The Role of SATB1 in Nuclear Organization

Special A-T rich Binding protein 1 (SATB1) is a nuclear matrix protein that binds DNA at matrix attachment regions (MARs) with a preference for A-T rich sequences. MARs are thought to spatially organize chromatin in the nucleus thus regulating transcription. SATB1 is predominantly expressed in T-cells and forms a cage-like structure in the nucleus. This structure might be an organizing scaffold for chromatin. SATB1 has also been shown to promote breast cancer growth, and metastasis. Yet, little is known about how SATB1 mediates these processes. We are interested in understanding the mechanism of how SATB1 regulates transcription, and how its characteristic nuclear structure contributes to this functional outcome. To this end we have performed a tandem affinity purification (TAP) to identify interacting proteins in an unbiased way. We have identified PARP-1 as a binding partner for SATB1 and are currently investigating how this interaction affects SATB1 function.

NGF Signaling Maintains HSV-1 Latency

Transmission of the neurotropic herpes simplex virus (HSV-1) frequently occurs through activation of a latent virus that exists in a silent form in peripheral neurons. Although many diverse stimuli can activate the latent virus, little is known about the cell surface receptor signaling mechanisms that allow the virus to escape latency. Here we have used an established primary culture model of sympathetic neurons to establish HSV-1 latency. We find that activation of the virus from neurons deprived of NGF is dependent upon TrkA tyrosine kinase signaling through the PI-3 kinase and PDK1 pathway, and not the MAP kinase pathway or from apoptosis signaling. The effect of TrkA tyrosine kinase signaling is specific since withdrawal of other growth factors, such as EGF, are unable to promote reactivation. The mechanism for the differential effect of NGF and EGF to maintain latency is due to the extent of receptor tyrosine kinase signaling. These results indicate that specific cell surface mechanisms are responsible for HSV-1 to maintain latency and to regulate its reactivation in the nervous system.

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Regulation of Immediate-early Genes Upon UV Damage and its Role in the DNA Damage Response

Cells from organisms have to safeguard their genome from exogenous and endogenous threats that can cause DNA damage. The resulting DNA lesions have to be repaired to prevent loss or incorrect transmission of genetic information to the daughter cells. After exposure of cells to diverse sources of DNA damage, sensor proteins are alerted to detect lesions on the genome and to activate cell cycle checkpoints that arrest or slow down cell cycle progression, leading to DNA repair, regulation of DNA synthesis, and/or apoptosis. All these processes are integrated in a complex signaling network called the DNA damage response (DDR), which can be promoted and sustained by transcriptional regulation of different genes.

A small group of transcription factors (TFs) belonging to the immediate-early gene family is upregulated following ultraviolet (UV) radiation. However, it is unclear how rapid activation of these TFs, including the c-Fos, c-Jun and Egr-1 genes, regulates DDR and cellular survival. My work is focused on characterizing the link between these immediate early genes and the DDR genes.

Molecular Pathogenesis of the Fanconi Anemia Cancer Susceptibility Pathway

Our lab is interested in understanding the molecular pathogenesis of the Fanconi anemia (FA) human genetic disorder. Cells derived from FA patients have abnormal cell cycle distribution, defects in DNA repair, genomic instability and hypersensitivity to DNA damaging agents. In normal cells, FA proteins are involved in a complex DNA repair pathway. Upon DNA damage, two key Fanconi proteins: FANCI and FANCD2 are monoubiquitinated enabling their localization at the site of DNA damage. The deubiquitining enzyme USP1 can antagonize the monoubiquitination of FANCI and FANCD2. However, it is unclear how USP1 activity is regulated during DNA damage, therefore my main research objective is to study the regulation of USP1 activity during DNA damage.

