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Browsing by Author "Skalnik, David"
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Item Identification of a Minimal Cis-element and Cognate Trans-factors Required for the Regulation of Rac2 Gene Expression during K562 Cell Differentiation(2009-03-18T18:48:49Z) Muthukrishnan, Rajarajeswari; Skalnik, David; Herring, B. Paul; Rhodes, Simon J.; Wek, Ronald C.This dissertation examines the molecular mechanisms regulating Rac2 gene expression during cell differentiation and identification of a minimal cis-element required for the induction of Rac2 gene expression during K562 cell differentiation. The Rho family GTPase Rac2 is expressed in hematopoietic cell lineages and is further up-regulated upon terminal myeloid cell differentiation. Rac2 plays an important role in many hematopoietic cellular functions, such as neutrophil chemotaxis, superoxide production, cytoskeletal reorganization, and stem cell adhesion. Despite the crucial role of Rac2 in blood cell function, little is known about the mechanisms of Rac2 gene regulation during blood cell differentiation. Previous studies from the Skalnik lab determined that a human Rac2 gene fragment containing the 1.6 kb upstream and 8 kb downstream sequence directs lineage-specific expression of Rac2 in transgenic mice. In addition, epigenetic modifications such as DNA methylation also play important roles in the lineage-specific expression of Rac2. The current study investigated the molecular mechanisms regulating human Rac2 gene expression during cell differentiation using chemically induced megakaryocytic differentiation of the human chronic myelogenous leukemia cell line K562 as the model system. Phorbol 12-myristate 13-acetate (PMA) stimulation of K562 cells resulted in increased Rac2 mRNA expression as analyzed by real time-polymerase chain reaction (RT-PCR). Luciferase reporter gene assays revealed that increased transcriptional activity of the Rac2 gene is mediated by the Rac2 promoter region. Nested 5’- deletions of the promoter region identified a critical regulatory region between -4223 bp and -4008 bp upstream of the transcription start site. Super shift and chromatin immunoprecipitation assays indicated binding by the transcription factor AP1 to three distinct binding sites within the 135 bp minimal regulatory region. PMA stimulation of K562 cells led to extensive changes in chromatin structure, including increased histone H3 acetylation, within the 135 bp Rac2 cis-element. These findings provide evidence for the interplay between epigenetic modifications, transcription factors and cis-acting regulatory elements within the Rac2 gene promoter region to regulate Rac2 expression during K562 cell differentiation.Item Loss of SIMPL increases TNFα sensitivity during hematopoiesis(2008-10) Benson, Eric Ashley; Harrington, Maureen A.; Goebl, Mark; Clapp, Wade; Skalnik, DavidThe innate and adaptive immune responses are critical for host survival. The TNFα/NF-κB signaling pathway is a major regulator of the immune response. The TNFα/NF-κB signaling pathway has also been proposed to play a role in the regulation of hematopoiesis. In the TNFα signaling pathway, full induction of NF-κB (specifically the p65 subunit) dependent transcription is regulated by a co-activator SIMPL. The biological significance of SIMPL in TNFα dependent responses is poorly understood. To study SIMPL in vitro and in vivo in mammalian cells, a knockdown system utilizing shRNA (short hairpin RNA) was used. Analysis of hematopoietic progenitor cells infected with a retrovirus encoding the SIMPL shRNA was used to study the role of SIMPL in hematopoiesis. The ability of progenitor cells lacking SIMPL to grow and differentiate was not compromised. In contrast in the progenitors cells lacking SIMPL, TNFα mediated inhibition of colony formation was significantly enhanced. These growth inhibitory effects of SIMPL were not due to an increase in apoptosis. The enhanced inhibitory affects were specific for TNFα and not found in other common hematopoietic inhibitors (TGF-β1 and IFNγ). Results of this work reveal that SIMPL is a component of the hematopoiesis that is required for TNFα dependent effects upon myeloid progenitors.Item The mechanism of triglyceride partitioning – how the ANGPTL3-4-8 system of proteins orchestrates tissue energy distribution(2020-12) Pottanat, Thomas