misexpression from cryptic promoters by concentrating Aly at active genes. Of the 1903 Aly
peaks identified with ChIP from wild-type testes,
the 248 Aly peaks that overlapped with strong
Kmg peaks showed via ChIP an overall reduction in enrichment of Aly from kmg KD testes as
compared with wild type (Fig. 6, A to C, case 1,
and fig. S14, A and B). In contrast, the Aly peaks
at cryptic promoters were more robust in kmg
KD testes than in wild type (Fig. 4G). In general,
over the genome 4129 new Aly peaks were identified by means of ChIP from kmg KD testes that
were absent or did not pass the statistical cutoff
in wild-type testes (Fig. 6, A to C, case 2, and fig.
S14A). More than 30% of the genomic regions
with new Aly peaks in kmg KD showed elevated
levels of RNA expression starting at or near the
Aly peak in kmg KD but not in wild-type testes
(Fig. 6D and fig. S14C), suggesting that misexpression of transcripts from normally silent
promoters in kmg KD testes is more widespread
than initially assessed with microarray. Together,
these findings raise the possibility that Kmg may
prevent misexpression of aberrant transcript by
concentrating Aly to active target genes in wild-type testes, preventing binding and action of Aly
at cryptic promoter sites.
Our results suggest that selective gene activation is not always mediated by a precise transcriptional activator but can instead be directed by
combination of a promiscuous activator and a gene-selective licensing mechanism (fig. S15A). Cryptic
promoters may become accessible as chromatin
organization is reshaped to allow expression of
terminal differentiation transcripts that were
tightly repressed in the progenitor state. We
posit that this chromatin organization makes a
number of sites that are accessible for transcription dependent on the testis-specific tMAC complex component Aly. In this context, activity of
Kmg and dMi-2 is required to prevent productive
transcript formation from unwanted initiation
sites, potentially by confining Aly to genes actively
transcribed in the testis and limiting the amount
of Aly protein acting at cryptic promoters.
The initiation of transcripts from cryptic promoters is reminiscent of loss of function of Ikaros,
a critical regulator of T and B cell differentiation
(21) and a tumor suppressor in the lymphocyte
lineage (22, 23). Like Kmg, Ikaros is a multiple–
zinc finger protein associated with Mi-2b, which
binds to active genes in T and B cell precursors
(16, 24). In T cell lineage acute lymphoblastic
leukemia (T-ALL) associated with loss of function
of Ikaros, cryptic intragenic promoters were acti-
vated, leading to expression of ligand-independent
Notch1 protein, contributing to leukemogenesis
(25). Thus, in addition to being detrimental for
proper differentiation, firing of abnormal tran-
scripts from normally cryptic promoters because
of defects in chromatin regulators may contribute
to tumorigenesis through generation of oncogen-
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We thank L. Di Stefano, H. White-Cooper, X. Chen, F. Port,
S. Bullock, A. Kuo, G. Crabtree, S. Park, S. Kim, J. Wysocka, and
G. Ramaswami for advice and generously sharing data and
reagents; members of the Fuller laboratory for discussions and
critical reading of the manuscript; the Stanford Cell Sciences
Imaging Facility for fluorescence microscopy (National Center for
Research Resources grants S10RR017959-01 and 1S10OD010580);
and the Stanford Functional Genomics Facility for high-throughput
sequencing. Genomic data are available under the National
Center for Biotechnology Information Gene Expression Omnibus
(GSE89506). J.K. was supported by the Anne T. and Robert
M. Bass Stanford Graduate Fellowship and the Bruce and Elizabeth
Dunlevie Bio-X Stanford Interdisciplinary Graduate Fellowship.
Research support was provided by Deutsche Forschungsgemeinschaft
grant TRR81 to A.B. and S.A., Kempkes Stiftung to S.A, NIH
grant 5R01GM061986, and the Reed-Hodgson Professorship in
Human Biology to M. T.F.
Materials and Methods
Figs. S1 to S15
Tables S1 to S8
1 November 2016; accepted 13 April 2017
Fig. 6. Kmg prevents promiscuous activity of Aly. (A) Genome browser screenshot showing
ChIP-seq results for Kmg and Aly in control and kmg KD testes and RNA-seq results from wild-type
testes. y axes are normalized read counts based on 1 million mapped reads per sample. (B and
C) Median profile of (B) Aly and (C) Kmg ChIP (solid lines) and corresponding input (dotted lines)
signals centered around (case 1, left) Aly peaks identified in wild-type testes (q < 10−10 and identified
in both replicates) that overlap with Kmg peaks, and (case 2, right) new Aly peaks identified in
kmg KD testes (q < 10−10 and identified in both replicates) that were not detected in wild-type testes.
For the 248 Aly peaks in case 1—because these peaks were present at the promoters of genes
with known strand information—read counts were plotted according to the direction of transcription
from 5′ to 3′. (D) Heatmap representation of normalized RNA-seq read counts centered around
4219 new Aly peaks that appeared in kmg KD (case 2). Darkest (black) color indicates read count
value at the 5th percentile. Brightest [yellow for reads mapped to Watson (+), light blue for Crick (–)
strands] colors indicate values at the 95th percentile among all values in kmg KD.