Biological Methylation: Post-translational Regulation of Intestinal Regeneration

Chronic diseases such as diabetes, asthma, or inflammatory bowel diseases are emerging in the western world and are affecting many patients world-wide. These chronic diseases are often accompanied by wounds that fail to heal properly, if at all. This may be an underlying cause for some of these diseases, while it is a consequence of disease for others. Of course, many aspects influence the progression of these illnesses such as diet, environment, and genetic components. In this project, we studied how healing of wounds in the intestine is regulated at a very basic and detailed level. In addition, we link wound healing to early-life development of the intestine, which occurs both pre- and postnatally. From earlier research, we had several clues that point toward certain genetic components that are important in intestinal wound healing. We used this funding to determine how these specific genetic components regulate intestinal wound healing. To do so, among other techniques, we are using organoid cell cultures in our work. Organoids are little organ-like structures that can grow in petri dishes. The use of organoids significantly contributes to the reduction of use of animals in biomedical research. In our case, we use intestinal epithelial organoids and treat those with specific drugs affecting the genetic components of our interest. Importantly, because these organoids are continuously healing or 'regenerating' themselves, they are an ideal method to study these processes. Ultimately, we hope our research will lead to the identification of new targets to treat chronic (inflammatory) diseases. However, our immediate goal is to decipher the basic regulatory mechanisms through which intestinal development is regulated, and how similar mechanisms are involved during wound healing and regeneration. We believe that understanding the way wounds normally heal is the key for drug development for chronic diseases in which wounds do not normally heal. In this project, we identified an important player in intestinal epithelial development after birth. Postnatally, many aspects of gut epithelial maturation take place, which of course co-occurs with a change in diet (from milk to solid foods). This early-life shift is characterized by the appearance of specific epithelial cells that help us deal with the microbes that we ingest through food. We have found a specific enzyme called LSD1 that is required for the appearance of specific gut epithelial cell lineages. Interestingly, a similar process happens after intestinal damage or during diseases such as inflammatory bowel diseases. In addition, we found that LSD1 expressed in epithelium is also responsible for the proper recruitment of immune cells to the gut. Finally, we continued studying the role of LSD1 in the intestinal epithelium and found that it is required for the defense against gut pathogens, likely due to a combination of affecting epithelium and immune cells. So, even though targeting LSD1 could be good in certain inflammatory conditions needing repair, it is a double-edged sword as it could be bad for treating infections or infection-induced damage. In summary, our discovery of how the early life intestinal development occurs will hopefully result in the potential targeting of this player in intestinal diseases.

Overarchingly, we achieved our primary objective by having identified a new molecular mechanism that is crucial for both early-life intestinal development as well as regeneration after damage, which is driven by the demethylase LSD1. Specifically, we have found that LSD1 is required for the maturation of epithelium during the postnatal stage. This is a time when normally our epithelium adjusts to the switch from milk to solid foods and when epithelium adjusts to the incoming and expanding microbiota. In addition, we identified a dual role for LSD1 during intestinal repair, either a positive or a negative effect dependning on the type of injury. Our work primarily impacts researchers. Indeed, the first two peer-reviewed manuscripts from this project have been well-cited in just the last few years after publication. For our society, acquiring and disseminating knowledge is beneficial as it builds on the knowledge economy. Furthermore, we hope future work will proof that the use of inhibitors of LSD1 could be beneficial for gastrointestinal diseases such as inflammatory bowel disease. However, there is still a long road before this can be expected to happen.

Many chronic inflammatory diseases are characterized by impaired wound healing or regeneration. Further, a wide set of patient groups would benefit significantly from enhanced wound healing to decrease recovery time after tissue damage. We currently lack the biological knowledge to address these issues. The intestinal epithelial model system is widely used in stem cell biology and regenerative medicine. Although the cellular abilities during regeneration have been elegantly described using cell-lineage tracing techniques, the molecular mechanisms behind these cellular processes are still not well defined. This project aims to establish a role for a druggable methylation signalling axis in intestinal development and regeneration. We present published and preliminary data showing that SETD7 and LSD1 counteract each other in the control of the Hippo and Wnt pathways, which both are very important in intestinal development and regeneration. This project is separated into three related aims. In the first aim we propose to use murine organoid cultures that genetically lack SETD7 or LSD1 combined with pharmacological inhibition of these enzymes. The combination of genetic and pharmacological intervention will answer fundamental questions about druggability, but also about "enzyme-specific" vs. "protein-specific" roles. The second aim focuses on the (intestinal) development of newly generated mouse strains that lack LSD1 in intestinal epithelial (stem) cells. Finally, the third aim will study the mice from Aim 2 in the context of intestinal inflammation and regeneration. Further, we will use human organoids to test the translational potential of our findings. Together, this project will determine the role for the SETD7-LSD1 methylation signalling axis in intestinal homeostasis and disease. If successful, these studies will highlight that methylation is an important post-translational modification within established signaling cascades.