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Supplementary Material
Genetic Inactivation of Cdk7 Leads to Cell Cycle Arrest and Induces Premature
Aging Due to Adult Stem Cell Exhaustion
Miguel Ganuza, Cristina Sáiz-Ladera, Marta Cañamero, Gonzalo Gómez, Ralph
Schneider, María A. Blasco, David Pisano, Jesús M. Paramio, David Santamaría and
Mariano Barbacid
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Figure S1. Generation of Cdk7lox and Cdk7mut alleles
(A) Schematic representation of the generation of the Cdk7lox and Cdk7mut alleles from
the Cdk7loxfrt allele present in ES clone D032B11 (German Gene Trap Consortium).
(Top) Representation of the Cdk7loxfrt null allele generated by the insertion of the
rFlipROSAbgeo gene-trap cassette within intron 2, 1.52 kbp upstream of exon 3. The
gene-trap cassette is in the active orientation, thus the endogenous Cdk7 transcript is
captured and prematurely terminated at the polyadenylation site (pA). (Middle)
Representation of the conditional Cdk7lox allele generated by Flpe-mediated
recombination of Cdk7loxfrt. The Flpe recombinase inverted the cassette to the inactive
orientation, reactivating normal splicing and allowing expression of the Cdk7 locus.
(Bottom) Representation of the null Cdk7mut allele generated by Cre-mediated
recombination of Cdk7lox. The Cre recombinase repositioned the gene-trap in the sense
orientation, thereby eliminating Cdk7 expression. Although this allele is null, we have
used Cdk7mut instead of the classical Cdk7– designation to indicate that no Cdk7
sequences have been ablated from this allele. Thick lines represent transcripts initiated
at the endogenous promoter. The frt (open triangles) and f3 (solid triangles) sequences
are target sequences for the Flpe recombinase. The loxP (red triangles) and 5171 (cian
triangles) sequences are recognized by the Cre recombinase. SA: splice acceptor; geo:
beta-galactosidase/neomycin phosphotransferase fusion gene; EV: EcoRV restriction
site. The expected genomic sizes upon EcoRV digestion are also indicated for each
allele. E2 and E3 represent the relative location of exons 2 and 3 within the Cdk7 locus.
Sizes are not drawn to scale.
(B) Representative Southern blot analysis of genomic DNA obtained from mice
carrying the Cdk7loxfrt, Cdk7lox and Cdk7mut alleles. DNAs were digested with EcoRV
and probed with a 409 bp fragment adjacent to the genomic EcoRV site. The size of the
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diagnostic bands corresponding to these alleles is indicated by arrows. The wild type
allele (17 kbp) is not depicted in the figure. A molecular weight marker (MW) is
included.
(C) Quantification of Cdk7 mRNA levels in Cdk7lox/lox primary MEFs (n=4) and
keratinocytes (n=4) seven days after infection with Ad-GFP (solid bars) or Ad-Cre
(empty bars). Data shown as mean ± s.d and normalized to Cdk7 mRNA levels for each
Ad-GFP paired control.
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Figure S2 Cdk7 is essential for cell proliferation and expression of E2F-dependent
genes but not RNA pol II-mediated global transcription in primary MEFs
(A) Proliferation of primary MEFs following infection with Ad-Cre (solid circles) or
Ad-GFP (empty circles) as a control. Data shown as mean ± s.d., n=3.
(B) Percentage of Cdk7lox/lox quiescent MEFs after infection with Ad-Cre (solid circles)
or Ad-GFP particles (empty circles) entering S-phase at the indicated time points
following addition of serum. Data shown as mean ± s.d., n=3.
(C) Immunoblot analysis of Cdk7, CycH and Mat1 in extracts of primary Cdk7lox/lox
MEFs infected with Ad-Cre or Ad-GFP. Samples from two independent experiments
are shown.
(D) Cdk7lox/lox MEFs were infected with Ad-Cre or Ad-empty controls and after seven
days subsequently infected with a lentiviral CMV-GFP reporter. The intensity of the
GFP signal was assessed by FACS after 72 hrs. A representative experiment (left)
including a GFP-negative control (lenti-CMV-empty) together with the quantification of
n=5 replicates (right) are shown. Data shown as mean ± s.d.
