Regulation of glucocorticoid receptor expression and downstream gene targets in murine lymphocytes

Glucocorticoids (GCs) are homeostatic steroid hormones with essential roles in the regulation of development, integrated metabolism, immune and neurological responses. GCs act via the widely expressed Glucocorticoid Receptor (GR), which is expressed from multiple untranslated exon 1s to yield 11 alternatively spliced transcripts in humans (1A-1H) and five in mice (1A-1E). These transcript isoforms are under the control of their own promoters which confers tissue specificity and a higher level of regulation to this transcription factor. In thymocytes activity from the GR1A promoter is implicated with increasing sensitivity to Glucocorticoid Induced Cell Death (GICD). CD4+CD8+ Double Positive (DP) cells and NKT cells in particular are hypersensitive to GICD. The main objectives of this study were to investigate further the molecular mechanisms involved in GICD, to examine more closely the role of GR in the development of T-lymphocytes, and to characterise the expression and regulation of the GR1A promoter in mouse tissues previously not examined. To explore the molecular pathway driving GICD in thymocytes we performed whole genome microarray analysis in mouse GR null thymocytes. Interesting direct GR targets included P21, Bim and Nfil3. Regulation of these targets by GCs was validated using qRT-PCR in WT thymocytes. Nfil3 in particular has been studied further. Previous studies demonstrated that GC-mediated up-regulation of Nfil3 is dependent on intracellular calcium levels, and correlates with GICD of GC-sensitive leukemic CEM cells. In silico promoter analysis revealed a putative Glucocorticoid Response Element in the Nfil3 5’UTR which was confirmed to interact with the GR by ChIP. Immunohistochemical staining of Nfil3 in whole thymus has localised NFIL3 protein primarily to the medullary region. Double labelling has co-localised NFIL3 to apoptotic cells and macrophages. Using siRNA technology we have shown that NFIL3 does in fact confer greater sensitivity to GICD in Ctll-2 cells. Previous to our studies transcripts initiating from the GR1A promoter had only been localised to the cortex of the brain and to T-lymphocytes. Using qRTPCR and in situ hybridisation we have detected transcripts initiating from the GR1A promoter in the anterior lobe of the pituitary. However, the role of the GR1A promoter activity in the brain is unknown. In the brain, particularly in the cortex, and also in the hypothalamus and pituitary (components of the Hypothalamic- Pituitary-Adrenal axis) GCs and their receptors have a key role in the response to stress. A 2.5 fold increase in the level of GR1A promoter usage in the pituitary was observed in response to treatment with the synthetic GC Dexamethasone. It is possible that a tissue/cell specific increase in activity of the GR1A promoter during periods of elevated levels of circulating GCs may help to make those cells more sensitive to these rising levels of GCs and serve as a fine tuning mechanism to aid in a rapid return to the normal state after stress. The significance of increased sensitivity of some T-cell lineages to GICD is controversial, with a proposed function being that it is involved in T-cell development. The direct role of the GR in development of T-cell populations and some specific lymphocyte lineages in T-cell specific GR-null mice (TGRKO) was examined using FACS. Major differences in CD8 CD4 cellularity was observed in spleen and liver and to a lesser extent in thymus. In Spleen NKT cellularity was reduced and TReg cell CD25 populations were altered. This study has shed light on some of the regulatory mechanisms and molecular interactions associated with GR function in the pathways of stress, T-cell development and GICD. We have identified rapidly induced GR target genes in GR null thymocytes and characterized the regulation of the transcriptional repressor Nfil3 by GR in normal mouse thymocytes. We have shown that NFIL3 is necessary for GICD in Ctll2-T cells. Additionally we have shown that the regulation of GR expression in the pituitary may occur via auto-regulation and maintenance of the GR1A promoter, an action which likely increases sensitivity to elevated GC levels and may account for the rapid return to a normal state following stress. Finally we have demonstrated T-cell development is subtly affected by loss of GR in T-cells

Regulation of glucocorticoid receptor expression and downstream gene targets in murine lymphocytes

