Bruce H. Howard, MD, Head, Human Genetics Section
Valya Russanova, PhD, Staff Scientist
Analia Porrás, MD, PhD, Research Fellow
Hariklia Dimitropoulos, PhD, Postdoctoral Fellow
Paraskevi Salpea, BS, Visiting Postbaccalaureate Fellow
Chris Heffelfinger, BS, Postbaccalaureate Fellow
Sarah Kozar, BS, Postbaccalaureate Fellow

The normal human lifespan is marked by a complex series of developmental transitions, relative stability during adulthood, and, ultimately, a gradual decline in viability. Biological clocks presumably underlie the developmental events that occur through childhood and adolescence, but the nature of those clocks has remained obscure. Progress in understanding biological clocks would be of considerable importance not only for our understanding of child development but also because instability in putative clock-like mechanisms may occur as part of the aging process. Such instability could compromise tissue function and contribute to many of the common degenerative diseases of later life.

Genome sampling, bioinformatics, and high-throughput approaches to map higher-order chromatin domains

Russanova, Howard, Porrás, Dimitropoulos, Salpea, Heffelfinger, Kozar

In previous studies, we mapped age-related chromatin remodeling across a region positioned 11 Mb from the human chromosome 4p terminus (4p16.1). This region, extending over 0.7 Mb, exhibits diminishing histone H4 acetylation over a period spanning fetal development to early childhood. Through comparisons between young and old adults, we found a second remodeling domain, less than 3 Mb from the 4q terminus (4q35.2). Despite marked time frame differences, chromatin changes in the two regions are broadly similar. These findings are novel and support the hypothesis that clock-like mechanisms can reside in chromatin-based epigenetic structures.

The past year has seen two major shifts in our research emphasis. First, cells studied earlier came from immortalized cell lines or tissue bank-derived non-immortal lineages. Protocols are now in place to investigate human tissues from several clinical sources. Peripheral blood monocytes are obtained from newborns (cord blood) through a collaboration with NICHD's Perinatology Branch while monocytes from adults are available through NIH's Department of Transfusion Medicine. Likewise, we have obtained human skin fibroblasts from newborns and adults, the latter under a new NICHD Institutional Review Board (IRB)-approved protocol that represents an essential step in the important goal of studying developmental and common age-related disease states.

Second, we have shifted the focus of our research from direct screens for chromatin remodeling to the use of RNA expression microarrays to search for instances of development- and age-related epigenetic change, including both chromatin- and DNA methylation-based mechanisms. It bears note that searches for epigenetic changes via alterations in RNA expression are more indirect than are direct chromatin screens. Nevertheless, given that a major goal of the laboratory is to link epigenetic mechanisms to specific genes and clinical disorders, the use of RNA expression arrays is likely the more efficacious approach.

We made substantial progress in advancing our new research directions. The differentiation program that transforms monocytes into antigen-presenting dendritic cells (triggered in response to IL4 and GM-CSF) revealed evidence of both developmental- and age-related change. Such change is highly selective, strongly affecting only a small number of genes, while global aspects of differentiation remain well preserved. Detailed studies now under way with selected genes will determine to what degree alterations in RNA levels reflect transcriptional or nuclear processing controls.

Results to date indicate that genes subject to both differentiation and developmental controls often exhibit a high degree of variance in expression levels during developmental- or age-related transitions. That this occurs, at least in part, through epigenetic mechanisms is supported by chromatin mapping data. In a general way, the well-studied combination of differentiation and developmental controls on fetal hemoglobin expression may prove to be an important paradigm for the developmental- and age-linked transitions observed in the current project.

Concurrent work with newborn and adult (young and old) skin fibroblasts is at a more preliminary stage than studies using the monocyte-dendritic system. Nonetheless, in early experiments, we observed striking differences in the responsiveness of cells from these respective sources to serum deprivation and refeeding. A long-term advantage of the fibroblast system is that it should make possible detailed in vitro mechanistic studies with the wide variety of gene transfer and gene knockdown tools now available.

COLLABORATORS

Keiko Ozato, PhD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda, MD
Roberto Romero, MD, Perinatology Research Branch, NICHD, Detroit, MI

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