UpSET Recruits HDAC Complexes and Restricts Chromatin Accessibility and Acetylation at Promoter Regions

Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Cell (Impact Factor: 32.24). 11/2012; 151(6). DOI: 10.1016/j.cell.2012.11.009
Source: PubMed


Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domain-containing protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin.


Available from: Jeffrey Delrow, Oct 13, 2015
UpSET Recruits HDAC Complexes
and Restricts Chromatin Accessibility
and Acetylation at Promoter Regions
Hector Rincon-Arano,
Jessica Halow,
Jeffrey J. Delrow,
Susan M. Parkhurst,
and Mark Groudine
Basic Sciences Division
Genomics Resource
Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
Department of Radiation Oncology, University of Washington School of Medicine, Seattle, WA 98109, USA
Developmental gene expression results from the
orchestrated interplay between genetic and epige-
netic mechanisms. Here, we describe upSET,a
transcriptional regulator encoding a SET domain-
containing protein recruited to active and in-
ducible genes in Drosophila. However, unlike other
Drosophila SET proteins associated with gene tran-
scription, UpSET is part of an Rpd3/Sin3-containing
complex that restricts chromatin accessibility and
histone acetylation to promoter regions. In the
absence of UpSET, active chromatin marks and
chromatin accessibility increase and spread to genic
and flanking regions due to destabilization of the
histone deacetylase complex. Consistent with this,
transcriptional noise increases, as manifest by acti-
vation of repetitive elements and off-target genes.
Interestingly, upSET mutant flies are female sterile
due to upregulation of key components of Notch
signaling during oogenesis. Thus UpSET defin es a
class of metazoan transcriptional regulators required
to fine tune transcription by prev enting the spread of
active chromatin.
Genome-wide analyses of chromatin accessibility, histone
modifications, and factor/cofactor recruitment have led to the
identification of regulatory elements and their associated protein
components (modENCODE Consortium et al., 2010; Filion et al.,
2010). For example, chromatin accessibility at promoters is
usually higher than in transcribing regions and sharply localized
to a few hundred base pairs from the transcriptional start site
(TSS). In addition, although transcribed regions are enriched
for histone marks such as H3K36m3 and H3K79m2, promoters
are enriched for other histone modifications, including acetyla-
tion (H3K9Ac and H4K16Ac) and methylation (H3K4m2 and
H3K4m3). Although the distribution of H3K4m2 is variable
among species, H3K4m3 and histone acetylation are conserved
landmarks for active promoters, and several reports suggest
that they are functionally linked (Cai et al., 2010). However, it is
still unclear whether these histone modifications are a cause
or consequence of promoter accessibility and how chromatin
accessibility and promoter remodeling are maintained in a local-
ized fashion (Henikoff and Shilatifard, 2011).
In contrast, the correlation between the proteins establish-
ing those modifications and transcriptional regulation is well
accepted. Promoter-associated H3K4m3 is mainly established
by SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain-con-
taining proteins including the Trithorax family of proteins (TrxG).
The SET domain is a 130 amino acid, evolutionarily conserved
sequence motif present in chromosomal proteins that function
in modulating gene activities from yeast to mammals (Dillon
et al., 2005). SET domain-containing proteins are part of large
complexes and are usually involved in establishing or affect-
ing posttranslational histone modifications that correlate with
chromatin remodeling, transcription initiation, and transcriptional
elongation (Eissenberg and Shilatifard, 2010). For example, the
yeast Set1 is part of a macromolecular H3K4 methyltransfer-
ase complex named COMPASS (Miller et al., 2001), and, in
Drosophila, there are three COMPASS-like complexes that
control H3K4me homeostasis (Set1, Trx, and Trr) with Set1
serving as the main H3K4 methyltransferase among these
complexes (Ardehali et al., 2011; Mohan et al., 2011; Hallson
et al., 2012; Eissenberg and Shilatifard, 2010). Proteomic anal-
ysis has revealed that histone acetyltransferases are critical
components of COMPASS complexes, supporting the notion
of a continuous crosstalk between histone acetylation and meth-
ylation (Schu
beler et al., 2004; Bernstein et al., 2005). Therefore,
it has been suggested that COMPASS complexes play a key role
in establishing promoter architecture.
