Consistent with numerous reports, 6 h following treatment of SW837 with 5 mM of the short chain fatty acid butyrate, cells in the G1
phase of the cell cycle increased by 40% (data not shown). Using sets of primers that assay exon 1, exon 1 to 2, exon 2, and exon 5 of the human cyclin D1 gene by qRT-PCR, steady state levels of expression of cyclin D1 mRNA were found to decrease progressively beginning at 2 h post-treatment, reaching approximately 20% of control value by 12 h (), leading to decreased cyclin D1 protein levels (). Similar results were seen for DLD1 cells, for which the increase of cells in G1
was 137% following butyrate treatment, again paralleled by a decrease in cyclin D1 steady state levels (not shown). Thus, these cell lines undergo standard cell cycle arrest and coincident decrease in cyclin D1 expression in response to butyrate. We have previously reported that steady state levels of c-myc mRNA and protein, also encoded by a Wnt target gene, also decreased in SW837 cells in response to butyrate. However, interrogation of the minor c-myc RNA fraction localized at the site of transcription in the interphase nucleus revealed that transcriptional attenuation was a mechanism that contributed substantially to this decrease in c-myc steady state levels (Wilson et al., 2002
Fig. 1 Decreased expression of cyclin D1 in SW837 cells treated with 5 mM butyrate. A: RNA was isolated from cultures and assayed by qRT-PCR for steady state of transcripts of the cyclin D1 gene using primers that amplified exon 1, exon 1/2, exon 2 and exon (more ...)
To investigate whether transcriptional attenuation was also triggered at the cyclin D1 locus by butyrate, we utilized the same fluorescence based method we previously reported to image nuclear transcription sites of the cyclin D1 gene within individual cells (Femino et al., 1998
; Levsky et al., 2002
; Wilson et al., 2002
). This method takes advantage of the fact that the highest concentration of any specific mRNA sequence is at the site of synthesis, and thus fluorescent probes hybridizing to these molecules emit a bright flash of light localized to the cognate gene in the nucleus. Oligonucleotide probes, which recognized exon 1 or exon 2 of the growing cyclin D1 RNA transcripts were labeled with two spectrally distinct fluorophores; these interrogated initiation of cyclin D1 transcription. The 3′ end of the RNA encoded by exon 5 was evaluated using probes labeled with a third distinct fluorophore. These exon 5 probes could only hybridize when full length, or almost completed transcripts, were present at the site of transcription. Therefore the detection of three colors at the nuclear site of transcription indicated the presence of full-length transcripts at the cyclin D1 locus, and the detection of a two-colored site indicated initiation, but attenuation, of cyclin D1 transcription. We have published images of such differentially labeled transcription sites (Wilson et al., 2002
), and an example of a single color and three color cyclin D1 transcription site is shown in . As described in Methods, the site showing only a single color would not have been scored as a true transcription site. shows the distribution of transcription sites detected in control, untreated SW837 or DLD-1 cells. For each cell line, we detected no active cyclin D1 transcription sites in approximately 70% of the nuclei examined, consistent with the fact that even expressed loci cycle between active transcription and no transcription, especially in an unsynchronized cell population. For SW837 cells, those active sites generating only initiated (i.e., two color) transcripts were approximately equivalent to those that generated full-length (three color) transcripts (). For DLD-1 cells, there was a bias towards a larger fraction of active transcription sites generating full-length transcripts ().
Fig. 3 FISH analysis for detection of active cyclin D1 transcription sites (A) FISH analysis of cyclin D1 transcription sites in untreated, growing SW837 cells. Sites were detected in nuclei using two differentially labeled probes complementary to the 5′ (more ...)