(E) Incorporation of [35S]methionine in Cdk7lox/lox MEFs infected with either Ad-Cre
(solid boxes) or Ad-GFP (empty boxes) and maintained in culture for one additional
week. In order to match the proliferation rates both cultures were shifted to low serum
(0.1% FBS) conditions for 72 hrs, incubated with [35S]methionine for the indicated
times, lysed and TCA precipitated. Incorporation of [35S]methionine was measured by
liquid scintillation counting. Data shown as mean ± s.d., n=3.
(F) Gene expression profiling of housekeeping and E2F target genes in Ad-Cre vs AdGFP infected Cdk7lox/lox MEFs. Representative examples of GSEA profiles
corresponding to the housekeeping (left), E2F total targets (middle) and E2F DNA
replication targets (right) gene-sets.
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(G) Immunoblot analysis of Cdk7, and Gapdh in extracts of primary Cdk7lox/lox MEFs
infected with Ad-Cre or Ad-GFP prepared at the time of RNA collection for gene
expression profiling. Samples from two independent experiments are shown.
(H) Summary of the gene expression profiling results. Status column: horizontal line
stands for no change, down-headed arrow represents reduced gene expression in Ad-Cre
infected samples compared to Ad-GFP controls. Differences are statistically significant
if FDR (q-value) is ≤ 0,25. Samples from eight independent experiments were analysed.
(I) Southern blot analysis of genomic DNA isolated from individual colonies of
Cdk7lox/lox MEFs infected with Ad-Cre and rescued by ectopic expression of cDNAs
encoding the indicated proteins. Genomic DNAs from Cdk7lox/lox MEFs infected with
Ad-Cre or Ad-GFP were included as controls for the migration of the Cdk7lox (4.0 kbp)
and Cdk7mut (2.4 kbp) alleles, respectively. A molecular weight marker (MW) was also
included.
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Figure S3 Characterization of defects present in Cdk7 null embryos.
(A) Immunochemical staining of consecutive sections of E5.5 deciduas obtained from
crosses among Cdk7+/loxfrt parentals. All embryos lacking Cdk7 positive staining showed
Active Caspase 3. Bar, 20 µm.
(B) Cdk7+/loxfrt and Cdk7loxfrt/loxfrt embryos were isolated at E2.5 and cultured in vitro for
5 days (E7.5 stage). Cdk7 was detected by immunofluorescence (green). Nuclei were
stained with Hoechst 33342 (blue). Bar, 100 µm.
(C) Quantification of the nuclear volume of individual trophoblast cells in the above
Cdk7+/loxfrt and Cdk7loxfrt/loxfrt E7.5 embryos on digital images upon Hoechst 33342
staining.
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Figure S4 Characterization of biochemical parameters upon wide spread
elimination of Cdk7 expression in adult mice
Cdk7+/lox;Ub-CreERT2+/T (solid bars) and Cdk7lox/lox;Ub-CreERT2+/T (open bars)
littermate controls were fed ad libitum with a tamoxifen diet and the indicated
parameters measured in plasma at 4 and 8 months of age. B.U.N., blood ureic nitrogen.
Data shown as mean ± s.d., n=4.
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Figure S5 Survival and age-related renal pathologies in adult mice lacking Cdk7
expression
(A) Analysis of Cre-mediated recombination of the Cdk7lox allele in a subset of
proliferating (PT) and non-proliferating (NPT) tissues from eight month old
Cdk7+/lox;Ub-CreERT2+/T animals exposed to a tamoxifen diet for seven months. DNAs
were digested with EcoRV and probed with a 409 bp fragment adjacent to the genomic
EcoRV site. The wild type allele (17 kbp) is not depicted in the figure. Samples from
two independent animals are shown. Tissues include kidney (Ki), liver (Li), brain (Br),
skin (Sk), colon (Co) and small intestine (In). Small intestine from a control (Ct)
Cdk7+/lox;Ub-CreERT2+/T littermate not exposed to the tamoxifen diet is included for
reference.
(B) Survival curve of Cdk7+/lox;Ub-CreERT2+/T (solid circles) and Cdk7lox/lox;UbCreERT2+/T (open circles) littermates fed ad libitum with a tamoxifen diet since
weaning.
(C) H&E stained sections of medulla and cortex regions of kidneys obtained from
eight-month old Cdk7+/lox;Ub-CreERT2+/T and Cdk7lox/lox;Ub-CreERT2+/T littermates
exposed to a tamoxifen diet for seven months. Bar, 50 µm.
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