Kathrin Muegge

Interleukin-7: physiological roles and mechanisms of action.

Interleukin-7 (IL-7), a product of stromal cells, provides critical signals to lymphoid cells at early stages in their development. Two types of cellular responses to IL-7 have been identified in lymphoid progenitors: (1) a trophic effect and (2) an effect supporting V(D)J recombination. The IL-7 receptor is comprised of two chains, IL-7R alpha and gamma(c). Following receptor crosslinking, rapid activation of several classes of kinases occurs, including members of the Janus and Src families and PI3-kinase. A number of transcription factors are subsequently activated including STATs, c-myc, NFAT and AP-1. However, it remains to be determined which, if any, previously identified pathway leads to the trophic or V(D)J endpoints. The trophic response to IL-7 involves protecting lymphoid progenitors from a death process that resembles apoptosis. This protection is partly mediated by IL-7 induction of Bcl-2, however other IL-7-induced events are probably also involved in the trophic response. The V(D)J response to IL-7 is partly mediated through increased production of Rag proteins (which cleave the target locus) and partly by increasing the accessibility of a target locus to cleavage through chromatin remodeling.

Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

ABSTRACT Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.

Lsh is involved in de novo methylation of DNA

Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in Lsh−/− embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA.

Lsh is required for meiotic chromosome synapsis and retrotransposon silencing in female germ cells

Lymphoid specific helicase (Lsh) is a major epigenetic regulator that is essential for DNA methylation and transcriptional silencing of parasitic elements in the mammalian genome. However, whether Lsh is involved in the regulation of chromatin-mediated processes during meiosis is not known. Here, we show that Lsh is essential for the completion of meiosis and transcriptional repression of repetitive elements in the female gonad. Oocytes from Lsh knockout mice exhibit demethylation of transposable elements and tandem repeats at pericentric heterochromatin, as well as incomplete chromosome synapsis associated with persistent RAD51 foci and γH2AX phosphorylation. Failure to load crossover-associated foci results in the generation of non-exchange chromosomes. The severe oocyte loss observed and lack of ovarian follicle formation, together with the patterns of Lsh nuclear compartmentalization in the germ line, demonstrate that Lsh has a critical and previously unidentified role in epigenetic gene silencing and maintenance of genomic stability during female meiosis.

Bax Deficiency Partially Corrects Interleukin-7 Receptor α Deficiency

The requirement for cytokines in hematopoiesis is partly attributable to the protection of cells from apoptosis. Since IL-7 is required for normal T cell development, we evaluated the role of Bax in vivo by generating mice deficient in both Bax and the IL-7 receptor α chain (IL-7R). Starting at birth, we observed complete recovery of all stages of αβ thymocyte development up to 4 weeks of age. However, by 12 weeks of age, thymic cellularity had reverted to that of mice deficient in IL-7R alone. The BH3 only proteins, Bad and Bim, were also part of the death pathway repressed by IL-7. Thus, in young mice, Bax emerges as an essential protein in the death pathway induced by IL-7 deficiency.

Lsh, a modulator of CpG methylation, is crucial for normal histone methylation

Methylation of histone tails and CpG methylation are involved in determining heterochromatin structure, but their cause and effect relationship has not been resolved as yet in mammals. Here we report that Lsh, a member of the SNF2 chromatin remodeling family, controls both types of epigenetic modifications. Lsh has been shown to be associated with pericentromeric heterochromatin and to be required for normal CpG methylation at pericentromeric sequences. Loss of Lsh, in Lsh‐deficient mice, results in accumulation of di‐ and tri‐methylated histone 3 at lysine 4 (H3‐K4me) at pericentromeric DNA and other repetitive sequences. In contrast, di‐ or tri‐methylation of H3‐K9 and distribution of HP1 appear unchanged after Lsh deletion, suggesting independent regulatory mechanisms for H3‐K4 or K9 methylation. Experimental DNA demethylation with 5′‐azacytidine results in a similar increase of H3‐K4me. These results support the model that loss of CpG methylation caused by Lsh deficiency antecedes elevation of H3‐K4me. Thus, Lsh is crucial for the formation of normal heterochromatin, implying a functional role for Lsh in the regulation of transcription and mitosis.

DNA methylation in early development

Development from separate parental germ cells through fertilization and proceeding to a fully functioning adult animal occurs through an intricate program of transcriptional and chromatin changes. Epigenetic alterations such as DNA methylation are an important part of this process. This review looks at the role of DNA methylation in early embryonic development, as well as how this epigenetic mark affects stem cell differentiation and tissue‐specific gene expression in somatic cells. Mol. Reprod. Dev. 77: 105–113, 2010. Published 2009 Wiley‐Liss, Inc.

Defective T-cell receptor γ gene rearrangement in interleukin-7 receptor knockout mice

Abstract T-cell receptor (TCR) genes need to be rearranged by a site specific-VDJ recombinase before they are expressed. This process, initiated in CD44 + 25 + thymocytes, takes place during the early stages of T-cell differentiation in the thymus. Interleukin-7 receptor α chain knockout (IL-7R −/− ) mice are severely deficient in B-lymphocytes and α β T-cells and completely lack the γ δ T-cell lineage. Thymocyte development is arrested at a very early stage (DN CD44 + CD25 − ). Because this arrest is earlier than in mice with a block in VDJ recombination, we examined the rearrangement status of TCR genes in thymocytes from IL-7R −/− mice. The TCR β locus showed a nearly normal pattern of VDJ rearrangements, consistent with the presence of α β T-cells in these mice. However, TCR γ locus rearrangement was absent or severely reduced for all the V γ genes analyzed (V γ 3, V γ 4, V γ 1.1, V γ 1.2 and V γ 2). In contrast, the δ locus showed little reduction in rearrangement. The defect in γ rearrangements in IL-7R −/− thymocytes is not simply due to an absence of mature γ δ T-cells, since TCR δ −/− mice, which also have only α β T-cells, had normal levels of γ and δ rearrangements. These findings indicate that one or both of the two known ligands of IL-7R, IL-7 and thymic stromal lymphopoietin (TSLP) serves as an extrinsic signal to specifically rearrange the TCR γ locus.

Cytokines and transcription factors

Transcriptional induction is mediated by transcription factors, which influence gene expression by interacting with specific elements in their regulatory regions. Here we discuss transcription factors involved in the regulation of cytokines and their receptors (interleukin-2, interleukin-2 receptor alpha chain, and interferon-beta), as well as transcription factors that are activated by cytokines (interleukin-1, tumor necrosis factor, interferons, and transforming growth factor beta).

Lsh, a SNF2 family member, is required for normal murine development

Lsh is a member of the SNF2 family of chromatin remodelers, that regulate diverse biological processes such as replication, repair and transcription. Although expression of Lsh is highly tissue specific in adult animals, Lsh mRNA is detectable in multiple tissues during embryogenesis. In order to determine the physiologic role of Lsh during murine development and to assess its unique function in adult mice, we performed targeted deletion of the Lsh gene using homologous recombination in murine embryonic stem cells. Lsh-/- embryos occurred with the expected Mendelian frequency after implantation and during embryogenesis. However, Lsh-/- mice died within a few hours after birth. Furthermore, newborn mice were 22% lower in weight in comparison with their littermates and showed renal lesions. Thus Lsh is a non-redundant member of the SNF2 family and is essential for normal murine development and survival.