Center for Molecular Medicine Cologne

Alvaro Rada-Iglesias - assoc. RG

Developmental Genomics: Transcriptional regulation in development and disease

The main interest of our laboratory is to uncover the genetic and epigenetic factors controlling the deployment of gene expression programs during vertebrate embryogenesis as well as how the disruption of these regulatory factors can lead to human congenital disease. Towards these goals, we are using a multidisciplinary approach that combines in vitro and in vivo models with biochemical, genomic and genetic engineering tools.

Introduction

Enhancers are compact (~200-500bp) DNA sequences that control the activity of their target gene over long distances and in an orientation-independent manner. The importance of enhancers during embryogenesis is well illustrated by enhancer deletions or mutations in both animal models  and humans  that can lead to severe congenital abnormalities. Despite the develop-mental and medical relevance of enhancers, our understanding of these distal regulatory elements was rather limited until recently. The lack of a stereotypical sequence com-position, limited sequence conservation and distal position with respect to target genes, had hist-orically made the discovery of enhancers a daunting task. In the last few years, epigenomic profiling and the use of chromatin signatures have emerged as powerful and universal tools to identify enhancers.

Our laboratory previously uncovered novel chro-matin signatures that enabled not only the global identification of enhancers but also their distinction into two major classes depending on their activity: active and poised. Importantly, these chromatin signatures were shown to be universal and have been subsequently used by us and others to identify enhancers in multiple cell types and organisms. In recent years, genome-wide asso-ciation studies (GWAS) have revealed that ~90% of genetic variants associated with common human diseases occur within the vast non-coding fraction of the human genome, preferentially within enhancers.

Moreover, it has been demonstrated that changes in the regulatory activity or topology of enhancers, due to genetic variation or genomic re-arrangements, respectively, can actually lead to human disease.

Functional and topological characterization of poised enhancers during embryonic stem cell differentiation.

We have previously described poised enhancers in embryonic stem cells (ESC) as a set of cis-regulatory elements characterized by the simultaneous presence of transcription factors, co-activators, H3K4me1 and H3K27me3. Our recent epigenomic and functional annotation of poised enhancers in mouse ESC suggested that, rather than being promiscuously used during the acquisition of somatic cell fates, poised enhancers were preferentially involved in the establishment of anterior neural identity.

Using CRISPR/Cas9 to delete poised enhancers in situ, we showed that these cis-regulatory elements are necessary for the induction of major forebrain regulators. Moreover, 4C-seq experiments revealed that poised enhancers established strong and specific physical interactions with their target genes already in the undifferentiated ESC state. Remarkably, additional 4C-seq experiments showed that such pre-existing topological conformations are polycomb dependent.

Together with additional characteri-zation of polycomb null ESC, we proposed that polycomb proteins confer poised enhancers with the appropriate regulatory topology and, thus, facilitate the timely induction of forebrain master regulators. Overall, our work illuminates a novel mechanism by which polycomb proteins might facilitate the proper establishment of developmental gene expression programs (Figure 1). This work was published in Cell Stem Cell.

Disruption of gene regulatory domains as a novel mechanism for human congenital disease.

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by Branchiooculofacial Syndrome (BOFS), a rare congenital disorder caused by hetero-zygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. We recently described a BOFS patient carrying a heterozygous inversion with one breakpoint located within a Topologically Associa-ting Domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCC).  Using patient-specific hiPSCs, we showed that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption or ectopic gains in gene expression. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expres-sion in patient hNCC. Therefore, our work highlights the power of hiPSCs differentiation to unveil the pathological mechanisms whereby structural variants can cause congenital abnormalities. This work was recently publihsed in Cell Stem Cell. Based on these recent findings, we are now dissecting the regulatory logic govering the compatibility and responsiveness between genes and enhancers, which we believe, includes genetic, epigenetic and structural factors. 

Perspectives

Susceptibility to most human diseases depends on the interactions between genetic, epigenetic and environmental factors. While disease-associated genetic variants frequently occur within enhancers, their potential consequences at these genomic locations remain poorly understood. Multiple re-dundant enhancers often control key regulatory genes, which, at least in invertebrate organisms, can provide transcriptional robustness against en-vironmental stress. We postulate that due to this enhancer redundancy the pathological effects of etiological enhancer variation can only be revealed under environmental stress. Using the neural crest to model congenital disease and teratogen sensi-tivity, we will use a multidisciplinary approach that combines genomics, computational modelling and genetic engineering to systematically evaluate the importance of enhancer redundancy. By considering enhancers as hubs of gene-environ-mental inter-actions, our approach can illuminate a novel and general etiological paradigm for human disease.

Selected publications 

1. Laugsch M, Bartusel M, Rehimi R, Alirzayeva H, Karaolidou A, Crispatzu G, Zentis P, Nikolic M, Bleckwehl T, Kolovos P, van Ijcken, W.F.J., Šarić T, Köhler K, Frommolt P, Lachlan K, Baptista J#, Rada-Iglesias, A#. Modelling the pathological long-range regulatory effects of human structural variation with patient-specific hiPSCs. Cell Stem Cell, 2019 May 2;24(5):736-752.

2. Rada-Iglesias A#, Grosveld FG, Papantonis A#. Forces driving the three-dimensional folding of eu-karyotic genomes. Mol Syst Biol. 2018 Jun 1;14(6):e8214. PMID: 29858282.

3. Rada-Iglesias A#. Is H3K4me1 at enhancers correlative or causative. Nat Genet. 2018 Jan;50(1):4-5. PMID:29273804.

4. Cruz-Molina S, Respuela P, Tebartz C, Kolovos P, Nikolic M, Fueyo R, van Ijcken W.F.J, Grosveld F, Frommolt P, Bazzi H, Rada-Iglesias A#. PRC2 facilitates the regulatory topology required for poised enhancer function during pluripotent stem cell differ-entiation. Cell Stem Cell. 2017 May 4;20(5):689-705. PMID:28285903.

5. Nikolić M, Papantonis A, Rada-Iglesias A#. GARLIC: A Bioinformatic Toolkit for Etiologically Connecting Diseases and Cell Type-Specific Regulatory Maps. Hum Mol Genet. 2017 Feb 15;26(4):742-752. PMID:28007912.

Dr. Alvaro Rada-Iglesias CMMC Cologne
Dr. Alvaro Rada-Iglesias

CMMC Affiliation C

assoicated RG (since 06/2018) Principal Investigator CMMC JRG VII (05/2013-06/2018)

+49 221 478 96988

+49 221 478 97902

CMMC Affiliation C

present address: Calle Albert Einstein 22

39011 Santander

http://zmmk-ari.uni-koeln.de/Rada-Iglesias_lab___Developmental_genomics/Home.html

CMMC Profile Page

Curriculum Vitae (CV)

Publications - Alvaro Rada-Iglesias

Link to PubMed

Group Members

Michaela Bartusel (doctoral student)
Tore Bleckwehl (doctoral student)
Tomas Pachano (doctoral student)
Rizwan Rehimi (postdoc)
Giuliano Crispatzu (postdoc)

Figure 1