Functional and topological characteristics of mammalian regulatory domains.
Symmons, O., Uslu, V.V., Tsujimura, T., Ruf, S., Nassari, S., Schwarzer, W., Ettwiller, L. & Spitz, F.
Genome Res. 2014 Jan 7.
Long-range regulatory interactions have an important role in shaping gene expression programs. However, the genomic features that organize these activities are still poorly characterized. We conducted a large operational analysis to chart the distribution of gene regulatory activities along the mouse genome, using hundreds of insertions of a regulatory sensor. We found that enhancers distribute their activities along broad regions and not in a gene-centric manner, defining large regulatory domains. Remarkably, these domains correlate strongly with the recently described TADs, which partition the genome into distinct self-interacting blocks. Different features, including specific repeats and CTCF-binding sites, correlate with the transition zones separating regulatory domains, and may help to further organize promiscuously distributed regulatory influences within large domains. These findings support a model of genomic organisation where TADs confine regulatory activities to specific but large regulatory domains, contributing to the establishment of specific gene expression profiles.
Efficient site-specific transgenesis and enhancer activity tests in medaka using PhiC31 integrase.
Kirchmaier, S., Hockendorf, B., Moller, E.K., Bornhorst, D., Spitz, F. & Wittbrodt, J.
Development. 2013 Oct;140(20):4287-95. doi: 10.1242/dev.096081. Epub 2013 Sep 18.
Established transgenesis methods for fish model systems allow efficient genomic integration of transgenes. However, thus far a way of controlling copy number and integration sites has not been available, leading to variable transgene expression caused by position effects. The integration of transgenes at predefined genomic positions enables the direct comparison of different transgenes, thereby improving time and cost efficiency. Here, we report an efficient PhiC31-based site-specific transgenesis system for medaka. This system includes features that allow the pre-selection of successfully targeted integrations early on in the injected generation. Pre-selected embryos transmit the correctly integrated transgene through the germline with high efficiency. The landing site design enables a variety of applications, such as reporter and enhancer switch, in addition to the integration of any insert. Importantly, this allows assaying of enhancer activity in a site-specific manner without requiring germline transmission, thus speeding up large-scale analyses of regulatory elements.
A switch between topological domains underlies HoxD genes collinearity in mouse limbs.
Andrey, G., Montavon, T., Mascrez, B., Gonzalez, F., Noordermeer, D., Leleu, M., Trono, D., Spitz, F. & Duboule, D.
Science. 2013 Jun 7;340(6137):1234167. doi: 10.1126/science.1234167.
Hox genes are major determinants of the animal body plan, where they organize structures along both the trunk and appendicular axes. During mouse limb development, Hoxd genes are transcribed in two waves: early on, when the arm and forearm are specified, and later, when digits form. The transition between early and late regulations involves a functional switch between two opposite topological domains. This switch is reflected by a subset of Hoxd genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently swing toward the centromeric domain, where they establish new contacts. This transition between independent regulatory landscapes illustrates both the modularity of the limbs and the distinct evolutionary histories of its various pieces. It also allows the formation of an intermediate area of low HOX proteins content, which develops into the wrist, the transition between our arms and our hands. This regulatory strategy accounts for collinear Hox gene regulation in land vertebrate appendages.
From remote enhancers to gene regulation: charting the genome's regulatory landscapes.
Symmons, O. & Spitz, F.
Philos Trans R Soc Lond B Biol Sci. 2013 May 6;368(1620):20120358. doi:10.1098/rstb.2012.0358. Print 2013.
Vertebrate genes are characterized by the presence of cis-regulatory elements located at great distances from the genes they control. Alterations of these elements have been implicated in human diseases and evolution, yet little is known about how these elements interact with their surrounding sequences. A recent survey of the mouse genome with a regulatory sensor showed that the regulatory activities of these elements are not organized in a gene-centric manner, but instead are broadly distributed along chromosomes, forming large regulatory landscapes with distinct tissue-specific activities. A large genome-wide collection of expression data from this regulatory sensor revealed some basic principles of this complex genome regulatory architecture, including a substantial interplay between enhancers and other types of activities to modulate gene expression. We discuss the implications of these findings for our understanding of non-coding transcription, and of the possible consequences of structural genomic variations in disease and evolution.