Aberrant Activation of mTOR in Peripheral Neuropathy

Demyelination results from dedifferentiation of Schwann cells and the consequent breakdown of their myelin sheaths. Although demyelination is a significant source of morbidity in nerve injuries and neuropathies, the mechanisms involved have remained elusive. Our lab has developed an experimental model of demyelination that involves addition of the Neuregulin-1 (NRG1) growth factor to myelinating cocultures. This results in the activation of the erbB receptors and their downstream signaling pathways followed by Schwann cell dedifferentiation and demyelination. Among the pathways activated by NRG1 is the mammalian target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational apparatus and integrates a number of upstream signals. We have found that inhibiting mTOR with rapamycin effectively prevents NRG1-induced demyelination. Recent results strongly suggest that mTOR is similarly aberrantly activated in the PMP22 overexpressing transgenic rat, a murine model of the demyelinating human neuropathy Charcot-Marie-Tooth 1A (CMT1A). This activation leads to Schwann cell dedifferentiation as inhibition of mTOR with rapamycin rescues myelination in a tissue culture model of CMT1A. Current studies seek to elucidate further the role of mTOR in Schwann cell dedifferentiation, and the mechanism by which alterations in gene dosage of PMP22 lead to its activation. In complementary studies, we will examine the potential of rapamycin as a novel therapeutic strategy for CMT1A in vivo.

A Sos-mediated Cross-talk Between Oncogenic and Wild-type Ras in Tumors

The RTK-Sos-Ras cascade functions as a central signaling pathway that controls the proliferative state of cells. We have identified a positive feedback loop for Sos-mediated Ras activation involving the binding of GTP-loaded Ras to an allosteric site on Sos. Since oncogenic forms of Ras are constitutively bound to GTP, the allosteric modulation of Sos may constitute a mechanism by which mutant Ras can potentiate the activity of wild-type Ras. To test this idea, we first analyzed the effects of the expression of K-RasV12 on the activation levels of wild-type H-, K- and N-Ras. All isoforms were potently activated in the presence of K-RasV12. This effect was cell-autonomous and abolished upon suppression of Sos expression. Similarly, in pancreatic cancer cell lines harboring K-Ras mutation, wild-type H-Ras was activated by mutant K-Ras and this activation was dependent on Sos. These observations suggest that in Ras-transformed cells the Ras signaling platform may involve both the oncogenic and wild-type forms through a Sos-mediated cross-talk. We have examined the effects of repression of Sos expression on the growth of pancreatic cancer cell lines. Loss of Sos expression led to attenuation of cell growth due to defects in cell cycle progression. Taken togther, these findings underscore the complementary functions of oncogenic and wild-type Ras in tumor cells and identify a potential new targeting strategy for Ras-driven tumors.

Elucidation of HSV-1 Reactivation Mechanism

Herpes simplex virus (HSV) is a viral pathogen infecting approximately 80% of the human population. Like all herpes viruses, HSV shows two modes of infection, lytic and latent infection. Selection of infection mode (lytic vs. latent) is dependent on host cell proliferation state. Latent HSV after exposure to cellular stresses, however, can switch into the lytic cycle. This process is called reactivation.

Several studies illustrate the role of HCF-1 (a cellular co-transcriptional regulator) in HSV infection. The viral tegument protein VP16 interacts with HCF-1 and translocates to nucleus where VP16, HCF-1 and Oct-1 initiate the immediate early gene (IE) transcription. In a provocative study by Kristie and colleagues reported that HCF-1 localizes to the cytoplasm in trigeminal ganglia from HSV infected animals. Cellular stress cause HCF-1 to translocate coincident with HSV reactivation.

In the past, reactivation studies have been extremely challenging due to limitations of the experimental system. With recent development of a superior cervical ganglion (SCG) tissue culture system from rat embryos in which HSV establishes a quiescent (‘latent-like’) state, I propose to investigate mechanism of HSV reactivation. I hypothesize that HCF-1 is a contributing factor in the transition from latency to lytic replication.

Rab5 Up-Regulation Induces Early Alzheimer Disease-related Endosomal Pathology

Abnormally enlarged endosomes were one of the earliest known cellular pathology of Alzheimer disease (AD). Rab5 is a small GTPase and a key regulator of early endosome, and its up-regulation is able to induce enlarged endosomes, possibly inducing abnormal endocytosis. Therefore, we hypothesize that Rab5 up-regulation induces AD-related endosomal pathology. To investigate this, we generated transgenic mice overexpressing Rab5. Transgenic animals recapitulated endosomal enlargements in neurons and showed defects in fear learning and memory along with electrophysiological changes. Furthermore, it was found that APP was able to activate Rab5 and induce enlargements and trafficking defects of early endosomes in neurites of cultured neurons. APP made a complex with other endocytic adaptors to activate Rab5. Finally, we generated APP/Rab5 double transgenic mice and found that fear learning and memory was defected in these mice. Thus, we suggest that APP-mediated Rab5 up-regulation is one of the crucial processes in AD pathogenesis.