Glucocorticoids (GCs) are homeostatic steroid hormones with essential roles in the regulation of development, integrated metabolism, immune and neurological responses. GCs act via the widely expressed Glucocorticoid Receptor (GR), which is expressed from multiple untranslated exon 1s to yield 11 alternatively spliced transcripts in humans (1A-1H) and five in mice (1A-1E). These transcript isoforms are under the control of their own promoters which confers tissue specificity and a higher level of regulation to this transcription factor. In thymocytes activity from the GR1A promoter is implicated with increasing sensitivity to Glucocorticoid Induced Cell Death (GICD). CD4+CD8+ Double Positive (DP) cells and NKT cells in particular are hypersensitive to GICD. The main objectives of this study were to investigate further the molecular mechanisms involved in GICD, to examine more closely the role of GR in the development of T-lymphocytes, and to characterise the expression and regulation of the GR1A promoter in mouse tissues previously not examined. To explore the molecular pathway driving GICD in thymocytes we performed whole genome microarray analysis in mouse GR null thymocytes. Interesting direct GR targets included P21, Bim and Nfil3. Regulation of these targets by GCs was validated using qRT-PCR in WT thymocytes. Nfil3 in particular has been studied further. Previous studies demonstrated that GC-mediated up-regulation of Nfil3 is dependent on intracellular calcium levels, and correlates with GICD of GC-sensitive leukemic CEM cells. In silico promoter analysis revealed a putative Glucocorticoid Response Element in the Nfil3 5’UTR which was confirmed to interact with the GR by ChIP. Immunohistochemical staining of Nfil3 in whole thymus has localised NFIL3 protein primarily to the medullary region. Double labelling has co-localised NFIL3 to apoptotic cells and macrophages. Using siRNA technology we have shown that NFIL3 does in fact confer greater sensitivity to GICD in Ctll-2 cells. Previous to our studies transcripts initiating from the GR1A promoter had only been localised to the cortex of the brain and to T-lymphocytes. Using qRTPCR and in situ hybridisation we have detected transcripts initiating from the GR1A promoter in the anterior lobe of the pituitary. However, the role of the GR1A promoter activity in the brain is unknown. In the brain, particularly in the cortex, and also in the hypothalamus and pituitary (components of the Hypothalamic- Pituitary-Adrenal axis) GCs and their receptors have a key role in the response to stress. A 2.5 fold increase in the level of GR1A promoter usage in the pituitary was observed in response to treatment with the synthetic GC Dexamethasone. It is possible that a tissue/cell specific increase in activity of the GR1A promoter during periods of elevated levels of circulating GCs may help to make those cells more sensitive to these rising levels of GCs and serve as a fine tuning mechanism to aid in a rapid return to the normal state after stress. The significance of increased sensitivity of some T-cell lineages to GICD is controversial, with a proposed function being that it is involved in T-cell development. The direct role of the GR in development of T-cell populations and some specific lymphocyte lineages in T-cell specific GR-null mice (TGRKO) was examined using FACS. Major differences in CD8 CD4 cellularity was observed in spleen and liver and to a lesser extent in thymus. In Spleen NKT cellularity was reduced and TReg cell CD25 populations were altered. This study has shed light on some of the regulatory mechanisms and molecular interactions associated with GR function in the pathways of stress, T-cell development and GICD. We have identified rapidly induced GR target genes in GR null thymocytes and characterized the regulation of the transcriptional repressor Nfil3 by GR in normal mouse thymocytes. We have shown that NFIL3 is necessary for GICD in Ctll2-T cells. Additionally we have shown that the regulation of GR expression in the pituitary may occur via auto-regulation and maintenance of the GR1A promoter, an action which likely increases sensitivity to elevated GC levels and may account for the rapid return to a normal state following stress. Finally we have demonstrated T-cell development is subtly affected by loss of GR in T-cells

Germline heterozygous mutations in Nxf1 perturb RNA metabolism and trigger thrombocytopenia and lymphopenia in mice

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export

The tumor suppressor Hic1 maintains chromosomal stability independent of Tp53

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability