title: Transcriptional activation of the enterocyte differentiation marker intestinal alkaline phosphatase is associated with changes in the acetylation state of histone H3 at a specific site within its promoter region in vitro.
abstract: Enterocyte differentiation is thought to occur through the transcriptional regulation of a small subset of specific genes. A recent growing body of evidence indicates that post-translational modifications of chromatin proteins (histones) play an important role in the control of gene transcription. Previous work has demonstrated that one such modification, histone acetylation, occurs in an in vitro model of enterocyte differentiation, butyrate-treated HT-29 cells. In the present work, we sought to determine if the epigenetic signal of histone acetylation occurs in an identifiable pattern in association with the transcriptional activation of the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). HT-29 cells were maintained under standard culture conditions and differentiated with sodium butyrate. The chromatin immunoprecipitation (ChIP) assay was used to compare the acetylation state of histones associated with specific regions of the IAP promoter in the two cell populations (undifferentiated vs. differentiated). Chromatin was extracted from cells and cleaved by sonication or enzymatic digestion to obtain fragments of approximately 200 to 600 base-pairs, as confirmed by polymerase chain reaction using primers designed to amplify the IAP segments of interest. The ChIP assay selects DNA sequences that are associated with acetylated histones by immunoprecipitation. Unbound segments represent DNA sequences whose histones are not acetylated. After immunoprecipitation, sequences were detected by radiolabeled polymerase chain reaction, and the relative intensity of the bands was quantified by densitometry. The relative acetylation state of histones at specific sites was determined by comparing the ratios of bound/unbound segments. We determined that in a segment of the IAP promoter between -378 and -303 base-pairs upstream from the transcriptional start site, the acetylation state of histone H3 increased twofold in the differentiated, IAP expressing cells, whereas that of histone H4 remained essentially constant. Additionally, at a distant site, between -1378 and -1303 base-pairs, the acetylation state of H3 and H4 did not change appreciably between the undifferentiated and differentiated cells. We conclude that butyrate-induced differentiation is associated with specific and localized changes in the histone acetylation state within the IAP promoter. These changes within the endogenous IAP gene may underlie its transcriptional activation in the context of the enterocyte differentiation program.
authors.affiliation: Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Grey-Bigelow 504, 55 Fruit Street, Boston, MA 02114, USA.
authors.forename: Brian F
authors.initials: BF
authors.lastname: Hinnebusch
authors.forename: J Welles
authors.initials: JW
authors.lastname: Henderson
authors.forename: Aleem
authors.initials: A
authors.lastname: Siddique
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Malo
authors.forename: Wenying
authors.initials: W
authors.lastname: Zhang
authors.forename: Mario A
authors.initials: MA
authors.lastname: Abedrapo
authors.forename: Richard A
authors.initials: RA
authors.lastname: Hodin
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000378:metabolism*; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D002843:Chromatin / Q000378:metabolism*; D020895:Enterocytes / Q000502:physiology*; D058851:GPI-Linked Proteins; D005819:Genetic Markers / Q000235:genetics; D019073:HT29 Cells; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D019901:Locus Control Region; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D015533:Transcriptional Activation
chemical_list: D000951:Antigens, Neoplasm; D002843:Chromatin; D058851:GPI-Linked Proteins; D005819:Genetic Markers; D006657:Histones; C063088:ALPI protein, human; D000469:Alkaline Phosphatase