In mammals, the Mixed Lineage Leukemia (MLL) family of
SET methyltransferase domain proteins includes at least six
members (MLL1-4, Set1A, Set1B) and are involved in gene acti-
vation (Ruthenburg et al., 2007). Biochemical analysis has re-
vealed that these proteins are also organized in COMPASS-like
complexes that can methylate H3K4 (Eissenberg and Shilatifard,
2010). Recently, human MLL5 (KMT2E) was added to the MLL
family because of its SET domain homology with MLL1 (Emerling
1214 Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc.
Page 1
et al., 2002). Interestingly, the MLL5 gene is located in a region
of chromosome 7 commonly deleted in a subset of leukemias
associated with poor prognosis (Emerling et al., 2002). Unlike
other MLL family members, MLL5 has a more centrally located
SET domain and a single PHD domain, but lacks AT-hook
domains. Putative MLL5 orthologs exist in other organisms,
including CG9007 in Drosophila and Set3 and Set4 in yeast
(Emerling et al., 2002; Eissenberg and Shilatifard, 2010).
Although the MLL5 SET domain is the primary region of conser-
vation among MLL5 orthologs, it lacks key catalytic amino acids,
bringing into question the catalytic activity of this SET domain
(Sebastian et al., 2009; Madan et al., 2009). However, O-linked
N-acetylglucosamine transferase (OGT)-mediated SET domain
glycosylation of a small human MLL5 isoform may be required
to mono- and dimethylate H3K4 (Fujiki et al., 2009). MLL5
knockout mice are viable and surprisingly exhibit only mild
hematological phenotypes and male sterility (Zhang et al.,
2009; Madan et al., 2009; Heuser et al., 2009; Yap et al., 2011),
which may reflect functional redundancy by a related gene
(SETD5) with high protein homology with MLL5. The yeast
MLL5 ortholog, Set3, lacks the proposed glycosylating residue
and displays no histone methyltransferase activity (Pijnappel
et al., 2001). Set3 is targeted to active genes and associated
with a histone deacetylase (HDAC) complex; however, its pre-
cise function once recruited is unclear (Kim and Buratowski,
2009; Govind et al., 2010; Buratowski and Kim, 2010).
Here we describe the Drosophila gene whose protein’s SET
domain most resembles MLL5. We have named this gene upSET
(CG9007) because of its genetic and biochemical features.
DamID chromatin profiling reveals preferential recruitment of
UpSET to transcriptionally active genes overlapping with RNA
pol II, TrxG proteins, and H3K4m3. UpSET interacts with a
dRpd3-containing HDAC complex and promotes its recruitment
to active regions. Knockdown experiments reveal an increase
in histone acetylation around the TSS, which can spread to
neighboring regions and correlates with increased chromatin
accessibility around UpSET bound regions. Knockdown cells
increase the expression of genes and repeat elements neigh-
boring regions of UpSET binding. Consistent with this, oogen-
esis is impaired by the lack of UpSET because of deregulated
Notch signaling, causing alterations in egg chamber formation
and anterior-posterior specification. This female sterility pheno-
type can be partially rescued by expressing the mouse MLL5
gene. In genetic interactions upSET enhances phenotypes asso-
ciated with PcG epigenetic repressors. Our results indicate that
UpSET is part of the machinery recruited to promoter regions to
regulate chromatin stability and transcription state by preventing
the spread of active chromatin.
