__Abstract__
Finding a cure for the human immunodeficiency virus type 1 (HIV-1) is extremely challenging. Development of highly active anti-retroviral therapy (HAART), transformed HIV-1 infection from an acute syndrome into chronic disease. Although using HAART results in suppression of viral replication, it is not curative. High rates of HIV-1 replication return in infected patients when HAART is interrupted. The presence of a long-lived latent reservoir for HIV-1 in resting CD4+ T cells allows the persistence of the virus despite therapy. This reservoir is composed of replication competent but transcriptionally silent virus, which is the major barrier toward HIV-1 eradication. Understanding the mechanisms that are involved in HIV-1 latency, and developing in vitro and in vivo models of latency have allowed the design and testing of therapies that target the latent reservoir. The current approach in the development a curative treatment for HIV-1 is called “Shock and Kill”. This approach entails reactivation of latent HIV-1 using latency reversing agents (LRAs) followed by elimination of the activated virus by an effective host immune system, cytolytic T lymphocytes (CTL) or viral cytopathic effects. Therefore, the first and promising step in eradication of viral reservoirs, is reactivation of latent HIV-1. This process constitutes pharmacological approaches to target different mechanisms and pathways that are involved in maintenance of HIV-1 latency and its reactivation. In this thesis I focus on the shock part of shock and kill strategies. This research aims to investigate the molecular mechanisms involved in HIV-1 post-integration latency establishment and maintenance, and reactivation of latent HIV-1.
In Chapter 2 we identified the mechanistic role of SWI/SNF in the establishment and maintenance of HIV-1 latency and its re-activation in latent HIV-1 infected cell models. SWI/SNF is one of the four ATP-dependent chromatin remodeling complex in mammals which contains two functionally distinct sub classes referred to as BAF and PBAF. We determined the nucleosome affinity of the HIV-1 LTR DNA sequence, and found a reverse correlation compared to the nucleosome positioning in vivo. Hypersensitive sites (DHS-1) displayed the highest nucleosome affinity, while the positioned nucleosomes displayed lower affinity for nucleosome formation. We examined the role of SWI/SNF in the nucleosome structure of the latent HIV-1 LTR. We found that establishment and maintenance of HIV-1 latency requires BAF, which removes a preferred nucleosome from DHS-1 to position the repressive nucleosome-1 over energetically unfavorable sequences. BAF depletion resulted in activation of the latent HIV-1. Our high resolution MNase nucleosomal mapping analysis showed a dramatic alteration in the LTR nucleosome profile after BAF depletion. Upon activation, BAF was lost from the HIV-1 promoter, while PBAF was specifically recruited by the HIV-1 transactivator protein TAT to facilitate HIV-1 transcription. Thus BAF and PBAF, recruited during different stages of the HIV-1 life cycle, display opposing function on the HIV-1 promoter. As a repressor of HIV-1, BAF may be an attractive therapeutic target to activate the latent reservoir in patients.
In Chapter 3 we explored the role of the Wnt signaling pathway in activation of transcription of latent HIV-1. We found that activation of the Wnt pathway via different mechanisms including treatment with natural Wnt ligand or small molecule inhibitors resulted in activation of latent HIV-1. Chromatin immunoprecipitation (ChIP) analysis showed that the Wnt molecular effectors LEF1 and β-catenin are recruited to the HIV-1 LTR after Wnt activation. Upon activation, we demonstrate that, nucleosomal structure on the latent HIV-1 LTR is dramatically restructured using high resolution MNase nucleosomal mapping. Lithium, one of the Wnt agonists in clinical use for treatment bipolar disease, activated HIV-1 transcription in latent HIV-1 infected primary CD4+ T cells. Using HDAC inhibitors such as Valporoic acid (VPA) and Vorinostat (SAHA), currently under clinical investigation, in combination with Wnt activators synergistically enhanced the activation of the latent HIV-1 LTR. Thus, targeting the Wnt pathway by small molecules and Wnt agonists may be an attractive strategy in a combinatorial therapy aimed at activation of latent HIV-1.
In Chapter 4 we investigated the mechanistic determinants of the functional specialization between the two TCF/LEF members, T cell factor1 (TCF1) and lymphoid enhancing factor 1 (LEF1), two molecular effectors of the Wnt signaling pathway, which are co-expressed in T cells. De-regulation of Wnt signaling is involved in formation of T cell leukemia. We found distinct isoform expression of TCF1 and LEF1 in CD4+ T-ALL leukemic cell lines, including both the Wnt responsive isoform as well as the dominant negative forms. We used immunoprecipitation-mass spectrometry analysis to find novel interacting partners for LEF1 and TCF1 in the presence or absence of Wnt signaling in leukemic cell lines. Using RNA sequencing analysis, we determined target genes which are regulated by TCF1 and LEF1 by siRNA depletion and examined differentially expressed genes in the presence or absence of Wnt signaling. ChIP assays on two novel target genes from RNA sequencing analysis, showed differential recruitment of Wnt molecular effectors to their regulatory regions on the targets in response to Wnt stimulation. Thus, our results provide a mechanistic basis to the in vivo observed functional specialization for TCF1 and LEF1 in T cell leukemia.
In Chapter 5 we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We showed that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during differentiation to macrophages. Our microarray analysis demonstrated that expression of HDAC7 interfered with the gene transcriptional program characteristic of macrophages during cell differentiation. We described that presence of HDAC7 blocked the expression of key genes for macrophage-mediated specific functions. Our co-immunoprecipitation and chromatin immunoprecipitation analysis gave insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells. We found specific interaction of HDAC7 with the transcription factor MEF2C in pre-B cells. HDAC7 was recruited to MEF2 binding sites located at the promoters of macrophage target genes. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes.
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