Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation, and adaptation processes in the mammalian intestinal epithelium. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.
transcriptome; intestinal mucosa; neoplasia; sucrase-isomaltase
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ARTICLE |
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THE INTESTINAL EPITHELIUM is a complex equilibrium system of multiple cell types undergoing continual renewal while maintaining precise interrelationships. The adult phenotype is established via a series of developmental transitions resulting from the interaction of visceral endoderm and mesoderm (9). During development and in the adult epithelium, cellular phenotypes are defined by the expression of specific sets of genes in individual cells. The sets of genes expressed in intestinal epithelial cells are principally determined by transcriptional initiation, although for many genes there is modulation after initiation, as well as protein modifications that affect function. The particular set of genes expressed in a single cell type has recently been referred to as the "transcriptome" (13). Intestinal epithelial cell transcriptomes shift in well-orchestrated patterns during development, differentiation, and adaptive processes of the intestinal mucosa. Thus understanding the molecular mechanisms that regulate transcription of cellular gene sets is necessary to achieve an understanding of developmental and differentiation events.
Current information on developmental gene expression in the intestine is limited to only a handful of genes, which are primarily highly expressed genes involved in differentiated cellular functions. Minimal information is available on the expression patterns of transcriptional regulatory factors and signaling proteins that may be involved in the regulation of cellular transcriptomes. In the future, methods of genome characterization combined with tissue microdissection will enable the assessment of the entire transcriptome of each of the multiple cell types in the intestinal mucosa. I predict that it will be possible to identify the relative levels of expression of all the genes in the genome in each cell type of the intestinal mucosa. Several outcomes are possible upon analysis of these data. First, a few cell-specific transcription factors may be identified that act as switches for cell-specific gene transcription. Second, there may be a number of cell-restricted transcription factors that act in combination to direct transcription, and changes in relative levels of this set of factors may alter the cellular transcriptome. However, it is unlikely that this analysis alone will be sufficient to understand the mechanisms of intestinal gene transcription and developmental and differentiation transitions in the intestinal epithelium.
Transcriptional initiation is an intricate biochemical process that involves the interaction of core nuclear machinery (basal transcriptional apparatus) with cell-specific DNA binding proteins. Coadaptor proteins serve to link the specific DNA binding proteins to portions of the basal apparatus. The coordinated assembly of a complex of many proteins at the transcriptional initiation site of a gene leads to modification of chromatin structure and initiation of RNA synthesis by RNA polymerase II. The biochemical interactions of these proteins are modulated by phosphorylation, a process that is connected to multiple cellular signaling pathways. Therefore, the regulatory networks of transcriptional proteins that control the expression of intestinal epithelial genes are modulated by many processes that extend beyond simple expression of transcription factors. Other important issues include the cellular localization of transcriptional proteins, posttranslational modifications of DNA binding proteins and coadapters, the presence or absence of intracellular ligands (e.g., nuclear hormone receptors), and the physical state of chromatin domains. Thus elucidation of transcriptional mechanisms of well-characterized genes will be required to understand how specific transcription factors function in a coordinated network of regulatory proteins in the cell nucleus.
Current understanding of transcriptional regulatory mechanisms in the intestinal epithelium is based on intense scrutiny of a few genes expressed in restricted cellular and developmental patterns. Our studies on the regulatory mechanisms of the sucrase-isomaltase (SI) gene have underscored the complexity of understanding intestinal gene expression during development and differentiation. SI mRNA is first expressed in the mouse at very low levels at the transition of stratified endoderm to columnar intestinal-type epithelium (11). Expression remains very low until the suckling-weaning transition, when there is induction of SI mRNA in enterocytes that occurs over the course of 1 day (11). After this time, SI mRNA is differentially expressed along the crypt-villus axis, with mRNA expression detected first in enterocytes of the upper third of the crypt near the crypt-villus junction, sustained through the midvillus, and decreased in villus tip enterocytes (4, 11).
Transgenic mouse experiments show that these patterns of SI gene expression are regulated by multiple functional cis-acting DNA elements (4, 11). Most importantly, the elements necessary to direct intestinal epithelial cell-specific expression are embodied in a 201-nucleotide, evolutionarily conserved 5'-flanking region of the gene (11). This mouse SI gene promoter directs expression of a reporter transgene to enterocytes in the proper developmental and differentiation-dependent patterns. Transgenes containing longer segments of the 5'-flanking region of the gene showed that there are also other important elements for modulating the expression of the SI gene. An unexpected finding was that all four epithelial cell types in the small intestinal mucosa expressed a long promoter transgene, suggesting that all epithelial cells appear to have the transcriptional machinery necessary to express the SI gene (4). A transcriptional silencing element located outside the limits of the SI genomic DNA analyzed might explain the discrepancy between expression of the transgene and the endogenous gene.