UpSET Is a Nuclear Protein and Localizes to Gene-Rich
Euchromatic Regions
upSET encodes a 330 kDa (3146 aa) protein with a single PHD
domain and centrally located H3K4 histone methyltransferase
SET domain (Figure 1A). UpSET coding sequence is highly
conserved among 12 Drosophila genomes, primarily over the 5
end of the gene (Figure 1A). The UpSET SET domain shares
high similarity among the Drosophilidae family (Figure S1A avail-
able online). UpSET shares restricted homology over the SET
domain with two mammalian proteins, MLL5 and SETD5 (Fig-
ure 1B). UpSET and MLL5 share 58% identity in their SET
domains and 75% identity in their adjacent PHD domains (Fig-
ure 1C). SETD5 SET domain similarly shares 60% identity with
UpSET’s SET domain, but lacks a PHD domain. Other than these
domain similarities, UpSET does not share significant homology
with MLL5, SETD5 or other SET-containing proteins (Figure 1B
and 1C and S1B). The presence of these chromatin regulatory
domains suggests that UpSET function is nuclear. Thus we
generated monoclonal antibodies to UpSET (Figures S1D) and
performed western (Figures 1D and S1C) and immunofluores-
cence (Figures 1E and 1E’) analyses in Drosophila Kc cells. As ex-
pected, UpSET is located mainly in the nucleus with a punctate
distribution. To establish whether UpSET interacts with chro-
matin in vivo, we examined binding of endogenous UpSET to
third instar larval salivary gland polytene chromosomes. Interest-
ingly, we detect roughly 1,000 strongly staining UpSET binding
sites on polytene chromosomes (Figures 1F–1H). These sites
are mainly in gene-rich euchromatic regions and generally ex-
cluded from DAPI-dense regions, supporting a global role of
UpSET in gene regulation.
A hallmark of SET domain-containing proteins is histone
methyltransferase (HMT) activity. Interestingly, the UpSET SET
domain lacks key amino acids required for this catalytic function
(Emerling et al., 2002). Therefore, we performed an in vitro HMT
assay to address whether UpSET’s SET domain is functional.
We bacterially expressed and purified the UpSET SET domain,
and found that by itself the SET domain does not have HMT
activity (Figure 1I). The mouse MLL5 SET domain also lacks these
key amino acids, however, whether it possesses catalytic activity
is controversial (Madan et al., 2009; Eissenberg and Shilatifard,
2010): it has been suggested that a short human MLL5 isoform
can exhibit HMT activity upon glycosylation of Threonine 440 (Fu-
jiki et al., 2009). Although this Threonine is conserved among
Drosophila species (Figures 1C and S1A), preincubation of the
UpSET SET domain with in vitro expressed Super sex combs
(Sxc; the main nuclear Drosophila OGT; Sinclair et al., 2009)or
nuclear extracts does not confer HMT activity (Figures 1I and
S1E). Thus, although UpSET shares structural SET domain ho-
mology with HMTs, it does not appear to be catalytically active. It
remains possible that the UpSET SET domain requires the con-
text of the full-length protein to function, but due to its large size
it has not yet been possible to express full-length UpSET in E. coli.
upSET Mutants Are Female Sterile
To address the biological functions of UpSET, we obtained an
existing allele (upSET
), in which a P-element is inserted in
the second intron of upSET upstream of the translation start
site (PBac{RB}CG9007[e00365]; Figure 1A). upSET
is a
protein null allele: UpSET protein is absent in upSET
mutants by western blot or on polytene chromosomes (Figures
1J and 1K). upSET
homozygous mutants are viable,
suggesting that UpSET is not required for survival. How-
ever, the resulting mutant females are sterile (98% of upSET
females do not lay eggs; Table 1) and display severe oogenesis
phenotypes: 80% of egg chambers do not progress beyond
Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc. 1215
Page 2
stage 8-9 and mature eggs are not produced (n = 215; Figures 1L
and 1M). Importantly, this female sterility can be rescued by
a BAC transgene containing the upSET locus (Table 1). Interest-
ingly, expression of the mouse MLL5 cDNA (similar to UpSET in
that it does not show HMT activity), also partially rescues female
sterility in 20% of upSET females (n = 50; Table 1, Figures S1F
Figure 1. UpSET Is a SET-Containing Nuclear Protein Required for Drosophila Oogenesis
(A) Schematic of the upSET locus on chromosome 3L. Location of the SET and PHD domains are highlighted. Arrowhead indicates P-element insertion in
. Graph shows the locus conservation in the Drosophilidae family.
(B) UpSET shares only 15% identity with mouse MLL5 in the SET domain. The mouse MLL5 SET domain shares 60% identity with that of mouse SETD5.
(C) Sequence alignment of the SET and PHD domains between Drosophila UpSET and mouse MLL5. Percent identity is indicated.