TRACER: a resource to study the regulatory architecture of the mouse genome.
Chen, C.K., Symmons, O., Uslu, V.V., Tsujimura, T., Ruf, S., Smedley, D. & Spitz, F.
BMC Genomics. 2013 Apr 2;14:215. doi: 10.1186/1471-2164-14-215.
BACKGROUND: Mammalian genes are regulated through the action of multiple regulatory elements, often distributed across large regions. The mechanisms that control the integration of these diverse inputs into specific gene expression patterns are still poorly understood. New approaches enabling the dissection of these mechanisms in vivo are needed. RESULTS: Here, we describe TRACER (http://tracerdatabase.embl.de), a resource that centralizes information from a large on-going functional exploration of the mouse genome with different transposon-associated regulatory sensors. Hundreds of insertions have been mapped to specific genomic positions, and their corresponding regulatory potential has been documented by analysis of the expression of the reporter sensor gene in mouse embryos. The data can be easily accessed and provides information on the regulatory activities present in a large number of genomic regions, notably in gene-poor intervals that have been associated with human diseases. CONCLUSIONS: TRACER data enables comparisons with the expression pattern of neighbouring genes, activity of surrounding regulatory elements or with other genomic features, revealing the underlying regulatory architecture of these loci. TRACER mouse lines can also be requested for in vivo transposition and chromosomal engineering, to analyse further regions of interest.
An integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape.
Marinic, M., Aktas, T., Ruf, S. & Spitz, F.
Dev Cell. 2013 Mar 11;24(5):530-42. doi: 10.1016/j.devcel.2013.01.025. Epub 2013Feb 28.
Fgf8 encodes a key signaling factor, and its precise regulation is essential for embryo patterning. Here, we identified the regulatory modules that control Fgf8 expression during mammalian embryogenesis. These enhancers are interspersed with unrelated genes along a large region of 220 kb; yet they act on Fgf8 only. Intriguingly, this region also contains additional genuine enhancer activities that are not transformed into gene expression. Using genomic engineering strategies, we showed that these multiple and distinct regulatory modules act as a coherent unit and influence genes depending on their position rather than on their promoter sequence. These findings highlight how the structure of a locus regulates the autonomous intrinsic activities of the regulatory elements it contains and contributes to their tissue and target specificities. We discuss the implications of such regulatory systems regarding the evolution of gene expression and the impact of human genomic structural variations.
Phenotypic impact of genomic structural variation: insights from and for human disease.
Weischenfeldt, J., Symmons, O., Spitz, F. & Korbel, J.O.
Nat Rev Genet. 2013 Feb;14(2):125-38. doi: 10.1038/nrg3373.
Genomic structural variants have long been implicated in phenotypic diversity and human disease, but dissecting the mechanisms by which they exert their functional impact has proven elusive. Recently however, developments in high-throughput DNA sequencing and chromosomal engineering technology have facilitated the analysis of structural variants in human populations and model systems in unprecedented detail. In this Review, we describe how structural variants can affect molecular and cellular processes, leading to complex organismal phenotypes, including human disease. We further present advances in delineating disease-causing elements that are affected by structural variants, and we discuss future directions for research on the functional consequences of structural variants.
Transcription factors: from enhancer binding to developmental control.
Spitz, F. & Furlong, E.E.
Nat Rev Genet. 2012 Sep;13(9):613-26. doi: 10.1038/nrg3207. Epub 2012 Aug 7.
Developmental progression is driven by specific spatiotemporal domains of gene expression, which give rise to stereotypically patterned embryos even in the presence of environmental and genetic variation. Views of how transcription factors regulate gene expression are changing owing to recent genome-wide studies of transcription factor binding and RNA expression. Such studies reveal patterns that, at first glance, seem to contrast with the robustness of the developmental processes they encode. Here, we review our current knowledge of transcription factor function from genomic and genetic studies and discuss how different strategies, including extensive cooperative regulation (both direct and indirect), progressive priming of regulatory elements, and the integration of activities from multiple enhancers, confer specificity and robustness to transcriptional regulation during development.
