Impact of genome architecture on the functional activation and repression of Hox regulatory landscapes

Background The spatial organization of the mammalian genome relies upon the formation of chromatin domains of various scales. At the level of gene regulation in cis, collections of enhancer sequences define large regulatory landscapes that usually match with the presence of topologically associating domains (TADs). These domains often contain ranges of enhancers displaying similar or related tissue specificity, suggesting that in some cases, such domains may act as coherent regulatory units, with a global on or off state. By using the HoxD gene cluster, which specifies the topology of the developing limbs via highly orchestrated regulation of gene expression, as a paradigm, we investigated how the arrangement of regulatory domains determines their activity and function.ResultsProximal and distal cells in the developing limb express different levels of Hoxd genes, regulated by flanking 3 and 5 ' TADs, respectively. We characterized the effect of large genomic rearrangements affecting these two TADs, including their fusion into a single chromatin domain. We show that, within a single hybrid TAD, the activation of both proximal and distal limb enhancers globally occurred as when both TADs are intact. However, the activity of the 3 ' TAD in distal cells is generally increased in the fused TAD, when compared to wild type where it is silenced. Also, target gene activity in distal cells depends on whether or not these genes had previously responded to proximal enhancers, which determines the presence or absence of H3K27me3 marks. We also show that the polycomb repressive complex 2 is mainly recruited at the Hox gene cluster and can extend its coverage to far-cis regulatory sequences as long as confined to the neighboring TAD structure.Conclusions We conclude that antagonistic limb proximal and distal enhancers can exert their specific effects when positioned into the same TAD and in the absence of their genuine target genes. We also conclude that removing these target genes reduced the coverage of a regulatory landscape by chromatin marks associated with silencing, which correlates with its prolonged activity in time.

Ectopic nuclear reorganisation driven by a Hoxb1 transgene transposed into Hoxd

Denis Duboule

The extent to which the nuclear organisation of a gene impacts on its ability to be expressed, or whether nuclear organisation merely reflects gene expression states, remains an important but unresolved issue. A model system that has been instrumental in investigating this question utilises the murine Hox gene clusters encoding homeobox- containing proteins. Nuclear reorganisation and chromatin decondensation, initiated towards the 3' end of the clusters, accompanies activation of Hox genes in both differentiation and development, and might be linked to mechanisms underlying colinearity. To investigate this, and to delineate the cis-acting elements involved, here we analyse the nuclear behaviour of a 3' Hoxb1 transgene transposed to the 5' end of the Hoxd cluster. We show that this transgene contains the cis-acting elements sufficient to initiate ectopic local nuclear reorganisation and chromatin decondensation and to break Hoxd colinearity in the primitive streak region of the early embryo. Significantly, in rhombomere 4, the transgene is able to induce attenuated nuclear reorganisation and decondensation of Hoxd even though there is no detectable expression of the transgene at this site. This shows that reorganisation of chromosome territories and chromatin decondensation can be uncoupled from transcription itself and suggests that they can therefore operate upstream of gene expression.

2008

An ancestral regulatory mechanism underlies Hoxd gene expression in both developing genitals and digits

Nicolas Lonfat

Coordinated expression of HoxD cluster genes is crucial for the emergence and patterning of both developing digits and external genitalia. As a shared evolutionary origin has been proposed for these secondary structures, which may have accompanied the transition from an aquatic towards a terrestrial life, we investigated whether the regulatory circuitries would also share similarities, indicating a common phylogenetic origin. We show that the transcription of posterior Hoxd genes in the distal part of these different appendages depends, in both cases, upon enhancer sequences located centromeric to the cluster, in a gene poor region, previously described as forming a regulatory archipelago in digit cells. Analysis of the epigenetic status and spatial conformation of the HoxD cluster and its centromeric neighborhood has identified several putative regulatory elements that interact with Hoxd genes via chromatin looping in the developing genital tubercle. In vivo, targeted deletions within the centromeric gene desert further revealed the requirement of these sequences for Hoxd genes transcription in this embryonic structure. We used lentivirus-mediated transgenesis to evaluate the enhancer activity of the identified regulatory elements. While some sequences were able to drive a reporter gene in both digits and genitalia, others were specific for either one of these two structures. We identified Cdx2 transcription factor as a putative interactor of the genitalspecific enhancers. These results suggest a common ancestral regulatory strategy for digits and genitals, yet with specific adaptations in each developmental context. This work should help understand the regulatory mechanism underlying Hoxd gene expression in the genital tubercle and digits, as well as the phylogenetic histories of such global regulations.

EPFL2013

Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states

Aleksandr Benke, Denis Duboule, Pierre Raymond Fabre, Suliana Manley

Chromatin condensation plays an important role in the regulation of gene expression. Recently, it was shown that the transcriptional activation of Hoxd genes during vertebrate digit development involves modifications in 3D interactions within and around the HoxD gene cluster. This reorganization follows a global transition from one set of regulatory contacts to another, between two topologically associating domains (TADs) located on either side of the HoxD locus. Here, we use 3D DNA FISH to assess the spatial organization of chromatin at and around the HoxD gene cluster and report that although the two TADs are tightly associated, they appear as spatially distinct units. We measured the relative position of genes within the cluster and found that they segregate over long distances, suggesting that a physical elongation of the HoxD cluster can occur. We analyzed this possibility by super-resolution imaging (STORM) and found that tissues with distinct transcriptional activity exhibit differing degrees of elongation. We also observed that the morphological change of the HoxD cluster in developing digits is associated with its position at the boundary between the two TADs. Such variations in the fine-scale architecture of the gene cluster suggest causal links among its spatial configuration, transcriptional activation, and the flanking chromatin context.

National Academy of Sciences2015