The Chromatin-Based Epigenome in Development and Aging

Bruce H. Howard, MD, Head, Section on Human Genetics
Valya Russanova, PhD, Staff Scientist
Hariklia Dimitropoulos, PhD, Postdoctoral Fellow
Sarah Kozar, BS, Postbaccalaureate Fellow

The normal human lifespan is marked by a complex series of developmental transitions, including relative stability during adulthood and, ultimately, a gradual decline in viability. Clock-like mechanisms presumably underlie the developmental events that occur through childhood and adolescence. Further, instabilities in such mechanisms are likely to be an integral part of the aging process and to contribute to many common degenerative diseases of later life. A breakthrough in the area of developmental transitions would have remarkable implications; thus, there is widespread interest in the prospect that epigenetics—and the rapidly evolving field of epigenomics—holds the key to further progress.

Linking gene expression to cis-acting epigenetic states

Russanova, Howard, Porrás,¹ Dimitropoulos, Kozar; in collaboratin with Romero

The past year has seen increasing refinement in studies that use human tissues from several clinical sources. We obtain peripheral blood monocytes from newborns (cord blood) through a collaboration with the Perinatology Branch, NICHD, while monocytes from adults are available through the NIH Department of Transfusion Medicine. Using the cytokines IL4 and GM-CSF, we induce these cells to differentiate in vitro into antigen-presenting dendritic cells. We procure human skin fibroblasts from newborns and adults, as needed, under a protocol approved by the NICHD Institutional Review Board. We induce the latter cells to enter a quiescent state by serum deprivation for six days and then examine them with respect to gene expression and chromatin structure upon serum stimulation.

With these monocyte- and fibroblast-based experimental systems, we employed RNA expression microarrays to identify several instances of developmental and age-related changes in gene regulation. Our work is thus shifting to studies on the mechanism(s) underlying such changes. Recognizing that cis-acting epigenetic states have several distinctive properties, we use several strategies to evaluate their presence or absence: (1) heterocellularity (variegation) in expression patterns—we apply RNA FISH and cytohistochemistry techniques; (2) allele independence—we use single-nucleotide polymorphisms (SNP) to search for examples of allelic skewing; and (3) memory of expression-state settings—we use heterokaryons between cells from newborns and adults or from young and old adults to determine the independence of development- and age-specific expression levels.

Complementary to these strategies are chromatin immunoprecipitation (ChIP) studies; we use both automated HPLC/fluorescence-based assays and ChIP-on-chip platforms. For the latter, we have developed bioinformatics tools for the custom design of large deoxynucleotide probe arrays and genome annotation, pattern recognition, and pattern comparison algorithms in order to analyze data and generate new hypotheses. Further, in the coming year, we plan to use single-nucleotide sequencing (ChIP-Seq), if possible. Of special interest will be results obtained from monozygotic (identical) twins. As part of an NICHD epigenomics initiative, we have received the first paired peripheral blood samples from such twins.

Results to date indicate that genes subject to both differentiation and developmental controls operate at least in part through the remodeling of higher-order chromatin structures. We will focus on large domains over which acetylation patterns are altered as well as on the boundary elements that limit these domains. The emerging goal is to generalize our paradigm to address a range of current problems in pediatrics and medicine. Based on the genes currently under study, we will most likely investigate deficiencies in the innate immune systems of newborns, peripheral insulin resistance and diabetes in adolescents and young adults, and a spectrum of neurodegenerative processes, including Parkinson’s and Alzheimer’s diseases, in the elderly.

Humphrey GW, Wang YH, Hirai T, Padmanabhan R, Panchision DM, Newell LF, McKay RDG, Howard BH. Complementary roles for histone deacetylases 1, 2 and 3 in differentiation of pluripotent stem cells. Differentiation 2007;76:348-356.
Porrás A, Kozar S, Russanova V, Salpea P, Hirai T, Sammons N, Mittal P, Kim JY, Ozato K, Romero R, Howard BH. Developmental and epigenetic regulation of the human TLR3 gene. Mol Immunol 2008;46:27-36.

¹ Analia Porrás, MD, PhD, former Research Fellow

Collaborators

Jonathan Epstein, MS, Scientific Software Support and Bioinformatics Core Facility, NICHD, Bethesda, MD
Ronald D.G. McKay, PhD, Laboratory of Molecular Biology, NINDS, Bethesda, MD
Roberto Romero, MD, Program in Perinatal Research and Obstetrics, NICHD, Detroit, MI

For further information, contact howard@helix.nih.gov.