Epigenetic control of gene transcriptional and behavioural responses to stress in the dentate gyrus

Lead Research Organisation: University of Bristol
Department Name: Henry Wellcome LINE


Stress affects the lives of both humans and animals in our society. Most debilitating for mental welfare and wellbeing in humans is psychological stress, for instance marital problems, job insecurity, and bullying. In farmed and companion animals, examples of psychological stress include overcrowding (e.g. chickens, pigs), long-lasting transports (e.g. cows, sheep, pigs) and abuse. Successful coping with such stressful events involves adaptive and cognitive processes in the brain that make the individual more resilient to repeated stress in the future. Some events, however, are so traumatic that the memories of them become a tremendous burden, leading to psychosomatic and behavioural disturbances and psychiatric diseases (anxiety and depression). To help people to cope with stress in their lives, to develop directives to reduce stress and to improve wellbeing of our companion, farmed and laboratory animals, we need to obtain better insight into how the brain processes stress and creates memories of psychologically stressful events. At present it is unclear how the healthy brain adapts to and learns from stressful events. We have recently discovered that after stress certain molecular processes ("epigenetic modifications") occur in the nucleus of nerve cells which are crucial for the expression of genes necessary for the adaptation of these cells to a stressful event. Our proposal aims to investigate the role of these epigenetic modifications in the regulation of gene expression and the identity of the genes affected by these modifications. Obtaining this information is crucial to understand how nerve cells "learn" from stressful events. Insight into these processes and the function of the genes involved will help to develop new ways to reduce the burden of stress-related disorders in humans and animals.

Technical Summary

Dealing with a psychologically stressful event requires, in addition to generation of the acute stress response, cognitive processing so that memories can be formed enabling appropriate responses in case of future recurrences. Insight into the molecular and cellular mechanisms underlying stress coping and stress processing in the brain is clearly vital for the development of strategies to improve the quality of life and maintaining (life-long) health in humans and animals. We have found that, in the case of a learned behavioural response to stress in rats and mice, this process involves (1) chromatin remodelling (driven by phosphorylation and subsequent acetylation of histone H3 (H3S10p-K14ac)) to facilitate transcriptional induction of immediate-early genes (IEGs; c-Fos, Egr-1) in sparse dentate gyrus (DG) neurons in the limbic brain and (2) the transcription factor CREB which is a main trans-activator of IEG gene transcription but found to be phosphorylated (pCREB) in all DG neurons. We hypothesise that 1. The psychological stress-induced H3S10p-K14ac epigenetic marks within gene promoters in DG neurons are a prerequisite for pCREB to gain access to CREs within the egr-1, c-fos and other promoters for activating gene transcription which is required for the consolidation of stress-related memory; and 2. Consequently, the CRE-dependent gene transcriptional response in DG neurons showing the H3S10p-K14ac histone marks will be distinct from the CRE-dependent transcriptional response in dentate neurons not presenting these marks. To test these hypotheses we will use state-of-the-art lentiviral-driven RNA interference technology, chromatin immuno-precipitation (ChIP), gene promoter identification using Illumina next generation sequencing (ChIPseq), and RNA Sequencing (RNA-seq) analyses in combination with neuroanatomical, immunohistochemical and behavioural analyses.

Planned Impact

Who will benefit from this research?

There are a number of beneficiaries for whom this research could be helpful in the longer term:

1. Owners of companion and farmed animals with stress-related behavioural disturbances
2. Patients suffering from stress-related disorders
3. Family and friends of such patients
4. The economy
5. The government and the National Health Service
6. Academia

How will they benefit from this research?

1. Owners of companion and farmed animals with stress-related behavioural disturbances. Stress is a common problem for companion and farmed animals. It can lead to behavioural (e.g. stereotypy, aggression), reproductive (e.g. infertility) and other disturbances. Our research will help to improve treatment of such animals which will benefit their health and wellbeing. Owners of companion animals will have a much more pleasant pet and farmers will have less economic loss. From a different perspective, our research will increase the awareness that stress has indeed a long-term impact on the animals' behaviour. Since (bad) experiences of the animals appear to become hardwired into their brain it may take substantial efforts to undo these changes and obtain a healthy animal again. This should motivate pet owners and farmers to treat their animals properly. Our research may also help to improve legislation.

