KAP1 regulates gene networks controlling mouse B-lymphoid cell differentiation and function.

Chromatin remodeling is fundamental for B-cell differentiation. In the present study, we explored the role of KAP1, the cofactor of KRAB-ZFP transcriptional repressors, in this process. B-lymphoid-specific Kap1-KO mice displayed reduced numbers of mature B cells, lower steady-state levels of Abs, and accelerated rates of decay of neutralizing Abs after viral immunization. Transcriptome analyses of Kap1-deleted B splenocytes revealed an up-regulation of PTEN, the enzymatic counteractor of PIK3 signaling, and of genes encoding DNA-damage response factors, cell-cycle regulators, and chemokine receptors. ChIP/seq studies established that KAP1 bound at or close to several of these genes and controlled chromatin status at their promoters. Genome wide, KAP1 binding sites lacked active B cell-specific enhancers and were enriched in repressive histone marks, further supporting a role for this molecule in gene silencing in vivo. Likely responsible for tethering KAP1 to at least some of these targets, a discrete subset of KRAB-ZFPs is enriched in B lymphocytes. Our results therefore reveal the role of KRAB/KAP1-mediated epigenetic regulation in B-cell development and homeostasis.

Liver-specific ablation of Krüppel-associated box-associated protein 1 in mice leads to male-predominant hepatosteatosis and development of liver adenoma.

UNLABELLED: The liver is characterized by sexually dimorphic gene expression translating into sex-specific differences in lipid, drug, steroid hormone, and xenobiotic metabolism, with distinct responses of males and females to environmental challenges. Here, we investigated the role of the Krüppel-associated box (KRAB)-associated protein 1 (KAP1) epigenetic regulator in this process. Liver-specific KAP1 knockout (KO) led to strikingly sexually dimorphic phenotypic disturbances, including male-predominant steatosis and hepatic tumors with up-regulation of protein kinase B and extracellular signal-related kinases 1/2 mitogen-activated protein kinase signaling. This correlated with the sex-specific transcriptional dysregulation of a wide range of metabolic genes, notably those involved in retinol and sex hormone processing as well as in detoxification. Furthermore, chromatin immunoprecipitation followed by deep sequencing indicated that a number of dysregulated genes are direct targets of the KRAB/KAP1 repression system. Those genes include sexually dimorphic cytochrome P 450 Cyp2d9, glutathione S-transferase π, Cyp2a, Cyp2b, and Cyp3a gene clusters. Additionally, we identified a male-restricted KAP1-binding site in the fat-specific protein 27 gene, correlating with its male-predominant up-regulation upon Kap1 deletion, suggesting that the latter might be an important trigger in the development of male-specific hepatosteatosis and secondary tumorigenesis. CONCLUSION: This work reveals KRAB/KAP1-mediated transcriptional regulation as a central event in metabolic control hormones, drugs, and xenobiotics in the liver and further links disturbances in these processes with hepatic carcinogenesis.

KAP1 regulates gene networks controlling T-cell development and responsiveness.

Chromatin remodeling at specific genomic loci controls lymphoid differentiation. Here, we investigated the role played in this process by Kruppel-associated box (KRAB)-associated protein 1 (KAP1), the universal cofactor of KRAB-zinc finger proteins (ZFPs), a tetrapod-restricted family of transcriptional repressors. T-cell-specific Kap1-deleted mice displayed a significant expansion of immature thymocytes, imbalances in CD4(+)/CD8(+) cell ratios, and altered responses to TCR and TGFß stimulation when compared to littermate KAP1 control mice. Transcriptome and chromatin studies revealed that KAP1 binds T-cell-specific cis-acting regulatory elements marked by the H3K9me3 repressive mark and enriched in Ikaros/NuRD complexes. Also, KAP1 directly controls the expression of several genes involved in TCR and cytokine signaling. Among these, regulation of FoxO1 seems to play a major role in this system. Likely responsible for tethering KAP1 to at least part of its genomic targets, a small number of KRAB-ZFPs are selectively expressed in T-lymphoid cells. These results reveal the so far unsuspected yet important role of KAP1-mediated epigenetic regulation in T-lymphocyte differentiation and activation.

Visualizing biochemical activities in living cells through chemistry.

The development of molecular probes to visualize cellular processes is an important challenge in chemical biology. One possibility to create such cellular indicators is based on the selective labeling of proteins with synthetic probes in living cells. Over the last years, our laboratory has developed different labeling approaches for monitoring protein activity and for localizing synthetic probes inside living cells. In this article, we review two of these labeling approaches, the SNAP-tag and CLIP-tag technologies, and their use for studying cellular processes.

Functional integration of "undead" neurons in the olfactory system.

