Sibley Lab In The Department of Pediatrics

Research Interests of the Sibley Lab

Overview

The mammalian gastrointestinal tract matures from a primitive tube into morphologically and functionally distinct regions during development. The mature small intestine functions in the digestion and absorption of ingested nutrients. Several of the hydrolases responsible for enzymatic nutrient digestion including lactase, sucrase-isomaltase, and maltase are expressed by the enterocyte, a specialized small intestinal epithelial cell derived from the gut endoderm cell lineage.

Maximal expression of several hydrolases is spatially restricted to distinct segments along the cephalo-caudal axis of the small intestine and is temporally regulated during postnatal maturation. Intestinal lactase, the hydrolase responsible for the digestion of lactose in milk, is expressed at highest levels in the jejunal segment of the small intestine shortly after birth and then declines dramatically just prior to weaning in most mammals.

Our research is directed towards defining the mechanisms regulating this spatial and temporal restriction of lactase gene expression during intestinal development. The overall goal is to relate these lactase control mechanisms to the broader pathways specifying acquisition of a small intestinal phenotype.

Transcriptional Regulation of Lactase during Intestinal Maturation

The normal maturational decline in lactase enzymatic activity is correlated with a decline in lactase messenger RNA levels and is likely to be transcriptionally regulated. We are interested in identifying maturation-specific lactase gene cis elements and in characterizing the nuclear proteins interacting with those elements.

We are carrying out a rigorous characterization of the 5' flanking region of the lactase gene in cell culture and in transgenic animals in order to map important maturational control elements and to subsequently isolate and characterize the function of their DNA-binding proteins. Data from our laboratory and others support a role for glucocorticoids and various growth hormones in regulating intestinal maturation. We intend, therefore, to incorporate hormonal responsiveness in our maturational analysis of lactase gene transcription control. Our goal is to define the interactions of the lactase gene elements and nuclear factors mediating the maturational decline in lactase transcription.

Spatial Restriction of Hydrolases along the Gut Cephalo-Caudal Axis

The intestine develops into a complex organ with specialized structures and functions unique to various spatial regions. Expression of several nutrient hydrolases, including lactase, is restricted to distinct segments along the small intestinal longitudinal axis. Disease or loss of a segment results in loss of nutrient hydrolases and symptoms of maldigestion and malabsorption as in the short bowel syndrome.

We are interested in defining the mechanisms regulating spatial restriction of lactase gene expression during development. Our approach is similar to that used to characterize maturational regulation of lactase transcription. We are interested in mapping gene elements essential for spatial restriction by mutational analysis in transgenic animals. Our goal is to identify nuclear factors interacting with those elements and to characterize their function in regulating lactase transcription spatially along the gut axis.

The same spatial control factors regulating lactase may be involved in restricting expression of other enterocyte proteins not only in the gut of mammals but other organisms as well. We are interested therefore in identifying conserved spatial cis regulatory elements in the genes of other nutrient hydrolases and enterocyte proteins and will extend our initial studies to examine conserved regulatory mechanisms. Identification of important spatial control factors will allow for future experimentation directed towards genetic manipulation of such factors, for instance, to induce a jejunal phenotype in an ileal segment. A long-term goal, therefore, is to apply the mechanisms regulating spatial restriction to design approaches for the induction of intestinal adaptation in such clinical problems as short bowel syndrome.

Enteric Gene Transfer

The intestine is a readily accessible organ and as such is an ideal target for gene transfer. The ability to target the intestine using gene transfer has widespread clinical implications. Such technology would allow gene therapy for intestinal enzyme or protein deficiency states including short bowel syndrome and congenital glucose-galactose transport deficiencies both of which present in infancy. In addition, the intestine could be targeted for production and systemic absorption of therapeutic products for non-intestinal diseases.

The digestive enzyme environment of the gastrointestinal tract, however, is a potential complicating factor for enteric gene transfer. Specifically, the gut lumen contains relatively high concentrations of nucleases, lipases, and proteases that are capable of degrading enterally administered DNA and transfer vehicles. Our laboratory is currently involved in a collaboration to study the uptake and expression of viral vectors in intestinal cells. We aim to devise means to deliver specific genes to the intestine which are capable of expressing proteins for experimental and therapeutic applications.

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