2. Patients suffering from stress-related disorders. Clearly, at present there is no satisfactory treatment for stress-related disorders such as psychosomatic disturbances (e.g. low-back pain, gastro-intestinal complaints) and psychiatric diseases (anxiety and major depressive disorders). The main reason for this situation is that the underlying neurobiology of these disorders is still unknown. Our research has the potential to lead to the development of new drugs for the treatment of stress-related disorders in the future. It should be noted that these disorders are extremely disruptive for the patient's life in personal terms (misery, suicide), social terms (divorce) and economic terms (job loss, poverty).

3. Family and friends of such patients. As mentioned, stress-related disorders can be very disruptive for someone's social life often leading to divorce and social isolation which is devastating for the partners, children and friends of the patient. Thus, the social environment of the patient would benefit greatly from a better treatment of the patient.

4. The economy. The economy in general would benefit because patients suffering from stress-related disorders are often unable to work. If skilled people drop out of the work force it can have a major negative impact on the management, development and productivity of companies. Research has shown that stress leads to the loss of over 15 million work days per year and many billions Pound Sterling in economic damages. A better treatment would doubtless be beneficial for the economy. The pharmaceutical industry would benefit as this research would spark new avenues in drug development.

5. The government and the National Health Service (NHS). It is logical that the social, economic and health problems of such patients are a great burden for the government and the NHS. Clearly, an improved treatment of these patients would alleviate this burden significantly. Better insight into the effects of stress on animals could be beneficial to improve legislation for a better treatment of our companion and farmed animals.

6. Academia. International academia in the fields of preclinical and clinical psychiatry, and molecular, cellular and behavioural neuroscience would greatly benefit from the scientific progress made by this research. This is underlined by the fact that our research output has been highly cited in the scientific literature (see CV Reul). Therefore, it may be anticipated that our results will have a major impact on academia (See also 'Academic Beneficiaries').


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Mifsud KR (2016) Acute stress enhances heterodimerization and binding of corticosteroid receptors at glucocorticoid target genes in the hippocampus. in Proceedings of the National Academy of Sciences of the United States of America

Description The aim of this research project was to investigate the role of signalling and epigenetic factors in the interaction of transcription factors with the neuronal genome in the hippocampus after stress as well as the implications of these interactions for gene transcription and adaptive behavioural responses. Our main findings:
-According to a widely held concept (based mainly on in vitro research and written in textbooks), for the induction of so-called immediate-early genes (IEGs) like Fos and Egr1, transcription factors (e.g. CREB) need to bind to the promoter region of such genes. We found however that under baseline (non-stress) conditions in vivo the promoter regions of IEGs in the rat hippocampus already show high binding of the transcription factor CREB despite the absence of any transcription of these genes. After stress, gene transcription is induced without any significant change in the (high) binding of the transcription factor.
-Further research discovered that certain epigenetic modifications (i.e. DNA de-methylation, histone phosphorylation and acetylation) of the genome are of critical importance for induction of IEG transcription in the hippocampus and associated adaptive behavioural responses after a stressful challenge (forced swimming). These findings show that induction of IEG transcription is controlled in a highly complex manner through transcription factor binding in conjunction with distinct epigenetic processes.
-Investigation of a glucocorticoid hormone binding transcription factor (MR) showed the reverse pattern: Based on previous research (largely by the PI) this transcription factor was expected to present high binding to gene promoters under baseline conditions and only slight increases after stress. It was found however that under baseline conditions MR binding at gene promoters was low but increased substantially after stress. Preliminary work suggests that the binding of this transcription factor is also governed by epigenetic factors as well as other proteins. These findings have prompted the adjustment of a long-standing concept on the role of this transcription factor in gene transcription and behaviour.
Thus, this research has led to the generation of new knowledge (published in several articles), improved research methods (e.g. Tandem chromatin immunoprecipitation (ChIP)), and opened up new research questions.
Exploitation Route Our findings are highly relevant for other scientists working in various disciplines (both academia and industry) on the effects of stress on the brain. Our findings are particularly relevant as they have led to changes in long-standing concepts on transcription factor-medicated control of gene transcription in vivo in relation to behavioural responses to stress. Our work has underlined the principal role of epigenetic regulation of gene transcription. Overall, the outcome of the research project has increased our understanding about the complexity of gene transcriptional control in vivo in relation to stress-evoked behavioural changes. The scientific community has welcomed our research findings as indicated by the acceptance of our scientific publications including two articles in the prestigious international journal Proceedings of the National Academy of Sciences USA in 2016.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description Dr Karen Mifsud, Senior Research Associate on this grant award, was selected to present her work to MPs and other interested parties in the House of Commons as part of STEM for Britain 2017 in London on March 13th, 2017. The title of her poster presentation in the Biological and Biomedical Sciences Session was "Understanding how the brain copes with stressful experience through the interaction of stress hormone receptors with the genome".
First Year Of Impact 2017
Sector Education,Healthcare,Government, Democracy and Justice
Impact Types Societal