Programmed cell death (PCD) is widespread during neurodevelopment, eliminating the surpluses of neuronal production. Using the Drosophila olfactory system, we examined the potential of cells fated to die to contribute to circuit evolution. Inhibition of PCD is sufficient to generate new cells that express neural markers and exhibit odor-evoked activity. These "undead" neurons express a subset of olfactory receptors that is enriched for relatively recent receptor duplicates and includes some normally found in different chemosensory organs and life stages. Moreover, undead neuron axons integrate into the olfactory circuitry in the brain, forming novel receptor/glomerular couplings. Comparison of homologous olfactory lineages across drosophilids reveals natural examples of fate change from death to a functional neuron. Last, we provide evidence that PCD contributes to evolutionary differences in carbon dioxide-sensing circuit formation in Drosophila and mosquitoes. These results reveal the remarkable potential of alterations in PCD patterning to evolve new neural pathways.

Gonadotropin-releasing hormone-II messenger ribonucleic acid and protein content in the mammalian brain are modulated by food intake.

GnRH-II is the most evolutionarily conserved member of the GnRH peptide family. In mammals, GnRH-II has been shown to regulate reproductive and feeding behaviors. In female musk shrews, GnRH-II treatment increases mating behaviors and decreases food intake. Although GnRH-II-containing neurons are known to reside in the midbrain, the neural sites of GnRH-II action are undetermined, as is the degree to which GnRH-II is regulated by energy availability. To determine whether GnRH-II function is affected by changes in food intake, we analyzed the levels of GnRH-II mRNA in the midbrain and GnRH-II protein in numerous target regions. Adult musk shrews were ad libitum fed, food restricted, or food restricted and refed for varying durations. Compared with ad libitum levels, food restriction decreased, and 90 min of refeeding reinstated, GnRH-II mRNA levels in midbrain and GnRH-II peptide in several target areas including the medial habenula and ventromedial nucleus. Refeeding for 90 min also reinstated female sexual behavior in underfed shrews. In male shrews, abundant GnRH-II peptide was present in all sites assayed, including the preoptic area, a region with only low GnRH-II in females. In contrast to females, food restriction did not affect GnRH-II protein in male brains or inhibit their mating behavior. Our results further define the relationship between GnRH-II, energy balance, and reproduction, and suggest that food restriction may inhibit female reproduction by reducing GnRH-II output to several brain nuclei. We postulate that this highly conserved neuropeptide functions similarly in other mammals, including humans, to fine-tune reproductive efforts with periods of sufficient energy resources.

Identification and molecular characterisation of Lausanne Institutional Biobank participants with familial hypercholesterolaemia - a proof-of-concept study.

AIMS: We aimed to identify familial hypercholesterolaemia mutation carriers among participants to the Lausanne Institutional Biobank (BIL). Our experimental workflow was designed as a proof-of-concept demonstration of the resources and services provided by our integrated institutional clinical research support platform. METHODS: Familial hypercholesterolaemia was used as a model of a relatively common yet often underdiagnosed and inadequately treated Mendelian disease. Clinical and laboratory information was extracted from electronic hospital records. Patients were selected using elevated plasma cholesterol levels (total cholesterol ≥7.5 mM or low-density lipoprotein cholesterol ≥5 mM), premature coronary artery disease status and age (18-60 yr) as main inclusion criteria. LDLR, APOB and PCSK9 were analysed by high-throughput DNA sequencing. The most relevant mutations were confirmed by Sanger sequencing. RESULTS: Of 23 737 patients contacted by the BIL, 17 760 individuals consented to participate and 13 094 wished to be recontacted if there were findings requiring clinical action. Plasma cholesterol records were available for 5111 participants, of whom 94 were selected for genetic screening. Twenty-five of the tested patients presented with premature coronary artery disease while 69 had no such diagnosis. Seven heterozygous carriers of eight rare coding missense variants were identified. Three mutations were pathogenic (APOB p.R3527Q) or likely pathogenic (LDLR p.C27W, LDLR p.P526S) for hypercholesterolaemia, while the others were either benign or of unknown significance. One patient was a double heterozygote for variants APOB p.R3527Q and LDLR p.P526S. CONCLUSION: This work illustrates how clinical and translational research can benefit from a dedicated platform integrating both a hospital-based biobank and a data support team.

The KRAB-ZFP/KAP1 system contributes to the early embryonic establishment of site-specific DNA methylation patterns maintained during development.

De novo DNA methylation is an essential aspect of the epigenetic reprogramming that takes place during early development, yet factors responsible for its instatement at particular genomic loci are poorly defined. Here, we demonstrate that the KRAB-ZFP-mediated recruitment of KAP1 to DNA in embryonic stem cells (ESCs) induces cytosine methylation. This process is preceded by H3K9 trimethylation, and genome-wide analyses reveal that it spreads over short distances from KAP1-binding sites so as to involve nearby CpG islands. In sharp contrast, in differentiated cells, KRAB/KAP1-induced heterochromatin formation does not lead to DNA methylation. Correspondingly, the methylation status of CpG islands in the adult mouse liver correlates with their proximity to KAP1-binding sites in ESCs, not in hepatocytes. Therefore, KRAB-ZFPs and their cofactor KAP1 are in part responsible for the establishment during early embryogenesis of site-specific DNA methylation patterns that are maintained through development.

