Agrawal et al.
ELECTRONIC SUPPLEMENTARY MATERIAL:
Compound mouse mutants of bZIP transcription factors Mafg and Mafk reveal a
regulatory network of non-crystallin genes associated with cataract
Smriti A. Agrawal1†, Deepti Anand1†, Archana D. Siddam1†, Atul Kakrana2,3, Soma
Dash1, David A. Scheiblin1, Christine A. Dang1, Anne M. Terrell1, Stephanie M. Waters1,
Abhyudai Singh2,4, Hozumi Motohashi5, Masayuki Yamamoto6, Salil A. Lachke1,2**
1
Department of Biological Sciences, University of Delaware, Newark, Delaware, USA
2
Center for Bioinformatics and Computational Biology, University of Delaware,
Newark, Delaware, USA
3
Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USA
4
Department of Electrical Engineering, University of Delaware, Newark, Delaware, USA
5
Department of Gene Expression Regulation, Institute of Development, Aging and
Cancer, Tohoku University, Sendai, Japan
6
Department of Medical Biochemistry, Graduate School of Medicine, Tohoku
University, Sendai, Japan
†
S. A. Agrawal, D. Anand, A. D. Siddam contributed equally.
Keywords: Cataract, Mafg, Mafk, lens, iSyTE, Gene Regulatory Network
**Corresponding author:
Salil A. Lachke, Ph.D.
Department of Biological Sciences
University of Delaware
Newark, DE 19716 USA
Tel.: (617) 959 9193
Fax: (302) 831 2281
E-mail: salil@udel.edu
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Agrawal et al.
LEGEND TO SUPPLEMENTARY FIGURES
Figure S1. Marker analysis of Mafg-/-:Mafk+/- mouse mutant lens. Immunostaining
with antibodies against marker proteins for lens epithelium (Foxe3, E-cadherin) and lens
fiber cells (Cryg) was performed for adult Mafg-/-:Mafk+/- mouse mutant and control
lens. No significant differences were observed between mutant and control for any of the
markers tested. Scale bar denotes 70 m; e, epithelium; f, fiber cells.
Figure S2. Cluster analysis for differentially regulated genes identified in Mafg-/:Mafk+/- mouse mutant lens. Following functional enrichment analysis with Database for
Annotation, Visualization and Integrated Discovery (DAVID, Bioinformatics Resources
6.7) database, cluster analysis was performed for genes that are found to be, A. downregulated, and B. up-regulated, in Mafg-/-:Mafk+/- mutant lens.
Figure S3. Expression of genes encoding small Maf binding co-regulatory proteins in the
lens. Mouse lens microarray datasets at various embryonic and postnatal developmental
stages were analyzed and compared to expression in the whole embryonic body (WB)
tissue reference data as per the iSyTE approach to examine the expression of small Maf
binding partners. While Bach1, Bach2, Nfe2l1, and Nfe2l2 are significantly expressed in
mouse lens Nfe2l3 and Nfe2 are absent. Moreover, Bach2, Nfe2l1 and in some stages,
Nfe2l2 were found to be lens-enriched. In microarray analysis, genes with values below
200 were not considered to have significant expression. Asterisk denotes p<0.002.
Figure S4. De novo motif identification reveals putative bZIP transcription factor binding
sites in genes differentially regulated in Mafg-/-:Mafk+/- mutant lens. Motif enrichment
analysis in the 2.5kb upstream region of Mafg-/-:Mafk+/- differentially regulated genes
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Agrawal et al.
identifies the presence of ARE, MARE and NF-E2 binding motifs, which closely match
their respective consensus sequences (left panel). Start and end locations of the sequence
are provided while their fold-change in mutant lens is indicated on right. Letters in grey
indicate divergent nucleotides from the consensus motif. Asterisk indicates genes
containing more than one motif.
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Figure S1. Marker analysis of Mafg-/-:Mafk+/- mouse mutant lens.
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Agrawal et al.
Figure S2. Cluster analysis for differentially regulated genes identified
in Mafg-/-:Mafk+/- mouse mutant lens.
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Figure S3. Expression of genes encoding small Maf binding co-regulatory
proteins in the lens.
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Figure S4.
De novo motif identification in differentially regulated genes in Mafg-/:Mafk+/- mutant lens.
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Agrawal et al.
SUPPLEMENTARY TABLE 1
Differentially regulated genes in Mafg-/-:Mafk+/- lens compared to control
Gene
Fold change in Mafg-/-:Mafk+/- lens
Dctn4
3.5
Ddit3
3
Fdps
2.7
A630042L21Rik
2.5
Hmox1
2.5
1700020N01Rik
2.4
Cyp51
2.3
Sqle
2.3
Chrng
2.3
Sc4mol
2
Xbp1
2
Gm106
2
Klhl9
1.9
BC032265
1.9
Tmem14c
1.9
Psmb5
1.8
Lyzl4
1.8
Cyp26a1
1.7
Hopx
1.7
Rps3
1.7
Hsd17b7
1.7
Creld2
1.6
Manbal
1.6
Cdk2ap2
1.6
Stard4
1.6
Kctd12
1.6
Dnajb9
1.6
4930511J11Rik
1.6
Arsi
1.6
Aacs
1.5
Plk3
1.5
Pnpla3
1.5
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Agrawal et al.