Title Tandem ChIP for brain tissue 
Description I have developed a chromatin immuno-precipitation (ChIP) method which allows to determine whether two transcription factors bind to the same element (e.g. a transcription factor response element) within the genome. As far as I know, I am first who has established this method for brain tissue. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Regulation of gene expression is controlled by multimeric protein complexes. With regular ChIP assays one can determine whether a particular protein is binding to genomic elements under certain circumstances. Tandem ChIP allows to investigate whether two factors are binding at the same time. This is important for transcription factors which are thought to homo- or hetero-dimerise at genomic elements. There is hardly any evidence that this is actually occurring in tissues in vivo. Concretely, I was able to demonstrate that the mineralocorticoid receptor and the glucocorticoid receptor (two ligand-dependent transcription factors) are indeed heterodimerizing at glucocorticoid-responsive elements in hippocampal neurons after stress in vivo. This work is presently written up for publication. This method can be applied to any transcription factor, enzyme, histone modification, chromatin remodelling factor for which there are high quality antibodies available. 
Description Future first career event for school children 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This was an assembly of 180 school children to share my (Dr Karen Mifsud's) experience in becoming a scientist. There were small group discussion about science as a career option.
Year(s) Of Engagement Activity 2017
Description Organisation of the Colston Research Society Public Lecture "Post-Traumatic Stress Disorder: Innovations in Treatment" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Together with Professor Astrid Linthorst, I organised the Colston Research Symposium 2015 with the title "Neurobiology, intervention and treatment of Post-traumatic Stress Disorder". Apart from a two-day scientific symposium this symposium included the so-called Colston Research Society Public Lecture "Post-Traumatic Stress Disorder: Innovations in Treatment", given by Professor Barbara Rothbaum from Emory University, Atlanta, USA. Professor Rothbaum is a renowned professor in psychiatry and specialises in research on the treatment of individuals with anxiety disorders, particularly focusing on post-traumatic stress disorder. This subject is of particular relevance for the general public in view of the high incidence of this mental disorder in war veterans and other victims of traumatic events. The public lecture was attended by approximately 250 people of the general public. The lecture sparked questions and discussion on this highly society-relevant subject afterwards.
Year(s) Of Engagement Activity 2015
Description Press releases associated with two articles in Proceedings of the National Academy of Sciences USA in 2016 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Based on two articles in Proceedings of the National Academy of Sciences USA two press releases were published. They were initially published on the University of Bristol's website and on the BBSRC's webpages. The main messages from these press releases were picked up by many other national and international websites, so the news of the outcome of our BBSRC funded research was spread around the globe.
Year(s) Of Engagement Activity 2016
Description Understand Animal Research presentation at Taunton School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This talk discussed how animals are used in scientific research. It was explained how we use experimental animals in our research on how stress affects the brain.
Year(s) Of Engagement Activity 2015
Description Understanding Animal Research presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This was a talk about how experimental animals are used scientific research in the UK as part of an assembly of 300 pupils. There were small group talks involving approximately 30 students on how we used animals in our research and how they have contributed to increase our understanding about how stress acts on the brain.
Year(s) Of Engagement Activity 2014