Cooperation of regulatory elements involved in the activation of the Hoxd-11 gene

We have used lacZ reporter gene constructs to study the cis regulation of the murine Hoxd-11 gene (previously Hox-4.6) in transgenic mice. We identified a genomic region, which was able to simulate important aspects of the developmental regulation of the gene. A short regulatory region, located 3' to the Hoxd-11 transcription unit, was required to mimic initial activation. This regulatory region contains two stretches of DNA, that are highly conserved in the chicken Hoxd-11 3' flanking region. A chimeric construct containing these short homologous regions from the chicken gene could replace the complete murine fragment thus demonstrating that the conserved domains carry the main regulatory elements involved in this activation. The first half of this bipartite regulatory region has positive regulatory activity, while the second half is required to restrict gene expression to the proper posterior domain in the somitic mesoderm. Our results suggest that the control of the Hoxd-11 promoter involves tissue-specific cooperations between regulatory elements.

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

Context-dependent enhancer function revealed by targeted inter-TAD relocation

Guillaume Andrey, Christopher Chase Bolt, Lucille Delisle, Denis Duboule, Aurélie Hintermann

Here the authors show that a strong enhancer sequence can be controlled by the chromatin environment provided by a topologically associated domain (TAD) located nearby. An enhancer relocated by homologous recombination takes all the hallmarks of its new neighboring enhancers located in the recipient TAD. The expression of some genes depends on large, adjacent regions of the genome that contain multiple enhancers. These regulatory landscapes frequently align with Topologically Associating Domains (TADs), where they integrate the function of multiple similar enhancers to produce a global, TAD-specific regulation. We asked if an individual enhancer could overcome the influence of one of these landscapes, to drive gene transcription. To test this, we transferred an enhancer from its native location, into a nearby TAD with a related yet different functional specificity. We used the biphasic regulation of Hoxd genes during limb development as a paradigm. These genes are first activated in proximal limb cells by enhancers located in one TAD, which is then silenced when the neighboring TAD activates its enhancers in distal limb cells. We transferred a distal limb enhancer into the proximal limb TAD and found that its new context suppresses its normal distal specificity, even though it is bound by HOX13 transcription factors, which are responsible for the distal activity. This activity can be rescued only when a large portion of the surrounding environment is removed. These results indicate that, at least in some cases, the functioning of enhancer elements is subordinated to the host chromatin context, which can exert a dominant control over its activity.

NATURE PORTFOLIO2022

Toward a mechanistic understanding of variable chromatin modules

Gerard Llimos Aubach

Our genome is a long sequence of DNA that contains all the information to be able to constitute a living organism like us, similarly to what the letters in a book do to create a story. This sequence, which is a stretch of molecules called nucleotides, is almost identical between organisms of the same species (>99.9% in humans), however it varies in a small percentage due to some nucleotides being different at specific positions of the genome. This variation in the DNA will therefore influence our traits and affect our susceptibility to a certain condition. Thus, their study is highly relevant in genetics and biomedicine since they allow not only to predict the predisposition to a disease, such as cancer, but also to understand the molecular and cellular mechanism behind a certain phenotype. The effect of a genetic variant on a phenotype is given by their capacity to modulate the expression of a gene, either by directly impacting its sequence and thus changing the protein, or by regulating its expression levels. Interestingly, more than 88% of the disease-associated genetic variants present in humans are located outside genes, on the so-called non-coding part of the genome. This poses a challenge to identify what gene the genetic variant affects and to understand how it does so. Genetic variants do not only influence gene expression but they also affect the epigenetics state of the DNA (i.e. DNA methylation, histone marks, transcription factor (TF) binding...) and its 3D conformation, consequently modifying the activity of gene regulatory elements like promoters and enhancers. Previously, it has been shown that some of these regulatory regions located in the same locus exhibit a high level of molecular coordination and interindividual variation, mostly affected by genetic variation or environmental effects. These regions were termed variable chromatin modules (VCMs) or cis-regulatory domains (CRDs) and are thought to be the manifestation of fine-grained regulatory units of the genome. Understanding how a VCM is formed and the molecular basis behind the cooperativity between regulatory elements is an outstanding question in the field. In this work, we aim at resolving part of this puzzle by mechanistically dissecting one VCM that is influenced by a non-coding germline variant in B cells. Using a set of tools such as genetic engineering, TF binding assays and chromosome conformation studies, we demonstrate that this variant creates a de novo binding site for the TF MEF2, which acts as a pioneering factor for dozens of other collaborating TFs. In turn, this actuates a cluster of enhancers spanning a region over 150 kilobases (kb) that regulates AXIN2 gene expression, and is associated with a global compaction of the chromatin. In addition, in line with the known tumor-suppressor properties of AXIN2, we found that this variant influences chronic lymphocytic leukemia (CLL) progression and predisposition. Together, the characterization of the AXIN2 VCM provides an unique example to the community of how a germline variant is able to switch on an entire genomic locus with high degree of cooperativity between regulatory elements, since it captures both the formation and transition behavior of a VCM. Furthermore, given the link with CLL, this focal variant could have translational potential by serving as a patient-stratifying or treatment diagnostic.

EPFL2021

Toward a mechanistic understanding of variable chromatin modules

Gerard Llimos Aubach

EPFL2021