G.; Berbari, Nicolas; Konrad, Robert; Perrin, Benjamin; Skalnik, DavidThe incidence of Metabolic Syndrome (MetS) is increasing worldwide and accompanied by elevated risks for cardiovascular disease (CVD) and other subsequent comorbidities. MetS is associated with increased circulating triglycerides. A key enzyme involved in triglyceride (TG) clearance is lipoprotein lipase (LPL) whose activity is modulated by a variety of factors. Recent literature has identified the importance of angiopoietin-like proteins (ANGPTL) as regulators of LPL activity and has hypothesized a model in which three of these proteins interact with LPL to regulate the partitioning of TG metabolism from adipose to skeletal muscle. The work detailed in this dissertation adds to the model of ANGPTL regulation of LPL by establishing how ANGPTL8 modulates the ability of ANGPTL3 and ANGPTL4 to inhibit LPL activity in the bloodstream and localized environments, respectively. In the updated model, elevated insulin concentrations result in increased hepatic ANGPTL3/8 secretion and increased ANGPTL4/8 in adipose tissue. ANGPTL3/8 works as an endocrine molecule to inhibit skeletal muscle LPL from hydrolyzing circulating TG. Simultaneously, ANGPTL4/8 works in a paracrine mechanism to bind LPL on the endothelial vasculature adjacent to adipose tissue to alleviate ANGPTL4-mediated LPL inhibition and also prevent ANGPTL3/8 inhibition of localized LPL. Thus, in the postprandial state free fatty acids (FFA) from the hydrolysis of TG are directed into adipocytes for storage. Under fasting conditions, ANGPTL8 production is decreased in adipocytes and hepatocytes. This decreased production results in diminished ANGPTL4/8 and ANGPTL3/8 secretion from their respective tissues. As a result, ANGPTL4 inhibits adipocyte localized LPL activity while ANGPTL3 at physiological concentrations has minimal effect on LPL activity. Furthermore, any ANGPTL3/8 which is produced has its LPL-inhibitory ability diminished by the circulating apolipoprotein ApoA5. LPL is more active in skeletal muscle compared to adipose tissue where energy is shunted towards utilization in the muscle and away from storage in adipose tissue. A complete understanding of LPL regulation by ANGPTL proteins can potentially provide therapeutics targets for MetS.Item Optimization of Marker Sets and Tools for Phenotype, Ancestry, and Identity using Genetics and Proteomics(2019-08) Wills, Bailey; Walsh, Susan; Picard, Christine; Skalnik, DavidIn the forensic science community, there is a vast need for tools to help assist investigations when standard DNA profiling methods are uninformative. Methods such as Forensic DNA Phenotyping (FDP) and proteomics aims to help this problem and provide aid in investigations when other methods have been exhausted. FDP is useful by providing physical appearance information, while proteomics allows for the examination of difficult samples, such as hair, to infer human identity and ancestry. To create a “biological eye witness” or develop informative probability of identity match statistics through proteomically inferred genetic profiles, it is necessary to constantly strive to improve these methods. Currently, two developmentally validated FDP prediction assays, ‘HIrisPlex’ and ‘HIrisplex-S’, are used on the capillary electrophoresis to develop a phenotypic prediction for eye, hair, and skin color based on 41 variants. Although highly useful, these assays are limited in their ability when used on the CE due to a 25 variant per assay cap. To overcome these limitations and expand the capacities of FDP, we successfully designed and validated a massive parallel sequencing (MPS) assay for use on both the ThermoFisher Scientific Ion Torrent and Illumina MiSeq systems that incorporates all HIrisPlex-S variants into one sensitive assay. With the migration of this assay to an MPS platform, we were able to create a semi-automated pipeline to extract SNP-specific sequencing data that can then be easily uploaded to the freely accessible online phenotypic prediction tool (found at https://hirisplex.erasmusmc.nl) and a mixture deconvolution tool with built-in read count thresholds. Based on sequencing reads counts, this tool can be used to assist in the separation of difficult two-person mixture samples and outline the confidence in each genotype call. In addition to FDP, proteomic methods, specifically