Expression and promoter analysis of a highly restricted integrin alpha gene in vascular smooth muscle

Abstract

Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5' promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity, indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArG-SRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region.

Figure 1. Tissue distribution of Itga8 mRNA

Kitchen et al, (A) RNase protection of Itga8 mRNA in indicated adult mouse tissues. Note the over-abundance of Itga8 mRNA in aorta versus other tissues as compared to Tagln1 (aka SM22). (B) qRT-PCR of Itga8 mRNA in independent mouse tissues (B) as well as aorta versus intestine (C). Note the differential expression of Itga8 mRNA between aorta and intestine versus that of Cnn1 and Myh11. (D) Northern blotting of comprehensive human tissues demonstrating similar enrichment of ITGA8 mRNA levels in aortic muscle tissue versus all other tissues. All expression data are representative of multiple experiments performed by independent investigators.

Figure 2. Comparative sequence and chromosomal analysis of ITGA8

Kitchen et al, (A) VISTA plot of ITGA8 and flanking sequences between mouse (M) versus rat (R) (top panels) and mouse versus human (H) (bottom panels). The green dashed boxed area is shown below in Figure 3. (B) Chromosome position of orthologous genes between human (Chr 10) and mouse (Chr 2). There is chromosomal synteny between species with ITGA8 in the centromeric direction (pointing right) but not in the telomeric direction (pointing left). Schematic generated from the UCSC Genome Browser (http://genome.ucsc.edu/). Numbers below ITGA8 represent the size of each transcribed gene.

Figure 2. Comparative sequence and chromosomal analysis of ITGA8

Kitchen et al, (A) VISTA plot of ITGA8 and flanking sequences between mouse (M) versus rat (R) (top panels) and mouse versus human (H) (bottom panels). The green dashed boxed area is shown below in Figure 3. (B) Chromosome position of orthologous genes between human (Chr 10) and mouse (Chr 2). There is chromosomal synteny between species with ITGA8 in the centromeric direction (pointing right) but not in the telomeric direction (pointing left). Schematic generated from the UCSC Genome Browser (http://genome.ucsc.edu/). Numbers below ITGA8 represent the size of each transcribed gene.

Figure 3. ITGA8 transcription start site mapping and conservation of genomic sequence

Kitchen et al, A VISTA plot (corresponding to the green dashed boxed region in Fig. 2A) of human versus mouse 5' ITGA8 is shown with the first two exons (in blue). The x-axis represents the human coordinates of ITGA8 and the y-axis indicates the percent homology between human and orthologous mouse sequences. The pink peaks represent intronic and upstream non-coding sequences with at least 75% of homology over 100 bp of sequence as in Figure 2A above. The bent arrow represents the mapped transcription start site (TSS, shown below in an RNase protection assay). Two conserved non-coding sequence modules (Mod1 and Mod2) are indicated by the arrows with corresponding sequence homology between mouse, rat, and human. A conserved CArG-like element is boxed (in red) in Mod1 (see below).

Figure 4. Itga8 promoter sequence and activity

Kitchen et al, (A) Nucleotide sequence of the mouse Itga8 promoter and 5' coding sequence (bold italics and underlined). The 5' end of several promoter construct used in luciferase assays is indicated by a triangle and the relative position to the TSS is indicated in parenthesis. Putative binding sites predicted by TRANSFAC and GCG software are indicated as is the mapped TSS. (B) Deletion constructs of the mouse Itga8 promoter corresponding to positions labeled in panel A were tested in the indicated cell lines and the luciferase activity normalized to a renilla reporter gene is shown. (C) Co-transfection experiments with either SRFVP16 or Myocardin (MYOCD_v3, (Imamura et al 2010) ) and either Mod1 of Itga8 fused to the −578 promoter or the SMC-restricted promoters SM22 or Calponin (Calp). All luciferase experiments were repeated multiple times in independent experiments and were statistically analyzed by one-way ANOVA with Tukey's posthoc test. Asterisks indicate p < 0.05 compared to corresponding empty vector control.

Figure 5. SRF and Myocardin effects on Itga8 promoter activity

Kitchen et al, Pac1 SMC were transfected with pGL3 Basic (vector) or the same luciferase backbone containing either −4,234, −1,218, or −578 nucleotides of the 5' promoter of Itga8 in the absence or presence of MYOCD_v3 (A) or SRFVP16 (B). A Leiomodin 1 (Lmod1) promoter construct known to be SRF and MYOCD dependent (Nanda and Miano 2012) was included as a positive control. Data are representative of two independent studies done in quadruplicate.

Figure 6. SRF and Myocardin effects on endogenous Itga8 expression

Kitchen et al, (A) Pac1 SMC were transduced with either shSRF or shEGFP adenovirus and endogenous Itga8 mRNA analyzed by qRT-PCR. The expression of Itga8 was normalized to a house-keeping gene (Ppia) that showed no changes in expression across treatments. (B) Mouse aortic tissue in which Srf was deleted through Tamoxifen (Tmx) induced Cre-mediated excision was analyzed 3 weeks post Tmx or oil treatment by qRT-PCR. Two independent aortic samples treated with Tmx are shown as is an oil control. Note obvious decrease in Srf as well as known SRF target gene, Cnn1, but no similar decrease in Itga8. (C) Conventional (gel) RT-PCR of ITGA8 mRNA in human coronary artery SMC (HCASM) transduced with 30 MOI of either Ad-lacZ (−) or Ad-MYOCD (+). This study is representative of at least three independent experiments on different isolates of HCASM. (D) Similar study as in panel C only HCASM were transduced with increasing titers (10, 30, or 100 MOI) of indicated viral constructs. This experiment was repeated once in HCASM and in several rat and mouse SMC lines (not shown).