Dr. Kaestner’s lab is employing modern genetic, genomic and epigenomic approaches (ChIP-Seq, RNA-Seq, gene targeting, tissue-specific and inducible gene ablation) to understand the molecular mechanisms of organogenesis and physiology of the liver, pancreas and gastrointestinal tract. Disease areas targeted by our research include diabetes and cancer.
Description of Research
Epigenomic rejuvenation of human pancreatic beta-cells.
The prevalence of Diabetes Mellitus has reached epidemic proportions world-wide, and is predicted to increase rapidly in the years to come, putting a tremendous strain on health care budgets in both developed and developing countries. There are two major forms of diabetes and both are associated with decreased beta-cell mass. No treatments have been devised that increase beta-cell mass in vivo in humans, and transplantation of beta-cells is extremely limited due to lack of appropriate donors. For these reasons, increasing functional beta-cell mass in vitro, or in vivo prior to or after transplantation, has become a “Holy Grail” of diabetes research. Our previous studies clearly show that adult human beta-cells can be induced to replicate, and – importantly - that cells can maintain normal glucose responsiveness after cell division. However, the replication rate achieved was still low, likely due in part to the known age-related decline in the ability of the beta-cell to replicate. We propose to build on our previous findings and to develop more efficacious methods to increase functional beta-cell mass by inducing replication of adult beta-cells, and by restoring juvenile functional properties to aged beta-cells. We will focus on mechanisms derived from studies of non-neoplastic human disease as well as age-related phenotypic changes in human beta-cells. In Aim 1, we will target the genes altered in patients with marked beta-cell hyperplasia, such as those suffering from Beckwith-Wiedemann Syndrome or Multiple Endocrine Neoplasia. Expression of these genes will be altered in human beta-cells via shRNA-mediated gene suppression and locus-specific epigenetic targeting. Success will be assessed in transplanted human islets by determination of beta-cell replication rate and retention of function. In Aim 2, we will determine the mechanisms of age-related decline in beta-cell function and replicative capacity, by mapping the changes in the beta-cell epigenome that occur with age. Selected genes will then be targeted as in Aim 1 to improve human beta-cell function, as assessed by glucose responsiveness. To accomplish these aims, we will use cutting-edge and emerging technologies that are already established or are being developed in our laboratories. The research team combines clinical experience with expertise in molecular biology and extensive experience in genomic modification aimed at enhancing beta-cell replication. By basing interventions on changes found in human disease and normal aging, this approach will increase the chances that discoveries made can be translated more rapidly into clinically relevant protocols.
Regulatory cascades in differentiation and proliferation of the gastrointestinal epithelium.
The mammalian gut epithelium is a highly organized and dynamic system which requires continuous controlled proliferation and differentiation throughout life. Proliferation, cell migration and cell adhesion all must be tightly controlled in order to prevent either inflammatory diseases or epithelial cancers. As with many other vertebrate organs, the digestive tract develops from heterogeneous embryonic origins. While the musculature and the connective tissue are derived from lateral plate mesoderm, the epithelium is derived from the endoderm. We have identified a novel member of the winged helix gene family termed Foxl1 which is expressed in the gut mesoderm and have begun its functional analysis in vivo through targeted mutagenesis in mice. Null mutations in the mesodermal transcription factor Foxl1 result in dramatic alterations in endoderm development, including epithelial hyperproliferation. We have now identified APC/Min and GKLF as downstream targets of Foxl1 and have begun the analysis of these genes in gastrointestinal differentiation by tissue-specific gene ablation.
Innovative Genetic Approaches for Hepatic Repopulation
A better understanding of the liver’s response to toxic injury, which includes hepatocyte proliferation, activation and differentiation of facultative hepatic stem cells (“oval cells”), and – unfortunately – an increased risk for hepatocellular carcinoma, is a prerequisite for the development of novel clinical treatments for chronic liver disease and improved cancer prevention. Likewise, cell replacement therapy, either through direct hepatocyte transplantation or in bio-artificial liver devices, needs to be improved in order to become a reliable alternative to liver transplantation. To date, investigations of hepatocyte proliferation have frequently focused on the partial hepatectomy paradigm, a “non¬injury” model that is not reflective of liver injury in humans and which has therefore failed to identify specific targets for either improved regeneration following toxic injury or for limiting proliferation in HCC in humans. In Specific Aim 1, we will determine which genes and gene combinations promote or repress hepatocyte repopulation following toxic liver injury using an innovative genetic approach. In Specific Aim 2, we will employ expression of key hepatic transcription factors to improve the differentiation of hepatic progenitor cells to functional hepatocytes. Together, these approaches will provide an improved understanding of the liver’s response to toxic injury, and facilitate the discovery of new cell replacement therapies to treat chronic liver disease and liver failure.
