The epigenetic basis for human immunodeficiencies that result from embryonic developmental defects, e.g. thymus hypoplasias, is poorly understood. This is due to sparse disease-relevant patient material available to study and limited rodent models that often differ in genomic organization and phenotype when compared to human. For example, DiGeorge syndrome (DGS), its associated thymus dysfunction, and ensuing immunodeficiency, have been linked to a dose-dependent haploinsufficiency of TBX1 during pharyngeal foregut development. The dose-sensitive regulation and the functional network it operates in remain largely elusive. Advances in the generation of disease-specific human pluripotent stem cells (PSCs) through reprogramming and gene targeting have led to opportunities for disease investigation and ultimately for the development of autologous cell replacement therapies. The primary goal of this project to understand the regulation of TBX1 expression and its dose-dependent function in human stem cell differentiation products relevant to the developmental defect and impending immunodeficiency seen in subjects with DiGeorge syndrome.
In aim 1 and 2 we will focus on characterizing factors that we identified to be involved in the regulation of TBX1 expression.
Aim 1 will focus on characterizing signaling pathways that control TBX1 expression in thymus developmental defect-relevant differentiation products. We have conducted an innovative combinatorial analysis of signaling pathways that influence TBX1 expression in DGS-relevant cell populations. We will characterize lead pathways for the ability to control cell differentiation and function.
In aim 2 we will characteriz epigenetic events that regulate transcriptional control of the tbx1 locus. We have previously identified specific chromatin modifications that are associated with activation of tbx1 gene expression. We will characterize the events and factors that are involved in activation and regulation of tbx1 gene expression. In combination, aim 1 and aim 2 will provide us with a comprehensive insights on how TBX1 expression is regulated. We expect that these mechanistic insights can be applied in vitro to target tbx1-dosage effects that are associated with the immunodeficiency phenotype as seen in subjects with DGS.
In aim 3 we will utilize reprogramming, gene targeting and regulated gene expression technology to address the molecular consequence of varying TBX1 expression levels. We expect that this study will not only identify key targets of TBX1 in developmental defect relevant cells, but will also provide unique insights on the functional consequence and the mechanism of tbx1 dosage-dependent activity. The detailed insights on the functional network TBX1 operates in DGS-associated immunodeficiency- relevant cell populations, and how this network is affected by dosage variations in cellular TBX1 levels, will elucidate the underlying mechanism of the developmental defect that results in immunodeficiency. Further, the insights can likely be applied to other dosage-dependent defects in organogenesis and will provide novel targets to overcome such defects in vitro for stem cell-based cell therapies. This work combines the expertise of Dr. Maehr in PSC differentiation, endoderm development and genome manipulation, of Dr. Rando in epigenetic profiling technology, and of Dr. Garber in integrating epigenetic and transcriptomic data to understand cell fate decisions.

Public Health Relevance

Due to lack of good model systems mechanistic insights on developmental defects leading to thymus dysfunction and associated immunodeficiencies are limited. Human pluripotent stem cells provide us with innovative opportunities for disease investigation and ultimately for the development of autologous cell replacement therapies. Herein, we propose applying a novel human pluripotent stem cell differentiation platform to elucidate the role of a disease-associated transcription factor, and the regulatory network it operates in, in normal and defective human development.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
High Priority, Short Term Project Award (R56)
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Special Emphasis Panel (ZRG1)
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Voulgaropoulou, Frosso
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University of Massachusetts Medical School Worcester
Other Basic Sciences
Schools of Medicine
United States
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Parsi, Krishna Mohan; Hennessy, Erica; Kearns, Nicola et al. (2017) Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells. Methods Mol Biol 1507:221-233
Genga, Ryan M; Kearns, Nicola A; Maehr, René (2016) Controlling transcription in human pluripotent stem cells using CRISPR-effectors. Methods 101:36-42