crop plants under a climate-change scenario will contribute to environmental protection and food security. More wide-ranging, longer term implications from improved food security such as improved national security and social cohesion follow from this.