(D) UpSET is mainly a nuclear protein. Western blot analysis of nuclear (NE) and cytopl asmic (Cyt) extracts from Kc cells probed with anti-UpSET N-end
monoclonal antibodies. Full-length UpSET protein is denoted by the arrow. Asterisk (*), nonspecific band.
(E and E’) UpSET forms foci in the nucleus. Confocal projection of 10 serial 0.2 mm optical sections of a Kc cell stained with a mix anti-UpSET NH3-terminus
monoclonal antibodies (green; E and E’) and DAPI to visualize DNA (E’).
(F–H) Third larval instar polytene chromosomes stained with a mix of antibodies to UpSET NH3- and COOH-terminus (green) and DNA counterstained with DAPI
(blue). Higher magnification view of X chromosom e (G). UpSET recruitment is enriched in euchromatic regions (H).
(I) UpSET does not exhibit histone methyltransferase activity (HMT). Radioactive HMT assays performed with bacterially purified GST or GST-UpSET SET domain
and purified histones as substrate (I). Recombina nt prot eins were incubated with Super Sex Combs (SXC) or nuclear extracts (NE). SET7 was used as positive
control. Loading controls for the histone methyltransferase assay are shown.
(J) Western blot analysis of larval nuclear extracts from wild-type and upSET
mutants blotted with a mix of the 5 antibodies against UpSET NH3- and COOH-
termini. Lamin Dm0 serves as loading control.
(K) Polytene chromosome from upSET
mutant third instar larva stained with a mix of the 5 UpSET monoclonal antibodies (green) and DNA counterstained
with DAPI (blue).
(L and M) Lack of UpSET disrupts oogenesis. Confocal projections of 5 serial 1 mm optical sections of ovaries from wild-type (L) and upSET
mutants (M) actin
stained with phalloidin.
Scale bars, 100 mm. See also Figure S1.
1216 Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc.
Page 3
and S1G). The partial rescue is likely due to the limited functional
conservation of UpSET with MLL5, as well as its similarity to the
other SET domain protein SETD5.
UpSET Binds to Transcriptionally Active Regions
Based on the upSET female sterile phenotype and its presence in
euchromatic regions, we addressed in more detail the genomic
regions targeted by UpSET. DamID chromatin profiling in Kc cells
(Greil et al., 2006; Orian et al., 2009) revealed that UpSET binds
to 3,500 regions genome-wide and overlaps with gene-rich
regions (Figures 2A–2C, and S2A), corresponding to 6,000
genes. These results are consistent with the broad presence of
UpSET observed on polytene chromosomes (Figures 1F–1H).
Recently, several different chromatin states have been charac-
terized in the Drosophila genome by profiling the binding of 53
different proteins (Filion et al., 2010), as well as histone modifica-
tions, transcription factor binding, and/or gene expression (mod-
ENCODE Consortium et al., 2010). To determine whether UpSET
associates with specific chromatin states, we examined the
UpSET DamID-signal distribution for each regulatory region
described by the modENCODE consortium. DamID signal distri-
bution over each chromatin state showed a preferential recruit-
ment of UpSET to TSS/promoter regions (p < 2.2 3 10
to a lesser extent, enhancer and transcribed regions (Figure 2D).
These enrichments are significant compared to other chromatin
states, as well as a control cohort of 6,000 random sequences
(Figure 2D; see Extended Experimental Procedures). Analyses of
UpSET enrichment in five chromatin types defined by Filion et al.
revealed a similarly high overlap with active regions (Figure S2B).
Thus, UpSET is preferentially targeted to active promoter regions.
To determine whether UpSET binding correlates with gene
expression, we performed an end analysis to correlate UpSET
binding relative to the 5
and 3
ends of genes arranged by
expression-based quintiles (Deal et al., 2010). Our results show
that a peak of UpSET binding mainly occurs 25–50 bp down-
stream of the TSS. This binding is dependent on gene expres-
sion, as quintiles with highly expressed genes have higher upSET
signal (Figure 2E). As active TSS regions are marked by active
chromatin marks (i.e., H3K4m3), as well as with high levels of
RNA pol II, we performed an end analysis of RNA pol II- and
H3K4m3-enriched sites from S2 cells (Muse et al., 2007). Consis-
tent with UpSET enrichment at active TSSs, we observed high
UpSET signal in regions enriched for RNA pol II and H3K4m3
(Figure 2F). This overlap is not due to polymerase pausing, as
promoters demonstrating high or low pausing indexes show
similar enrichment for UpSET (Figure S2C) (Gilchrist et al.,
2010). Together, our results suggest that UpSET targets pro-
moter/TSS regions of transcriptionally active genes indepen-
dently of the pausing features of the promoters.