A regulatory archipelago controls hox genes transcription in digits.
Montavon, T., Soshnikova, N., Mascrez, B., Joye, E., Thevenet, L., Splinter, E., de Laat, W., Spitz, F. & Duboule, D.
Cell. 2011 Nov 23;147(5):1132-45.
The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations. PAPERFLICK:
Large-scale analysis of the regulatory architecture of the mouse genome with a transposon-associated sensor.
Ruf, S., Symmons, O., Uslu, V.V., Dolle, D., Hot, C., Ettwiller, L. & Spitz, F.
Nat Genet. 2011 Mar 20;43(4):379-86. doi: 10.1038/ng.790.
We present here a Sleeping Beauty-based transposition system that offers a simple and efficient way to investigate the regulatory architecture of mammalian chromosomes in vivo. With this system, we generated several hundred mice and embryos, each with a regulatory sensor inserted at a random genomic position. This large sampling of the genome revealed the widespread presence of long-range regulatory activities along chromosomes, forming overlapping blocks with distinct tissue-specific expression potentials. The presence of tissue-restricted regulatory activities around genes with widespread expression patterns challenges the gene-centric view of genome regulation and suggests that most genes are modulated in a tissue-specific manner. The local hopping property of Sleeping Beauty provides a dynamic approach to map these regulatory domains at high resolution and, combined with Cre-mediated recombination, allows for the determination of their functions by engineering mice with specific chromosomal rearrangements.
KAP1 controls endogenous retroviruses in embryonic stem cells.
Rowe, H.M., Jakobsson, J., Mesnard, D., Rougemont, J., Reynard, S., Aktas, T., Maillard, P.V., Layard-Liesching, H., Verp, S., Marquis, J., Spitz, F., Constam, D.B. & Trono, D.
Nature. 2010 Jan 14;463(7278):237-40.
More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.
Uncoupling time and space in the collinear regulation of Hox genes.
Tschopp, P., Tarchini, B., Spitz, F., Zakany, J. & Duboule, D.
PLoS Genet. 2009 Mar;5(3):e1000398. Epub 2009 Mar 6.
During development of the vertebrate body axis, Hox genes are transcribed sequentially, in both time and space, following their relative positions within their genomic clusters. Analyses of animal genomes support the idea that Hox gene clustering is essential for coordinating the various times of gene activations. However, the eventual collinear ordering of the gene specific transcript domains in space does not always require genomic clustering. We analyzed these complex regulatory relationships by using mutant alleles at the mouse HoxD locus, including one that splits the cluster into two pieces. We show that both positive and negative regulatory influences, located on either side of the cluster, control an early phase of collinear expression in the trunk. Interestingly, this early phase does not systematically impact upon the subsequent expression patterns along the main body axis, indicating that the mechanism underlying temporal collinearity is distinct from those acting during the second phase. We discuss the potential functions and evolutionary origins of these mechanisms, as well as their relationship with similar processes at work during limb development.
Analysis of mammalian gene batteries reveals both stable ancestral cores and highly dynamic regulatory sequences.
Ettwiller, L., Budd, A., Spitz, F. & Wittbrodt, J.
Genome Biol. 2008 Dec 16;9(12):R172.
ABSTRACT: BACKGROUND: Changes in gene regulation are suspected to be one of the driving forces for evolution. To address the extent of cis-regulatory changes and how they impact on global regulatory networks across eukaryotes, we systematically analysed the evolutionary dynamics of target gene batteries controlled by different transcription factors. RESULTS: We found that gene batteries show variable conservation within vertebrates, with slow and fast evolving modules. Hence, while a key gene battery associated with the cell cycle is conserved throughout metazoans, the POU5F1 (Oct4) and SOX2 batteries in ES cells show strong conservation within mammals, with the striking exception of rodents. Within the genes composing a given gene battery, we could identify a conserved core that likely reflects the ancestral function of the corresponding transcription factor. Interestingly, we showed that the association between a transcription factor (TF) and its target genes is conserved even when we excluded conserved sequence similarities of their promoter regions from our analysis. This supports the idea that turnover, either of the core TF binding site or its direct neighbouring sequence, is a pervasive feature of proximal regulatory sequences. CONCLUSION: Our study reveals the dynamics of evolutionary changes within metazoan gene networks - including both the composition of gene batteries and the architecture of target gene promoters. This variation provides the playground required for evolutionary innovation around conserved ancestral core functions.
