Periodic Reporting for period 2 - ENHPATHY (Molecular basis of human enhanceropathies)
Okres sprawozdawczy: 2022-03-01 do 2024-02-29
The primary goal of ENHPATHY was to provide ESRs with a multidisciplinary training program in which cutting-edge genomics approaches are combined with in vitro and in vivo disease models to enable understanding of the molecular basis of human enhanceropathies. Together with our private partners, we explored the translation of our molecular findings into new diagnostic and therapeutic avenues for patients.
We divided our research program into 3 complementary scientific work packages (WP) to address the following key questions:
WP1: How do genetic and epigenetic features control enhancer activity? We studied how transcription factor binding and genetic/epigenetic variation impact on enhancer activity.
WP2: How do enhancers function within complex regulatory landscapes? We studied the mechanisms by which enhancers control gene regulation.
WP3: How does enhancer deregulation impact human diseases? We identified disease-causing enhancer alterations and studied their pathological consequences.
In parallel, we implemented an interdisciplinary and intersectoral program to train a future generation of highly skilled scientists in state-of-the-art research (WP4) and transferable skills (WP5), along with streamlined communication and outreach strategies through our specific STED and EnhancerInArt programs (WP6).
We divided our research program into 3 complementary scientific work packages (WP) to address the following key questions:
WP1: How do genetic and epigenetic features control enhancer activity? We studied how transcription factor binding and genetic/epigenetic variation impact on enhancer activity.
WP2: How do enhancers function within complex regulatory landscapes? We studied the mechanisms by which enhancers control gene regulation.
WP3: How does enhancer deregulation impact human diseases? We identified disease-causing enhancer alterations and studied their pathological consequences.
In parallel, we implemented an interdisciplinary and intersectoral program to train a future generation of highly skilled scientists in state-of-the-art research (WP4) and transferable skills (WP5), along with streamlined communication and outreach strategies through our specific STED and EnhancerInArt programs (WP6).
ESR9. We have combined experimental and computational approaches to investigate the role of extragenic transcription generated by enhancers or promoters.
ESR12. We have set up multiome (joint RNA and chromatin) single cell sequencing using 10X technology to understand how leukemic stem cells may remodel the bone marrow niche cells in myelodysplastic syndrome patients.
ESR13: We have generated and analyzed 5 full genomes of Congenital Heart Disease (CHD) patients using SuRE. We identified >10,000 SNPs that appear to be affecting regulatory element activity which we have further prioritize based on allele frequency, parental zygosity state and proximity to known CHD genes. Ten such variants are now being tested in transgenic mice
ESR1: To directly assess the contribution of Epromoters to the regulation of type I interferon response, we performed paralleled experiments to measure gene expression (RNA-seq) and enhancer activity of gene promoters (CapSTARR-seq).
ESR8: To investigate the role of enhancers in the PPARG locus encoding the master regulator of adipogenesis, we employed CRISPR/Cas9 to excise individual enhancers. These studies have revealed key promoter proximal enhancers as well as an interacting lead enhancer in downstream super enhancers.
ESR5: We analyzed thousands of high-throughput epigenomic assays in seven human cell types. We used existing and custom-made computational approaches to define several thousands of genomic regions where cis-regulatory elements exhibit high levels of coordinated variation, referred to as variable chromatin modules.
ESR7: The β-globin locus consists of 5 globin genes, which are controlled by a strong, distant enhancer, the locus control region (LCR). We have shown that deleting the intervening sequence between an enhancer and a silenced reporter, integrated in a repressive genomic context, we can stably reactivate its expression in K562 cells.
ESR10: Our aim is to develop and test the concept of the structural epigenomic landscape of regulatory elements around promoter regions for selected cell types and the different human individuals. We propose a biophysical method to construct probabilistic ensembles of three-dimensional conformations at several genomic domains scales.
