Alvaro Rada-Iglesias - JRG VII

Genomic context and mechanisms of transcriptional repression during early patterning of the mammalian embryo

The main interest of our laboratory is to uncover the genetic and epigenetic factors controlling the deployment of gene expression programs during vertebrate embryogenesis. Towards this goal, we are using a multidisciplinary approach that combines in vitro and in vivo models with biochemical, genomic and genetic engineering tools. Moreover, inspired by our previous and recent work on vertebrate embryogenesis, we are applying similar experimental approaches to uncover the molecular basis of human congenital diseases. More specifically, using the human neural crest a model, we are investigating how genetic variants and genomic re-arrangements can lead to human disease through the disruption of gene regulatory landscapes.

Introduction

The execution of developmental gene expression programs depends on regulatory networks consisting of cis-regulatory sequences, such as enhancers, and the transcription factors (TFs) that bind to them and influence their activity. The importance of enhancers during embryogenesis is well illustrated by enhancer deletions or mutations that can lead to severe congenital abnormalities. Similarly, the role of TFs as major determinants of cellular identity and developmental fidelity is supported by the numerous mutations within these genes that can lead to congenital and mendelian diseases. Moreover, the medical relevance of enhancers and other non-coding regulatory sequences is further highlighted by the fact that ~90% of genetic variants associated with common human diseases occur within the vast non-coding fraction of the human genome. Given the abundance and functional relevance of enhancers, we and others have speculated that a significant fraction of this disease-associated genetic variation might occur within cis-regulatory elements, potentially altering their activity and ultimately leading to quantitative changes in gene expression and human disease (Rada-Iglesias et al., 2014). In agreement with this hypothesis, recent reports have confirmed that changes in the regulatory activity or topology of enhancers, due to genetic variation or genomic re-arrangements, respectively, can lead to human disease. Therefore, the elucidation and in depth characterization of the regulatory networks (i.e. enhancers and TFs) controlling vertebrate and human embryogenesis has far-reaching medical implications.

Functional dissection of cellular heterogeneity

During vertebrate embryogenesis, developing tissues display high cellular heterogeneity as they contain multiple progenitor and differentiating cellular states that are critical for tissue formation and function. Similarly, the homeostasis and regenerative potential of adult epithelial tissues depends on heterogeneous and distinct stem cell populations residing within such tissues. Therefore, understanding the molecular basis of cellular heterogeneity can provide major insights into tissue formation and homeostasis, which are typically altered in congenital and age-related diseases, respectively. Using spinal neural tube dorsoventral patterning and hair follicle stem cell diversity as models of biologically relevant cellular heterogeneity, we have recently implemented a novel, simple and broadly applicable epigenomic strategy that allows the functional dissection of cellular heterogeneity in various biological and pathological contexts (Rehimi et al., submitted).

Genetic and epigenetic regulation of the exit from pluripotency

Using mouse embryonic stem cells (mESC) as a model, our laboratory is interested in uncovering the regulatory principles orchestrating the exit from pluripotency. We recently uncovered FOXD3 as a novel regulator that, acting as a transcriptional repressor, promotes the transition from naïve to primed pluripotency. Mechanistically, FOXD3 executes this important developmental role through the decommissioning of active enhancers necessary for the expression of key naïve pluripotency/germline genes. Subsequently, silencing of Foxd3 is a necessary event during specification of the germline (Respuela et al., 2016).

On the other hand, we previously proposed that poised enhancers represented a unique set of regulatory elements that could facilitate the activation of somatic gene expression programs upon ESC differentiation. However, the functional relevance of poised enhancers remained untested. Using Crispr/Cas9 technology to generate poised enhancer deletions, we recently demonstrated that these regulatory elements are necessary for the induction of major anterior neural regulators. Interestingly, we also uncovered that poised enhancers establish physical interactions with their target genes in ESC in a Polycomb repressive complex 2 (PRC2) dependent manner. Moreover, we showed that loss of PRC2 lead to neither the activation of poised enhancers nor the induction of their putative target genes in undifferentiated ESC. In contrast, loss of PRC2 severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Hence, our work illuminates a novel function for polycomb proteins as topological facilitators of neural induction (Cruz-Molina et al., submitted).

Genetic and structural disruption of regulatory landscapes in human congenital disease

Non-coding genetic and structural variation has recently emerged as a major cause of human disease. Assuming that disease-associated genetic variants frequently occur within cis-regulatory sequences, we have recently developed an in silico toolkit to etiologically link human diseases and cell types. Moreover, once a given disease and a cell type are linked, potential disease-causative genetic variants overlapping relevant cis-regulatory elements can be identified and prioritized for further experimental assessment (Nikolic et al., Submitted).

Using this novel in silico toolkit, we became interested in the TFAP2A locus, as it contained several non-coding genetic variants overlapping enhancers active in human neural crest cells (hNCC) (Rada-Iglesias et al., 2012) and associated with orofacial clefts. Moreover, TFAP2A haploinsufficiency due to mutations or deletions within this gene leads to BOFS, a rare congenital disease characterised by facial, ocular and hearing abnormalities. Using ChIP-seq and 4C-seq technologies, we performed an in depth epigenomic and topological analysis of the TFAP2A locus in hNCC. We are currently using CRISPR/Cas9 technology to mechanistically and functionally characterize the regulatory landscape and topological organization of the TFAP2A locus. We anticipate that, once completed, our work will illustrate how the genetic and/or structural disruption of gene regulatory topologies can be a frequent yet largely unexplored cause of human congenital disease

Perspectives

Inspired by the multidisciplinary and translational research environment of the CMMC and the University of Cologne, we have started to apply our basic biological knowledge towards the elucidation of human disease molecular mechanisms. Our major and general aim for the future is to investigate the pathological consequences of the disruption of transcriptional regulatory networks.

Selected publications 2016

Respuela P, Nikolic M, Tan M, Frommolt P, Zhao Y, Wysocka J, and Rada-Iglesias A (2016). Foxd3 promotes exit from naive pluripotency through enhancer decommissioning and inhibits germline specification. Cell Stem Cell 18, 118-133.

Selected publications 2014

Rada-Iglesias A (2014). Genetic variation within transcriptional regulatory elements and its implications for human disease. Biol Chem 395, 1453-6009.


Dr. rer. nat. Alvaro Rada-Iglesias

Center for Molecular Medicine Cologne

Dr. rer. nat. Alvaro Rada-Iglesias

Principal Investigator - CMMC JRG VII

aradaigl@uni-koeln.de

Publications - Alvaro Rada-Iglesias

Link to PubMed

Group Members

Patricia Respuela (PostDoc)
Rizwan Rehimi (PostDoc)
Magdalena Laugsch (PostDoc)
Rodrigo Osorno (PostDoc)
Sara de la Cruz Molina (doctoral student)
Michaela Bartusel (doctoral student)
Milos Nikolic (doctoral student)
Tore Bleckwehl (doctoral student)
Jan Appel (Lab Manager).

Figure 1

Figure 1. Epigenomic strategy to predict spinal neural tube dorsoventral patterning regulators based on the combination of RNA-seq and H3K27me3 ChIP-seq data.

Figure 2

Figure 2. Foxd3fl/fl and Foxd3-/- ESC were differentiated into PGCLC and the number of PGCLC was quantified by FACS.