Genotypic features of lentivirus transgenic mice.
Sauvain, M.O., Dorr, A.P., Stevenson, B., Quazzola, A., Naef, F., Wiznerowicz, M., Schutz, F., Jongeneel, V., Duboule, D., Spitz, F. & Trono, D.
J Virol. 2008 Jul;82(14):7111-9. Epub 2008 May 7.
Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.
Characterization of mouse Dactylaplasia mutations: a model for human ectrodactyly SHFM3.
Friedli, M., Nikolaev, S., Lyle, R., Arcangeli, M., Duboule, D., Spitz, F. & Antonarakis, S.E.
Mamm Genome. 2008 Apr;19(4):272-8. Epub 2008 Apr 5.
SHFM3 is a limb malformation characterized by the absence of central digits. It has been shown that this condition is associated with tandem duplications of about 500 kb at 10q24. The Dactylaplasia mice display equivalent limb defects and the two corresponding alleles (Dac ( 1j ) and Dac ( 2j )) map in the region syntenic with the duplications in SHFM3. Dac ( 1j ) was shown to be associated with an insertion of an unspecified ETn-like mouse endogenous transposon upstream of the Fbxw4 gene. Dac ( 2j ) was also thought to be an insertion or a small inversion in intron 5 of Fbxw4, but the breakpoints and the exact molecular lesion have not yet been characterized. Here we report precise mapping and characterization of these alleles. We failed to identify any copy number differences within the SHFM3 orthologous genomic locus between Dac mutant and wild-type littermates, showing that the Dactylaplasia alleles are not associated with duplications of the region, in contrast with the described human SHFM3 cases. We further show that both Dac ( 1j ) and Dac ( 2j ) are caused by insertions of MusD retroelements that share 98% sequence identity. The differences between the nature of the human and mouse genomic abnormalities argue against models proposed so far that either envisioned SHFM3 as a local trisomy or Dac as a mutant allele of Fbxw4. Instead, both genetic conditions might lead to complex alterations of gene regulation mechanisms that would impair limb morphogenesis. Interestingly, the Dac ( 2j ) mutation occurs within a highly conserved element that may represent a regulatory sequence for a neighboring gene.
Global control regions and regulatory landscapes in vertebrate development and evolution.
Spitz, F. & Duboule, D.
Adv Genet. 2008;61:175-205. doi: 10.1016/S0065-2660(07)00006-5.
During the course of evolution, many genes that control the development of metazoan body plans were co-opted to exert novel functions, along with the emergence or modification of structures. Gene amplification and/or changes in the cis-regulatory modules responsible for the transcriptional activity of these genes have certainly contributed in a major way to evolution of gene functions. In some cases, these processes led to the formation of groups of adjacent genes that appear to be controlled by both global and shared mechanisms.
Transgenic analysis of Hoxd gene regulation during digit development.
Gonzalez, F., Duboule, D. & Spitz, F.
Dev Biol. 2007 Jun 15;306(2):847-59. Epub 2007 Mar 23.
In tetrapods, posterior Hoxd genes (from groups 10 to 13) are necessary to properly pattern the developing autopods, including the number and identities of digits. Their coordinated expression is achieved by sharing a global control region (GCR), which was isolated and localized 200 kb 5' (centromeric) of the gene cluster. However, in transgenic assays, the GCR was unable to fully recapitulate all aspects of the endogenous Hoxd expression patterns during distal limb development. In this paper, we further analyze the regulatory potential of this locus and report the characterization of Prox, a second enhancer element that contributes to the transcriptional activity of posterior Hoxd genes in developing distal limb buds. We show that the GCR and Prox elements complement each other and work in combination to correctly establish the late phase of Hoxd genes expression. Based on DNA sequence conservation and transgenic assays, we discuss the functions of these regulatory regions as well as a potential evolutionary scheme accounting for their emergence along with the evolution of tetrapod limbs.
