IU Indianapolis ScholarWorks :: Browsing by Author "Skalnik, David Gordon"

Browsing by Author "Skalnik, David Gordon"

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Analysis of differentiation capacity of Cfp1 null embyronic stem cells
(2014) Bowen, Tamara R.; Skalnik, David Gordon; Marrs, James; Chang, Hua-Chen
Epigenetics is defined as “the study of stable, often heritable, changes that influence gene expression that are not mediated by DNA sequence” (Fingerman et al., 2013). Epigenetic marks such as covalent histone modifications and DNA methylation are important for maintaining chromatin structure and epigenetic inheritance. Several proteins have been found to bind and/ or regulate epigenetic marks. One such protein, CXXC finger protein 1 (Cfp1) is an important chromatin regulator that binds to unmethylated CpG islands. It has been found to be essential for mammalian development. Mice lacking Cfp1 exhibit an embryonic- lethal phenotype. However, the function of Cfp1 can be studied using Cfp1 Null mouse ES cells, which are viable. Thus far, Cfp1 has been shown to be important for cell growth, cytosine methylation, histone modifications, subnuclear localization of Set1A histone H3K4 methyltransferase, and cellular differentiation. When Cfp1 Null ES cells are induced to differentiate by removal of Leukemia Inhibitory Factor (LIF), the cells are not able to turn off pluripotency markers such as Oct4 and alkaline phosphatase and fail to express differentiation markers such as Gata4 and Brachyury. In this study, we used established protocols to further examine the differentiation capacity of Cfp1 Null cells. Specifically, we tested the ability of Cfp1 Null ES cells to retain stem cell properties in the absence of LIF, differentiate into cardiomyocytes in the presence of TGF-β2 and differentiate into neuron precursors in the presence of retinoic acid (RA). While the differentiation effects of RA were inconclusive, Null cells were able to start differentiating in the absence of LIF, either as individual cells or EBs, and the presence of TGF-β2 when seeded on gelatin coated tissue culture dishes. However, no difference was seen between cells treated without LIF and those treated with TGF-β2. In both conditions, only a small portion of cells were able to differentiate, while the majority of the cell population retained stem cell characteristics. Cell growth and the differentiation capacity of Cfp1 Null cells were also compromised in comparison to WT cells. Thus, further supporting the need for the correct epigenetic patterns maintained by Cfp1 during cellular differentiation.
BMP Pathway and Reactive Retinal Gliosis
(2013-03-06) Dharmarajan, Subramanian; Belecky-Adams, Teri; Skalnik, David Gordon; Zhang, Xin; Atkinson, Simon
Reactive gliosis is known to have a beneficial and a degenerative effect following injury to neurons. Although many factors have been implicated in reactive gliosis, their role in regulating this change is still unclear. We investigated the role of bone morphogenetic proteins in reactive gliosis in vivo and in vitro. In vivo, IHC analysis indicated reactive gliosis in the 6 week Ins2Akita mouse and WPK rat retinas. Expression of BMP7 was upregulated in these models, leading to an increase in the phosphorylation of downstream SMAD1. In vitro, treatment of murine retinal astrocyte cells with a strong oxidizing agent such as sodium peroxynitrite regulated RNA levels of various markers, including GFAP, CSPGs, MMPs and TIMPs. BMP7 treatment also regulated RNA levels to a similar extent, suggesting reactive gliosis. Treatment with high glucose DMEM and BMP4, however, did not elicit increase in levels to a similar degree. Increase in SMAD levels and downstream targets of SMAD signaling such as ID1, ID3 and MSX2 was also observed following treatment with sodium peroxynitrite in vitro and in the 6 week Ins2Akita mouse retinas in vivo. These data concur with previously established data which show an increase in BMP7 levels following injury. It also demonstrates a role for BMP7 in gliosis following disease. Further, it suggests SMAD signaling to play a role in initiating reactivity in astrocytes as well as in remodeling the extracellular matrix following injury and in a disease condition.
