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

In Vivo Studies on the Control of Gene Expression and Protein Degradation

Cellular homeostasis is maintained by tightly regulated gene networks, controlled notably at the levels of transcription, mRNA processing, and protein post-translational modification and turnover. This thesis focuses on two of these regulatory steps, namely gene transcription and protein stability. I first explored the impact of the KRAB-ZFP/KAP1 gene regulation system on liver function. KRAB-ZFPs constitute the largest family of transcription factors encoded by mouse and humans, yet their physiological roles and gene targets remain mostly elusive. Conversely, their molecular mechanism of action has been rather well characterized, as they can recognize DNA in a sequence-specific manner and recruit the universal co-factor KAP1 (also known as TRIM28), which in turn serves as a scaffold for various heterochromatin-inducing effectors. Several studies have shed some light on the roles played by this system in embryonic tissues, but its functions in the adult remain almost completely unknown. As chromatin regulation has been shown to be a major factor in the control of metabolism, we decided to investigate the role of KRAB-ZFP/KAP1 in the liver, an organ central to this process. For this, we generated conditional liver-specific Kap1 KO mice, the study of which revealed a strikingly sex-specific phenotype, with early-onset steatosis and age-related tumorigenesis restricted to males, contrasting with the mild metabolic disturbances recorded in Kap1-deleted females. Underlying this phenotype, our combined transcriptome and chromatin analyses revealed that KAP1 controls sexually dimorphic genes notably involved in hormone, drug and xenobiotic metabolism. Finally, by using a recently developed technique for direct RNA quantification, we established the range of KRAB-ZFPs expressed in the liver and likely responsible for at least part of the observed effects. This study thus demonstrates the central role of the KRAB/KAP1 system in control of sexual dimorphism, responses to xenobiotic stress and metabolic control in the liver, and further links disturbances in these processes with hepatic carcinogenesis. In parallel, in an effort to pursue a multidisciplinary training combining skills in biology and chemistry, I worked towards the development of a method to study the half-life of proteins in vivo. Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Capitalizing on an in vitro technology previously developed in Kai Johnsson's laboratory, I demonstrated how chemical labeling can be used to measure the half-life of resident intracellular and extracellular proteins in living mice. Our approach is based on labeling of SNAP-tag-fusion proteins with fluorescent probes. First, we verified that SNAP-tag substrates have wide bio-availability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then applied near-infrared probes to perform non-invasive imaging of in vivo labeled tumors. Finally, we used SNAP-mediated chemical pulse-chase labeling to measuring in vivo the half-life of different extra- and intracellular proteins, including KAP1. Importantly, this tagging technology can be applied to any protein amenable to expression as a fusion partner, whether cell-associated or secreted. Furthermore, because the SNAP-tag chemical labeling allows for a large array of probes also suited for experiments aimed at manipulating and monitoring protein function in vivo, such as cross-linking, pull-down or FRET, this methodology opens broad perspectives for studying protein function in living animals

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

DNA methylation by CcrM contributes to genome maintenance in the Agrobacterium tumefaciens plant pathogen

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric genome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and inhibits gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. We thus propose that methylation by CcrM stimulates RepABC-dependent chromosomal origins, uncovering a novel and original connection between CcrM-dependent DNA methylation and genome maintenance in an Alphaproteobacterial pathogen

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

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

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 KRAB-associated protein 1 (KAP1) epigenetic regulator in this process. Liver-specific KAP1 knockout led to strikingly sexually dimorphic phenotypic disturbances, including male-predominant steatosis and hepatic tumors with upregulation of AKT and ERK1/2 mitogen-activated protein kinase signaling. This correlated with 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 Cyp2d9, Gst\u3d5, Slp Cyp2a, Cyp2b and Cyp3a gene clusters. Additionally, we identified a male-restricted KAP1 binding site in the fsp27 (fat specific protein 27) gene, correlating with its male-predominant upregulation 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 the metabolic control hormones, drugs and xenobiotics in the liver, and further links disturbances in these processes with hepatic carcinogenesis

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

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 TGFβ 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