Christophe Verthuy

Polycomb Mediated Epigenetic Silencing and Replication Timing at the INK4a/ARF Locus during Senescence

Background The INK4/ARF locus encodes three tumor suppressor genes (p15Ink4b, Arf and p16Ink4a) and is frequently inactivated in a large number of human cancers. Mechanisms regulating INK4/ARF expression are not fully characterized. Principal Findings Here we show that in young proliferating embryonic fibroblasts (MEFs) the Polycomb Repressive Complex 2 (PRC2) member EZH2 together with PRC1 members BMI1 and M33 are strongly expressed and localized at the INK4/ARF regulatory domain (RD) identified as a DNA replication origin. When cells enter senescence the binding to RD of both PRC1 and PRC2 complexes is lost leading to a decreased level of histone H3K27 trimethylation (H3K27me3). This loss is accompanied with an increased expression of the histone demethylase Jmjd3 and with the recruitment of the MLL1 protein, and correlates with the expression of the Ink4a/Arf genes. Moreover, we show that the Polycomb protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. Finally, we demonstrate that Polycomb proteins and associated epigenetic marks are crucial for the control of the replication timing of the INK4a/ARF locus during senescence. Conclusions We identified the replication licencing factor CDC6 as a new partner of the Polycomb group member BMI1. Our results suggest that in young cells Polycomb proteins are recruited to the INK4/ARF locus through CDC6 and the resulting silent locus is replicated during late S-phase. Upon senescence, Jmjd3 is overexpressed and the MLL1 protein is recruited to the locus provoking the dissociation of Polycomb from the INK4/ARF locus, its transcriptional activation and its replication during early S-phase. Together, these results provide a unified model that integrates replication, transcription and epigenetics at the INK4/ARF locus.

Definition of a T-cell receptor beta gene core enhancer of V(D)J recombination by transgenic mapping.

ABSTRACT V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activatingcis-recombination events. A striking example is the T-cell receptor β-chain (TCRβ) gene enhancer (Eβ), which in the mouse consists of at least seven nuclear factor binding motifs (βE1 to βE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Eβ required for recombination enhancer activity. The Eβ core is comprised of a limited set of motifs (βE3 and βE4) and an additional previously uncharacterized 20-bp sequence 3′ of the βE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the βE1 through βE4 motifs but not the 3′ sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Eβ recombinational activity. Our results emphasize the multifunctionality of the TCRβ enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.

T Cell Development in TCRβ Enhancer-Deleted Mice: Implications for αβ T Cell Lineage Commitment and Differentiation

T cell differentiation in the mouse thymus is an intricate, highly coordinated process that requires the assembly of TCR complexes from individual components, including those produced by the precisely timed V(D)J recombination of TCR genes. Mice carrying a homozygous deletion of the TCRβ transcriptional enhancer (Eβ) demonstrate an inhibition of V(D)J recombination at the targeted TCRβ locus and a block in αβ T cell differentiation. In this study, we have characterized the T cell developmental defects resulting from the Eβ−/− mutation, in light of previously reported results of the analyses of TCRβ-deficient (TCRβ−/−) mice. Similar to the latter mice, production of TCRβ-chains is abolished in the Eβ−/− animals, and under these conditions differentiation into cell-surface TCR−, CD4+CD8+ double positive (DP) thymocytes depends essentially on the cell-autonomous expression of TCRδ-chains and, most likely, TCRγ-chains. However, contrary to previous reports using TCRβ−/− mice, a minor population of TCR γδ+ DP thymocytes was found within the Eβ−/− thymi, which differ in terms of T cell-specific gene expression and V(D)J recombinase activity, from the majority of TCR−, αβ lineage-committed DP thymocytes. We discuss these data with respect to the functional role of Eβ in driving αβ T cell differentiation and the mechanism of αβ T lineage commitment.