Identification of putative targets of Nkx2-5 in Xenopus laevis using cross-species annotation and microarray gene expression analysis
(2011-10) Breese, Marcus R.; Edenberg, Howard J.; Hurley, Thomas D., 1961-; Rhodes, Simon J.; Skalnik, David Gordon
The heart is the first organ to form during development in vertebrates and Nkx2-5 is the first marker of cardiac specification. In Xenopus laevis, Nkx2-5 is essential for heart formation, but early targets of this homeodomain transcription factor have not been fully characterized. In order to discover potential early targets of Nkx2-5, synthetic Nkx2-5 mRNA was injected into eight-cell Xenopus laevis embryos and changes in gene expression measured using microarray analysis. While Xenopus laevis is a commonly used model organism for developmental studies, its genome remains poorly annotated. To compensate for this, a cross-species annotation database called CrossGene was constructed. CrossGene was created by exhaustively comparing UniGene transcripts from Homo sapiens, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans using the BLAST family of algorithms. Networks were then assembled by recursively combining reciprocal best matches into groups of orthologous genes. Gene ontology annotation from all organisms could then be applied to all members of the reciprocal group. In this way, the CrossGene database was used to augment the existing genomic annotation of Xenopus laevis. Combining cross-species annotation with differential gene expression analysis of Nkx2-5 overexpression led to the discovery of 99 potential targets of Nkx2-5.
In vivo analysis of human LHX3 enhancer regulation
(2013-03) Park, Soyoung; Rhodes, Simon J.; Day, Richard N.; Harrington, Maureen A.; Herring, B. Paul; Skalnik, David Gordon
The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.
In Vivo Analysis of Human LHX3 Gene Regulation
(2011-02) Mullen, Rachel D.; Rhodes, Simon J.; Herring, B. Paul; Skalnik, David Gordon; Thurmond, Debbie C.; Walvoord, Emily C.
LHX3 is a transcription factor important in pituitary and nervous system development. Patients with mutations in coding regions of the gene have combined pituitary hormone deficiency (CPHD) that causes growth, fertility, and metabolic problems. Promoter and intronic elements of LHX3 important for basal gene expression in vitro have been identified, but the key regulatory elements necessary for in vivo expression were unknown. With these studies, I sought to elucidate how LHX3 gene expression is regulated in vivo. Based on sequence conservation between species in non-coding regions, I identified a 7.9 kilobase (kb) region 3' of the human LHX3 gene as a potential regulatory element. In a beta galactosidase transgenic mouse model, this region directed spatial and temporal expression to the developing pituitary gland and spinal cord in a pattern consistent with endogenous LHX3 expression. Using a systematic series of deletions, I found that the conserved region contains multiple nervous system enhancers and a minimal 180 base pair (bp) enhancer that direct expression to both the pituitary and spinal cord in transgenic mice. Within this minimal enhancer, TAAT/ATTA sequences that are characteristic of homeodomain protein binding sites are required to direct expression. I performed DNA binding experiments and chromatin immunoprecipitation assays to reveal that the ISL1 and PITX1 proteins specifically recognize these elements in vitro and in vivo. Based on in vivo mutational analyses, two tandem ISL1 binding sites are required for enhancer activity in the pituitary and spine and a PITX1 binding site is required for spatial patterning of gene expression in the pituitary. Additional experiments demonstrated that these three elements cannot alone direct gene expression, suggesting a combination of factors is required for enhancer activity. This study reveals that the key regulatory elements guiding developmental regulation of the human LHX3 gene lie in this conserved downstream region. Further, this work implicates ISL1 as a new transcriptional regulator of LHX3 and describes a possible mechanism for the regulation of LHX3 by a known upstream factor, PITX1. Identification of important regulatory regions will also enable genetic screening in candidate CPHD patients and will thereby facilitate patient treatment and genetic counseling.
