Laurent M. Sachs

CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

A role for cofactor–cofactor and cofactor–histone interactions in targeting p300, SWI/SNF and Mediator for transcription

Transcriptional activation from chromatin by nuclear receptors (NRs) requires multiple cofactors including CBP/p300, SWI/SNF and Mediator. How NRs recruit these multiple cofactors is not clear. Here we show that activation by androgen receptor and thyroid hormone receptor is associated with the promoter targeting of SRC family members, p300, SWI/SNF and the Mediator complex. We show that recruitment of SWI/SNF leads to chromatin remodeling with altered DNA topology, and that both SWI/SNF and p300 histone acetylase activity are required for hormone‐dependent activation. Importantly, we show that both the SWI/SNF and Mediator complexes can be targeted to chromatin by p300, which itself is recruited through interaction with SRC coactivators. Furthermore, histone acetylation by CBP/p300 facilitates the recruitment of SWI/SNF and Mediator. Thus, our data indicate that multiple cofactors required for activation are not all recruited through their direct interactions with NRs and underscore a role of cofactor–cofactor interaction and histone modification in coordinating the recruitment of multiple cofactors.

Non-viral gene transfer: Applications in developmental biology and gene therapy

The main limitation of non-viral gene transfer methods is their relatively low efficiency in vivo. However, a number of approaches can be taken to improve their performances, whether the aim is studying gene function during development or employing these techniques for gene therapy. Three non-viral delivery systems that we have been particularly involved in in developing are described: the cationic lipid, dioctadecylamidoglycylspermine (DOGS), the cationic polymer polyethylenimine (PEI) and free DNA. The application of each of these methods to different in vivo situations is presented: the use of DOGS for transfecting embryos and the developing mammalian nervous system; the recent application of PEI to the nervous system; and how naked DNA can be employed for transfecting different muscles and brain. The relative efficiencies are compared on the basis of luciferase reporter gene expression assessed in each tissue with the most appropriate vector system. Finally, the perspectives for constructing composite vectors combining safety and efficiency are considered briefly.

Unliganded thyroid hormone receptor is essential for Xenopus laevis eye development

Thyroid hormone receptors generally activate transcription of target genes in the presence of thyroid hormone (T3) and repress their transcription in its absence. Here, we investigated the role of unliganded thyroid hormone receptor (TR) during vertebrate development using an amphibian model. Previous studies led to the hypothesis that before production of endogenous T3, the presence of unliganded receptor is essential for premetamorphic tadpole growth. To test this hypothesis, we generated a Xenopus laevis TR β mutant construct ineffective for gene repression owing to impaired corepressor NCoR recruitment. Overexpression by germinal transgenesis of the mutant receptor leads to lethality during early development with numerous defects in cranio‐facial and eye development. These effects correlate with TR expression profiles at these early stages. Molecular analysis of transgenic mutants reveals perturbed expression of genes involved in eye development. Finally, treatment with iopanoic acid or NH‐3, modulators of thyroid hormone action, leads to abnormal eye development. In conclusion, the data reveal a role of unliganded TR in eye development.

Metamorphic T3‐response genes have specific co‐regulator requirements

Thyroid hormone receptors (TRs) have several regulatory functions in vertebrates. In the absence of thyroid hormone (T3; triiodothyronine), apo‐TRs associate with co‐repressors to repress transcription, whereas in the presence of T3, holo‐TRs engage transcriptional coactivators. Although many studies have addressed the molecular mechanisms of T3 action, it is not known how specific physiological responses arise. We used T3‐dependent amphibian metamorphosis to analyse how TRs interact with particular co‐regulators to differentially regulate gene expression during development. Using chromatin immunoprecipitation to study tissue from pre‐metamorphic tad‐poles, we found that TRs are physically associated with T3‐responsive promoters, whether or not T3 is present. Addition of T3 results in histone H4 acetylation specifically on T3‐response genes. Most importantly, we show that individual T3‐response genes have distinct co‐regulator requirements, the T3‐dependent co‐repressor‐to‐coactivator switch being gene‐specific for both co‐regulator categories.

Specific Histone Lysine 4 Methylation Patterns Define TR-Binding Capacity and Differentiate Direct T3 Responses

The diversity of thyroid hormone T(3) effects in vivo makes their molecular analysis particularly challenging. Indeed, the current model of the action of T(3) and its receptors on transcription does not reflect this diversity. Here, T(3)-dependent amphibian metamorphosis was exploited to investigate, in an in vivo developmental context, how T(3) directly regulates gene expression. Two, direct positively regulated T(3)-response genes encoding transcription factors were analyzed: thyroid hormone receptor β (TRβ) and TH/bZIP. Reverse transcription-real-time quantitative PCR analysis on Xenopus tropicalis tadpole brain and tail fin showed differences in expression levels in premetamorphic tadpoles (lower for TH/bZIP than for TRβ) and differences in induction after T(3) treatment (lower for TRβ than for TH/bZIP). To dissect the mechanisms underlying these differences, chromatin immunoprecipitation was used. T(3) differentially induced RNA polymerase II and histone tail acetylation as a function of transcriptional level. Gene-specific patterns of TR binding were found on the different T(3) -responsive elements (higher for TRβ than for TH/bZIP), correlated with gene-specific modifications of H3K4 methylation (higher for TRβ than for TH/bZIP). Moreover, tissue-specific modifications of H3K27 were found (lower in brain than in tail fin). This first in vivo analysis of the association of histone modifications and TR binding/gene activation during vertebrate development for any nuclear receptor indicate that chromatin context of thyroid-responsive elements loci controls the capacity to bind TR through variations in histone H3K4 methylation, and that the histone code, notably H3, contributes to the fine tuning of gene expression that underlies complex physiological T(3) responses.

