Identification and Characterization of a Gene Encoding a Gut-enriched Krüppel-like Factor Expressed during Growth Arrest

Janiel M Shields,Robert J Christy,Vincent W Yang

doi:10.1074/jbc.271.33.20009

Abstract

A cDNA clone, named gut-enriched Krüppel-like factor (GKLF), was isolated from an NIH 3T3 library using a probe encoding the zinc finger region of the immediate-early transcription factor zif/268. The deduced GKLF amino acid sequence contains three tandem zinc fingers that are related to members of the Krüppel family of transcription factors. By indirect immunofluorescence, GKLF is localized to the cell nucleus. In cultured fibroblasts, GKLF mRNA is found in high levels in growth-arrested cells and is nearly undetectable in cells that are in the exponential phase of proliferation. The growth-arresting nature of GKLF is demonstrated by an inhibition of DNA synthesis in cells transfected with a GKLF-expressing plasmid construct. In the mouse, GKLF mRNA is present in select tissues and is most abundant in the colon, followed by the testis, lung, and small intestine. In situ hybridization experiments indicate that GKLF mRNA is enriched in epithelial cells located in the middle to upper crypt region of the colonic mucosa. Taken together, these results suggest that GKLF is potentially a negative regulator of cell growth in tissues such as the gut mucosa, where cell proliferation is intimately coupled to growth arrest and differentiation.

Highlights

A cDNA clone, named gut-enriched Kruppel-like factor (GKLF), was isolated from an NIH 3T3 library using a probe encoding the zinc finger region of the immediate-early transcription factor zif/268
Cloning of Mouse GKLF cDNA—To identify transcription factors that are involved in growth regulation, a cDNA library generated with mRNA from NIH 3T3 cells that were rendered quiescent and stimulated with serum for 3 h (Lanahan et al, 1992) was screened under reduced stringency conditions with a DNA probe containing the zinc finger region of the immediateearly transcription factor zif/268 (Christy et al, 1988)
Analysis of GKLF cDNA and Deduced Amino Acid Sequences—The GKLF cDNA contains a 311-bp 5Ј-untranslated region, a single open reading frame (ORF) of 1449 bp, and a 977-bp 3Ј-untranslated region that is trailed by a poly(A) tail

Summary

EXPERIMENTAL PROCEDURES

Materials—Restriction endonucleases and modifying enzymes were purchased from New England Biolabs Inc. (Beverly, MA). Following washing with PBS, rabbit anti-GKLF serum (1:2000 dilution) was added to the coverslips at room temperature for 15 min, after which they were washed with PBS. Hybridizations and washings were performed under high stringency conditions as described by Oliva et al (1993) using a radioactively labeled GKLF cDNA probe. When RNA from fractionated colonic tissue was used, the inner surface of an everted piece of freshly obtained mouse colon was gently scraped with a razor blade, and RNA was isolated as described above This method yields a highly enriched population of cells of epithelial origin as noted before (Oliva et al, 1993). In Situ Hybridization—A 575-bp PflMI-KpnI restriction endonuclease fragment of the GKLF cDNA was subcloned into pBluescript, and the recombinant plasmid was used to generate 35S-labeled antisense or sense RNA probe by in vitro transcription using T7 or T3 RNA polymerase, respectively. Slides were counterstained with hematoxylin and eosin and viewed under dark-field microscopy

RESULTS

RSDHLTT

DISCUSSION