Protein phosphatase 2A (PP2A) holoenzymes regulate death associated protein kinase (DAPK) in ceramide-induced anoikis
(2010-05-03T19:42:36Z) Widau, Ryan Cole; Gallagher, Patricia J.; Herring, B. Paul; Rhodes, Simon J.; Skalnik, David Gordon
Modulation of sphingolipid-induced apoptosis is a potential mechanism to enhance the effectiveness of chemotherapeutic drugs. Ceramide is a pleiotropic, sphingolipid produced by cells in response to inflammatory cytokines, chemotherapeutic drugs and ionizing radiation. Ceramide is a potent activator of protein phosphatases, including protein phosphatase 2A (PP2A) leading to dephosphorylation of substrates important in regulating mitochondrial dysfunction and apoptosis. Previous studies demonstrated that death associated protein kinase (DAPK) plays a role in ceramide-induced apoptosis via an unknown mechanism. The tumor suppressor DAPK is a calcium/calmodulin regulated serine/threonine kinase with an important role in regulating cytoskeletal dynamics. Auto-phosphorylation within the calmodulin-binding domain at serine308 inhibits DAPK catalytic activity. Dephosphorylation of serine308 by a hitherto unknown phosphatase enhances kinase activity and proteasomal mediated degradation of DAPK. In these studies, using a tandem affinity purification procedure coupled to LC-MS/MS, we have identified two holoenzyme forms of PP2A as DAPK interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Serine308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK protein levels by enhancing proteasomal-mediated degradation of the kinase, as a means to attenuate prolonged kinase activation. These studies also demonstrate that ceramide causes a caspase-independent cell detachment in HeLa cells, a human cervical carcinoma cell line. Subsequent to detachment, these cells underwent caspase-dependent apoptosis due to lack of adhesion, termed anoikis. Overexpression of wild type DAPK induced cell rounding and detachment similar to cells treated with ceramide; however, this effect was not observed following expression of a phosphorylation mutant, S308E DAPK. Finally, the endogenous interaction of DAPK and PP2A was determined to be required for ceramide-induced cell detachment and anoikis. Together these studies have provided exciting and essential new data regarding the mechanisms of cell adhesion and anoikis. These results define a novel cellular pathway initiated by ceramide-mediated activation of PP2A and DAPK to regulate inside-out signaling and promote anoikis.
The role of DNA methylation in regulating LHX3 gene expression
(2013-07) Malik, Raleigh Elizabeth; Rhodes, Simon J.; Harrington, Maureen A.; Mirmira, Raghavendra G.; Skalnik, David Gordon; Day, Richard N.
LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.
Setd1 Histone 3 Lysine 4 Methyltransferase Complex Components in Epigenetic Regulation
(2011-03-16) Pick-Franke, Patricia A.; Skalnik, David Gordon; Chun, Kristin; Rhodes, Simon J.
Setd1 histone 3 lysine 4 methyltransferases are critical for epigenetic regulation and gene expression. Setd1a is multiprotein complex comprised of several critical subunits including wdr82, which is essential for embryonic development, and cfp1, critical for regulation of both activation and repression of transcriptional programs required in basic and developmental cellular processes.
Soypeptide lunasin in cytokine immunotherapy for lymphoma
(2014-08-01) Lewis, David; Chang, Hua-Chen; Skalnik, David Gordon; Watson, John C., 1953-; Atkinson, Simon
Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We previously reported that chemotherapy-treated lymphoma patients acquire a deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNy production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer cells (NKCs). Peripheral blood mononucleated cells (PBMCs) from healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNy production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines have higher tumoricidal activity than those stimulated with cytokines alone using in vitro tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation at target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.
Structure-function analysis of CXXC finger protein 1
(2009-04) Tate, Courtney Marie; Skalnik, David Gordon; Bigsby, Robert M.; Dynlacht, Joseph R.; Wek, Ronald C.
This dissertation describes structure-function studies of CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, in order to determine the functional significance of Cfp1 protein domains and properties. Cfp1 is an important regulator of chromatin structure and is essential for mammalian development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1-/-) are viable but demonstrate a variety of defects, including hypersensitivity to DNA damaging agents, reduced plating efficiency and growth, decreased global and gene-specific cytosine methylation, failure to achieve in vitro differentiation, aberrant histone methylation, and subnuclear mis-localization of Setd1A, the catalytic component of a histone H3K4 methyltransferase complex, and tri-methylated histone H3K4 (H3K4me3) with regions of heterochromatin. Expression of wild-type Cfp1 in CXXC1-/- ES cells rescues the observed defects, thereby providing a convenient method to assess structure-function relationships of Cfp1. Cfp1 cDNA expression constructs were stably transfected into CXXC1-/- ES cells to evaluate the ability of various Cfp1 fragments and mutations to rescue the CXXC1-/- ES cell phenotype. These experiments revealed that expression of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) is sufficient to rescue the hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and differentiation defects. These results reveal that Cfp1 contains redundant functional domains for appropriate regulation of cytosine methylation, histone methylation, and in vitro differentiation. Additional studies revealed that a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the 1-367 fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1A and Setd1B histone H3K4 methyltransferase complexes ablates the rescue activity of the 361-656 Cfp1 fragment. In addition, introduction of both point mutations (C169A and C375A) ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either DNA-binding or Setd1 association of Cfp1 is required to rescue hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and in vitro differentiation. In contrast, confocal immunofluorescence analysis revealed that full-length Cfp1 is required to restrict Setd1A and histone H3K4me3 to euchromatic regions.

Browsing by Author "Skalnik, David Gordon"

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