arms in metaphase spreads and were significantly
reduced by the inhibitor (Fig. 1, E to V, and fig. S6,
A and B). Chromosome arm transcription was
also detected in BJ fibroblasts (fig. S6, C to U),
further confirming that mitotic transcription is
not limited to the centromere and occurs in non-transformed cells.
We used quantitative reverse transcription polymerase chain reaction to independently assess
transcripts that were called to be mitotically expressed or not, using intron-directed primers.
Primer sets were confirmed to detect nascent
transcripts because treatment with triptolide,
an RNAP2 inhibitor, diminished their signals
but did not decrease signals for primer sets to
glyceraldehyde-3-phosphate dehydrogenase mRNA
(fig. S7A). All three mitotically expressed primary
transcripts were detected in mitotic cells (fig. S7B)
and were triptolide-sensitive (fig. S7C), demonstrating the dependence of their expression on
To test for expression of mitotically expressed
genes in naturally occurring mitotic cells, we performed RNA fluorescence in situ hybridization
in asynchronous cells (Fig. 2, A to L). Exon and
intron probes were used because colocalization,
together with chromatin, is indicative of a transcriptional event. We detected a significant occurrence of primary transcripts in mitosis for all three
genes tested (fig. S7D). We also found 789 transcripts (484 genes) that were higher in mitosis
than in asynchronous cells (Fig. 2M and table S7).
The genes were enriched for those involved in
extracellular structure and transcription (Fig. 2N
and table S8) and were not specific to G2 or other
nonmitotic phases (table S9) ( 23).
To assess whether there is a hierarchy of reactivation during mitotic exit, all transcripts expressed in asynchronous cells were parsed by the
time at which their FPKM first increased 1.5-fold
over that in mitosis (Fig. 3A, fig. S8A, and tables
S10 to S15). The largest number of transcripts
first increased at 80 min (fig. S8B and table S11)
and were reactivated with the largest amplitude (fig. S9A), as seen previously ( 11, 17). Of the
transcripts first activated at 80 min in hepatoma
cells, 55% maintained their transcription rate for
the duration of mitotic exit (fig. S9B), similar to
that seen in erythroblasts (fig. S9C) ( 17). Yet the
sensitivity of our approach allowed for the identification of additional waves of reactivation after
the initial burst (Fig. 3 and tables S12 to S14).
EU-labeling affords the sensitivity to detect 927
transcripts that first increase at only 40 min
(Fig. 3 and table S10), well before bulk transcription reactivation seen by EU-fluorophore
labeling or RNAP2 antibody staining and chromatin immunoprecipitation ( 11, 17).
The first genes to increase have functions in
lumen and envelope formation and translation
(Fig. 4A and tables S16 and S17) ( 24, 25). Therefore, genes that reconstitute basic cell structure
and growth are prioritized immediately after mitosis, expanding on the ribosomal and metabolic
genes seen elsewhere ( 26). The next wave of reactivation is enriched for adhesion genes, which
is consistent with the epithelial nature of HUH7
time of first increase of nearest TSS-associated gene
number of unique genes
FPKM n.s. n.s.
600 400 200
Fig. 4. Basic cell functions are prioritized over cell specificity during mitotic exit. (A) Representative
GO categories for 40 to 80 min. (B) FPKMs of liver-specific genes. Bar, mean; whiskers, quartiles; P <
0.001 from 300 min to asynchronous; n = 149 liver-specific genes. (C) Increase in eRNAs ( 29) within
100 kb of transcription start site (TSS) during mitotic exit. Bar, mean; whiskers, quartiles; n.s., not significant;
blue, comparison to mitosis; red, comparison to previous time point; *P < 0.05, ***P < 0.001.
minutes of release from mitosis
20 kb 5
- 5 FPM
Fig. 3. Transcription reactivation during mitotic exit. (A) z scores of transcripts (rows) that
first increase ≥1.5-fold over mitosis, rank ordered within each time point (columns). (B) Liver genes
during mitotic exit. y axis, FPM. Colored bars to the left indicate mitotic or mitotic exit stages
generally observed at each time point.