The frequency of cell nuclei containing active cyclin D1 transcription sites (either 2- or 3-colored) remained constant following butyrate treatment of SW837 cells (28 ± 3%—not shown). However, the ratio between cells with cyclin D1 loci generating full length transcripts in SW837 cells to those in which transcription was initiated, but not completed, decreased as a function of time. The ratio of full-length to initiated transcription sites decreased by 30% relative to untreated cells within 1 h of butyrate stimulation (). This decrease reached 50% relative to control by 3 h (P < 0.05), with a maximal decrease of 78% at 6 h, and a recovery thereafter (). Similar results were seen for DLD-1 cells, although the decrease appeared to be more rapid (~50% decrease by 1 h), reaching a 75% decrease in the ratio of full length to initiated sites at 6 h post-butyrate treatment, relative to control cells ().
Treatment of colon carcinoma cells with 10−7
M 1α, 25-dihydroxyvitamin D3
also induces a cell cycle arrest (Diaz et al., 2000
). illustrates that vitamin D3
also decreased the ratio of full length to initiated transcription sites within an hour in both SW837 and DLD-1 cells, respectively, with a maximum decrease again at 6 h, although again, the decrease appeared to be more rapid in DLD-1 cells than in SW837.
Transcriptional attenuation of the cyclin D1 gene therefore makes a substantial contribution to the reduced steady state levels of cyclin D1 that accompany the cell cycle arrest of colon carcinoma cells induced by butyrate or 1α, 25-dihydroxyvitamin D3.
We also investigated sequence elements of the primary transcript of the cyclin D1 gene in relation to the transcriptional attenuation. Using the pre-computed multiple species alignments provided by the UCSC Genome Browser (Karolchik et al., 2003
), we found two regions of pronounced sequence conservation between the human and mouse genome in the 4 kb long intron 3 of the mouse cyclin D1 gene. We then used the Zuker's Mfold program to predict the most favorable secondary structure (Zuker, 2003
). The first sequence (encircled area, ) forms a longer hairpin structure that is highly conserved between human, mouse, rat, and dog cyclin D1 loci, although for the dog, the sequence is present in intron 2 rather than intron 3, which is the longest intron in the dog, as is intron 3 in the other species () (Altschul et al., 1997
). qRT-PCR analysis shows that this sequence is transcribed, and, like exons 1–5, is decreased in expression by butyrate (not shown). However, although it has a secondary structure similar to known microRNA precursors (Bartel, 2004
), and its high conservation across species suggests functional significance, we have not found evidence that it exists as an independent non-coding small RNA molecule.
Fig. 4 A: Predicted secondary structure of a sequence in intron 3 (1,260–1,300 bp) of the human cyclin D1 gene, a portion of which is highly conserved between the human and mouse genome; (B) sequence homologies within intron 3 (human, mouse, rat) and (more ...)
In conclusion, we have now demonstrated that transcriptional attenuation triggered by butyrate or vitamin D3
plays an important role in the down regulation of two key genes that regulate colonic cell maturation and transformation—c-myc and cyclin D1 (Wilson et al., 2002
), and data above—therefore eliminating the increased steady state levels of c-myc and cyclin D1 which might be expected from the elevation in Wnt signaling. Recent data (Daroqui and Augenlicht, unpublished work) demonstrate that this mechanism of transcriptional attenuation appears to be involved in regulating the expression of a significant percent of genes for which expression is altered in the extensive reprogramming initiated by butyrate (Mariadason et al., 2000
). In this regard, it has been reported recently that a high percentage of developmentally regulated sequences in embryonic stem cells are initiated in transcription but fail to produce full length transcripts by a mechanism associated with a unique pattern of histone acetylation (Guenther et al., 2007
), which likely influences chromatin structure and interaction with other transcription factors. It is important to note that butyrate, an HDAC inhibitor, is present at very high concentrations in the lumen of the colon, where SCFAs are generated by fermentation of fiber and serve as the principal energy source for colonic epithelial cells. Therefore, butyrate, like vitamin D3
, is a physiological agent that provides an extrinsic signal important in establishing and maintaining colonic mucosal homeostasis. Thus, transcriptional attenuation may be a common mechanism involved in normal, well integrated, finely balanced programs of cell maturation in both embryogenesis and in response to physiological inducers of cell maturation in the adult, such as butyrate and vitamin D3
in the intestine.