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Establishment of a 3D Culture System of Mouse Primary Pancreatic Duct Epithelial Cells (PDEC) to Study Pancreatic Tumorigenesis

Oncogenic forms of KRas are highly prevalent in pancreatic cancer and are thought to play a critical role in disease development. It has been shown that oncogenic Ras expression can trigger premature senescence through the upregulation of p16INK4A, p19ARF and p53. Since the biological outcomes of Ras signaling may vary depending on signaling intensity and cell type, we have studied the effects of endogenous expression of oncogenic KRas (KRasG12D) on senescence in primary pancreatic duct epithelial cells (PDEC). Wild-type PDEC undergo premature senescence in culture, during the course of which p16INK4A is induced, but the levels of p19ARF, p53, and p21 remain unchanged. By contrast, endogenous expression of KRasG12D protects PDEC from undergoing senescence through the suppression of p16INK4A induction. The abrogation of p16INK4A induction by KRasG12D is mediated by Twist, a bHLH transcription factor. Senescence is considered a critical barrier to cancer development. Our findings suggest that KRasG12D may promote pancreatic tumorigenesis by antagonizing a permanent cell growth arrest triggered by a variety of cellular stresses.

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NSDs Family Histone Methyltransferase

Nuclear receptor binding SET Domain-containing (NSD) family proteins, including NSD1, NSD2 and NSD3 are a group of histone methyltransferases involved in diseases such as childhood developmental diseases, acute myeloid leukemia and multiple myeloma.  There have been wide discrepancies regarding which histone residues the NSDs modify. NSD1 has been reported to methylate histone H3K36 and H4K20; NSD2, H3K4, H3K27 and H4K20; NSD3, H3K4 and H3K27.  Using recombinant NSD1-3 SET domain proteins , I found that NSD1-3 are very specific H3K36 di-methylase on recombinant nucleosomes, however, they target multiple residues on histone octamers  include at least K36 and K18 on H3 and some unknown sites on H4 besides K20. NSD1 has been found to interact with RAR, RXR, TR and AR and may act as a nuclear receptor co-regulator.   To study the mechanism of how NSDs work, I purified NSD2 interacting proteins from NSD2 stable cell line and am ready to send them for Mass spectrometry analysis. To identify genome wide target genes of the NSD family proteins by ChIP-Seq and study how NSDs regulate their transcription I have raised antibodies against NSD1, NSD2 and SET2 and verified their specificity by RNAi and tested them for Western blot and immunoprecipitation. 

Cellular and Molecular Response of the Epithelial Cells to Distinct Forms of Death

Experimental evidence supports a prominent role for necrotic cell death in tumor initiation and progression. However, the mechanisms that underlie the influence of necrosis on tumorigenesis remain largely unresolved. Previous studies on necrosis have focused on the interplay between inflammatory and necrotic cell populations. Since epithelial tumorigenesis is often associated with a progressive switch from predominantly apoptotic to necrotic cell death, we sought to establish an experimental in vitro system that will allow us to study the direct impact of necrotic cell death on the neighboring epithelial monolayer. Using a combination of live imaging, immunofluorescence and microinjection techniques, we are conducting a comparative analysis between the cellular and molecular responses of the epithelial cells to apoptotic versus necrotic death of their neighboring cells.

