Coordinated Hoxd genes expression during embryogenesis is dependent on a strict disposition of genes and polarized regulators within the HoxD cluster

HOX transcription factors determine the identity of body regions along the rostro-caudal axis during bilaterian embryogenesis. In vertebrates Hox genes distinctively lie organized in dense clusters, each typically composed of a dozen paralogous transcription units spread over 100 kb of genomic DNA. In every Hox cluster the Hox genes are arranged following a complete 5'-3' transcriptional polarization. Even though Hox are found across the whole animal kingdom, this ordered physical arrangement distinctly characterizes vertebrate Hox clusters. This organization, and the attendant collinearity phenomena have been proposed to be a crucial feature to Hox genes' co-option in several structures organized along secondary body axes, as exemplified by the patterning of various tetrapod-specific structures (e.g. limbs, dig-its and external genitalia). The aim of this thesis is to investigate to what extent Hox transcriptional polarization results from constraints imposed by their close linear proximity when arranged in clusters. Therefore, we challenged the murine HoxD cluster by engineering inversions inside the cluster using CRISPR/Cas9 mutagenesis approaches in vivo. We produced two novel alleles bearing inversions of either Hoxd11 or Hoxd12. Complementarily, we also reanalyzed extensively a larger targeted inversion encompassing Hoxd11-d12 loci in a variety of embryonic organs and tissues. Together, the comparative analysis of these alleles by a combination of approaches (WISH, RNAseq, ChIPseq and 4Cseq) illuminated different types of outcomes resulting from the disruption of HoxD transcriptional polarity and the inversion of non-coding regulatory elements. From our work, we developed the view that engineered inversions of particular Hox genes are tolerated by this genetic system and does not result systematically in major functional disruption of the affected cluster. This result therefore demonstrates that Hox transcriptional polarity is not a feature arising from an absolute developmental constraint. This conclusion is nevertheless nuanced by two instances where intra-HoxD inversion disrupts the regulation and function of the HoxD cluster. In one case, the inversion of Hoxd11 affects the transcriptional output of its posterior neighbor Hoxd12. This perturbation is likely implying interference mechanism related to the transcriptional leakage emitted onto Hoxd12 by the inverted Hoxd11 locus. In the second case, the inversion of the Hoxd11-d12 loci in a Hoxd13lacZ background results in the specific gain-of-expression of this reporter gene in a variety of embryonic structures. Of interest, we present here diverse pieces of evidence that this dysregulation may not be directly related to the disruption of Hox gene transcriptional polarity but instead caused by the translocation and inversion of a CTCF binding site. This observation supports the hypothesis that CTCF dependent topological organization of the chromatin plays a role in the regulatory insulation of Hoxd13. It appears therefore that spontaneous inversions within polarized Hox clusters might affect the function and regulation of the Hox patterning mechanism in different functional contexts. This conclusion leads us to conjecture that once disposed in dense clusters, the developmental expression of Hox genes is extremely sensitive to genetic perturbations, due notably to the presence of several conserved polarized regulatory elements interspersed between the individual Hox genes i

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

Studies on KRAB/KAP1-Mediated Transcriptional Repression

Anna Claire Groner

The sequencing of the human genome revealed that the C2H2-zinc finger proteins (ZFPs) are the largest family of human transcription factors. Around 300 members of this family contain, in addition to their zinc finger motifs, a KRAB domain that has been implicated in transcriptional repression. It is thought that KRAB-ZFPs mediate this effect through the recruitment of the corepressor KAP1, which induces facultative heterochromatin through the activity of several associated chromatin-modifying factors. Our studies focused on the elucidation of the molecular mechanisms of transcriptional repression induced by KRAB-ZFPs and KAP1. For this, we made use of the conditional binding activity of the KRAB-containing repressor tTRKRAB. This ectopic protein serves as a paradigm for endogenous KRAB-ZFP functions, as it depends on cellular KAP1 for transcriptional repression. tTRKRAB only binds to heterologous TetO sites in the absence of doxycycline, thus making repressor activity drug-regulated. When we targeted the repressor to euchromatic sites modified with TetO, we found that it induced increased levels of H3K9me3 and HP1β at the genes it bound to. We also monitored the effect of repressor binding on transcription by assessing the activity of TetO-containing reporter genes that were driven by endogenous promoters and that were initially used to create euchromatic tTRKRAB binding sites. This analysis revealed that KRAB/KAP1 mediated long-range heterochromatin spreading, which only resulted in transcriptional repression if repressive marks reached the promoter and impaired transcriptional initiation. The efficiency of this process was highest in an environment of very strong gene activity, as indicated by the levels of active histone modifications. We found that KRAB-ZFP genes themselves are probably targets of KRAB/KAP1-mediated repression and elucidated their transcriptional regulation and chromatin structure. KAP1 was localized to the promoter and to the 3' end of KRAB-ZFP genes, suggesting self-regulatory loops. However, only KAP1 localized to the 3' end of KRAB-ZFP genes mediated increased levels of H3K9me3 and HP1β along the gene body, without affecting the promoter. Therefore, KAP1 is involved in controlling the spread of heterochromatin at these endogenous sites, largely without regulating transcription. The function of this heterochromatic chromatin landscape may lie in the protection from chromosomal rearrangements between highly homologous KRAB-ZFP genes. Despite this repressive structure, KRAB-ZFP genes are still expressed, as high levels of transcription-associated H3K36me3 accompany H3K9me3 throughout the gene bodies. Other projects described in this thesis include the elucidation of the role of KAP1 in behavior and the assessment of KRAB/KAP1-mediated repression in the context of episomal structures. Both of these areas are of scientific and biomedical interest and address basic principles of the molecular mechanisms of KRAB/KAP1-mediated transcriptional repression.

EPFL2011

Studies on KRAB/KAP1-Mediated Transcriptional Repression

Anna Claire Groner

EPFL2011