UpSET Interacts with Sin3A/Rpd3 Histone Deacetylase
The histone deacetylase Rpd3 and its accessory subunit Sin3
can be recruited to transcriptionally active regions (Filion et al.,
Table 1. upSET Mutants Are Female Sterile, Increase Polycomb Phenotypes, and Reduce Trithorax-Associated Homeotic
Lack of UpSET causes female sterility
Female Male Sterile females (%) No. sterile females (n = 50) Re scue (%) No. egg
+/+ +/+ 0 0 n/a 1536
+/+ 0 0 n/a 1330
+/+ 98 49 n/a 5
; upSET
+/+ 26 13 72 1035
; upSET
+/+ 5 3 93 1231
; upSET
+/+ 78 39 20 408
upSET enhances Pc phenotypes
Percent with enhanced phenotype
e00365 f
MB8950 f,h
+/+; +/
0 (350) 0 (303) 0 (239)
/+; +/
40 (403) 89 (495) 76 (184)
/+; +/
33 (320) 75 (256) 75 (122)
/+; +/
56 (465) 9 (327) 4 (409)
n/a, not applicable.
BAC1X= w; P[acman](attA CH322.187J08 attB)/CyO.
BAC2X= w; P[acman](attA CH322.187J08 attB)/P[acman](attA CH322.187J08 attB).
mMLL5= w;UAS_mMLL5/ActGAL4.
For the Pc alleles Pc
and Pc
, males of the genotypes indicated in the first column were scored for the presence of a sex comb on the second and/or
third leg (usually found only on the first leg).
was scored for the presence of an apical bristle on the third leg (usually found on the second leg).
‘*’ refers to +, upSET
as indicated.
Wild-type is OregonR.
See also Figure S7A.
Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc. 1217
Page 4
2010). The proposed yeast ortholog of UpSET, Set3, has also
been shown to interact with an HDAC complex over transcribing
regions, however its role once recruited is still unclear (Kim and
Buratowski, 2009; Govind et al., 2010; Buratowski and Kim,
2010). Interestingly, the histone deacetylase Rpd3 and its
accessory subunit Sin3 have recently been shown to be re-
cruited to transcriptionally active regions (Filion et al., 2010). To
determine whether UpSET and the HDAC machinery occupy
similar genomic regions, we compared the published Sin3 and
Rpd3 chromatin profiles to the 6,000 UpSET bound promoter
regions (Filion et al., 2010). This analysis revealed a high enrich-
ment for Sin3 and Rpd3 in UpSET bound regions compared to
6,000 random sequences (Figures 3A and 3B). This correlation
is specific to these two corepressors because repressors
present in other chromatin states (i.e., CtBP) are not enriched
(Figure 3A). UpSET bound regions overlap with 50% of Rpd3
and Sin3 bound sites (Figure 3C). Consistent with this, costaining
of polytene chromosomes with UpSET and Sin3 or Rpd3 anti-
bodies shows a high degree of overlap (Figures 3D and 3G).
This overlap is not generalized because some regions are bound
by UpSET but not by the other two proteins and vice versa, sug-
gesting that UpSET likely participates in additional protein
To determine whether UpSET interacts with the Sin3 and Rpd3
deacetylase machinery directly in vivo, Kc cell nuclear extracts
were incubated with antibodies against Sin3 and Rpd3, and
then blotted with UpSET antibodies. As shown in Figure 3H,
UpSET physically associates with both Sin3a and Rpd3.
Lack of UpSET Increases Histone Acetylation around
Promoter Regions and Causes a Redistribution of Active
A recent genome-wide analysis revealed that the HDAC
machinery binds at both active and inactive genes (Wang et al.,
2009), consistent with results of DamID-based chromatin
profiling of Sin3 and Rpd3 in insect cells (Filion et al., 2010).