ESR14: In order to study chromatin organization and interactions of regulatory sequences, we have developed a new versatile, low-cost droplet microfluidic platform with high throughput. It achieves a high degree of monodispersity as well as precise control over droplet size for encapsulation and barcoding of regulatory complexes.
ESR6 (new ESR started in February 2022)
ESR2: Control, ZIC2+/- and ZIC2-/- ESC lines have been differentiated into neural progenitors with and without retinoic acid (RA) and gene expression changes have been evaluated by RNA-seq. Preliminary results support an interaction between Zic2 dosage and RA exposure.
ESR3: We employed a mouse model of chronic multifocal osteomyelitis, a chronic inflammation autoinflammatory disease, and investigated how it affects hematopoietic stem maintenance and fate. In this project, we demonstrated that chronic inflammation has a detrimental effect on hematopoietic stem cells (HSCs) and we demonstrated that gene deregulation was the mechanistic caused. These results were recently published (Grusanovic et al, EMBO Rep, 2023). Next, we analyzed the susceptibility of mice suffering from chronic inflammation to leukemia development. We observed that mice suffering from chronic inflammation exhibited an accelerated progression to transformation. The results of this part of the project were recently published (Burocziova et al, Exp Hem, 2023). Finally, in collaboration with the laboratory of Prof. Dariusz Plewczyński (CeNT, University of Warsaw, Poland), supervisor of ESR10, we analyzed the transcriptional and genomic accessibility changes that occur to mice which undergo an acute inflammatory challenge. We demonstrated that HSCs are subjected to a lymphoid to myeloid switch at early stages of the challenge, and that changes in gene accessibility were the underlying cause. These results were published (Vanickova et al, EMBO J, 2023).
ESR4: A computational workflow to integrate CAGE with chromatin accessibility (ATAC-seq) has been set up in an R package alongside methods for mapping enhancers and their activities. Further, we have established a collaboration with Rigshospitalet to study enhancer dysregulation in pediatric AML patients.
ESR11: We processed vascular endothelial tissue biopsies taken from the ascending aorta of ten patients who underwent open heart surgery at the Karolinska Hospital. We performed HiCap experiments for all samples.
ESR15 project: Using CHIP-seq and luciferase-reporting assays methods they have identified drug-responsive enhancers participating in ALDH-associated resistance mechanisms
ESR12. We have set up multiome (joint RNA and chromatin) single cell sequencing using 10X technology to understand how leukemic stem cells may remodel the bone marrow niche cells in myelodysplastic syndrome patients.
ESR13: We have generated and analyzed 5 full genomes of Congenital Heart Disease (CHD) patients using SuRE. We identified >10,000 SNPs that appear to be affecting regulatory element activity which we have further prioritize based on allele frequency, parental zygosity state and proximity to known CHD genes. Ten such variants are now being tested in transgenic mice
ESR1: To directly assess the contribution of Epromoters to the regulation of type I interferon response, we performed paralleled experiments to measure gene expression (RNA-seq) and enhancer activity of gene promoters (CapSTARR-seq).
ESR8: To investigate the role of enhancers in the PPARG locus encoding the master regulator of adipogenesis, we employed CRISPR/Cas9 to excise individual enhancers. These studies have revealed key promoter proximal enhancers as well as an interacting lead enhancer in downstream super enhancers.
ESR5: We analyzed thousands of high-throughput epigenomic assays in seven human cell types. We used existing and custom-made computational approaches to define several thousands of genomic regions where cis-regulatory elements exhibit high levels of coordinated variation, referred to as variable chromatin modules.
ESR7: The β-globin locus consists of 5 globin genes, which are controlled by a strong, distant enhancer, the locus control region (LCR). We have shown that deleting the intervening sequence between an enhancer and a silenced reporter, integrated in a repressive genomic context, we can stably reactivate its expression in K562 cells.
ESR10: Our aim is to develop and test the concept of the structural epigenomic landscape of regulatory elements around promoter regions for selected cell types and the different human individuals. We propose a biophysical method to construct probabilistic ensembles of three-dimensional conformations at several genomic domains scales.
