ENHPATHY

Promoter

Studying enhancer function to understand human disease Enhancers play an important role in determining how genes are expressed in different cells in the body. The goal of the ENHPATHY consortium is to identify these enhancers and how they regulate gene expression. Knowledge about enhancer function may eventually lead to new treatments against diseases associated with enhancer dysfunction, as Dr Salvatore Spicuglia explains.

In multicellular organisms, spatial and temporal regulation of gene expression is crucial for development, cellular differentiation, and homeostasis. Cell-type-specific regulation of gene expression is achieved through the interaction between regulatory elements that are either proximal (promoters) or distal (enhancers) to genes. Enhancers, which primarily function in a cell type-specific manner, play an important role in determining where and when genes are expressed in different cells in the body.

These enhancers are short regions of DNA that contain binding sites for transcription factors which can increase the expression of nearby target genes. Enhancers can work in a synergistic way to direct a specific pattern of expression for a given gene and regulate different genes at different time points. “These factors help to determine how genes are expressed in the human body,” says Dr Salvatore Spicuglia, Director of Research at the TAGC Institute in Marseilleand scientific coordinator of ENHPATHY. Enhancers can be altered in different ways, such as through structural variation or point mutations, which can increase the susceptibility of the carrier to a particular condition. “Point mutations can lead to alterations in gene expression, which is a factor that contributes for instance to the development of malformations. There can also be an increased risk of complex genetic disorders, like diabetes, obesity and cancer,” explains Dr Spicuglia. “Point mutations in enhancers influence the way genes are expressed, and that will increase the risk of developing a disease.”

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The major genetic cause of human disease has historically been thought to be mutations within coding genes; however, there has been a shift in perspective over recent years, with more attention now being paid to the role of the non-coding regions in the genome. This is a highly complex area of research, as identifying and characterizing mutations on DNA regulatory sequences is a technically challenging task. “Mutations on DNA regulatory sequences are difficult to interpret,” says Dr Spicuglia. These

ENHPATHY consortium As the coordinator of the EU-funded ENHPATHY project, Dr Spicuglia is part of a multi-disciplinary team investigating the molecular basis of these human enhanceropathies. The consortium operates as an Innovative Training Network (ITN), in which 15 Early Stage Researchers (ESRs) are investigating several different topics around the role of enhancers in health and disease. “We are training a new generation of ESRs with multdisciplinary skills, involving genetics, molecular biology and bioinformatics.

Figure 2: Enhancer dysfunction in disease. Mutations of genetic variations in non-coding regions such as enhancers will modify the efficiency of transcription factor binding and subsequence regulation of gene expression. This might results in either loss-of-function (reduced gene expression = less arrows) or gain-of- function (increased gene expression = more arrows). The altered gene expression will results in divers types of disease referred as enhanceropathies

Health

Point mutations at enhancers can lead to alterations in gene expression, which is a factor in the development of malformations for example. There can also be an increased risk of complex genetic disorders, like diabetes, obesity and cancer. mutations are known to be involved in certain genetic disorders, as well as many types of cancer. “We know that the oncogenes that are involved in cancer progression are driven mainly by dysregulation of regulatory elements, that drive very strong expression of these oncogenes in the cancer cell,” continues Dr Spicuglia. “They can also be involved in drug resistance, by inducing the expression of some genes that are known to be involved in these processes. These alterations in genes can lead to diseases called enhanceropathies.”

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We want to understand how genes are regulated in normal individuals, and also in the pathological context,” outlines Dr Spicuglia.

The aim of this research is to build a deeper understanding of the mechanisms underlying enhanceropathies, with ESRs pursuing research organised into three workpackages. “The first is about understanding the features that control enhancer activity, and how a specific mutation can alter the function,” says Dr Spicuglia. “The second focuses on unravelling how enhancers work within complex regulatory cascades.

Enhancers can be located at different distances

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protein that transports oxygen around the body.

“The team led by Wouter de Laat at the Hubrecht Institute in the Netherlands is working on the beta-globin locus, which is often deleted or mutated in people with thalassaemia. Normally the foetal haemoglobin is active before it declines and then the adult haemoglobin starts to be expressed. In thalassaemia the adult haemoglobin gene is mutated and not properly expressed anymore,” explains Dr Spicuglia.