Pyrophosphate Removal in Escherichia coli mRNA degradation

Bacteria live in a constantly changing environment, and therefore have a continuous need to modulate their protein expression. Three processes control protein synthesis in those microorganisms: transcription, translation, and mRNA degradation. It had long been thought that mRNA degradation in Escherichia coli begins with endonucleolytic cleavage by RNase E. However, recent work has shown that RNase E cleavage can be triggered by a prior step: the removal of pyrophosphate from the 5’ terminus of primary transcripts. An enzyme that catalyzes this initial event (RppH) has been discovered in E. coli. E. coli also have exonucleases that can degrade transcripts from the 3’ end. Canonical transcripts bear a protective stem-loop at this end. Previous evidence has suggested that elements at the 5’ end, including the phosphorylation state, affect exonucleolytic degradation through these stem-loops. Using a novel RNA I have shown that pyrophosphate removal by RppH can influence efficient RNA degradation from the 3’ end of transcripts in vivo.

Nucleolin is a Multifunctional Protein Involved in Ribosome Biogenesis, Angiogenesis, and the Cellular Stress Response. It is Phosphorylated During Interphase by Casein Kinase 2 (CK2)

In this study, I have addressed the role of this phosphorylation event in the dynamics, location, stability and proliferative activities of nucleolin. Mutation of the CK2 phosphorylation sites to alanines resulted in a greater mobility of the mutant compared with the wild-type protein as measured by fluorescence recovery after photobleaching (FRAP). Notably, the nucleolin mutations were found to reduce the stability of the protein via proteosomal degradation. Treatment of cells with CK2 inhibitors resulted in a decrease in nucleolin protein levels, further supporting the mutagenesis data.  Moreover, mutation of the CK2 sites reduces nucleolin RNA binding activity. These results provide evidence that phosphorylation at these sites reduces the affinity of nucleolin for nucleolar components and implicate CK2 in regulating the proliferative activities of nucleolin

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The Polycomb Group Protein Ezh2 Regulates Pancreas Regeneration

Pancreatic cancer is the 4th leading cause of cancer related mortality in the United States with an annual incidence of 34,000 cases.  The prognosis for pancreatic cancer is dismal with a mean survival time of 6 months following diagnosis and a five-year survival rate of 5%. Chronic pancreatitis, a disease characterized by recurrent cycles of pancreatic injury and regeneration, is a major risk factor for pancreatic cancer.  Often chronic inflammation precedes the development of pancreatic cancer, but little is known regarding mechanistic links between chronic inflammation in the pancreas and the development of pancreatic cancer.

Ezh2 is a polycomb group protein with histone methyltransferase activity that is responsible for the tri-methylation of histone H3 lysine 27, a histone modification that is involved in transcriptional silencing of developmental genes allowing appropriate tissue specific gene expression.  We found that Ezh2 protein levels are increased following chronic pancreatic inflammation. Chromatin immunoprecipitation experiments demonstrated that Ezh2 is recruited to Polycomb responsive promoters following inflammation.  To explore this novel role of Ezh2 we used a cre-lox system to engineer a pancreas specific Ezh2 knockout mouse (Ezh2-/-).  Ezh2-/- mice were viable with normal pancreas development.  When subject to chronic inflammation, Ezh2-/- mice have a severe inflammatory phenotype and delayed recovery from injury.  This delay was associated with a decrease in proliferative capacity and impaired regeneration of fully differentiated acinar cells following injury.  Our results suggest that Ezh2 is required for effective regeneration following pancreatic injury and inflammation

Role of Necl-4 Complex in PNS Myelination

Interactions between axons and Schwann cells in the peripheral nervous system induce myelination. Nectin-like 4 (Necl-4) is expressed on myelinating Schwann cells and binds specifically to Necl-1 expressed on axons. These proteins mediate heterophilic interactions at the internode. Knock down of Necl-4 inhibits Schwann cell differentiation and subsequent myelination. A key question is how Necl-4 promotes myelination. Recent data has identified Par-3, a PDZ-domain containing protein of the Par-aPKC polarity complex, as required for Schwann cell myelination. Our preliminary results indicate that Necl-4, through its PDZ-binding domain, interacts with Par-3 directly. Necl-4 also interacts with the 4.1G protein via its FERM-binding domain. Together these findings suggest a model where Par-3 is recruited by Necl-4 to the adaxonal membrane of the Schwann cell upon axon-glial contact, generating anterior-posterior polarity and the formation of a leading edge in the Schwann cell that drives spiral wrapping of the axon.

