Jean Lozach

Molecular determinants of crosstalk between nuclear receptors and toll-like receptors

Nuclear receptors (NRs) repress transcriptional responses to diverse signaling pathways as an essential aspect of their biological activities, but mechanisms determining the specificity and functional consequences of transrepression remain poorly understood. Here, we report signal- and gene-specific repression of transcriptional responses initiated by engagement of toll-like receptors (TLR) 3, 4, and 9 in macrophages. The glucocorticoid receptor (GR) represses a large set of functionally related inflammatory response genes by disrupting p65/interferon regulatory factor (IRF) complexes required for TLR4- or TLR9-dependent, but not TLR3-dependent, transcriptional activation. This mechanism requires signaling through MyD88 and enables the GR to differentially regulate pathogen-specific programs of gene expression. PPARgamma and LXRs repress overlapping transcriptional targets by p65/IRF3-independent mechanisms and cooperate with the GR to synergistically transrepress distinct subsets of TLR-responsive genes. These findings reveal combinatorial control of homeostasis and immune responses by nuclear receptors and suggest new approaches for treatment of inflammatory diseases.

Opposing LSD1 complexes function in developmental gene activation and repression programmes

Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1—a component of the CoREST-CtBP co-repressor complex—is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.

Histone H2A Monoubiquitination Represses Transcription by Inhibiting RNA Polymerase II Transcriptional Elongation

Solving the biological roles of covalent histone modifications, including monoubiquitination of histone H2A, and the molecular mechanisms by which these modifications regulate specific transcriptional programs remains a central question for all eukaryotes. Here we report that the N-CoR/HDAC1/3 complex specifically recruits a specific histone H2A ubiquitin ligase, 2A-HUB/hRUL138, to a subset of regulated gene promoters. 2A-HUB catalyzes monoubiquitination of H2A at lysine 119, functioning as a combinatoric component of the repression machinery required for specific gene regulation programs. Thus, 2A-HUB mediates a selective repression of a specific set of chemokine genes in macrophages, critically modulating migratory responses to TLR activation. H2A monoubiquitination acts to prevent FACT recruitment at the transcriptional promoter region, blocking RNA polymerase II release at the early stage of elongation. We suggest that distinct H2A ubiquitinases, each recruited based on interactions with different corepressor complexes, contribute to distinct transcriptional repression programs.

Mechanisms establishing TLR4-responsive activation states of inflammatory response genes.

Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses.

Interleukin-4 induction of the CC chemokine TARC (CCL17) in murine macrophages is mediated by multiple STAT6 sites in the TARC gene promoter

BackgroundMacrophages (Mθ) play a central role in the innate immune response and in the pathology of chronic inflammatory diseases. Macrophages treated with Th2-type cytokines such as Interleukin-4 (IL-4) and Interleukin-13 (IL-13) exhibit an altered phenotype and such alternatively activated macrophages are important in the pathology of diseases characterised by allergic inflammation including asthma and atopic dermatitis. The CC chemokine Thymus and Activation-Regulated Chemokine (TARC/CCL17) and its murine homologue (mTARC/ABCD-2) bind to the chemokine receptor CCR4, and direct T-cell and macrophage recruitment into areas of allergic inflammation. Delineating the molecular mechanisms responsible for the IL-4 induction of TARC expression will be important for a better understanding of the role of Th2 cytokines in allergic disease.ResultsWe demonstrate that mTARC mRNA and protein are potently induced by the Th2 cytokine, Interleukin-4 (IL-4), and inhibited by Interferon-γ (IFN-γ) in primary macrophages (Mθ). IL-4 induction of mTARC occurs in the presence of PI3 kinase pathway and translation inhibitors, but not in the absence of STAT6 transcription factor, suggesting a direct-acting STAT6-mediated pathway of mTARC transcriptional activation. We have functionally characterised eleven putative STAT6 sites identified in the mTARC proximal promoter and determined that five of these contribute to the IL-4 induction of mTARC. By in vitro binding assays and transient transfection of isolated sites into the RAW 264.7 Mθ cell-line, we demonstrate that these sites have widely different capacities for binding and activation by STAT6. Site-directed mutagenesis of these sites within the context of the mTARC proximal promoter revealed that the two most proximal sites, conserved between the human and mouse genes, are important mediators of the IL-4 response.ConclusionThe induction of mTARC by IL-4 results from cooperative interactions between STAT6 sites within the mTARC gene promoter. Significantly, we have shown that transfer of the nine most proximal mTARC STAT6 sites in their endogenous conformation confers potent (up to 130-fold) IL-4 inducibility on heterologous promoters. These promoter elements constitute important and sensitive IL-4-responsive transcriptional units that could be used to drive transgene expression in sites of Th2 inflammation in vivo.

Upregulation of NRG-1 and VAMP-1 in human brain aggregates exposed to clozapine.

