Nadine Zangger

KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading.

Krüppel-associated box domain-zinc finger proteins (KRAB–ZFPs) are tetrapod-specific transcriptional repressors encoded in the hundreds by the human genome. In order to explore their as yet ill-defined impact on gene expression, we developed an ectopic repressor assay, allowing the study of KRAB–mediated transcriptional regulation at hundreds of different transcriptional units. By targeting a drug-controllable KRAB–containing repressor to gene-trapping lentiviral vectors, we demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases (kb) away from their DNA binding sites, with an efficiency which is generally higher for promoters located within 15 kb or less. Silenced promoters exhibit a loss of histone H3-acetylation, an increase in H3 lysine 9 trimethylation (H3K9me3), and a drop in RNA Pol II recruitment, consistent with a block of transcriptional initiation following the establishment of silencing marks. Furthermore, we reveal that KRAB–mediated repression is established by the long-range spreading of H3K9me3 and heterochromatin protein 1 β (HP1β) between the repressor binding site and the promoter. We confirm the biological relevance of this phenomenon by documenting KAP1–dependent transcriptional repression at an endogenous KRAB–ZFP gene cluster, where KAP1 binds to the 3′ end of genes and mediates propagation of H3K9me3 and HP1β towards their 5′ end. Together, our data support a model in which KRAB/KAP1 recruitment induces long-range repression through the spread of heterochromatin. This finding not only suggests auto-regulatory mechanisms in the control of KRAB–ZFP gene clusters, but also provides important cues for interpreting future genome-wide DNA binding data of KRAB–ZFPs and KAP1.

Long-Term Antiretroviral Treatment Initiated at Primary HIV-1 Infection Affects the Size, Composition, and Decay Kinetics of the Reservoir of HIV-1-Infected CD4 T Cells

ABSTRACT Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

24 Hours in the Life of HIV-1 in a T Cell Line

HIV-1 infects CD4+ T cells and completes its replication cycle in approximately 24 hours. We employed repeated measurements in a standardized cell system and rigorous mathematical modeling to characterize the emergence of the viral replication intermediates and their impact on the cellular transcriptional response with high temporal resolution. We observed 7,991 (73%) of the 10,958 expressed genes to be modulated in concordance with key steps of viral replication. Fifty-two percent of the overall variability in the host transcriptome was explained by linear regression on the viral life cycle. This profound perturbation of cellular physiology was investigated in the light of several regulatory mechanisms, including transcription factors, miRNAs, host-pathogen interaction, and proviral integration. Key features were validated in primary CD4+ T cells, and with viral constructs using alternative entry strategies. We propose a model of early massive cellular shutdown and progressive upregulation of the cellular machinery to complete the viral life cycle.

FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.

Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

ABSTRACT One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.

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 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. (HEPATOLOGY 2012)

A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing

BackgroundKRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.MethodTo investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.ResultsWe found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.ConclusionTogether, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.

RIP4 inhibits STAT3 signaling to sustain lung adenocarcinoma differentiation

Loss of epithelial differentiation and extracellular matrix (ECM) remodeling are known to facilitate cancer progression and are associated with poor prognosis in patients with lung cancer. We have identified Receptor-interacting serine/threonine protein kinase 4 (RIP4) as a regulator of tumor differentiation in lung adenocarcinoma (AC). Bioinformatics analyses of human lung AC samples showed that poorly differentiated tumors express low levels of RIP4, whereas high levels are associated with better overall survival. In vitro, lung tumor cells expressing reduced RIP4 levels showed enhanced activation of STAT3 signaling and had a greater ability to invade through collagen. In contrast, overexpression of RIP4 inhibited STAT3 activation, which abrogated interleukin-6-dependent induction of lysyl oxidase, a collagen cross-linking enzyme. In an autochthonous mouse model of lung AC initiated by Kras(G12D) expression with loss of p53, Rip4 knockdown tumors progressed to a poorly differentiated state marked by an increase in Hmga2, reduced Ttf1, and enrichment of genes regulating extracellular remodeling and Jak-Stat signaling. Tail vein injections of cells overexpressing Rip4 showed a reduced potential to invade and form tumors, which was restored by co-expression of Stat3. Altogether, our work has identified that loss of RIP4 enhances STAT3 signaling in lung cancer cells, promoting the expression of ECM remodeling genes and cancer dedifferentiation.

Caloric dose-responsive genes in blood cells differentiate the metabolic status of obese men

We have investigated the postprandial transcriptional response of blood cells to increasing caloric doses of a meal challenge to test whether the dynamic response of the human organism to the ingestion of food is dependent on metabolic health. The randomized crossover study included seven normal weight and seven obese men consuming three doses (500/1000/1500 kcal) of a high-fat meal. The blood cell transcriptome was measured before and 2, 4, and 6 h after meal ingestion (168 samples). We applied univariate and multivariate statistics to investigate differentially expressed genes in both study groups. We identified 624 probe sets that were up- or down-regulated after the caloric challenge in a dose-dependent manner. These transcripts were most responsive to the 1500 kcal challenge in the obese group and were associated with postprandial insulin and oxidative phosphorylation. Furthermore, the data revealed a separation of the obese group into individuals whose response was close to the normal weight group and individuals with a transcriptional response indicative of a loss of metabolic flexibility. The molecular signature provided by the postprandial transcriptomic response of blood cells to increasing caloric doses of a high-fat meal challenge may represent a sensitive way to evaluate the qualitative impact of food on human health.

RANKL Signaling Sustains Primary Tumor Growth in Genetically Engineered Mouse Models of Lung Adenocarcinoma

Introduction: NSCLC is the leading cause of cancer mortality. Recent retrospective clinical analyses suggest that blocking the receptor activator of NF‐&kgr;B (RANK) signaling pathway inhibits the growth of NSCLC and might represent a new treatment strategy. Methods: Receptor activator of NF‐&kgr;B gene (RANK) and receptor activator of NF‐&kgr;B ligand gene (RANKL) expression in human lung adenocarcinoma was interrogated from publicly available gene expression data sets. Several genetically engineered mouse models were used to evaluate treatment efficacy of RANK‐Fc to block RANKL, with primary tumor growth measured longitudinally using microcomputed tomography. A combination of RANKL blockade with cisplatin was tested to mirror an ongoing clinical trial. Results: In human lung adenocarcinoma data sets, RANKL expression was associated with decreased survival and KRAS mutation, with the highest levels in tumors with co‐occurring KRAS and liver kinase B1 gene (LKB1) mutations. In KrasLSL‐G12D/WT, KrasLSL‐G12D/WT; Lkb1Flox/Flox and KrasLSL‐G12D/WT; p53Flox/Flox mouse models of lung adenocarcinoma, we monitored an impaired progression of tumors upon RANKL blockade. Despite elevated expression of RANKL and RANK in immune cells, treatment response was not associated with major changes in the tumor immune microenvironment. Combined RANK‐Fc with cisplatin revealed increased efficacy compared with that of single agents in p53‐ but not in Lkb1‐deficient tumors. Conclusions: RANKL blocking agents impair the growth of primary lung tumors in several mouse models of lung adenocarcinoma and suggest that patients with KRAS‐mutant lung tumors will benefit from such treatments.