Researchers are trying to modify the regulatory sequence of the foetal haemoglobin so that it can be expressed again, and Dr Spicuglia says early results are promising. “Our partners have shown that this approach works with primary cells. They can reposition the haemoglobin regulatory sequence, and rescue or recover haemoglobin expression,” he outlines.

This is a good example of how a deeper understanding of regulatory mechanisms can lead to more effective therapeutic strategies, which is a central part of the project’s overall agenda. While a lot of attention in the project is focused on investigating the underlying mechanisms behind enhanceropathies, Dr Spicuglia and his colleagues also aim to translate their findings into new therapies and improved diagnostic techniques, work which is ongoing. “There is a lot of interest in building on this research to develop improved treatments against enhanceropathies, and a further project is planned,” he continues.

Expression

Enhancer

dysregulation and disease. With diseases like diabetes or obesity, mutations are already present in our DNA and differences in cells will accumulate over time; Dr Spicuglia says mutations in cancer cells are different. “There you have somatic mutations, which may arrive at a certain point in the lifecourse. These somatic mutations may alter enhancer function, leading to the over-expression of some genes that will then become oncogenes,” he explains. As part of his own role in the project, Dr Spicuglia is collaborating with the Advanced BioDesign company and a team led by Meritxel Alberich Jordà from the Institute of Molecular Genetics in Prague to co-supervise an ESR conducting research into acute myeloid leukemia (AML), a form of cancer that affects white blood cells.

In this research, Dr Spicuglia and his colleagues are investigating the reasons behind resistance to certain drugs in AML patients, and significant progress has been made in this respect. “We have identified enhancers that get activated in cells when exposed to a drug used in chemotherapy, contributing to the development of resistance to the treatment,” he outlines. “Next, our aim is to prevent cells from developing resistance.”

Researchers in the project are also exploring therapeutic possibilities against other enhanceropathies. For example, researchers are working on new approaches to treating thalassaemia, an inherited condition that affects the body’s ability to produce haemoglobin, a

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Mutation or Genetic Variation

loss of function

Gene

Cell differentiation Activation Stress response

kbs to Mb

from their target genes, but a given gene can also be regulated by many different enhancers.

The third is about identifying disease-causing enhancer alterations and setting the ground for the development of new enhancer-based diagnostic and therapeutic tools.”

The question here is how enhancers work together to regulate a given gene, and why they don’t regulate other genes in their vicinity. A gene may be regulated by a specific enhancer in a given tissue, while in another tissue it may be regulated by a different enhancer, part of a complex overall picture. “It’s very important to try and identify all the enhancers that are active in the different tissues, but also in different pathological contexts,” says Dr Spicuglia. The latter topic is being addressed in the third workpackage (interview with Jorge Ferrer overleaf), which Dr Spicuglia says is focused more on disease. “We aim to identify enhancers and try to understand how they can affect the regulation of genes in certain disease contexts,” he explains. “One way of identifying enhancers is by looking for epigenetic marks on the genome. We can also use high-throughput reporter assays, where we essentially clone these pieces of DNA in front of a reporter gene, then we look in parallel at how these sequences will activate the reporter gene in different cell types or cellular contexts.”

This will help researchers build a deeper picture of the relationship between enhancer

Altered

Figure 1: Enhancers are essential players for the regulation of gene expression. Enhancers are cis-regulatory elements that are located far from the genes they regulate (from few kilobases to Megabase distances). Upon cell differentiation, activation or stress response, they come close to their target genes and activate the upstream promoters to initiate gene transcription (arrows).

Enhanceropathies

Mendelian diseases Increased risk in complex genetic disorders Cancer Drug resistance

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www.euresearcher.com

gain of function

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ENHPATHY

Molecular Mechanisms of Diabetes

Molecular basis of human enhanceropathies

Project Objectives

ENHPATHY aims to identify key deregulated enhancers and regulatory mechanisms, and provide new biomarkers and therapeutic avenues for enhanceropathies. To achieve this goal, ENHPATHY has set up an innovative, integrated and disease-focused research programme that brings together European leaders in enhancer biology, computational biology and human genetics.

