Transcriptional profiling of mRNA expression in the mouse distal colon

Abstract

Background & aims: Intestinal epithelial cells and the myenteric plexus of the mouse gastrointestinal tract contain a circadian clock-based intrinsic time-keeping system. Because disruption of the biological clock has been associated with increased susceptibility to colon cancer and gastrointestinal symptoms, we aimed to identify rhythmically expressed genes in the mouse distal colon.

Methods: Microarray analysis was used to identify genes that were rhythmically expressed over a 24-hour light/dark cycle. The transcripts were then classified according to expression pattern, function, and association with physiologic and pathophysiologic processes of the colon.

Results: A circadian gene expression pattern was detected in approximately 3.7% of distal colonic genes. A large percentage of these genes were involved in cell signaling, differentiation, and proliferation and cell death. Of all the rhythmically expressed genes in the mouse colon, approximately 7% (64/906) have been associated with colorectal cancer formation (eg, B-cell leukemia/lymphoma-2 [Bcl2]) and 1.8% (18/906) with various colonic functions such as motility and secretion (eg, vasoactive intestinal polypeptide, cystic fibrosis transmembrane conductance regulator).

Conclusions: A subset of genes in the murine colon follows a rhythmic expression pattern. These findings may have significant implications for colonic physiology and pathophysiology.

Figure 1

(A) Gene tree of colonic transcripts. Increased transcript abundance is presented in red and decreased abundance in green. (B) Cluster patterns. For each cluster, a trace is shown indicating the average profile of all oscillating transcripts in each respective cluster.

Figure 2

Array data (solid line) and quantitative PCR (dotted line) for a subset of clock genes. Data represent mean fold change ± SE as percentage of maximum expression.

Figure 3

Pie chart of functional categories of rhythmically expressed genes in mouse colon.

Figure 4

Subset of rhythmically expressed genes. (A) Genes involved in colon cancer formation, (B ) genes involved in inflammation, and (C) genes involved in other gastrointestinal functions such as motility and secretion. Data represent mean fold change ± SE as percentage of maximum expression.

Figure 5

Daily profile of gene mRNA levels and corresponding fitted cosinor curve under constant darkness with ad libitum access to food. mRNA levels are expressed as means ± SE. PR, percentage rhythms; P value, probability from zero-amplitude (no rhythm) test.

Figure 6

Daily profile of colonic clock gene mRNA levels and corresponding fitted cosinor curve under constant darkness and fasting conditions. mRNA levels are expressed as means ± SE. PR, percentage rhythms; P value, probability from zero-amplitude (no rhythm) test.

Figure 7

(A) Western blotting for proteins (BCL2, CFTR, and nNOS) in mice with ad libitum access of food under a regular light/dark cycle. (B) Quantitative analysis of protein expression and corresponding fitted cosinor curve. Protein levels are expressed as means ± SE. PR, percentage rhythms; P value, probability from zero-amplitude (no rhythm) test.