Transgenesis procedures in Xenopus.

Stable integration of foreign DNA into the frog genome has been the purpose of several studies aimed at generating transgenic animals or producing mutations of endogenous genes. Inserting DNA into a host genome can be achieved in a number of ways. In Xenopus, different strategies have been developed which exhibit specific molecular and technical features. Although several of these technologies were also applied in various model organizms, the attributes of each method have rarely been experimentally compared. Investigators are thus confronted with a difficult choice to discriminate which method would be best suited for their applications. To gain better understanding, a transgenesis workshop was organized by the X‐omics consortium. Three procedures were assessed side‐by‐side, and the results obtained are used to illustrate this review. In addition, a number of reagents and tools have been set up for the purpose of gene expression and functional gene analyses. This not only improves the status of Xenopus as a powerful model for developmental studies, but also renders it suitable for sophisticated genetic approaches. Twenty years after the first reported transgenic Xenopus, we review the state of the art of transgenic research, focusing on the new perspectives in performing genetic studies in this species.

Mechanisms of thyroid hormone receptor action during development: lessons from amphibian studies.

BACKGROUND Thyroid hormone (TH) receptor (TR) plays critical roles in vertebrate development. However, the in vivo mechanism of TR action remains poorly explored. SCOPE OF REVIEW Frog metamorphosis is controlled by TH and mimics the postembryonic period in mammals when high levels of TH are also required. We review here some of the findings on the developmental functions of TH and TR and the associated mechanisms obtained from this model system. MAJOR CONCLUSION A dual function model for TR in Anuran development was proposed over a decade ago. That is, unliganded TR recruits corepressors to TH response genes in premetamorphic tadpoles to repress these genes and prevent premature metamorphic changes. Subsequently, when TH becomes available, liganded TR recruits coactivators to activate these same genes, leading to metamorphic changes. Over the years, molecular and genetic approaches have provided strong support for this model. Specifically, it has been shown that unliganded TR recruits histone deacetylase containing corepressor complexes during larval stages to control metamorphic timing, while liganded TR recruits multiple histone modifying and chromatin remodeling coactivator complexes during metamorphosis. These complexes can alter chromatin structure via nucleosome position alterations or eviction and histone modifications to contribute to the recruitment of transcriptional machinery and gene activation. GENERAL SIGNIFICANCE The molecular mechanisms of TR action in vivo as revealed from studies on amphibian metamorphosis are very likely applicable to mammalian development as well. These findings provide a new perspective for understanding the diverse effects of TH in normal physiology and diseases caused by TH dysfunction. This article is part of a Special Issue entitled Thyroid hormone signalling.

The co‐chaperone XAP2 is required for activation of hypothalamic thyrotropin‐releasing hormone transcription in vivo

Transcriptional control of hypothalamic thyrotropin‐releasing hormone (TRH) integrates central regulation of the hypothalamo‐hypophyseal‐thyroid axis and hence thyroid hormone (triiodothyronine (T3)) homeostasis. The two β thyroid hormone receptors, TRβ1 and TRβ2, contribute to T3 feedback on TRH, with TRβ1 having a more important role in the activation of TRH transcription. How TRβ1 fulfils its role in activating TRH gene transcription is unknown. By using a yeast two‐hybrid screening of a mouse hypothalamic complementary DNA library, we identified a novel partner for TRβ1, hepatitis virus B X‐associated protein 2 (XAP2), a protein first identified as a co‐chaperone protein. TR–XAP2 interactions were TR isoform specific, being observed only with TRβ1, and were enhanced by T3 both in yeast and mammalian cells. Furthermore, small inhibitory RNA‐mediated knockdown of XAP2 in vitro affected the stability of TRβ1. In vivo, siXAP2 abrogated specifically TRβ1‐mediated (but not TRβ2) activation of hypothalamic TRH transcription. This study provides the first in vivo demonstration of a regulatory, physiological role for XAP2.

Xenopus laevis as a model for studying thyroid hormone signalling : From development to metamorphosis

Amphibian metamorphosis is a well-established model for dissecting the mechanisms underlying thyroid hormone (TH) action. How the pro-hormone, T(4), the active form, T(3), the deiodinases and the nuclear receptors (TRs) contribute to metamorphosis in Xenopus has been extensively investigated. Our recent work has concentrated on two key ideas in TH signalling in Xenopus: first, that there could be active roles for both liganded and unliganded receptors, and second, that ligand availability is a determining factor orchestrating these actions and is tightly controlled in target tissues. Recently, we addressed these questions at stages preceding metamorphosis, i.e. during embryogenesis, before differentiation of a functional thyroid gland. We show that repression by unliganded TR is essential to craniofacial and eye development during early development and that at these stages all three deiodinases are active. These results open new perspectives on the potential roles of TH signalling during embryogenesis.