Project Funding

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 860002.

Project Partners

https://www.enhpathy.eu

Contact Details

Project Coordinator, Salvatore Spicuglia Inserm – Aix-Marseille University U1090 163, Avenue de Luminy 13288 MARSEILLE cedex 09 T: +33(0)4 91 82 87 E: salvatore.spicuglia@inserm.fr W: https://www.enhpathy.eu/ Zaugg JB, Sahlén P, Andersson R, Alberich-Jorda M, de Laat W, Deplancke B, Ferrer J, Mandrup S, Natoli G, Plewczynski D, Rada-Iglesias A, Spicuglia S. (2022) Current challenges in understanding the role of enhancers in disease. Nat Struct Mol Biol. 2022 Dec;29(12):1148-1158. doi: 10.1038/ s41594-022-00896-3

Salvatore Spicuglia

Jorge Ferrer

Professor Jorge Ferrer is coordinator of the Medical Genomics Transversal programme at the Centre for Genomic Regulation in Barcelona. He leads workpackage 3 within the Enhpathy network, in which he is supervising research into the molecular mechanisms behind different forms of diabetes.

EU Researcher: What is the main focus of your research?

Professor Jorge Ferrer: The major focus of our genetic studies is on less common single-gene disorders, so called monogenic or Mendelian disorders. Many patients with monogenic diabetes do not have mutations in DNA sequences of genes that encode for proteins. We are looking for mutations in enhancers and other types of regulatory elements that cause these sub-types of diabetes.

For example, people born without a pancreas sometimes have a mutation in an enhancer that is necessary to switch on pancreas development. We’ve modelled these mutations in mice and human stem cells, and we’ve learned that this is a special enhancer, what we call a lead enhancer.

EUR: Does that enhancer function as part of a team in a way?

Professor Ferrer: Yes, there are clusters Salvatore Spicuglia is a Director of Research from the French National Institute of Medical Research (INSERM) and group leader in Functional Genomics at the Theories and Approaches of Genome Complexity laboratory (TAGC, Marseille). His team is devoted to genome-wide studies to decipher epigenetic and gene regulation mechanisms at play during normal and pathological contexts. The team has revealed novel type of cis-regulatory elements and shed lights on mechanisms involved in epigenetic deregulation in Leukemia.

Jorge Ferrer leads a team that has revealed genetic mechanisms underlying different forms of diabetes. He is Senior Group Leader and Coordinator of The Computational Biology and Health Genomics Programme in the Centre for Genomic Regulation in Barcelona, and CIBER de diabetes y enfermedades metabolicas, as well as Professor in Imperial College London.

of enhancers that regulate a single gene.

Enhancer clusters have built-in redundancy – there are multiple enhancers, and if one fails then there are others which can be called up. However, a lead enhancer kicks in before all the rest and activates them, which makes that enhancer more essential. The question then is; how many other lead enhancers are there?

There are different ways of addressing this question, such as experimentally with genome-editing tools and using screening permutations. We’re working

to identify rare mutations in enhancers that cause relatively rare forms of diabetes in young people.

EUR: Are you also conducting research into other forms of diabetes?

Professor Ferrer: Yes, we’re also looking at type-2 diabetes for example, the genetics of which is very different to the monogenic forms. Type-2 diabetes is a polygenic disease – hundreds of genetic variants have a tiny effect on an individual’s susceptibility for developing the disease.

We’ve made maps of enhancers and pancreatic islets, looking to understand which genes are regulated by these variants, and how that affects susceptibility to type-2 diabetes. Some people have such a high number of these type-2 diabetes susceptibility variants that they develop the disease, even if they’re not particularly obese or sedentary, which are known to be important risk factors.

EUR: What implications does your research hold for the diagnosis of diabetes? Could your research lead to more precise, earlier diagnosis of diabetes?

Professor Ferrer: Yes. We’re combining information from enhancer variants and coding variants to try to develop diagnostics, particularly for young people with the disease. Genetic testing will play a key role in diagnosing diabetes in future.

ENHPATHY’s stage in Marseille: Event VI & EMBO workshop meeting, 15-20 October 2023.

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