Rotation Projects for 2014
(subject to change at a moment’s notice):
1. ChIP-Seq analysis. Chromatin immunoprecipitation using various transcription factor or modified histone antibodies. Library construction, ultra-high throughput sequencing and computational analysis of target sequences.
2. Molecular, histological and metabolic analysis of mouse models of diabetes, hypoglycemia, and GI cancer.
3. Analysis of complex genomic and epigenomic data sets
Reina Aoki, Graduate Student
Vasumati Kameswaran, Graduate Student
Ellen Elliott, Graduate Student
Rinho Kim, Graduate Student
Julia Kieckhaefer, Graduate Student
Diana Bernstein, Graduate Student
Aryel Heller, Graduate Student
Dr. Monica Teta, Postdoc
Dr. Natalie Terry, Posdoc
Dr. Michal Shoshkes, Postdoc
Dr. Karyn Sheaffer, Postdoc
Dr. Jia Zhang, Postdoc
Dr. Soona Shin, Postdoc
Dr. Kirk Wangenstein, Postdoc
Dr. Amanda Misfeldt, Postdoc
Dr. Julia Wang, Postdoc
Dr. Catherine Lee, Research Associate
Dr. Adam Zahm, Research Associate
Dr. Jonathan Schug, Technical Director, Functional Genomics Core
Dr. Shilpa Rao, Programmer/Analyst
Tia Bernard, Research Specialist
Olga Smirnova, Research Specialist
Haleigh Zillges, Research Specialist
Christina Theodorou, Research Specialist
Goutham Nadendla, Programmer/Analyst
Wangensteen KJ, Zhang S, Greenbaum LE, Kaestner KH.: A genetic screen reveals Foxa3 and TNFR1 as key regulators of liver repopulation. Genes Dev 29(9): 904-9, May 2015.
Elliott EN, Sheaffer KL, Schug J, Stappenbeck TS, Kaestner KH.: Dnmt1 is essential to maintain progenitors in the perinatal intestinal epithelium. Development Epub ahead of print, May 2015.
Bernstein, D.A., Le Lay, J.E., Ruano, E.G. and Kaestner, K.H.: TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts. J. Clin. Invest. 125(5): 1998-2006, May 2015.
Sheaffer, K.L., Kim, R., Aoki, R., Elliott, E.N., Schug, J., Burger, L., Schubeler, D., and Kaestner, K.H.: DNA methylation is required for the control of stem cell differentiation in the small intestine. Genes Dev 28(6): 652-664 March 2014.
Li, Z, Gadue, P, Chen, K, Jiao, Y, Tuteja, G, Schug, J, Li, W, and Kaestner, KH: Foxa2 and H2A.Z Mediate Nucleosome Depletion during Embryonic Stem Cell Differentiation. Cell 151(7): 1608-16, December 2012.
Li, Z., Tuteja, G., Schug, J. and Kaestner, K.H.: Foxa1 and Foxa2 are Essential for Sexual Dimorphism in Liver Cancer Cell 148: 72-83, January 2012.
Bramswig, N., Evertt, L., Schug, J., Dorrell, C., Liu, C., Luo, Y., Streeter, P., Naji, A., Grompe, M., And Kaestner, K.H.: Epigenomic plasticity enables human pancreatic α to β cell reprogramming. J. Clin. Invest. 123(3): 1275-84, March 2013.
Li Z, Schug J, Tuteja G, White P, Kaestner KH.: The nucleosome map of the mammalian liver. Nat Struct Mol Biol 18(6): 742-6, June 2011.
Shin S, Walton G, Aoki R, Brondell K, Schug J, Fox A, Smirnova O, Dorrell C, Erker L, Chu AS, Wells RG, Grompe M, Greenbaum LE, Kaestner KH: Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential. Genes Dev 25(11): 1185-1192, June 2011.
Gao, N, Kaestner, KH: Cdx2 regulates endo-lysosomal function and epithelial cell polarity. Genes & Development 24(12): 1295-1305, JUN 15 2010.
McKenna, LB, Schug, J, Vourekas, A, McKenna, JB, Bramswig, NC, Friedman, JR, Kaestner, KH: MicroRNAs Control Intestinal Epithelial Differentiation, Architecture, and Barrier Function. Gastroenterology 139(5): 1654, NOV 2010.
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Last updated: 06/11/2015
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