This is an online supplement to: COTRASIF: conservation-aided transcription factor binding site finder
For Type I interferons the dominating signal transduction pathway is well-studied - namely the Jak-STAT pathway, which results in the activation of ISGF3 transcription factor (interferon-stimulated gene factor), which binds specifically to ISRE (interferon-stimulated response element). Thus, searching for ISRE will yield the most probable genes of the primary response to interferon treatment.
We did the search for genes using HMM finder tool. Matrix and sequences used for search are present as examples on COTRASIF HMM task submission page:
1 12 0 0 0 0 0 7 1 1 0 0 0 2 1 8 0 0 0 0 0 13 1 7 0 0 3 8 7 8 2 1 13 0 0 0 0 1 2 0 0 0 0 2 3 2 0 0 13 13 13 0 4 3 12 13 10 5 2 1
TAGTTTCACTTTCCC CAGTTTCGGTTTCCC GAGTTTCATTTTCCC CAGTTTCATTTTCCC TGGTTTCGTTTCCTC AAGTTTCTATTTCCT TAGTTTCAGTTTCAT CAGTTTCTGTTTTGC
Search was conducted in the Rattus norvegicus promoters extracted from Ensembl release 52.
We found 743 putative ISREs in 707 probable IFN Type I rat primary response genes, and 1292 putative ISREs in 1163 mouse genes.
- HMM search result for Rattus norvegicus, as generated by COTRASIF (gene ID list)
- HMM search result for Mus musculus, as generated by COTRASIF (gene ID list)
- conservation filtering result, rat (target) vs mouse (reference) (list of 162 unique rat gene IDs, used as list #1 in FatiGO)
- all 17725 rat protein-coding genes, which have their promoters in COTRASIF internal database (list #2 in FatiGO)
- first, we performed GO enrichment analysis of 707 rat genes versus the 17725 protein-coding rat genes using FatiGO. At adjusted pvalue < 0.05, 5 GO categories were found to be enriched:
Term List #1 vs #2 p-value Adjusted p-value GO molecular function at level 6 serine-type endopeptidase activity (GO:0004252) 75.27%, 24.73% 6.2*10-5 0.0382 GO molecular function at level 7 tissue kallikrein activity (GO:0004293) 93.62%, 6.38% 1.4*10-3 0.0402 GO cellular component at level 7 MHC protein complex (GO:0042611) 83.59%, 16.41% 3.76*10-4 0.0177 GO cellular component at level 8 MHC class I protein complex (GO:0042612) 86.64%, 13.36% 8.52*10-5 0.00554 GO molecular function at level 5 serine-type peptidase activity (GO:0008236) 74.91%, 25.09% 5.05*10-5 0.02611
- using FatiGO, we also performed GO enrichment analysis of 162 rat genes obtained after applying conservation filter; the only enriched GO category this time was immune response GO:0006955, with p-value 1.23*10-4 and adjusted p-value 8.52*10-3; list of 9 genes
- these 9 genes are:
- Mbl1, mannose-binding protein A precursor (MBP-A); no known correlations of Mbl1 expression and interferon treatment
- LOC687510, ENSRNOG00000029191; this rat gene is not annotated, but is in 1-to-1 orthologous relation to Mus musculus GBP6 gene; murine GBP6 was recently shown to be IFN-inducible (Degrandi,D., Konermann,C., Beuter-Gunia,C., Kresse,A., Wurthner,J., Kurig,S., Beer,S. and Pfeffer,K. (2007) Extensive Characterization of IFN-Induced GTPases mGBP1 to mGBP10 Involved in Host Defense. J Immunol, 179, 7729-7740.)
- Mx1 and Mx2, interferon-induced GTP-binding proteins
- Cxcl4, platelet factor 4 precursor (PF-4, C-X-C motif chemokine 4); no known expression correlation with interferon treatment
- Igsf4a, immunoglobulin superfamily, member 4A (NECL-2); no correlation with interferon found
- Gzma, granzyme A; is regulated by interferon alpha (Zhang,B., Zhang,J. and Tian,Z. (2008) Comparison in the effects of IL-2, IL-12, IL-15 and IFNalpha on gene regulation of granzymes of human NK cell line NK-92. Int. Immunopharmacol., 8, 989-996, and Li,S., Xia,X., Zhang,X. and Suen,J. (2002) Regression of tumors by IFN-alpha electroporation gene therapy and analysis of the responsible genes by cDNA array. Gene Ther., 9, 390-397.)
- Oasl2, 2'-5' oligoadenylate synthetase-like 2 is a well-known target of IFN
- GBP4_predicted, ENSRNOG00000028768; similar to the LOC687510, murine GBP4 ortholog is IFN-inducible (Degrandi,D., Konermann,C., Beuter-Gunia,C., Kresse,A., Wurthner,J., Kurig,S., Beer,S. and Pfeffer,K. (2007) Extensive Characterization of IFN-Induced GTPases mGBP1 to mGBP10 Involved in Host Defense. J Immunol, 179, 7729-7740.)
- as a conclusion: of 9 genes, 4 are well-known IFN type I targets, 2 genes have mouse orthologs which are known to be IFN type I and type II regulated (see references above), and for 3 genes there is no accumulated evidence of IFN type I regulation.
Previously (for Ensembl release 49, and COTRASIF version 0.14), to verify the usability of COTRASIF's tools, and to illustrate possible uses, we performed a sample investigation of the genes of the primary response to Type I interferons.
The Ensembl gene ID overlap between the search results is 83.9%. That is, 271 genes from the Ensembl-49 323-gene-long search results file are also present in 707-gene-long Ensembl-52 search results file. Elongation of the results file roughly correlates with the increase of promoter sizes from 800+ to 2000+ bp (2000/800=2.5, 707/323=2.19); the 16.1% non-overlap is due to the changes in HMM threshold automatic calculation we introduced since COTRASIF version 0.14, and (to a much lesser extent) due to Ensembl annotation changes between release version 49 and 52.
Matrix and sequences used were exactly as above.
We found 338 ISREs in 323 rat genes. Literature suggests that there are up to 100-300 genes of interferon response in mammalian cells; based on this estimate, these search results were not further processed using conservation filter.
- HMM result, as generated by COTRASIF
- same result in Excel format, sorted by similarity score (best-worst)
- same list - gene IDs only; it was used as "4. file of interesting gene list" in GOTM
- all rat protein-coding genes (for GOTM input field 5b)
- trimmed-down [3MiB] (or original [7.5MiB]) GO tree output ("export GOTree" in GOTM)
- 24 enriched categories: tree view
- 24 enriched categories: DAG view (as an image)
- list of genes for each GO category found to be enriched
- 34 genes from enriched categories, best-scoring model-similarity first
We performed enrichment analysis of 323 rat genes using GOTM. The following 24 categories were found to be enriched (13 known interferon-related underlined):
In biological processpositive regulation of signal transduction
myeloid leukocyte differentiation
positive regulation of protein biosynthesis
immune system process
immune effector process
leukocyte mediated immunity
lymphocyte mediated immunity
B cell mediated immunity
adaptive immune response
adaptive immune response (sensu Gnathostomata)
innate immune response
negative regulation of transferase activity
negative regulation of protein kinase activity
response to biotic stimulus
response to other organism
response to virus
In molecular functionmonosaccharide binding
serine-type endopeptidase activity
enzyme activator activity
phospholipid transporter activity
In cellular componentMHC protein complex
MHC class I protein complex