DNA regulatory elements and associated DNA binding proteins have been
carefully assessed within the evolutionarily conserved promoter region
(6, 10, 12). There are at least three groups of transcriptional
proteins involved in SI promoter transcription, including hepatocyte
nuclear factor 1 (HNF-1) (12), caudal-related homeodomain proteins
(Cdx) (6), and nuclear proteins that interact with a GATA binding site,
which are as yet poorly characterized. What does the pattern of
expression of these proteins in the epithelium during development tell
us about their roles in SI gene transcription? Cdx1 and Cdx2 are first
expressed at high levels in the intestine just before the
endoderm-intestinal transition and are expressed continuously from this
late fetal period throughout adulthood, even though SI is only
expressed at very low levels until weaning. Moreover, both Cdx1 and
Cdx2 are expressed in small intestinal crypt cells and colonocytes,
where there is little or no SI mRNA expression. HNF-1 and HNF-1
are expressed from very early in endoderm development through adulthood
and are also expressed in crypt cells and colonocytes. Finally, from
the available information, the known intestinal GATA factors (GATA 4, 5, and 6) are also expressed early in endoderm development. Thus there
is little correlation of SI gene transcription with the expression of
these transcription factors.
There are many possible explanations for this lack of coordination between expression patterns of putative regulatory transcription factors and transcription of the SI gene. 1) It is possible that differences in the relative levels of transcriptional proteins in an individual cell may have a marked effect on SI gene transcription. To explore this possibility, there must be a more careful analysis of the relative levels of each transcriptional protein as well as the localization of SI in cell types and along the crypt-villus axis of each transcriptional protein. 2) Transcription factor interaction with the promoter may be modulated by localization in the cell, the physical state of the chromatin, or protein phosphorylation. In fact, it is possible that certain proteins may not be associated with the DNA regulatory elements in the nucleus even though binding proteins are detected using DNA-protein interaction assays. 3) There may be different factors binding to the identified regulatory sites. One can never be certain that a minor binding protein as assayed by interaction experiments might not have profound effects in vivo. 4) There may be other DNA binding proteins that interact with other sites in the promoter that have not been identified using cell lines and isolated intestinal epithelial cell nuclear extracts. 5) Finally, there may be coactivator proteins that are required for linking factors bound to the promoter elements with the basal transcriptional apparatus. These coactivator proteins might be differentially expressed in cells and thereby modulate transcription of the promoter. Our laboratory has obtained indirect evidence that such interacting proteins may play an important role in some functional aspects of transcriptional activation by Cdx2 (8). Each of these possibilities requires exploration to develop hypotheses to explain the patterns of SI gene transcription.
From the available data, we hypothesize that the temporal and spatial patterns of SI gene transcription in the intestinal epithelium are dependent on coordinated interactions of multiple DNA binding proteins that are linked to the basal transcriptional apparatus by coadaptor proteins. Modulation of these complex DNA-protein and protein-protein interactions may occur via phosphorylation of specific proteins. Protein modifications are likely linked to cellular signaling processes that are activated by extracellular cues such as matrix, growth factors, or adjacent cells. Continued analysis of the SI gene promoter and its function during development will provide the framework to build a model of these complex interactions that regulate gene transcription.
Is it possible that such a detailed description of the transcriptional regulatory mechanisms for a single intestinal gene will provide insight into the pathogenesis of disease? There is evidence to suggest the answer to this question is yes. After the discovery that Cdx2 had a functional role in transcription of SI, it was found to have a profound effect on proliferation, morphogenesis, and gene expression in intestinal epithelial cells (7). Subsequently it has been shown that expression of both Cdx2 (2, 3) and Cdx1 (3, 5) is markedly diminished in human colon adenomas and carcinomas. Finally, it was recently shown that mice harboring one null allele for Cdx2 develop colorectal neoplasms early in life (1). Thus diminished expression of Cdx2, and possibly Cdx1, may be involved in the pathogenesis of colorectal neoplasms. Because the SI promoter is a natural target for Cdx protein function, it will be helpful in dissecting the functional domains of the proteins. Moreover, as the other pieces of the transcriptional puzzle of the SI gene are assembled there will be new information to generate hypotheses on the relationship of transcriptional mechanisms to intestinal disease. Finally, a structural understanding of these mechanisms will provide targets for novel therapeutic agents to modulate proliferation and differentiation programs.
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FOOTNOTES |
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* Fifth in a series of invited articles on Epithelial Cell Growth and Differentiation.
Address reprint requests to Dept. of Medicine, 100 Centrex, Hospital of the Univ. of Pennsylvania, 34th and Spruce St., Philadelphia, PA 19104.
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