Its possible their cis elements are far away from the promoter region.

Which gene are you talk about, it would be nice to see check the cis-regulatory elements for this gene. Saiyu is right. It is possible that the regulatory regions are far from the genes, which can form loop during transcription factor interaction. As we know, regulatory elements such as transcription factor binding sties should be

I think distant enhancer elements are not that common in Arabidopsis. It is quite possible that a relatively short intergenic region contains an active promoter. For instance, light-responsive regulatory elements in photosynthesis-associated genes are located within 300 bp upstream the start codon. Also, some introns can contain cis-elements to regulate gene expression.

I would suggest:

1. Look for the homologous genes from plants with completely sequenced genome. That is quite easy to do using Phytozome. Check for the size of the intergenic region, to see if that is a conserved feature.

2. Get the sequence of the intergenic region for the ortologs, and look for conserved cis-elements. If you use databases such as ATCOECIS or AGRIS, that will show you only those elements previously reported for other genes. Try SCOPE or Melina II, that way you'll get de novo potential regulatory elements evolutionarily conserved among the ortologs (and thus potentially functional). This analysis can also show conservation of combinatorial interactions of cis-elements.

3. Perform a gain of function experiment. That would be the experimental evidence to show the intergenic region confers the regulatory characteristics observed with RT-PCR or microarrays. You should be able to see the same response from the transcriptional fusion, otherwise, that would indicate additional elements outside the intergenic region are necessary.

You can check from my publications:

-Sugar and ABA responsiveness of a minimal RBCS light-responsive unit is mediated by direct binding of ABI4

-Functional analysis of the Arabidopsis PLDZ2 promoter reveals an evolutionarily conserved low-Pi-responsive transcriptional enhancer element

Agree with Gustavo. Arabidopsis genome is quite condensed comparing with human genome. A lot promoters are very short. I recently cloned a promoter, just around 350bp before first codon, still showed specific promoter activity.

For a gene X it does not matter a priori what other functions there are in the flanking regions, meaning that you can also ignore the annotation and regard a region of 1-2 kb upstream a a potential promoter. This is the view of the plant on its genome anyway, because it cannot look it up at TAIR in Seqviewer or similar databases. Nevertheless, a neighboring or even overlapping gene can potentially interfere and compete with the accessibility and transcription of gene X (and vice versa).

But: most regions of the genome are anyway transcribed intensely, even in both directions, etc....

So, in the old-fashioned way it may still be fine to stick to a 1 or 2 kb fragment if you want to bioinformatically or expereimentally analyze a promoter.

Three possibilities are there for gene pairs transcribing tandemly.

1) The short intergeneic region is sufficient to regulate the expression of downstream present gene.

2) first do check the expression for both the genes. If two genes are showing differential expression (spatial and temporal), it is quite possible that the downstream gene is using coding sequence of upstream gene as promoter element to get transcribed.

3) obviously some enhancers could act to regulate the expression of gene pairs

What do you mean by very close, where the basal approximal and distal promoter regions are from the transcription start site? Anyway, the machinery of transcription (regulatory elements ans their transcription factors mechanisms is no easy to understand in slico and you need experimental assays. So, it is better to give an example about these genes and how close they are by size so we can help you much better

As Wilco said, a lot of plant genes have regulatory elements in the first intron, that would be a good place to start looking too and complement your search.

But I'm also wondering if the few regulatory elements in your genes would be enough to drive specific expression in your conditions.

Yes, it would be necessary to know a bit more about the structures of the genes that you are referring to. Wilco (above) makes an excellent point that some regulatory elements can be found within introns, assuming the genes you are referring to have introns. Another point to make is that we are still learning about plant promoters and even with sophisticated bioinformatics, we can only know about something for which there is precedent (and the bioinformatic data for making such predictions). Finally, within your "shared promoter" do you find nucleosome depleted regions (NDRs). You can determine this using a predictive algorithm (see Field, Y., Kaplan, N., Fondufe-Mittendorf, Y., Moore, I.K., Sharon, E., Lubling, Y., Widom, J., and Segal, E. (2008). Distinct Modes of Regulation by Chromatin Encoded through Nucleosome Positioning Signals. PLoS Comput Biol 4, e1000216). If there are clear NDRs (characterized by poly(dA:dT) tracts, you may be dealing with a promoter that has an alternative type of activation element. Such promoters are well known in yeast (but even in yeast there is a gray area).

Thanks everyone for your interest in this. The gene set consists of At1g32940, At1g32950, At1g32960, At1g32970 and At1g32980. The five are evolutionarily related. Here is how they are positioned in the chromosome:

At1g32940 from 11937576 to 11940956, forward

462 bases

At1g32950 from 11941418 to 119447440, forward

547 bases

At1g32960 from 11945287 to 11948630, forward

----

At1g32970 from 11948701 to 11951962, backward

At1g32980 from 11954258 to 11955342, backward

Data collected from various microarray experiments show differential expression. We are studying specific conditions where At1g32940, At1g32950 and At1g32960 differ by qualitative RT-PCR (close to all-or-none situations, not just degree of expression). The last two are not expressed under these specific conditions. The pattern for At1g32950 is the same as other members of the same gene family on other chromosomes, not its neighbors on the genome. I was thinking I might find some recognizable cis elements in common among those with similar expression, different from others in the family of 56 genes. As suggested, I will look in the intergenic regions of At1g32940. Other suggestions are welcome.

Hello Anna,

I think promoter analysis can give you some clues about the regulation of your genes. Here I am attaching a very fast and preliminary analysis of the intergenic regions of AT1G32940, AT1G32950 and AT1G32960 using SCOPE. There you can see the cis-elements predicted in the promoters of the first and last genes are the same, some of them even in the same arrangement (suggesting conserved combinatorial interaction). AT1G32950, on the other hand, shows only a couple of these elements.

If you know other genes in the family co-expressed with AT1G32950, you can perform a similar analysis to find cis-elements potentially involved in that particular regulation, or cluster your genes based on their expression patterns and perform this type of analysis to relate particular cis-elements or their arrangements to the transcriptional behavior of the genes.

As I mentioned before, the advantage of using SCOPE or similar tools is that you can identify candidate motifs de novo, specific for the particular regulation of your genes. ATCOECIS or AGRIS only look for those motifs already reported, which is very limiting. Besides, it is quite easy to use, just need to write the AGI list and that's it!

http://genie.dartmouth.edu/scope/

Hope this is helpful. Good luck with your project!

Thanks for pointing me to a new promoter analysis site. I will try it out. I noticed that I can examine just a fixed number of bases upstream or I can examine intergenic regions. In case cis elements for the gene of interest might be in the intergenic region of the previous one as Wilco and others have suggested, I will try both situations.