Mechanism of Epigenetic Marks

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Negative Regulation of the Drosophila Apoptosome

The Drosophila Apoptosome is a cell-death triggering holoenzyme formed by the Drosophila Apaf-1 (dapaf-1, hac-1, ark) homolog and the Drosophila initiator caspase Dronc. Dronc has previously been shown to be a target of ubiquitylation by the E3 ubiquitin ligase Diap-1 (Drosophila inhibitor of apoptosis). We have shown that Apaf-1 is unstable, and Apaf-1’s instability is also dependant on a Diap-1. We have shown that the Apaf-1 and Dronc are coupled in degradation, as well as in activation. We seek to determine other regulators of Apaf-1 stability using an inducible RNAi strategy to screen known and predicted components of the ubiquitin proteasome system in vivo.

Epigenetic Regulators of Gene Expression

Characterization of the Novel H3K27me1 in EED null ES cells

Polycomb proteins (PcG) are an evolutionarily conserved family of chromatin regulators which play a role in establishing and maintaining epigenetic memory during development. Polycomb Repressive complex 2 (PRC2) consists of three core components: enhancer of zeste 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12). The EZH2 is a SET domain-containing methyltransferase that catalyzes the formation of the H3K27me3 mark, which forms the recruiting mark for Polycomb Repressive Complex 1 (PRC1) thought to be the effector of PcG-mediated long-term epigenetic memory. In Drosophila, loss of EZH or ESC/ESCL (EED homologue) functions results in loss of monomethylation, dimethylation, and trimethylation of H3K27, indicating that it is the only K27 methyltransferase activity. However, in mammals, loss of the EZH2 eliminates only dimethylation and trimethylation, implying that monomethylation of H3K27 is carried out by a different complex. Moreover, recent studies have shown that H3K27me1 was correlated with gene activation rather than gene repression. We are interested in characterizing the new H3K27 mono-methyltransferase in mammal and understanding the mechanism of how H3K27me1 marker influences gene expression. To identify H3K27me1, we are processing the conventional chromatography using EED null embryonic stem cells where we can rule out the in vitro PRC2 methyltransferase activity on H3K27. 

The Role of K-Ras at the Endoplasmic Reticulum

Previous work in the lab has shown that K-Ras becomes phosphorylated at Serine 181 in response to PKC agonists. This phosphorylation event activates a "farnesyl electrostatic switch", which causes the translocation of K-Ras from the plasma membrane to the ER and mitochondria. Surprisingly, this translocation event caused cells to undergo cell death in a Bcl-XL-dependent manner. Targeting K-Ras to the ER (but not mitochondria) was able to recapitulate the death phenotype of phosphomimetic K-Ras, suggesting that the ER was the platform for the pro-death signal. At the ER, GTP-bound phospho-K-Ras forms a complex with IP3R and Bcl-XL. This complex causes changes in Ca2+ flux through IP3R and affects calcium homeostasis, ultimately leading to cell stress and cell death.

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Identification and Characterization of a p56lck-interacting Protein Expressed in CD8+ Tumor Infiltrating T Cells

The immune system can recognize protein products of genetic and epigenetic changes in transformed cells. However cancers grow, implying that antitumor immune responses are either not sufficiently vigorous to eliminate the tumor or that antitumor immunity is suppressed. Our lab studies the phenotype of CD8+ antitumor T cells that infiltrate tumor tissue ('TIL') wherein we have shown that lytic function of CD8+ antitumor TIL is defective and is correlated with blockade of proximal TCR-mediated signal transduction. When purified and briefly cultured in the absence of tumor cells TIL recover both proximal signaling and lytic function. This suggests the involvement of a fast acting ‘switch’ in the regulation of TIL function and also that the inhibitory signal is tumor-derived. We have analyzed the function of p56lck and determined that p56lck kinase function is inactivated by rapid contact-dependent dephosphorylation of the p56lck activation motif (by Shp-1) thus accounting for both defective signal transduction and effector phase function of TIL. In the course of those studies we have identified an interacting partner of p56lck, a 115kDa protein tentatively identified as Protocadherin 18 (Pcdh18). RNA studies have shown that Pcdh18 is transcriptionally regulated in memory CD8+ T cells induced by infection by Listeria monocytogenes as well as CD8+ TIL in an activation dependent manner. Moreover, similar to freshly isolated TIL, expression of Pcdh18 in naïve CD8+ T-cells activated with a-CD3e causes apoptosis, the inability to proliferate and regulate main effector cytokines. We hypothesize that tumor-induced defective p56lck activity in CD8+ tumor infiltrating T cells may be mediated by a novel p56lck-binding protein, Protocadherin-18

