Notch: From fly wings to human hematological tumors

Notch history begins in 1919 with Thomas Hunt Morgan studies on fruit fly mutants. From then, this gene aroused lively interest in the scientific community since it is involved in a wide variety of processes, including morphogenesis, tissue homeostasis, and stem cell maintenance. Deregulation of Notch signaling characterizes several human tumors. Hematopoietic system is affected by mutations of Notch receptors, Notch ligands, and proteins controlling their stability. Approximately 60% T acute lymphoblastic leukemia (T-ALL) patients carry activating Notch1 mutations prompting blasts growth. In addition, multiple myeloma is characterized by Notch signaling hyper-activation due to an abnormal expression of the Jagged2 ligand; this affects not only myeloma cells, but also their interaction with bone marrow microenvironment, influencing tumor burden and bone disease. These findings make Notch a rational target of a therapeutic approach. Inhibitors of the Notch activating enzyme, ?-Secretase, have been successfully used in vitro and in vivo and are currently under clinical trials for T-ALL and breast cancer. Yet a wide use of these inhibitors is prevented by frequently occurring drug resistance. To elucidate the mechanism underlying this phenomenon, a number of pathways have been identified mediating Notch biological effects: AKT and c-Myc are frequently deregulated in leukemic patients and account for resistance to ?-Secretase inhibitors by acting downstream Notch receptor. Therefore, the interaction of Notch with other cancer-associated proteins should be clarified to predict the biological outcome of a Notch targeted therapy and possibly, to exploit combined treatments against the key deregulated elements in Notch-associated cancers

MeCP2/H3meK9 are involved in IL-6 gene silencing in pancreatic adenocarcinoma cell lines

The aim of the present study was to analyse the molecular mechanisms involved in the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our results demonstrate that TNF-α, a major IL-6 inducer, is able to induce IL-6 only in three out of six cell lines examined. 5-aza-2′-deoxycytidine (DAC), but not trichostatin A (TSA), activates the expression of IL-6 in all cell lines, indicating that DNA methylation, but not histone deacetylation, plays an essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a methylation status that correlates with IL-6 expression and binds MeCP2 and H3meK9 only in the non-expressing cell lines. Our results suggest that critical methylations located from positions –666 to –426 relative to the transcription start site of IL-6 may act as binding sites for MeCP2

Regulation of transcription factor binding specificity: from binding motifs to local DNA context

Regulation of transcription factor (TF) binding specificity lies at the heart of transcriptional control which governs how cells divide, differentiate, and respond to their environments. TFs are known to bind to DNA in a sequence specific manner, and such short sequence is known as transcription factor binding site (TFBS). However, the in vivo TF bound regions do not always contain a TFBS, and additionally, there are often excessive non-functional TFBSs with binding potential in the regulatory regions that are unbound for a given TF. This dissertation focuses on understanding the principles of TF binding specificity and is divided into two chapters: 1) developing a novel high throughput method that would facilitate the study of TF binding regulations and the resulting functional output; 2) analyzing the roles of local DNA context around TFBS in specifying TF localization. In the first chapter of this dissertation, we report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and energetic cost of 1.37 KBT for sites that are positioned “out of phase”. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p, and Gcr2p, that act together as a “TF collective”, an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function. In the second chapter of this dissertation, we seek out to investigate if predictive information is embedded in local DNA context (LDC) on a large collection of TFs in Saccharomyces cerevisiae. We identify there is a general preference for TFs to bind at CG rich sequences; we then analyze whether such preference is linked to intrinsic nucleosome binding preference and found the CG preference in LDC for TF binding was independent of nucleosome regulation. We next examine the possible mechanism by which LDC influence TFs binding site selection, through recruiting ‘licensing’ factors or kinetically assisting TF search for a target site. We show high CG LDC is preferred by TFs in vitro condition, which suggests such preference only involves TFs and DNA and directs us to TF search kinetics mechanism. CG rich feature in LDC may act as an energetical funnel to facilitate TF recognizing a target binding site, and we verify the theoretical validity of this hypothesis with Gillespie simulation. In the end, we reveal CG preference was also present in a large group of human TFs, indicating the usage of LDC is a general mechanism for TF binding specificity

Novel Mechanisms In Dendritic Cells That Promote Th2 and Th17 But Not Th1 Responses In The Lung

Dendritic cells (DCs) are integral to differentiation of T helper cells into Th1, Th2 and Th17 subsets. We have dissected two novel pathways in DCs that specifically regulate CD4 T cell responses. The first is the role of the c-Kit-Phosphatidyl inositol 3 kinase (PI3 kinase)-interleukin-6 (IL-6) axis and the second that of vascular endothelial growth factor (VEGF). IL-6 plays a central role in regulating CD4 T cell immune responses by limiting a Th1 response and promoting Th2 and Th17 responses. We investigate pathways in DCs that promote IL-6 production and show that the allergen house dust mite or the mucosal adjuvant cholera toxin but not Th1-inducing adjuvant, CpG oligodeoxynucleotide (ODN) promote cell surface expression of c-kit and its ligand, stem cell factor (SCF), in DCs. This dual upregulation of c-kit and SCF results in sustained PI3-kinase signaling promoting IL-6 secretion. Intranasal administration of antigen into c-kit mutant mice or neutralization of IL-6 blunted Th2 and Th17 but promoted Th1 responses in lung-draining lymph nodes. DCs lacking functional c-kit elicit diminished allergic airway inflammation when adoptively transferred into mice. Expression of the Notch ligand, Jagged-2, which has been associated with Th2 differentiation, was reduced in DCs from c-kit mutant mice. DCs generated from mice expressing a catalytically inactive form of the p110ƒÔ (p110D910A) subunit of PI3-kinase secrete lower levels of IL-6 upon stimulation with CT. These results collectively highlight the importance of the c-kit-PI3-kinase-IL-6 signaling axis in DCs in regulating T cell responses.We also investigated mechanisms underlying the production of VEGF, which has been recently shown to be a Th2-skewing cytokine and to promote allergic asthma. We found that CT-stimulated DCs secrete high levels of VEGF while LPS induces minimal VEGF production. Activation of iNOS, NF-ƒÛB and PI3 kinase enhanced production of VEGF in DCs whereas IL-12, a Th1-skewing cytokine, inhibited VEGF production. This mechanism highlights a critical but previously unknown role for DC-derived VEGF. Taken together, these findings broaden our understanding of diverse mechanisms in DCs that enable T cell polarization and offer novel targets for therapeutic interventions