MOF maintains Transcriptional Programs
regulating Cellular Stress Response
Bilal N. Sheikh1#, Wibke Bechtel-Walz2#, Jacopo Lucci1#, Oleksandra Karpiuk1, Iris
Hild2, Björn Hartleben2, Julia Vornweg2, Martin Helmstädter2, Abdullah H. Sahyoun1,
Vivek Bhardwaj1, Thomas Stehle1, Sarah Diehl1, Oliver Kretz2, Anne K. Voss3,4, Tim
Thomas3,4, Thomas Manke1, Tobias B. Huber2,5 and Asifa Akhtar1 *
Supplementary Information
Contents of Supplementary Information
1. Supplementary Figures and Supplementary Figure legends
2. Supplementary Table legends
1
Supplementary Figure 1
Analysis of Mof knockout MEFs
(a) DNA genotyping gel depicting the recombined Mof allele in Moffl/fl;Cre-ERT2T/+
samples after 96 hours of 4-hydroxy tamoxifen (4OHT) treatment. Control samples
did not show any recombination. Gender of embryos used to establish individual MEF
lines were determined through the presence of the Y-linked gene Sry. (b)
2
Quantification of Mof mRNA levels after Mof-deletion and in controls. Mof mRNA
was reduced to undetectable levels in Moffl/fl;Cre-ERT2T/+ samples after 4OHT
treatment. Control samples continued to express normal levels of Mof mRNA. (c)
Growth curves of control MEFs. The presence of Cre-ERT2 or treatment with
tamoxifen alone had no affect on the growth characteristics of MEFs. (d,e) Mofdeleted and control MEFs stained for β-galactosidase activity with the β-galactosidase
substrate C12FDG. Data were analyzed by flow cytometry. (d) Senescent cells showed
an approximately 6.8-fold increase in GFP signal, which represents processed
C12FDG. (e) Histograms showing C12FDG expression. No differences in βgalactosidase activity were observed between MEFs lacking Mof and control cells. (f)
Expression of senescence markers Ink4a, Arf and p21. Expression levels were
normalized to housekeeping genes Hsp90ab1 and Gapdh. A modest increase was
observed in Ink4a, but not in Arf or p21 expression upon Mof deletion.
Data are presented as mean ± s.e.m. Data were analyzed using ANOVA, followed by
a student t-test. Asterisks denote statistical significance at * p < 0.05. 4OHT – 4hydroxy tamoxifen.
3
Supplementary Figure 2
Mof deletion before RNA-sequencing analysis
Recombination of the Mof allele in Moffl/fl;Cre-ERT2T/+ samples was induced by
treating MEFs with 4OHT for 3.5 days. Control Moffl/fl cells were also treated with
4OHT as a control. (a) Quantification of the wild type Mof allele at the genomic level.
The wild type Mof allele was depleted in Moffl/fl;Cre-ERT2T/+ samples but not in
control Moffl/fl cells after 4OHT treatment. (b) Quantification of the knockout Mof
allele at the genomic level. The deleted Mof allele was detected in Moffl/fl;CreERT2T/+ MEFs but not in Moffl/fl controls after 3.5 days of treatment with 4OHT. (c)
Quantification of Mof mRNA after 4OHT treatment. Mof mRNA was reduced by
more than 95% in Moffl/fl;Cre-ERT2T/+ cells compared to controls, both in the
presence and absence of Adriamycin.
Data are presented as mean ± s.e.m. Data were analyzed using ANOVA, followed by
a student t-test. Asterisks denote statistical significance at *** p < 0.001. 4OHT – 4hydroxy tamoxifen.
4
Supplementary Figure 3
RNA-sequencing analysis of Mof-deleted and control MEFs
(a) Number of genes bound by MOF in human CD4 cells. Genes upregulated and
downregulated in Mof-deleted MEFs are separately presented. (b) The top 50 Broad
Institute datasets represented as a bubble-plot. The full list can be found in
Supplementary Table 23. The top 50 datasets were all under-expressed in Moffl/fl;CreERT2T/+ MEFs and were most highly enriched for datasets involved in “cell cycle”,
“DNA repair” and “nuclear” genes. The size of the bubble represents the number of
genes in each of the Broad Institute datasets. The significance of correlation is
presented on the y-axis.
5
Supplementary Figure 4
(a) Microarray analysis of gene expression in MEFs following Mof deletion. Heat
maps displaying the absolute intensities measured in microarray experiments for 92
cell cycle related
upon Mof deletion (left panel) and the relevant log-2 fold change (right panel).
Colored boxes in the first column associate the relevant gene to the phase of cell cycle
in which it is know to achieve the highest expression level. Black boxes in the last
column describe whether the promoter of the relevant gene is scored as bound in
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CD4+ cells. (b) Doughnut chart displaying differential expression of all cell cycle
related genes in MOF depleted MEFs. A pool of genes displaying a logFC≤ -1 (n=234
in females, n= 128 in males, 87 in common) was analyzed. WT – wild type (Moffl/fl),
KO – Moffl/fl;Cre-ERT2T/+ MEFs treated with 4OHT. MOF ChIP profiles human
CD4+ cells were obtained from Ref. 1. n = 3 independent MEF lines per genotype for
each sex.