Insig1
1.5
E130203B14Rik
1.5
Kdelr2
1.5
Grpel1
1.5
Hsp90b1
1.5
Supt16h
1.5
Klhl22
1.5
2400003C14Rik
1.5
Tmem55a
1.5
Calr
1.5
Vegfb
1.5
Apoe
-1.5
Smad1
-1.5
Tmem218
-1.5
Calr4
-1.5
Lrrc66
-1.5
Mgst1
-1.5
C1qtnf2
-1.5
1110036O03Rik
-1.5
Tfpi
-1.5
Sspn
-1.5
Gluld1
-1.5
Uchl1
-1.5
Igfbp5
-1.5
Txnip
-1.5
Pcbd2
-1.5
Pcbd1
-1.5
Hdgf
-1.5
Gsto1
-1.5
Mapk14
-1.5
Crygf
-1.5
4632417K18Rik
-1.5
Dgat2
-1.5
Stambpl1
-1.5
Mdk
-1.5
Aldh1a3
-1.6
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Agrawal et al.
Deadc1
-1.6
Metrn
-1.6
Mxd4
-1.6
Nudt7
-1.6
Ccdc53
-1.6
Ubr5
-1.6
Wfdc1
-1.6
Serping1
-1.6
Hspb1
-1.6
Mt1
-1.6
Ccdc23
-1.6
Serpina3h
-1.6
Ccng1
-1.7
5730507A09Rik
-1.7
Abcc5
-1.7
Fn3k
-1.7
Gp2
-1.7
Vamp8
-1.8
2610024G14Rik
-1.8
Sfrs14
-1.8
Bgn
-1.8
Itgb1bp1
-1.8
Pbld
-1.9
Trex1
-2
Alox5ap
-2
OTTMUSG0000000
5065
OTTMUSG0000000
1070
Lims2
-2.1
Mafg
-3.7
Aldh3a1
-3.8
Ttc27
-5.1
-2.2
-2.4
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SUPPLEMENTARY TABLE 2
DAVID analysis for differentially regulated lens in Mafg-/-:Mafk+/- mutant lens.
SUPPLEMENTAL TABLE 2
DAVID analysis for genes up-regulated in Mafg-/-:Mafk+/- mutant lens
Category
Term
GOTERM_BP_FAT
GO:0008610~lipid biosynthetic process
GOTERM_BP_FAT
GO:0016126~sterol biosynthetic process
GOTERM_BP_FAT
GO:0006457~protein folding
SP_PIR_KEYWORDS Chaperone
GOTERM_MF_FAT
GO:0051082~unfolded protein binding
GOTERM_BP_FAT
GO:0006979~response to oxidative stress
GOTERM_BP_FAT
GO:0043433~negative regulation of transcription factor activity
Count
6
5
4
4
3
3
2
% PValue
15.4 3.08E-04
12.8 4.41E-07
10.3
0.00244
10.3 0.003124
7.69 0.008819
7.69 0.014708
5.13 0.045951
DAVID analysis for genes down-regulated in Mafg-/-:Mafk+/- mutant lens
Category
Term
Count % PValue
SP_PIR_KEYWORDS acetylation
14
28.6 0.005749
GOTERM_CC_FAT
GO:0005576~extracellular region
10
20.4 0.017632
GOTERM_MF_FAT
GO:0030414~peptidase inhibitor activity
4
8.16 0.010956
GOTERM_BP_FAT
GO:0045597~positive regulation of cell differentiation
3
6.12 0.078149
BIOCARTA
m_arenrf2Pathway:Oxidative Stress Induced Gene Expression Via Nrf2
2
4.08
0.09368
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Genes
CYP51, INSIG1, FDPS, PNPLA3, HSD17B7, SC4MOL
CYP51, INSIG1, FDPS, HSD17B7, SC4MOL
GRPEL1, HSP90B1, DNAJB9, CALR
GRPEL1, HSP90B1, DNAJB9, CALR
HSP90B1, DNAJB9, CALR
PSMB5, HMOX1, DDIT3
HMOX1, DDIT3
Genes
MAFG, PCBD2, PCBD1, UCHL1, FN3K, HDGF, SMAD1, ALDH3A1, VAMP8, ALDH1A3, MT1, HSPB1, GSTO1, MGST1
METRN, BGN, C1QTNF2, GP2, HDGF, TFPI, WFDC1, SERPING1, MDK, IGFBP5
SERPINA3H, TFPI, WFDC1, SERPING1
METRN, MAPK14, SMAD1
MAFG, MAPK14