Growing genetic evidence has implicated a role for neuregulin-1 (NRG-1) in schizophrenia pathogenesis as well as alterations in SNAP receptor (SNARE) proteins at both gene and protein levels in post-mortem investigations. In relation to a potential therapeutic mechanism for atypical antipsychotic medications, clozapine has been shown to increase both NRG-1 levels and synaptic markers in rodents. As evidence continues to mount for a potential restoration in connectivity by antipsychotic medications being a mode of efficacy we chose to examine the effects of the atypical antipsychotic clozapine and the typical antipsychotic haloperidol on NRG-1 and SNARE protein transcripts in human brain aggregates exposed to plasma levels chronically for a period of three weeks. At the end of this exposure period we performed quantitative real-time PCR to investigate the mRNA levels of NRG-1, VAMP-1 and SNAP-25. Overall we found that clozapine had the ability to upregulate NRG-1 (+3.58 fold change) and VAMP-1 (+1.92) while SNAP-25 remained unchanged. Changes for haloperidol exposed aggregates were below our cut-off of +1.5. Overall the results of our investigation lend further support to atypical antipsychotic medications having the potential to increase levels of neurotrophic and synaptic markers such as NRG-1 and VAMP-1, the former being a strong candidate susceptibility gene for schizophrenia. In the absence of frank neuronal loss in schizophrenia, restoration of neuronal and synaptic functions by atypical antipsychotics in the brains of schizophrenics maybe a key mechanism of therapeutic efficacy by re-establishing normal connectivity and functioning.

Mnt-deficient mammary glands exhibit impaired involution and tumors with characteristics of myc overexpression.

The proto-oncogene c-Myc plays a central role in cell growth and the development of human tumors. c-Myc interacts with Max and Myc-Max complexes bind to E-box and related sequences to activate transcription. Max also interacts with Mnt but Mnt-Max complexes repress transcription when bound to these sequences. MNT maps to human chromosome 17p13.3, a region frequently deleted in various human tumors, including mammary gland tumors. Consistent with the possibility that Mnt functions as a Myc antagonist, Mnt-deficient fibroblasts exhibit many of the hallmark characteristics of cells that overexpress Myc, and conditional (Cre/Lox) inactivation of Mnt in mammary gland epithelium leads to adenocarcinomas. Here, we further characterize mammary gland tissue following conditional deletion of Mnt in the mammary gland. We show that loss of Mnt severely disrupts mammary gland involution and leads to hyperplastic ducts associated with reduced numbers of apoptotic cells. These findings suggest that loss of Mnt in mammary tissue has similarities to Myc overexpression. We tested this directly by using promoter array analysis and mRNA expression analysis by oligonucleotide arrays. We found that Mnt and c-Myc bound to similar promoters in tumors from MMTV-c-Myc transgenic mice, and mRNA expression patterns were similar between mammary tumors from MMTV-Cre/Mnt(KO/CKO) and MMTV-c-Myc transgenic mice. These results reveal an important role for Mnt in pregnancy-associated mammary gland development and suggest that mammary gland tumorigenesis in the absence of Mnt is analogous to that caused by Myc deregulation.

HIV downregulates interferon-stimulated genes in primary macrophages

HIV is able to outpace the innate immune response, including that mediated by interferon (IFN), to establish a productive infection. Primary macrophages, however, may be protected from HIV infection by treatment with type I IFN before virus exposure. The ability of HIV to modulate the type I IFN-mediated innate immune response when it encounters a cell that has already been exposed to IFN remains poorly defined. The optimal pretreatment time (12 h) and the most potent HIV-inhibitors (e.g., IFN-α2 and -ω) were identified to investigate the ability of HIV to modulate an established type I IFN response. Gene expression at the level of the entire transcriptome was then compared between primary macrophages treated with type I IFNs, as opposed to treated with IFNs and then infected with HIV. Although HIV was not able to establish a robust infection, the virus was able to downregulate a number of IFN-stimulated genes (ISGs) with a fold change greater than 1.5 (i.e., AXL, IFI27, IFI44, IFI44L, ISG15, OAS1, OAS3, and XAF1). The downregulation of OAS1 by the presence of HIV was confirmed by real-time quantitative polymerase chain reaction. In conclusion, even though HIV replication is significantly inhibited by IFN pretreatment, the virus is able to downregulate the transcription of known antiviral ISGs (e.g., IFI44, ISG15, and OAS1).

Gene expression before HAART initiation predicts HIV-infected individuals at risk of poor CD4+ T-cell recovery.

Objective: To identify a pre-HAART gene expression signature in peripheral blood mononuclear cells (PBMCs) predictive of CD4+ T-cell recovery during HAART in HIV-infected individuals. Design: This retrospective study evaluated PBMC gene expression in 24 recently HIV-infected individuals before the initiation of HAART to identify genes whose expression is predictive of CD4+ T-cell recovery after 48 weeks of HAART. Methods: The change in CD4+ T-cell count (ΔCD4) over the 48-week study period was calculated for each of the 24 participants. Twelve participants were assigned to the ‘good’ (ΔCD4 ≥ 200 cells/μl) and 12 to the ‘poor’ (ΔCD4 < 200 cells/μl) CD4+ T-cell recovery group. Gene expression profiling of the entire transcriptome using Illumina BeadChips was performed with PBMC samples obtained before HAART. Gene expression classifiers capable of predicting CD4+ T-cell recovery group (good vs. poor), as well as the specific ΔCD4 value, at week 48 were constructed using methods of Class Prediction. Results: The expression of 40 genes in PBMC samples taken before HAART predicted CD4+ T-cell recovery group (good vs. poor) at week 48 with 100% accuracy. The expression of 22 genes predicted a specific ΔCD4 value for each HIV-infected individual that correlated well with actual values (R = 0.82). Predicted ΔCD4 values were also used to assign individuals to good vs. poor CD4+ T-cell recovery groups with 79% accuracy. Conclusion: Gene expression in PBMCs can be used as biomarkers to successfully predict disease outcomes among HIV-infected individuals treated with HAART.