in hair protein analysis, opens doors and possibilities for forensic investigations when standard DNA profiling methods come up short. Here, we analyzed 233 genetically variant peptides (GVPs) within hair-associated proteins and genes for 66 individuals. We assessed the proteomic methods ability to accurately infer and detect genotypes at each of the 233 SNPs and generated statistics for the probability of identity (PID). Of these markers, 32 passed all quality control and population genetics criteria and displayed an average PID of 3.58 x 10-4. A population genetics assessment was also conducted to identify any SNP that could be used to infer ancestry and/or identity. Providing this information is valuable for the future use of this set of markers for human identification in forensic science settings.Item Regulation of SRF Activity by the ATP-dependent Chromatin Remodeling Enzyme, CHD8(2009-03-18T18:39:57Z) Rodenberg, Jennifer Marie; Herring, B. Paul; Gallagher, Patricia J.; Pavalko, Fredrick M.; Skalnik, DavidUnder normal conditions, smooth muscle cells do not replicate, or proliferate, and provide a means of contraction for many internal organs, including blood vessels and the gut. However, under abnormal or disease conditions, such as congenital heart disease and cancer, smooth muscle cells acquire the ability to replicate, to make extracellular matrix proteins and to migrate. Thus, determining how smooth muscle cells regulate these processes is crucial to understanding how the cells can switch between normal and diseased states. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation and migration. It is generally accepted that one way that SRF can distinguish between these functions is through pathway-specific co-factor interactions. A novel SRF co-factor, chromodomain helicase DNA binding protein 8 (CHD8), was originally isolated from a yeast two-hybrid assay. CHD8 is widely expressed in adult tissues including smooth muscle. Data from in vitro binding assays indicate that the N-terminus of CHD8 can interact directly with the MADS domain of SRF. Co-immunoprecipitation assays verified the ability of these two proteins to interact within cells. Adenoviral-mediated shRNA knockdown of CHD8 in smooth muscle cells resulted in statistically significant 10-20% attenuation of expression of SRF-dependent, smooth muscle-specific genes. Similar experiments revealed that knockdown of CHD8 did not affect the SRF-dependent induction of immediate early genes required to promote proliferation. In contrast, knockdown of CHD8 in A10 vascular smooth muscle cells resulted in a marked induction in of apoptosis, characterized by increases in apoptotic markers such as phospho-H2A.X, cleaved PARP and activated caspase-3. These data suggest that CHD8 may play a specific role in modulating SRF’s activity toward anti-apoptotic genes, thereby regulating smooth muscle cell survival.Item Uhrf1 regulates active transcriptional marks at bivalent domains in pluripotent stem cells through Setd1a(Nature Publishing Group, 2018-07-03) Kim, Kun-Yong; Tanaka, Yoshiaki; Su, Juan; Cakir, Bilal; Xiang, Yangfei; Patterson, Benjamin; Ding, Junjun; Jung, Yong-Wook; Kim, Ji-Hyun; Hysolli, Eriona; Lee, Haelim; Dajani, Rana; Kim, Jonghwan; Zhong, Mei; Lee, Jeong-Heon; Skalnik, David; Lim, Jeong Mook; Sullivan, Gareth J.; Wang, Jianlong; Park, In-Hyun; Biology, School of ScienceEmbryonic stem cells (ESCs) maintain pluripotency through unique epigenetic states. When ESCs commit to a specific lineage, epigenetic changes in histones and DNA accompany the transition to specialized cell types. Investigating how epigenetic regulation controls lineage specification is critical in order to generate the required cell types for clinical applications. Uhrf1 is a widely known hemi-methylated DNA-binding protein, playing a role in DNA methylation through the recruitment of Dnmt1 and in heterochromatin formation alongside G9a, Trim28, and HDACs. Although Uhrf1 is not essential in ESC self-renewal, it remains elusive how Uhrf1 regulates cell specification. Here we report that Uhrf1 forms a complex with the active trithorax group, the Setd1a/COMPASS complex, to maintain bivalent histone marks, particularly those associated with neuroectoderm and mesoderm specification. Overall, our data demonstrate that Uhrf1 safeguards proper differentiation via bivalent histone modifications.