TCRα enhancer activation occurs via a conformational change of a pre‐assembled nucleo‐protein complex

The TCR α enhancer (Eα) has served as a paradigm for studying how enhancers organize trans‐activators into nucleo‐protein complexes thought to recruit and synergistically stimulate the transcriptional machinery. Little is known, however, of either the extent or dynamics of Eα occupancy by nuclear factors during T cell development. Using dimethyl sulfate (DMS) in vivo footprinting, we demonstrate extensive Eα occupancy, encompassing both previously identified and novel sites, not only in T cells representing a developmental stage where Eα is known to be active (CD4+CD8+–DP cells), but surprisingly, also in cells at an earlier developmental stage where Eα is not active (CD4−CD8−–DN cells). Partial occupancy was also established in B‐lymphoid but not non‐lymphoid cells. In vivo DNase I footprinting, however, implied developmentally induced changes in nucleo‐protein complex topography. Stage‐specific differences in factor composition at Eα sequences were also suggested by EMSA analysis. These results, which indicate that alterations in the structure of a pre‐assembled nucleo‐protein complex correlate with the onset of Eα activity, may exemplify one mechanism by which enhancers can rapidly respond to incoming stimuli.

Assessing the role of the T cell receptor β gene enhancer in regulating coding joint formation during V(D)J recombination

To assess the role of the T cell receptor (TCR) β gene enhancer (Eβ) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRβ rearrangement of Eβ-deleted (ΔEβ) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with ΔEβ, ΔEβ Bcl-2 thymocytes show a small accumulation of TCRβ standard recombination products, including coding ends, that involves the proximal Dβ-Jβ and Vβ14 loci but not the distal 5′ Vβ genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Eβ, by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Eβ-dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRβ allelic exclusion depending on the position within the locus.

Normal Immune System Development in Mice Lacking the Deltex-1 RING Finger Domain

ABSTRACT The Notch signaling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1Δ/Δ mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular those from a fourth Deltex gene identified during the course of this study, are also discussed.

Duality of Enhancer Functioning Mode Revealed in a Reduced TCRβ Gene Enhancer Knockin Mouse Model

The TCRβ gene enhancer (Eβ) commands TCRβ gene expression through the lifespan of T lymphocytes. Genetic and molecular studies have implied that in early thymocytes, Eβ directs chromatin opening over the Dβ-Jβ-Cβ domains and triggers initial Dβ-Jβ recombination. In mature T cells, Eβ is required for expression of the assembled TCRβ gene. Whether these separate activities rely on distinct Eβ regulatory sequences and involve differing modes of activation is unclear. Using gene targeting in mouse embryonic stem cells, we replaced Eβ by a conserved core fragment (Eβ169). We found that Eβ169-carrying alleles were capable of sustaining β gene expression and the development of mature T cells in homozygous knockin mice. Surprisingly, these procedures and underlying molecular transactions were affected to a wide range of degrees depending on the developmental stage. Early thymocytes barely achieved Dβ-Jβ germline transcription and recombination. In contrast, T cells displayed substantial though heterogeneous levels of VDJ-rearranged TCRβ gene expression. Our results have implications regarding enhancer function in cells of the adaptive immune system and, potentially, TCRβ gene recombination and allelic exclusion.

αβ T-cell development is not affected by inversion of TCRβ gene enhancer sequences: polar enhancement of gene expression regardless of enhancer orientation

V(D)J recombination and expression of the T‐cell receptor β (TCRβ) gene are required for the development of the αβ T lymphocyte lineage. These processes depend on a transcriptional enhancer (Eβ) which acts preferentially on adjacent upstream sequences, and has little impact on the 5′ distal and 3′ proximal regions of the TCRβ locus. Using knock‐in mice, we show that αβ T‐cell differentiation and TCRβ gene recombination and expression are not sensitive to the orientation of Eβ sequences. We discuss the implication of these results regarding the mode of enhancer function at this locus during T lymphocyte development.