Measuring in vivo protein half-life.

Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Here, we demonstrate how chemical labeling of SNAP-tag fusion proteins can be exploited to measure the half-life of resident intracellular and extracellular proteins in living mice. First, we demonstrate that SNAP-tag substrates have wide bioavailability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then apply near-infrared probes to perform noninvasive imaging of in vivo-labeled tumors. Finally, we use SNAP-mediated chemical pulse-chase labeling to perform measurement of the in vivo half-life of different extra- and intracellular proteins. These results open broad perspectives for studying protein function in living animals.

Critical periods of susceptibility to short-term energy challenge during pregnancy: Impact on fertility and offspring development.

In female mammals, reproduction is tightly regulated by energy status and food availability. Although acute energetic challenges inhibit female reproductive behavior and gonadotropin secretion, less attention has been given to the effects of short-term energetic challenge on pregnancy and gestation. Furthermore, species differences in pregnancy physiology necessitate more detailed analyses of specific pregnancy models. Here, we studied musk shrews, which display induced ovulation and obligate delayed implantation, and whose reproductive physiology is tightly linked to metabolic status. We tested whether acute energetic challenges of varied degrees given at specific pregnancy stages (including before and after delayed implantation) have different effects on gestational outcome and offspring postnatal development. We found that 48 h of either 40% or 50% food restriction, which reduced body weight and strongly inhibited sexual behavior, had minimal effects on pregnancy success and litter dynamics when administered early in gestation (pre-implantation). However, <30% of females experiencing short-term food restriction later in gestation successfully gave birth (versus > or =70% of ad-libitum fed controls), and the pups of these food-restricted females exhibited a 30% slower postnatal growth trajectory. Interestingly, although pregnancy success and litter dynamics were unaffected by food restriction before implantation, gestation length was increased by metabolic challenges experienced at this time, indicating that energy status may regulate the timing of implantation. We conclude that 1) there are critical periods of pregnancy, particularly after implantation, when short-term, mild energetic challenges have significant impacts on fertility and offspring postnatal development, and 2) delayed implantation may have evolved, in part, as a buffering mechanism to prevent pregnancy failure during impaired energy balance in early gestation.

Neuropeptide Y influences acute food intake and energy status affects NPY immunoreactivity in the female musk shrew (Suncus murinus).

Neuropeptide Y (NPY) stimulates feeding, depresses sexual behavior, and its expression in the brain is modulated by energetic status. We examined the role of NPY in female musk shrews, a species with high energetic and reproductive demands; they store little fat, and small changes in energy can rapidly diminish or enhance sexual receptivity. Intracerebroventricular infusion of NPY enhanced acute food intake in shrews; however, NPY had little affect on sexual receptivity. The distribution of NPY immunoreactivity in the female musk shrew brain was unremarkable, but energy status differentially affected NPY immunoreactivity in several regions. Similar to what has been noted in other species, NPY immunoreactivity was less dense in brains of ad libitum shrews and greater in shrews subjected to food restriction. In two midbrain regions, both of which contain high levels of gonadotropin releasing hormone II (GnRH II), which has anorexigenic actions in shrews, NPY immunoreactivity was more sensitive to changes in food intake. In these regions, acute re-feeding (90-180 min) after food restriction reduced NPY immunoreactivity to levels noted in ad libitum shrews. We hypothesize that interactions between NPY and GnRH II maintain energy homeostasis and reproduction in the musk shrew.

Overexpression of heme oxygenase-1 in murine melanoma: increased proliferation and viability of tumor cells, decreased survival of mice.

Heme oxygenase-1 (HO-1), a cytoprotective enzyme, can be induced in tumors in response to anti-cancer therapies. We investigated the role of HO-1 in B16(F10), S91, and Sk-mel188 melanoma cells. Overexpression of HO-1 after transduction with adenoviral vectors increased cell proliferation, resistance to oxidative stress generated by H2O2, and angiogenic potential as determined by induction of endothelial cell divisions. Likewise, cells stably transfected with HO-1 cDNA (B16-HO-1) showed higher proliferation, stress resistance, and angiogenic activity than the wild-type line (B16-WT). HO-1 overexpression in tumors significantly shortened survival of mice after subcutaneous injection of cancer cells (38 and 22 days for B16-WT and B16-HO-1, respectively; P=0.017). This also resulted in development of more packed tumors, with more melanoma cells, and reduced inflammatory edemas. Mice injected with B16-HO-1 had lower levels of tumor necrosis factor and higher serum concentrations of its soluble receptor tumor necrosis factor-RI, whereas tumors overexpressing HO-1 displayed augmented vascularization and stronger production of vascular endothelial growth factor. Finally, B16-HO-1 cells injected intravenously formed more metastases in lungs. Thus, HO-1 overexpression increased viability, proliferation, and angiogenic potential of melanoma cells, augmented metastasis, and decreased survival of tumor-bearing mice, suggesting that induction of HO-1 may be detrimental in anti-cancer therapy of melanoma.