David Jones - US grants

DESCRIPTION (provided by applicant): Mutations in APC, or in its regulatory target, beta-catenin, are thought to cause colon neoplasms by promoting proliferation and preventing proper differentiation of colonocytes. However, our understanding of the mechanisms that control colonocyte differentiation is limited. Recent studies have shown that human colon adenomas and carcinomas show a profound deficiency of retinoic acid biosynthetic enzymes. Furthermore, re-introduction of wild type APC into an APC-deficient colon cancer cell line induced retinol dehydrogenase L and increased retinoic acid production. These observations suggest a novel model wherein APC promotes enterocyte differentiation by controlling retinoic acid biosynthesis. The studies outlined in this proposal will examine the genetic relationship between APC and retinoic acid biosynthesis in normal enterocytes using zebrafish as a model system. Preliminary data show that morpholino knockdown of either zAPC or zRDHB in zebrafish embryos results in defects in structures known to require retinoic acid, consistent with a role for these genes in RA-dependent pathways. In addition, APC or zRDHB morphant fish develop intestines that lack columnar epithelial cells and fail to express the differentiation marker intestinal fatty acid binding protein (i-FABP). Treatment of either APC or zRDHB morphant embryos with retinoic acid rescued the defective phenotypes, for the first time placing zAPC upstream of RA and implicating RA in intestinal differentiation. These preliminary data, therefore, strongly support a critical role for retinoic acid in zebrafish enterocyte development and provide genetic evidence placing retinoic acid and hox genes downstream of APC. The long term goal of this project is to facilitate the development of new preventive measures for colon adenoma formation by understanding the earliest cellular perturbations that follow APC mutation.

The project, a partnership between the University of Nebraska-Lincoln and Nebraska's six community colleges, is increasing the number of students that are successfully pursuing and obtaining baccalaureate degrees in engineering. The overall goal is to develop and institutionalize an effective pathway enabling community college students to complete several freshman and sophomore engineering courses and transfer seamlessly into the University's College of Engineering. Academic, financial, and social support is being provided to transfer students to ensure retention and encourage completion of a baccalaureate engineering degree in the traditional timeframe. The project's objectives are: (1) increasing the number of students transferring into engineering so that the percentage of transfer students choosing engineering is equal to the College's percentage of total students enrolled; (2) increasing by 100% the number of underrepresented minority students transferring into the College each year; (3) increasing by 100% the number of women transferring into the College each year; (4) retaining at least 80% of all transfer students; and (5) graduating at least 65% of all transfer students after three years of enrollment in the College. These increases in enrollment and graduation rates are being achieved through an expanded physical and administrative infrastructure that supports students transferring from community college programs to engineering. Specifically, the project is working to: (1) develop and implement four new introductory community college engineering courses along with corresponding articulation agreements; 2) establish a complementary set of student support activities that facilitate mentoring, community building, and retention, particularly among women and minority transfer students; and (3) facilitate paid internship opportunities to complement the high quality instruction students are receiving. Internal and external advisory boards are guiding the project and an evaluation team is in place. Broader impacts include an increase in the participation of students from underrepresented groups, the redefinition of the engineering transition process from community colleges to the University, and a broad dissemination of the project's results.

DESCRIPTION (provided by applicant): We will create a resource for the investigation of chromatin structure, epigenetic modifications, and transcriptional regulators in early zebrafish development. The resource will be named CZECH, the Center for Zebrafish Epigenetics and CHromatin. Chromatin regulates gene expression, and impacts development by influencing the transcriptional programs that drive cell growth and fate decisions. DNA methylation and other chromatin marks (defined as the epigenome) strongly influence the timing and strength of expression of particular genes during development, but a systematic study has not been performed in any organism. Furthermore, misregulation of transcription via chromatin underlies many developmental defects and certain cancers. Thus, we must understand how chromatin dynamics help guide development. The Center will address this issue using a combination of genomic and genetic approaches, will provide detailed protocols for genomic approaches, and will deploy and manage a database for the community to view and submit datasets. Here, we will define the basic epigenome of germ cells and the early embryo of the zebrafish, both before and during organ specification. We will combine chromatin immunoprecipitation (ChIP) with high-density genome tiling arrays (to examine chromatin), and also perform gene expression profiling. We will initially limit our examination to the chromatin modifications most closely associated with transcriptional repression (DNA methylation, H3K9me3, and H3K27me3), or transcriptional activation (H3K4me3), which include the `bivalent'modifications important for poising genes in stem cells. Notably, our preliminary studies suggest the poising of early developmental regulators in sperm chromatin by DNA hypomethylation. In later stage embryos, we will use FACS to isolate developing tissues and then determine their epigenome. PUBLIC HEALTH RELEVANCE: This project addresses how chromosomes and their resident genes are packaged in germ cells and then change their packaging following fertilization to regulate gene expression during early development. This process is central to understanding how cells differentiate, how organs develop, and how cell growth is regulated. The misregulation of chromosome and gene packaging leads to problems such as birth defects, retardation, or cancer, depending on the particular chromosome(s) or gene(s) affected.