7
Supplementary Figure 5
Isolation and culture of podocytes from 6-week old mice
(a)
Schematic
representation
of
glomeruli
isolation
protocol.
(b)
Immunohistochemistry of kidney sections isolated from 8 week old Moffl/fl;Nphs2CreT/+;tomatofl/+>eGFPT/+ mice. Tomato expressing cells represent the cell population
with inactive Cre recombinase, while GFP-positive cells represent podocytes with
active Nphs2-Cre transgene. Scale bars equal 20 µm (left image) and 5 µm (right
image). (c) Sorting strategy for the isolation of primary podocytes by FACS.
Podocytes were sorted based on their GFP expression.
8
Supplementary Figure 6
Mof mRNA levels after knockdown
Mof mRNA levels in differentiated MPC5 podocytes after infection with a lentivirus
containing a Mof shRNA construct. Infected cells were selected over four days using
puromycin.
Data are presented as mean ± s.e.m. Data were analyzed using ANOVA, followed by
a student t-test. Asterisks denote statistical significance at *** p < 0.001.
9
Supplementary Figure 7
Standardization of Adriamycin and Puromycin dose
(a) Images depicting the effects of Adriamycin on differentiated MPC5 podocytes and
MEFs. The ideal dose of Adriamycin was determined by treating cells over a 24-hour
period. The highest dose, which induced little to no cell death, was used. For
differentiated MPC5 podocytes, this was determined to be 0.25 g/ml, which is the
same as used in other studies2-4. For MEFs, which are a proliferative cell population
and therefore more likely to be sensitive to Adriamycin, the ideal dose was
determined to be 0.10 g/ml. (b) Determination of Puromycin dosage for selection
after infection with Mof and Scramble shRNA constructs. The lowest concentration
that killed the vast majority of uninfected MPC5 podocytes within a 24-hour period
was selected (2 g/ml).
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11
Supplementary Figure 8
Downregulated genes in the lysosome pathway
Genes in the KEGG annotated lysosome pathway that were downregulated in Mofknockdown MPC5 podocytes after Adriamycin treatment. Downregulated genes are
indicated with red stars. Analyses were carried out using the DAVID platform5,6.
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Supplementary Figure 9
Downregulated genes in the endocytosis pathway
Genes in the KEGG annotated endocytosis pathway that were downregulated in Mofknockdown MPC5 podocytes after Adriamycin treatment. Genes downregulated are
indicated with red stars. Analyses were carried out using the DAVID platform5,6.
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Supplementary Figure 10
Gene expression changes and MOF, NSL and MSL binding in pathways affected
by the loss of Mof
The heatmaps represent gene expression changes in stressed Mof-knockdown versus
Scramble control podocytes associated with the (a) KEGG extracellular matrix
(ECM) pathway, (b) KEGG lysosome pathway, (c) KEGG endocytosis pathway, and
(d) genes present in the gene ontology category vacuole. Genes were included in
these analyses if they showed a FDR of less than 0.05 in the Mof-knockdown versus
Scramble control comparison. Gene lists from the KEGG and gene ontology
(biological process) databases were obtained through the DAVID platform5,6. Boxes
on the left-hand side of each heatmap represent binding of MOF (black), NSL
complex members (red) and MOF-MSL complex members (green). The MOF-NSL
complex bound a significant number of deregulated genes in the lysosome,
endocytosis and vacuole pathways.
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Supplemental Table Legends
Supplementary Table 1
Number of reads per library made from Moffl/fl;Cre-ERT2T/+ and control Moffl/fl MEFs.
Supplementary Table 2
Genes differentially expressed in Moffl/fl;Cre-ERT2T/+ MEFs compared to Moffl/fl
controls after Mof recombination using 4-hydroxy tamoxifen (4OHT). A cut-off of
FDR < 0.05 and an absolute log2 fold change of more than 0.5 was used.
Supplementary Table 3
GO term analysis (biological processes) for genes differentially expressed in
Moffl/fl;Cre-ERT2T/+ MEFs compared to Moffl/fl controls treated with 4OHT. Analyses
were carried out using the DAVID platform5,6. N = 3 MEF lines per genotype. All
cells were treated with 4OHT.
Supplementary Table 4
GO term analysis (cellular component) for genes differentially expressed in
Moffl/fl;Cre-ERT2T/+ MEFs compared to Moffl/fl controls treated with 4OHT. Analyses
were carried out using the DAVID platform5,6. N = 3 MEF lines per genotype. All
cells were treated with 4OHT.
Supplementary Table 5
Number of reads per library from Mof knockdown and Scramble controls in MPC5
cell line, each group with Adriamycin and vehicle treatments. N = 3 per group.
