Periodic Reporting for period 1 - ENHPATHY (Molecular basis of human enhanceropathies)
Okres sprawozdawczy: 2020-03-01 do 2022-02-28
The primary goal of ENHPATHY is to provide ESRs with a multidisciplinary training program in which cutting-edge genomics and genome editing approaches are combined with in vitro and in vivo disease models to enable understanding the molecular basis of human enhanceropathies. Moreover, together with our private partners, we will explore the translation of our molecular findings into new diagnostic and therapeutic avenues for patients.
We have divided our research program into three highly complementary and interconnected scientific work packages (WP) to address the following key questions:
> WP1 - How do genetic and epigenetic features control enhancer activity? We will study how transcription factor binding and genetic/epigenetic variation impact on enhancer activity.
> WP2 - How do enhancers function within complex regulatory landscapes? We will study the mechanisms by which individual enhancers control distal gene regulation as well as the precise role of individual enhancers within clusters of enhancers.
> WP3 - How does enhancer deregulation impact human diseases? We will identify disease-causing enhancer alterations, develop approaches to discern pathogenic enhancer variants from neutral variants, study their pathological consequences, and set the ground for the development of new enhancer-based diagnostic and therapeutic tools.
To tackle such extraordinarily complex problems of scientific, economic and societal relevance, we have 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 program (WP6).
We have divided our research program into three highly complementary and interconnected scientific work packages (WP) to address the following key questions:
> WP1 - How do genetic and epigenetic features control enhancer activity? We will study how transcription factor binding and genetic/epigenetic variation impact on enhancer activity.
> WP2 - How do enhancers function within complex regulatory landscapes? We will study the mechanisms by which individual enhancers control distal gene regulation as well as the precise role of individual enhancers within clusters of enhancers.
> WP3 - How does enhancer deregulation impact human diseases? We will identify disease-causing enhancer alterations, develop approaches to discern pathogenic enhancer variants from neutral variants, study their pathological consequences, and set the ground for the development of new enhancer-based diagnostic and therapeutic tools.
To tackle such extraordinarily complex problems of scientific, economic and societal relevance, we have 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 program (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 a SuRE library of a Congenital Heart Disease (CHD) affected child. We identified >1000 SNPs that appear to be affecting regulatory element activity. Further, we are now annotating wrt allele frequency, parental zygosity state and proximity to known CHD genes.
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. These results have been submitted for publication and are currently undergoing revision.
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 a SuRE library of a Congenital Heart Disease (CHD) affected child. We identified >1000 SNPs that appear to be affecting regulatory element activity. Further, we are now annotating wrt allele frequency, parental zygosity state and proximity to known CHD genes.
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. These results have been submitted for publication and are currently undergoing revision.
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. However, most of the ESRs have started around 1 year ago, therefore their scientific impact is not yet made effective in terms of peer-reviewed publications. But 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 arised as a consequence of the frequent interactions among ENHPATHY members (see Table 1 of the Technical Report).
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 arised as a consequence of the frequent interactions among ENHPATHY members (see Table 1 of the Technical Report).