Transcript Poster - dbZach - Michigan State University

Identification of Positionally Conserved Dioxin Response Elements in
Orthologous Human, Mouse and Rat Genes
Burgoon, L.D., Cabunoc, A.E., Zacharewski, T.R.
Department of Biochemistry & Molecular Biology, National Food Safety & Toxicology Center
and the Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824
Abstract
Computational identification and prediction of dioxin response elements (DREs)
within gene regulatory regions is one method to identify novel, putative aryl
hydrocarbon receptor (AhR) binding locations. This aids the phenotypic
anchoring of toxicogenomic data to the AhR-signaling mechanism, as well as
facilitates cross-species comparisons and more comprehensive microarray data
interpretation. However, computationally identified putative DREs have a high
false positive rate, which is further confounded by the frequent occurrence of the
DRE core element (GCGTG) in the genome. The identification of positionally
conserved DREs (the core plus flanking sequences) within orthologous human,
mouse, and rat genes partially addresses this limitation by increasing the
probability of identifying functional DREs. We use a position weight matrix
(PWM) to identify putative DREs within regulatory regions (-10,000bp relative to
the transcription start site through the 5’-UTR) of species-conserved and speciesspecific dioxin-responsive genes. Agglomerative hierarchical clustering of the
DRE sequences across the human, mouse, and rat is then used to identify
conserved DREs. In addition, an empirical distance threshold decreases the
false positive clustering rate, and a map of the DREs across the three
orthologous genes illustrates the positionally conserved orthologous DREs.
Maps for conserved dioxin responsive genes, such as Cyp1a1, Cyp1b1, and
Aldh3a1 identify the location of positionally conserved DREs in human, mouse
and rat regulatory regions. This approach is being expanded to prioritize speciesspecific dioxin-responsive genes that warrant further validation. This method in
combination with the identification of other regulatory elements may facilitate the
elucidation of the regulatory grammar required for AhR-mediated gene regulation.
Funded by NIEHS Superfund grant P42 ES04911.
The AhR Gene Battery of Bona Fide Dioxin Response Elements
Gene
Site
DRE Sequence (5'-3')
Cyp1a1
A
CAAGCTCGCGTGAGAAGCG
Cyp1a1
B
CCTGTGTGCGTGCCAAGCA
Cyp1a1
C
GAGGCTAGCGTGCGTAAGC
Cyp1a1
D
CGGAGTTGCGTGAGAAGAG
Cyp1a1
E
CCAGCTAGCGTGACAGCAC
Cyp1a1
F
CGGGTTTGCGTGCGATGCT
Cyp1a1
XRE 1 CGGAGTTGCGTGAGAAGAG
Cyp1a1
XRE 2 CCAGCTAGCGTGACAGCAC
Cyp1b1
XRE 5 CCCCCTTGCGTGCGGAGCT
Aldh3a1
XRE
TGATGGGGCGTGCATTAGT
Nqo1
XRE
TCCCCTTGCGTGCAAAGGC
Sod1
XRE
GAGGCCTGCGTGCGCGCCT
Gsta2
XRE
GCATGTTGCGTGCATCCCT
Ugt1a6
XRE
AGAATGTGCGTGACAAGGT
Table 1: List of Bona Fide
Genes and DREs
This list represents bona fide and
genome database cross-referenced
2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) responsive genes and their
dioxin response elements (DREs)
reported in the literature.
A
Species-Specific TCDD Responses
Dioxin Response Element Alignment Method
Pak1
B
C
Site
Sequence Difference
DRE Distance
(kb)
1
0
59
2
0
18
3
2.65
68
4
3
107
5
2
60
6
2.24
4
7
0
59
8
3.32
6930
9
3.32
58
10
3.61
156
11
0
59
12
4.12
81
Figure 2: Identification of Putative Positionally Conserved Orthologous DREs in the
Human, Mouse, and Rat Cyp1a1 Gene
A) The PWM-based search of the Cyp1a1 gene regulatory region (-10,000 relative to the TSS
through the 5’-UTR) identified 12 putative positionally conserved orthologous DREs. B)
Putative DRE (matrix similarity score > 0.80) sequences were clustered by agglomerative
hierarchical clustering using Euclidean distance to evaluate the degree of sequence similarity
(sequence difference). Highly similar putative DREs are defined as having a Euclidean
distance less than 4.20 units with scores of 0 representing identical sequences. C) Sequence
differences, and the distance (in kb) between the highly similar putative DREs. 11 of these
sites (1-7, 9-12) are identified as putative positionally conserved orthologous DREs.
Lpl
Figure 4: Species-Specific TCDD Responsive Genes Lack Strong Correlation with
Positionally Conserved DREs
The rat-specific Pak1, and the mouse-specific Lpl, lack positionally conserved DREs. Generally,
species-specific TCDD responsive genes have few positionally conserved DREs. These cases
warrant further investigation using ChIP techniques and reporter gene assays to investigate the
role of the AhR and the computationally identified putative in these responses.
The mouse Lpl core DRE at -329 (arrow) narrowly missed the putative DRE matrix similarity
score cut-off (matrix similarity score = 0.7997).
Confirmation of In Vivo Species-Specific Responses by cDNA Microarray
Lpl
Pak1
Species-Conserved TCDD Responses
Aldh3a1
Cyp1b1
Literature and Genome Reconciled PWM
Figure 5: Gene Expression Confirms the Species-Specificity of the TCDD Responses
The rat-specific Pak1 and the mouse-specific Lpl both show species-specific expression. Pak1
is expressed at early time points, with levels returning to baseline (vehicle) levels by 8hrs. Lpl
is expressed at mid and late time points within the mouse (18-168hrs), with increasing
expression as a function of time.
Summary
• Combining the PWM search with agglomerative hierarchical clustering identified putative
positionally conserved DREs.
• Species-conserved TCDD responsive genes from the AhR gene battery tended to show more
positional conservation between mouse and rat, than compared to the human genes.
Nqo1
• The number of positionally conserved putative DREs tended to be fewer in the species-specific
responsive genes.
• This suggests that species-specific responsive genes may be regulated by species-specific
regulons, or through mechanisms secondary to AhR activation.
Figure 1: Weblogo of the New Position Weight Matrix (PWM)
This PWM was constructed based on the bona fide DREs, and the -2kb DRE of
Aldh3a1. The new PWM differs from the previous one in that it is consistent with the
more widely accepted consensus sequence of TNGCGTG.
Figure 3: Positionally Conserved DREs in Species-Conserved TCDD Responsive Genes
Several positionally conserved DREs were identified in the species-conserved responsive
genes. Nqo1 showed the fewest positionally conserved DREs.
This work supported in part by the Superfund Basic Research Program: P42 ES04911
● E-mail: [email protected]
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