Analysis of long-range gene regulation at the HoxD locus

Hoxd genes are essential for the development of the various body axes in vertebrates and hence the underlying regulatory mechanisms are of paramount importance. Among these various mechanisms are long-range acting enhancers, which are located in the two adjacent regulatory landscapes. Analyses of chromatin architecture at this gene cluster has revealed the existence of two topologically associating domains (TADs) flanking the cluster and encompassing these regulatory landscapes. However, the dynamics of such regulatory regions as well as the stability and functional contribution of specific enhancer-promoter interactions during development remains to be established. In this work, we analysed the 3D chromatin organization and transcription profile at the HoxD locus, at different time points during genital tubercle (GT) development, and observe that the 3D conformation of this regulatory region predates the embryonic emergence of the GT. Along with this tissue development, we observe a reduction in transcript levels correlating with a decrease in enhancer-promoter chromatin loops within the adjacent gene desert. This decrease occurs while maintaining a subset of CTCF/Cohesin associated contacts, which are preserved independently from the transcriptional status of the gene cluster. To further explore the functional contribution of this regulatory landscape, we used CRISPR-Cas9 technology to generate mice carrying partial deletions of this region, as well as targeted deletions of both transient (enhancer associated) and constitutive (CTCF/Cohesin associated) contacts. We observe that single deletions of both transient and constitutive contacts displayed little if any effect on Hoxd genes expression in the GT. On the contrary, the single deletion of a previous characterized Hoxd enhancer, the Prox element, or deletions comprising several enhancers, result in the reduction of Hoxd genes expression levels. Overall our results suggest that not all enhancer elements within a complex regulatory landscape have the same functional strength, and highlight the existence of a dynamic yet robust system to tightly regulate Hoxd genes expression.

Histone Modifications in Development and Malignancy

Elisa Oricchio

Non-Hodgkin lymphoma (NHL) development is driven by the accumulations of multiple genetic, epigenetic, and chromosomal alterations. These lesions can lead to modifications of the chromatin architecture. To identify novel oncogenic interactions driven by modifications of the chromatin 3D organization, we combined high-throughput chromatin conformation capture data (Hi-C) in lymphoma cells with whole genome sequencing (WGS) and epigenetic and transcriptional profiles of primary patient samples and cell lines. Recently, we found that a significant interplay exists between the compartmentalization of the genome into topologically associating domains (TADs) and epigenetic and transcriptional changes mediated by mutated EZH2. Indeed, EZH2 mutations drive aberrant increase of H3K27me3 within specific TADs, resulting in loss of promoter-promoter interactions, synergistic silencing of multiple tumor suppressors and, thus, increased B-cell proliferation and tumor aggressiveness1. Now, we are further investigating how chromosomal amplifications and translocations forms new long-range interactions between enhancers and promoters to support oncogene expression. Importantly, we observed that epigenetic editing of enhancers and pharmacological depletion of H3K27Ac reduces the number of interactions between specific enhancers and promoters and this loss of interactions directly affects gene expression and cell differentiation. These results indicate that is possible to block the oncogenic effect of chromosomal alterations by targeting epigenetic modification in enhancer regions and preventing the formation of oncogenic chromatin interactions. Overall, our results demonstrate that it is important to contextualize the effects of genomic alterations in the 3D organization of the chromatin, to uncover new oncogenic mechanisms and improve the design of new therapeutic approaches.

AMER SOC HEMATOLOGY2019

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

In mammals the circadian clock drives daily behavioural and physiological changes that resonate with environmental cues, which can be observed, for example, in the intricate timing of rest during the night and activity during the day in humans. The circadian clock consists of a network of hierarchical clocks in which the central pacemaker is located in the suprachiasmatic nucleus, which is sensitive to external light and propagates time to the peripheral organ-based clocks. Within organs, clocks tick in each individual cell and are molecularly encoded in the genome as a network of dynamic feedback loops. By mediating the expression of the appropriate gene products at the correct moment of the day, the molecular clocks op-timize physiological functions of virtually every cell in our body, allowing us to synchronize with daily fluc-tuations in the environment. In mammalian cells the chromatin organization in the nucleus consists of a topological network, in which chromatin interactions between distal regulatory elements such as enhancers and target genes are essen-tial to regulate gene expression. Important questions remain: is the complex chromatin folding dynamic and if so on which genomic and temporal scales? In fact, dynamic rearrangements of the chromatin have been reported on multiple genomic and time scales, from the entire genome to enhancer-promoter con-tacts, across developmental transitions, cellular differentiation, and transcription cycles. With this per-spective in mind, the circadian oscillator provides a unique model to study the dynamics of genomic inter-actions in the context of fluctuating transcription. Here, we explored chromatin contacts of core-clock and clock-controlled gene promoters in the mouse. We aimed at evaluating the putative dynamics of chromatin contacts, at estimating their conservation across tissues, and at exploring the contribution of the circadian clock to this interaction network. To achieve this goal, we performed 4C-sequencing assays at two circadian time points in the liver and kidney of WT and clock-deficient animals. Overall, we observe that chromatin contacts are largely conserved across circadian time points and genetic backgrounds, and are tissue-specific. Our experiments reveal that the promoters of core-clock and clock-controlled genes contact distal genomic regions containing en-hancer chromatin signatures, suggesting a functional role of these distal sites in target gene regulation. Our data also suggest a clock-driven module of rhythmic genes that are colocalized in the nucleus. However, we also observe dynamic contacts between oscillating gene promoter and enhancer-like ge-nomic regions. In this thesis, we discuss in detail two examples of dynamic chromatin looping that are ob-served at the Cry1 and Gys2 loci. Our data reveal not only a dynamic coupling between the chromatin loop and the transcriptional state but also that the dynamic of chromatin looping depends on a functional clock. Furthermore, genome editing experiments using CRISPR-Cas9 tools reveal that the Cry1 distal site is essential for the transcriptional regulation of Cry1 and contributes to the circadian clock robustness both in-vivo and in cultured cells. These results demonstrate how local chromatin rearrangements take place in a 24-hour time-scale and set chromatin looping between gene promoters and distal regions as a new reg-ulatory layer for circadian gene expression.

EPFL2017

EZH2 oncogenic mutations drive epigenetic, transcriptional, and structural changes within chromatin domains

Elena Battistello, Giovanni Ciriello, Maria Christine Donaldson, Kyle Michael Douglass, Natalya Katanayeva, Suliana Manley, Marco Mina, Elisa Oricchio, Timo Henry René Rey, Stephanie Jocelyne Sungalee

Chromatin is organized into topologically associating domains (TADs) enriched in distinct histone marks. In cancer, gain-of-function mutations in the gene encoding the enhancer of zeste homolog 2 protein (EZH2) lead to a genome-wide increase in histone-3 Lys27 trimethylation (H3K27me3) associated with transcriptional repression. However, the effects of these epigenetic changes on the structure and function of chromatin domains have not been explored. Here, we found a functional interplay between TADs and epigenetic and transcriptional changes mediated by mutated EZH2. Altered EZH2 (p.Tyr646* (EZH2Y646X)) led to silencing of entire domains, synergistically inactivating multiple tumor suppressors. Intra-TAD gene silencing was coupled with changes of interactions between gene promoter regions. Notably, gene expression and chromatin interactions were restored by pharmacological inhibition of EZH2Y646X. Our results indicate that EZH2Y646X alters the topology and function of chromatin domains to promote synergistic oncogenic programs.

2019

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

EPFL2017