Genomics and development: Taking developmental biology to new heights.
Spitz, F. & Furlong, E.E.
Dev Cell. 2006 Oct;11(4):451-7.
The 2006 Arolla meeting brought together scientists from around the globe to discuss how genomic scale analyses can enhance progress in understanding developmental biology.
Inversion-induced disruption of the Hoxd cluster leads to the partition of regulatory landscapes.
Spitz, F., Herkenne, C., Morris, M.A. & Duboule, D.
Nat Genet. 2005 Aug;37(8):889-93. Epub 2005 Jul 3.
The developmental regulation of vertebrate Hox gene transcription relies on the interplay between local and long-range controls. To study this complex genomic organization, we designed a strategy combining meiotic and targeted recombinations to induce large chromosomal rearrangements in vivo without manipulating embryonic stem cells. With this simple approach (called STRING), we engineered a large 7-cM inversion, which split the Hoxd cluster into two independent pieces. Expression analyses showed a partition of global enhancers, allowing for their precise topographic allocation on either side of the cluster. Such a functional organization probably contributed to keeping these genes clustered in the course of vertebrate evolution. This approach can be used to study the relationship between genome architecture and gene expression, such as the effects of genome rearrangements in human diseases or during evolution.
Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression.
Zuniga, A., Michos, O., Spitz, F., Haramis, A.P., Panman, L., Galli, A., Vintersten, K., Klasen, C., Mansfield, W., Kuc, S., Duboule, D., Dono, R. & Zeller, R.
Genes Dev. 2004 Jul 1;18(13):1553-64. Epub 2004 Jun 15.
The mouse limb deformity (ld) mutations cause limb malformations by disrupting epithelial-mesenchymal signaling between the polarizing region and the apical ectodermal ridge. Formin was proposed as the relevant gene because three of the five ld alleles disrupt its C-terminal domain. In contrast, our studies establish that the two other ld alleles directly disrupt the neighboring Gremlin gene, corroborating the requirement of this BMP antagonist for limb morphogenesis. Further doubts concerning an involvement of Formin in the ld limb phenotype are cast, as a targeted mutation removing the C-terminal Formin domain by frame shift does not affect embryogenesis. In contrast, the deletion of the corresponding genomic region reproduces the ld limb phenotype and is allelic to mutations in Gremlin. We resolve these conflicting results by identifying a cis-regulatory region within the deletion that is required for Gremlin activation in the limb bud mesenchyme. This distant cis-regulatory region within Formin is also altered by three of the ld mutations. Therefore, the ld limb bud patterning defects are not caused by disruption of Formin, but by alteration of a global control region (GCR) required for Gremlin transcription. Our studies reveal the large genomic landscape harboring this GCR, which is required for tissue-specific coexpression of two structurally and functionally unrelated genes.
A global control region defines a chromosomal regulatory landscape containing the HoxD cluster.
Spitz, F., Gonzalez, F. & Duboule, D.
Cell. 2003 May 2;113(3):405-17.
During limb development, coordinated expression of several Hoxd genes is required in presumptive digits. We searched for the underlying control sequences upstream from the cluster and found Lunapark (Lnp), a gene which shares limb and CNS expression specificities with both Hoxd genes and Evx2, another gene located nearby. We used a targeted enhancer-trap approach to identify a DNA segment capable of directing reporter gene expression in both digits and CNS, following Lnp, Evx2, and Hoxd-specific patterns. This DNA region showed an unusual interspecies conservation, including with its pufferfish counterpart. It contains a cluster of global enhancers capable of controlling transcription of several genes unrelated in structure or function, thus defining large regulatory domains. These domains were interrupted in the Ulnaless mutation, a balanced inversion that modified the topography of the locus. We discuss the heuristic value of these results in term of locus specific versus gene-specific regulation.