Forces driving the three-dimensional folding of eukaryotic genomes

The last decade has radically renewed our understanding of higher order chromatin folding in the eukaryotic nucleus. As a result, most current models are in support of a mostly hierarchical and relatively stable folding of chromosomes dividing chromosomal territories into A- (active) and B- (inactive) compartments, which are then further partitioned into topologically associating domains (TADs), each of which is made up from multiple loops stabilized mainly by the CTCF and cohesin chromatin-binding complexes. Nonetheless, the structure-to-function relationship of eukaryotic genomes is still not well understood. Here, we focus on recent work highlighting the biophysical and regulatory forces that contribute to the spatial organization of genomes, and we propose that the various conformations that chromatin assumes are not so much the result of a linear hierarchy, but rather of both converging and conflicting dynamic forces that act on it

Identification of candidate regulatory SNPs by combination of transcription-factor-binding site prediction, SNP genotyping and haploChIP

Disease-associated SNPs detected in large-scale association studies are frequently located in non-coding genomic regions, suggesting that they may be involved in transcriptional regulation. Here we describe a new strategy for detecting regulatory SNPs (rSNPs), by combining computational and experimental approaches. Whole genome ChIP-chip data for USF1 was analyzed using a novel motif finding algorithm called BCRANK. 1754 binding sites were identified and 140 candidate rSNPs were found in the predicted sites. For validating their regulatory function, seven SNPs found to be heterozygous in at least one of four human cell samples were investigated by ChIP and sequence analysis (haploChIP). In four of five cases where the SNP was predicted to affect binding, USF1 was preferentially bound to the allele containing the consensus motif. Allelic differences in binding for other proteins and histone marks further reinforced the SNPs regulatory potential. Moreover, for one of these SNPs, H3K36me3 and POLR2A levels at neighboring heterozygous SNPs indicated effects on transcription. Our strategy, which is entirely based on in vivo data for both the prediction and validation steps, can identify individual binding sites at base pair resolution and predict rSNPs. Overall, this approach can help to pinpoint the causative SNPs in complex disorders where the associated haplotypes are located in regulatory regions. Availability: BCRANK is available from Bioconductor (http://www.bioconductor.org/)

Imputation of Orofacial Clefting Data Identifies Novel Risk Loci and Sheds Light on the Genetic Background of Cleft Lip ± Cleft Palate and Cleft Palate Only.

Abstract Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signalswithin this high-density datasetare enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinementapproach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation

Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting transcriptional memory

Metastasis arises from disseminated tumour cells (DTCs) that are characterized by intrinsic phenotypic plasticity and the capability of seeding to secondary organs. DTCs can remain latent for years before giving rise to symptomatic overt metastasis. In this context, DTCs fluctuate between a quiescent and proliferative state in response to systemic and microenvironmental signals including immune-mediated surveillance. Despite its relevance, how intrinsic mechanisms sustain DTCs plasticity has not been addressed. By interrogating the epigenetic state of metastatic cells, we find that tumour progression is coupled with the activation of oncogenic enhancers that are organized in variable interconnected chromatin domains. This spatial chromatin context leads to the activation of a robust transcriptional response upon repeated exposure to retinoic acid (RA). We show that this adaptive mechanism sustains the quiescence of DTCs through the activation of the master regulator SOX9. Finally, we determine that RA-stimulated transcriptional memory increases the fitness of metastatic cells by supporting the escape of quiescent DTCs from NK-mediated immune surveillance. Overall, these findings highlight the contribution of oncogenic enhancers in establishing transcriptional memories as an adaptive mechanism to reinforce cancer dormancy and immune escape, thus amenable for therapeutic intervention

Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores

[Background]: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. [Methods]: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein–protein-interactions), were used for final prioritization. [Conclusion]: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.The present study was supported by the German Research Foundation (DFG)-Grants BE 3828/8-1, LU 1944/2-1, MA 2546/5-1, and LU1944/3-1

Uncovering Enhancer Functions Using the α-Globin Locus

Over the last three decades, studies of the α- and β-globin genes clusters have led to elucidation of the general principles of mammalian gene regulation, such as RNA stability, termination of transcription, and, more importantly, the identification of remote regulatory elements. More recently, detailed studies of α-globin regulation, using both mouse and human loci, allowed the dissection of the sequential order in which transcription factors are recruited to the locus during lineage specification. These studies demonstrated the importance of the remote regulatory elements in the recruitment of RNA polymerase II (PolII) together with their role in the generation of intrachromosomal loops within the locus and the removal of polycomb complexes during differentiation. The multiple roles attributed to remote regulatory elements that have emerged from these studies will be discussed

Orphan CpG islands dictate enhancer-gene compatibility

Trabajo presentado en el Seminario del Centro Nacional de Biotecnología CNB-CSIC, celebrado en modalidad virtual el 19 de febrero de 2021

Compatibility between genes and enhancers in development and congenital disease

Trabajo presentado en los Seminarios de Stanford University, celebrado en modalidad virtual el 4 de marzo de 2020

Orphan CpG islands as prevalent regulators of enhancer activity across vertebrates

Trabajo presentado en el EMBO Workshop: The evolution of animal genomes, celebrado en modalidad virtual del 13 al 17 de septiembre de 2021