Multiple Enhancers Regulate Hoxd Genes and the Hotdog LncRNA during Cecum Budding

Hox genes are required for the development of the intestinal cecum, a major organ of plant-eating species. We have analyzed the transcriptional regulation of Hoxd genes in cecal buds and show that they are controlled by a series of enhancers located in a gene desert flanking the HoxD cluster. The start site of two opposite long noncoding RNAs (lncRNAs), Hotdog and Twin of Hotdog, selectively contacts the expressed Hoxd genes in the framework of a topological domain, coinciding with robust transcription of these genes during cecum budding. Both lncRNAs are specifically transcribed in the cecum, albeit bearing no detectable function in trans. Hedgehogs have kept this regulatory potential despite the absence of the cecum, suggesting that these mechanisms are used in other developmental situations. In this context, we discuss the implementation of a common "budding toolkit" between the cecum and the limbs.

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

Regulatory Switches Underlie Hoxd Gene Activation in Tetrapod Limbs

Guillaume Andrey

Accurate spatio-temporal control of Hox cluster genes expression is critical for the proper organization of body structures during vertebrate embryogenesis. In the limbs, Hoxd genes are critical in setting up the proximodistal and anteroposterior axes. Several studies have shown that long-range global regulatory elements are required to establish Hoxd genes expression territories in this tissue, via the co-regulation of several genes at once. Limb buds take their origin from the lateral plate mesoderm where the HoxD cluster is controlled by cues from the main body axis. During limb outgrowth Hoxd genes are then subjected to two subsequent activation waves: an early phase patterning proximal limb structures, arms and forearms and a late one instructing digits. As the late activation of Hoxd genes is known to rely upon a large regulatory archipelago located centromeric to the gene cluster, the control of the early phase remains poorly described (Montavon et al., 2011). In this work we show that the early activation domain requires a different, telomeric-located, regulatory landscape. This one megabase (Mb) large domain holds at least three regulatory regions contributing to Hoxd gene activation in early forelimb and hindlimb buds. To our surprise the abrogation of most of the early telomeric enhancers induced a dramatic decrease of central Hoxd transcription in forelimbs but only removed 50% of the transcripts in hindlimbs. This discrepancy most likely arises from the difference in the epigenetic status of the HoxD cluster in anterior and posterior limb fields where forelimb and hindlimb respectively take their origin. As the cluster does not possess the same fraction of active genes in these progenitors it most likely does not respond to early cues in the same way. In the second part of this work we noticed an unexpected decrease of the late expression phase in all mutants lacking early enhancers. This observation shows that digit progenitors have to implement the early phase in order to gain competence toward the late centromeric regulation. This transition between early and late regulations involves a set of central Hoxd genes, i.e. Hoxd11 to Hoxd9. To implement both regulations, these loci switch between two topological domains containing the centromeric and telomeric regulatory landscapes respectively (Dixon et al., 2012). The ability to switch from one domain to another depends on the relative position of genes towards the boundary between both domains and correlates with their respective expression. Moreover, the switch between both topological domains mechanically induces an intermediate, Hoxd-free zone, in-between the two expression phases that will later produce the mesopodium, the articulation between our arms and our hands.

EPFL2013

The HoxD cluster is a dynamic and resilient TAD boundary controlling the segregation of antagonistic regulatory landscapes.

Leonardo Beccari, Lucille Delisle, Denis Duboule, Pierre Raymond Fabre, Edgardo Rodriguez Carballo

The mammalian HoxD cluster lies between two topologically associating domains (TADs) matching distinct enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of Hoxd genes in forearm cells, whereas the centromeric TAD subsequently regulates more posterior Hoxd genes in digit cells. Therefore, the TAD boundary prevents the terminal Hoxd13 gene from responding to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryos, we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts of this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient, and only a 400-kb deletion, including the whole-gene cluster, was eventually able to merge the neighboring TADs into a single structure. In this unified TAD, both proximal and distal limb enhancers nevertheless continued to work independently over a targeted transgenic reporter construct. We propose that the whole HoxD cluster is a dynamic TAD border and that the exact boundary position varies depending on both the transcriptional status and the developmental context.