ESR14: In order to study chromatin organization and interactions of regulatory sequences, we have developed a new versatile, low-cost droplet microfluidic platform with high throughput. It achieves a high degree of monodispersity as well as precise control over droplet size for encapsulation and barcoding of regulatory complexes.
ESR6 (new ESR started in February 2022)
ESR2: Control, ZIC2+/- and ZIC2-/- ESC lines have been differentiated into neural progenitors with and without retinoic acid (RA) and gene expression changes have been evaluated by RNA-seq. Preliminary results support an interaction between Zic2 dosage and RA exposure.
ESR3: We employed a mouse model of chronic multifocal osteomyelitis, a chronic inflammation autoinflammatory disease, and investigated how it affects hematopoietic stem maintenance and fate. In this project, we demonstrated that chronic inflammation has a detrimental effect on hematopoietic stem cells (HSCs) and we demonstrated that gene deregulation was the mechanistic caused. These results were recently published (Grusanovic et al, EMBO Rep, 2023). Next, we analyzed the susceptibility of mice suffering from chronic inflammation to leukemia development. We observed that mice suffering from chronic inflammation exhibited an accelerated progression to transformation. The results of this part of the project were recently published (Burocziova et al, Exp Hem, 2023). Finally, in collaboration with the laboratory of Prof. Dariusz Plewczyński (CeNT, University of Warsaw, Poland), supervisor of ESR10, we analyzed the transcriptional and genomic accessibility changes that occur to mice which undergo an acute inflammatory challenge. We demonstrated that HSCs are subjected to a lymphoid to myeloid switch at early stages of the challenge, and that changes in gene accessibility were the underlying cause. These results were published (Vanickova et al, EMBO J, 2023).
ESR4: A computational workflow to integrate CAGE with chromatin accessibility (ATAC-seq) has been set up in an R package alongside methods for mapping enhancers and their activities. Further, we have established a collaboration with Rigshospitalet to study enhancer dysregulation in pediatric AML patients.
ESR11: We processed vascular endothelial tissue biopsies taken from the ascending aorta of ten patients who underwent open heart surgery at the Karolinska Hospital. We performed HiCap experiments for all samples.
ESR15 project: Using CHIP-seq and luciferase-reporting assays methods they have identified drug-responsive enhancers participating in ALDH-associated resistance mechanisms
The successful recruitment of 15 ESRs within the ENHPATHY project will have a strong research and innovation impact at researcher, organization and system levels across Europe, ensuring society profits from the knowledge and training provided to excellent life scientists. Given the scientific progress described in sections 1.2-3 of the Technical Report, we are confident that their work will have a strong impact in the field of Enhancer biology.
The consortium has already published 5 papers directly related to the ENHPATHY’s objectives. The majority of the studies were published in high impact journals.
To increase the impact and visibility of the network, we have implemented three major dissemination actions (see section 2 of the Technical Report): 1) The EMBO Workshop Enhanceropathies: understanding enhancer function to understand human disease”; 2) A Perspective review to be published in Nature journal and 3) A special Issue in the journal BioEssays "Enhancer biology in normal and pathological conditions". The impact of the ENHPATHY network is also measured by a number of new collaborations aroused as a consequence of the frequent interactions among ENHPATHY members.
The consortium has already published 5 papers directly related to the ENHPATHY’s objectives. The majority of the studies were published in high impact journals.
To increase the impact and visibility of the network, we have implemented three major dissemination actions (see section 2 of the Technical Report): 1) The EMBO Workshop Enhanceropathies: understanding enhancer function to understand human disease”; 2) A Perspective review to be published in Nature journal and 3) A special Issue in the journal BioEssays "Enhancer biology in normal and pathological conditions". The impact of the ENHPATHY network is also measured by a number of new collaborations aroused as a consequence of the frequent interactions among ENHPATHY members.