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Supplementary Table 6
Genes differentially expressed in Mof knockdown samples, compared to Scramble
controls, after vehicle treatment. A cut-off of FDR < 0.05 and an absolute log2 fold
change of more than 0.5 was used. N = 3 per group.
Supplementary Table 7
KEGG pathway analysis for genes downregulated in Mof knockdown MPC5 cells
compared to Scramble controls, after vehicle treatment. Analyses were carried out
using the DAVID platform5,6. N = 3 per group.
Supplementary Table 8
GO-term analyses (biological processes) for genes downregulated in Mof knockdown
MPC5 cells compared to Scramble controls, after vehicle treatment. Analyses were
carried out using the DAVID platform5,6. N = 3 per group.
Supplementary Table 9
GO-term analyses (cellular compartment) for genes downregulated in Mof knockdown
MPC5 cells compared to Scramble controls, after vehicle treatment. Analyses were
carried out using the DAVID platform5,6. N = 3 per group.
Supplementary Table 10
KEGG pathway analysis for genes upregulated in Mof knockdown MPC5 cells
compared to Scramble controls, after vehicle treatment. Analyses were carried out
using the DAVID platform5,6. N = 3 per group.
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Supplementary Table 11
GO-term analyses (biological processes) for genes upregulated in Mof knockdown
MPC5 cells compared to Scramble controls, after vehicle treatment. Analyses were
carried out using the DAVID platform5,6. N = 3 per group.
Supplementary Table 12
GO-term analyses (cellular compartment) for genes upregulated in Mof knockdown
MPC5 cells compared to Scramble controls, after vehicle treatment. Analyses were
carried out using the DAVID platform5,6. N = 3 per group.
Supplementary Table 13
Genes differentially expressed in Mof shRNA treated samples compared to Scramble
controls, after Adriamycin treatment. A cut-off of FDR < 0.05 and a log2(fold change)
of more than log2 | 0.5 | was used. N = 3 per group.
Supplementary Table 14
GO-term enrichment analyses (biological processes) for genes downregulated in Mof
knockdown MPC5 cells compared to Scramble controls, after Adriamycin treatment.
Analyses were carried out using the DAVID platform5,6. N = 3 per group.
Supplementary Table 15
GO-term enrichment analyses (cellular compartment) for genes downregulated in Mof
knockdown MPC5 cells compared to Scramble controls, after Adriamycin treatment.
Analyses were carried out using the DAVID platform5,6. N = 3 per group.
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Supplementary Table 16
KEGG pathway analysis for genes upregulated in Mof knockdown MPC5 cells
compared to Scramble controls, after Adriamycin treatment. Analyses were carried
out using the DAVID platform5,6. N = 3 per group.
Supplementary Table 17
GO-term enrichment analyses (biological processes) for genes upregulated in Mof
knockdown MPC5 cells compared to Scramble controls, after Adriamycin treatment.
Analyses were carried out using the DAVID platform5,6. N = 3 per group.
Supplementary Table 18
GO-term enrichment analyses (cellular compartment) for genes upregulated in Mof
knockdown MPC5 cells compared to Scramble controls, after Adriamycin treatment.
Analyses were carried out using the DAVID platform5,6. N = 3 per group.
Supplementary Table 19
Number of reads per library generated from Moffl/fl;Cre-ERT2T/+ and control Moffl/fl
MEFs RNA after Adriamycin treatment. N = 3 independent MEF cell lines, each
generated from a different embryo, per genotype.
Supplementary Table 20
Genes differentially expressed in Moffl/fl;Cre-ERT2T/+ MEFs compared to Moffl/fl
controls after 4OHT and 24 hours of Adriamycin treatment. A cut-off of FDR
< 0.05 and an absolute log2 fold change of more than 0.5 was used. N = 3
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independent MEF cell lines per genotype, each generated from a different
embryo.
Supplementary Table 21
GO-term enrichment analyses (biological processes) for gene expression changes in
Moffl/fl;Cre-ERT2T/+ MEFs compared to Moffl/fl controls after 4OHT treatment
followed by 24 hours of Adriamycin treatment. Analyses were carried out using the
DAVID platform5,6. N = 3 independent MEF cell lines, each generated from a
different embryo, per genotype.
Supplementary Table 22
GO-term enrichment analyses (cellular compartment) for gene expression changes in
Moffl/fl;Cre-ERT2T/+ MEFs compared to Moffl/fl controls after 4OHT treatment,
followed by 24 hours of Adriamycin treatment. Analyses were carried out using the
DAVID platform5,6. N = 3 independent MEF cell lines, each generated from a
different embryo, per genotype.
Supplementary Table 23
Datasets curated by the Broad Institute that were highly enriched in Moffl/fl;CreERT2T/+ MEFs compared to Moffl/fl controls (vehicle treated). A significance cut-off
of 0.01 was applied.
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