The Visualization and Quantification of Liver Phagocytic Immune Cell Uptake of L. Monocytogenes Early in Infection

Listeria Monocytogenes is a facultative, gram-positive, intra-cytosolic pathogen that causes severe infections in immunocompromised individuals. During innate immune responses to infections with Listeria, resident liver macrophages, Kupffer cells, capture and present Listeria circulating in the blood to CD8+ and CD4+ T cells, promoting clearance from the body and long term immunity. At the same rate natural killer cells, neutrophils and infiltrating marcrophages are trafficked to the site of infection and are involved in the initial containment of Listeria and the activation and recruitment of effector cells by their release of cytokines, IFN-γ, CSF-1 and MCP-1, respectively. The primary goal of this thesis project is to characterize and visualize the efficiency of resident liver antigen presenting cells and infiltrating lymphocytes and granulocytes response to bacterial infections using intravital microscopy. This efficiency will be based on the migratory patterns, cell-cell and host-pathogen interactions, and the rate of phagocytosis that lead to clearance of bacteria from the body and the priming T cells for long-term immunity. From results gathered during the intravital imaging of murine liver, the rate and efficiency of Listeria uptake by kupffer cells has been determined at both high and sublethal challenge dosages, allowing for the direct visualization of host and pathogen interactions, while presenting physiologically relevant events. The effector functions of neutrophils and monocytes have also been characterized based on trafficking patterns and cell-cell interactions. Based on these and future experiments, the molecular basis for innate immune responses in the liver will be further characterized.

Characterization of Rap1 and Riam signaling in T-cells

Rap1 is a small GTPase that regulates the inside-out signaling of integrins. Rap1 is localized to the plasma membrane, to the nuclear envelope, and to paranuclear vesicles, but it is only in the active form at the plasma membrane. Riam (Rap1 GTP Interacting Adaptor Molecule) is a recently discovered effector that is necessary for Rap1 induced adhesion. It contains an RA domain, a PH domain, and several polyproline domains. Riam was shown to be in a stable complex with ADAP and Skap55 while associating with Talin in a Rap1 GTP dependent manner. Riam, ADAP, Skap55, and Talin translocate to the plasma membrane during T-cell activation and have all been shown to be necessary for Rap1 induced integrin activation. Experiments using a fluorescently tagged truncation of Riam containing the RA and PH domains revealed the selective recruitment of Riam to the plasma membrane by active Rap1. Further investigation has shown that a functional PH domain is necessary for PM recruitment. This work suggests a model whereby active Rap1 cooperates with an unknown phosphoinositide to recruit Riam to the PM. This recruitment then allows Riam to bring Talin to the PM so that Talin can directly activate integrins.

A Novel Membrane-binding Domain in Son of Sevenless

The duration and intensity of Ras-MAPK signaling determines various cellular outcomes. SOS, a predominantly cytosolic protein, couples the RTKs to the Ras-MAPK pathway by mediating a signal dependent activation of Ras, which is a membrane-bound protein. A complex interplay of incompletely understood inter/intra-molecular interactions keep Sos in the cytoplasm as well as autoinhibited. The N-terminus of SOS presents an unique Histones fold (HF) domain of unknown function. We have identified that HF binds to Phosphatidic Acid (PA), a membrane lipid, with high affinity through a basic motif. Mutation of this motif leads to a decreased membrane translocation of an isolated GFP-tagged HF construct. In the context of the FL-Sos, mutation of the PA-binding motif leads to an abrogation of Sos activity. Using Sos constructs that target constitutively to the membrane; we show that the HF-PA interaction causes the release of Sos autoinhibition at the membrane.