Abstracts
2002 Digestive Disease Week

# 107458 Abstract ID: 107458 Differentiation-Induced Transcriptional Activation of Intestinal Alkaline Phosphatase Is Associated with an Increase in Histone H3 Acetylation in a Specific Promoter Region In-Vitro.
Brian F Hinnebusch, Joseph W Henderson IV, Madhu S Malo, Richard A Hodin, Boston, MA

Although the exact mechanisms are poorly understood, enterocyte differentiation is thought to occur through the transcriptional regulation of a small subset of specific genes. Recent findings have suggested that transcription can be regulated by a number of post-translational modifications to the DNA-packaging proteins known as histones. These modifications may comprise an epigenetic code which plays an important role in transcriptional regulation. A common model of enterocyte differentiation, butyrate-treated HT-29 cells, results in histone acetylation. We sought to determine if this modification was localized to specific DNA sequences in the promoter region of an enterocyte differentiation marker, intestinal alkaline phosphatase (IAP). HT-29 cells were maintained under standard culture conditions and differentiated with butyrate. The novel technique of chromatin immunoprecipitation (ChIP) was used to compare the acetylation state of histones within two regions of the IAP promoter in undifferentiated versus differentiated cells. The ChIP assay selects DNA sequences that are associated with acetylated histones by immunoprecipitation. The unbound segments represent sequences that are not associated with acetylated histones. Sequences are detected with radiolabeled PCR and the relative acetylation state of the histones at that site is determined by comparing the ratio of unbound to bound fractions. Bands were quantified by densitometry and all data were analyzed using Student?s paired t-test, with p<0.05 considered significant. Chromatin was immunoselected with antibodies to acetylated histone H3 or acetylated histone H4. We found that in a segment of the IAP promoter between -1378 and -1303 bp upstream from the transcriptional start site, the acetylation state of histones H3 and H4 did not change significantly (ratios of 1.21 vs. 1.84, p=0.28 and 0.87 vs. 1.23, p=0.1 respectively). However, at a more proximal site, between -378 and -303 bp, the acetylation state of histone H3 increased by 2 fold (p<0.05). The acetylation state of histone H4 at this site appeared to actually decrease slightly, although this result did not reach statistical significance (p=0.08). We conclude that butyrate-induced differentiation is associated with specific and localized changes in histone acetylation state rather than a global histone hyperacetylation.