SUPPLEMENTARY DATA FILE LEGENDS
Supplementary Figure 1. Specificity of E2f3 antibody. Total protein from neurospheres at stage
E14.5 from wild-type, E2f3-/- or E2f3a-/- mice were extracted and subjected to Electrophoretic
Mobility Shift Assay, as previously described9, to measure interaction with an E2F binding
motif-containing radiolabelled probe. Addition of specific antibodies identifies the banding
pattern of individual E2f proteins due to altered migration of protein-DNA complexes in the
presence of bound antibody. The location of bands corresponding to E2f4, E2f5, pRb and p107
are labeled as previously identified62. ‘Cold competitor’ is an EMSA experiment run in the
presence of 100-fold excess unlabeled DNA probe. Among the bands corresponding to free E2fs
bound to DNA, the E2f3 antibody super-shifts only the band corresponding to E2f3&1. The band
intensity is reduced but not eliminated (due to the presence of E2f1 complexes), and is similar to
that observed in the E2f3-/- neurospheres.
Supplementary Figure 2. Specificity of the E2f4 antibody. Total protein from telencephalon of
wild-type or E2f4-/- mice at stage E12.5, or from wild-type NPCs (cultured neurospheres) at
E14.5, were extracted, run on SDS-PAGE and probed by western blot for E2f4 using the sc-866
antibody. The predominant band at approximately 65 kDa is undetectable in E2f4-/- tissue.
Supplementary Figure 3. Distribution of E2f3 and E2f4 bound loci relative to TSSs across
mouse chromosome 7
Pilot ChIP-chip experiments were performed to identify E2f3 and E2f4 binding sites across the
entire mouse chromosome 7 and to determine their distribution with regards to TSS. E2f bound
loci were ranked according to their distance to the nearest TSS, and were binned into 500bp to
5kb regions. The percent of all E2f peaks within each bin range was calculated. The vast
majority of loci bound by E2f3 (A) and E2f4 (B) are located within 3.5kb upstream and 1.5kb
downstream of the nearest TSS.
Supplementary Figure 4. Anti-E2f3 ChIP-chip signal in wild-type and in E2f3a-/- or E2f3b-/neurospheres
(A) The microarray signal at each probe is represented as the moving average score (MAS) in the
anti-E2f3 experiments, in wild-type cells (x-axis) and in E2f3a-/- cells (left) and in E2f3b-/(right). Green spots represent probes on the microarray that belong to any set of E2f3 peaks,
while red spots are those that belong to peaks unique to E2f3 in WT cells. Probes for peaks
unique to E2f3 in wild-type cells tend to have lower score values, in both WT and mutant cells.
This result suggests that regions that appear uniquely bound in WT cells do show some
enrichment in mutant cells as well, and are probably not called as true peaks because the peak
calling criteria are slightly too stringent.
(B) Similar representation of the data in the anti-E2f4 experiment, as a function of the signal in
the anti-E2f3 experiment in WT cells.
Supplementary Figure 5. In NPCs, E2fs bind the TSS of genes that are marked by histone
modifications correlated with active gene transcription
Promoter regions were generated by obtaining sequences lying 1000bp on either side of the TSS
of all known mouse genes. The percentage of promoter regions bound by E2f factors in our
experiments that also contained the chromatin marks H3K4me3, H3K27me3, or both (bivalent)
in NPCs48 was calculated. The vast majority of E2f-bound sites, regardless of the category of
E2f-bound genes, also possess the activating H3K4me3 modification. The percentage of all
promoters surveyed in our experiments containing these chromatin marks was included as a
comparison.
Supplementary Figure 6. Expression profile of E2f3 target genes across multiple tissues
Expression profiles were extracted for all E2f3 target genes discovered in NPCs that are
represented in the mouse Gene Atlas GNF3 study. Data (genes and tissue/cell types) were
normalized and clustered using k-means. Predominant clusters are indicated along the right hand
side (genes found in neuronal and testis tissues). The legend indicates the colour profile related
to normalized gene expression values, in log base 2. ESCs = embryonic stem cells.
Supplementary Table 1. Complete list of E2f-bound loci and assigned target genes
Excel workbook providing the coordinates (mm9 genome assembly) of all E2f peaks that passed
our peak-calling threshold and associated target genes. Includes E2f3-bound peaks in WT,
E2f3a-/- and E2f3b-/- NPCs, and E2f4-bound peaks in WT cells. TSS, transcriptional start site;
TES, transcriptional end site.
Supplementary Table 2. Complete list of loci and associated target genes common and unique
to E2f3 and E2f4
Excel workbook giving the coordinates (mm9 genome assembly) and associated target genes of
all peaks common or unique to both E2f3 and E2f4, and those unique to either E2f factor.
Supplementary Table 3. Complete list of loci and associated target genes common and unique
to E2f3 isoforms
Excel workbook giving the coordinates (mm9 genome assembly) and associated target genes of
all peaks common or unique to E2f3a and E2f3b. Also includes loci, and associated genes, bound
by E2f3 in WT cells only.
Supplementary Table 4. Select genes corresponding to key biological processes overrepresented among E2f3 or E2f4 targets
Select gene ontology classifications enriched among E2f target genes in NPCs, with examples of
target genes both common and unique to E2f3 and E2f4.
Supplementary Table 5. Quantification of select gene ontology processes among genes targeted
by E2f3a and E2f3b
Table displaying the percent and number of genes bound by E2f3 in each group (in WT, E2f3a-/or E2f3b-/- NPCs), belonging to particular GO annotations, as indicated by GREAT
bioinformatics analysis.
Supplementary Table 6. List of genes whose promoters are bound by E2f3 and/ or Ctcf
Excel workbook showing all genes surveyed in our ChIP-chip experiments whose promoters are
bound by E2f3 alone, Ctcf alone, or by both E2f3 and Ctcf (Ctcf data from E14.5 brain tissue).
Supplementary Table 7. Correlation between number of EPUs and gene ontology
All EPUs identified in E14.5 brain were assigned target genes, and the genes were clustered into
broad categories based on the number of EPUs formed. The percentage of genes assigned to
specified GO categories, as indicated by GREAT bioinformatics analysis, is shown for each
cluster.
Supplementary Table 8. Oligonucleotide primers used in ChIP assays
List of all primer sequences used in ChIP assays in this study.
Supplementary Table 9. Microarray probes from mouse gene atlas GNF3 in the ‘neuronal high’
cluster of E2f3 targets
Simple text file giving the identity of genes contained within the cluster of genes that exhibited
generalized increased expression levels in neuronal tissues compared to other tissues were
selected. Shown is the Affymetrix probe ID and gene symbol for each gene in the cluster, and the
relative expression level is shown for each gene in all tissues examined. The table is in cdt
format and can be opened in Java Treeview.
Supplementary Table 10. Quantification of select gene ontology processes among E2f3 target
genes in neural and muscle precursors
Table displaying the percent and number of genes bound by E2f3 in each group (NPCs only,
MBs only, or both NPCs and MBs), belonging to particular GO annotations, as indicated by
GREAT bioinformatics analysis.
Supplementary Text 1. Supplementary materials, methods, results and references