Analytic Core B provides support for a

We have shown that human colon adenomas and carcinomas show a profound deficiency of retinoic acid biosynthetic enzymes and that APC controls intestinal cell differentiation by controlling the expression of retinol dehydrogenases. These findings suggest a novel model wherein APC promotes enterocyte differentiation by controlling retinoic acid biosynthesis and indicates that the functions of APC are not limited to its well-established role in regulating canonical WNT signaling. Mechanistically, we have identified the transcriptional co-repressor, C-terminal binding protein (CtBP), as a novel, APC-regulated protein that suppresses retinol dehydrogenases and intestinal cell differentiation. Consistent with APC control of CtBP, human adenomas taken from FAP patients showed robust staining for nuclear CtBP in comparison to adjacent, uninvolved tissues. Surprisingly, however, these same sections showed little evidence of nuclear B-catenin suggesting that accumulation of CtBP may precede nuclear accumulation of B-catenin and that nuclear accumulation of B-catenin may require events in addition to loss of APC. Consistent with this possibility are a number of reports showing that activation of (B-catenin signaling in small versus large adenomas appears to parallel activating mutations in the k-ras oncogene. Further, work in mice carrying a mutation typical of those found in human FAP has shown that activation of k-ras increased polyp size and number. These data suggest that k-ras activation permits B-catenin-stimulated intestinal cell proliferation during the formation of a large adenoma. Indeed our preliminary data show that APC loss alone is insufficient to promote intestinal cell proliferation in zebrafish. Furthermore, oncogenic k-ras promotes the accumulation of nuclear B-catenin and subsequent proliferation in both human cells and zebrafish. Our access to both FAP and undefined high-risk families, as well as our expertise in using zebrafish as a genetic model system to study APC, provides us a unique opportunity to evaluate the contribution of CtBP and k-ras in the initiation and progression of colon adenomas.

PROJECT SUMMARY/ABSTRACT The human ether-a-go-go gene (hERG) and KCNQ1 genes encode proteins that conduct the cardiac repolarizing currents IKr and IKs, respectively. Impaired repolarization increases the risk of potentially lethal ventricular arrhythmias and is a hallmark of long QT syndrome (LQTS) and heart failure. Treatments for impaired repolarization are limited largely due to their high incidence of off-target effects. hERG cytoplasmic domains interact to negatively modulate IKr, making them potential targets to treat diseases of repolarization. The preliminary data for this grant demonstrate that small antibody peptide fragments targeting distinct regions of the cytoplasmic hERG Per-Arnt-Sim (PAS) domain selectively increase IKr and shorten action potential duration (APD). As a therapeutic, such fragments would potentially shorten APD and restore impaired repolarization in LQTS and heart failure. Additionally, antibody fragments directed toward the PAS and other domains would be useful tools to characterize how specific gating processes regulate cellular behavior and overall cardiac physiology. In the mentored phase of this project, I will characterize the mechanisms by which these antibody fragments modify hERG gating using electrophysiological and spectroscopy techniques. I will also characterize antibody fragment hERG modulation in healthy human stem cell-derived cardiomyocytes (iPSC-CMs) and iPSC-CMs obtained from LQTS patients using scFvs delivered intracellularly via the recording pipette. In the independent phase of this project, I will combine the scFv antibodies with two intracellular delivery techniques: (1) the cell-penetrating Cardiac Targeting Peptide, CTP, and (2) the adeno-associated virus serotype 9 (AAV9). I will introduce antibody-CTP fusion proteins into iPSC-CMs and monitor the resulting biophysical and physiological effects with patch clamp and micro-electrode techniques. To develop an approach leading to a longer-term treatment, I will use the viral AAV9 to produce stable transfer of the antibody-encoding sequence into mammalian cardiomyocytes. These experiments will generate novel tools to probe the mechanistic role of distinct channel processes in native physiology. Additionally, these experiments will act as a proof-of-concept for future experiments designed to develop anti-hERG antibody fragments into treatments for impaired repolarization. This proposal is designed to fulfill my short-term goals of expanding my skills in cardiac electrophysiology and transitioning into the independent phase of my career. This will ultimately allow me to obtain my long-term goal of studying translational cardiac electrophysiology research by using patient-derived iPSC-CMs to explore triggers and treatments for cardiac arrhythmia.