The recruitment of HDACs to active genes would be expected
to control histone acetylation at promoters and transcribed
regions. Therefore, we hypothesized that the lack of UpSET
should increase acetylation levels at UpSET-regulated genes.
Figure 2. UpSET Is Preferentially Recruited to Active Genes
(A) Sites of UpSET recruitment based on DamID. The black vertical lines represent the relative position on the chromosomes of the 14,000 Drosophila genes.
Blue dots represent single peaks (3.5–6 kb) and the red dots (>6 kb) UpSET binding sites.
(B and C) UpSET chromatin profile over a 600 kb region of chromosome 3R and a 11 kb region of chromosome 2L. Single peaks and regions are presented in blue
and red, respectively. The mean signal was smoothed + whiskers. Coding regions are shown in blue (B) and black (C).
(D) UpSET is mainly enriched in promoter (TSS) regions. Box plots of the UpSET DamID-based chromatin profile over the nine different chromatin states reported
by the modENCODE Consortium. TSS, transcriptional start sites; Exon, elongating regions; Enh, Enhancers; OC, open chromatin; MXC, male X genes; Pc,
Polycomb; HC, constitutive heterochromatin; HLC, heterochromatin-like in euchromatin; IEC, basal, intergenic euchromatin. Random: set of 6,000 random
sequences. *p < 2.2 3 10
(E) Preferential association of UpSET over transcribed genes. End analysis of the UpSET chromatin profile to the 5
and 3
end of genes ranked by expression
quintiles from high (Q1) to low (Q5) gene expression.
(F) End analysis of UpSET DamID signal to RNA Pol II (black) and H3K4m3 (red) enriched regions.
See also Figure S2.
1218 Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc.
Page 5
We knocked down UpSET in Kc cells (Figure 4A) and performed
chromatin immunoprecipitation (ChIP) with antibodies recog-
nizing the H3-tetraAc, H3K9Ac and H4K16Ac histone marks
(modENCODE Consortium et al., 2010). Primers targeting several
promoter regions (±250 bp around TSS) of UpSET bound genes
were used to evaluate acetylation levels. Although UpSET-regu-
lated genes exhibit acetylation marks due to their active tran-
scription state, the absence of UpSET results in increased
H3K9Ac and H4K16Ac levels (Figures 4B, S3A and S3B). This
increase is not due to an increase in nucleosome density, as
ChIP of histone H4 or H3 remains constant (Figures S3C and
S3D). Our results reveal that upon UpSET depletion, acetylation
levels increase at active promoter regions targeted by UpSET.
As UpSET is required for controlling histone acetylation at
promoter regions, UpSET may be acting as a structural bridge
to stabilize Rpd3-containing complexes at promoter/transcribed
regions. To test this hypothesis, we performed a ChIP analysis
to investigate the presence of the Rpd3-containing complex at
UpSET targeted promoters in UpSET knockdown cells. We
find that Rpd3 and Sin3 are detected at most of the promoters
tested, albeit enriched to different amounts (Figures 4C and
4D). Upon UpSET depletion, Rpd3 levels are reduced at several
UpSET regulated promoters (five out of six) but not in the C15
gene, which is targeted by Rpd3 and Sin3 but not UpSET
(Figures 4C and S3A) (Schwartz et al., 2006). SIN3 is also de-
tected in low levels over UpSET-regulated promoters (Figure 4D).
Figure 3. UpSET Interacts with an HDAC Complex
(A) UpSET-bound regions are enriched for the Histone deacetylase Rpd3 and its associated cofactor Sin3. Box plot analysis of Rpd3 and Sin3 DamID signal
overlapping with UpSET bound regions.
(B) Browser view of chromosome 2L highlighting the overlapping DamID signal for UpSET, Rpd3, and Sin3.
(C) Venn diagram showing the overlap among regions bound by UpSET, Rpd3, and Sin3. UpSET overlaps with only half of the loci cobound by Rpd3 and Sin3.
(D–G) UpSET colocalizes with Sin3 and Rpd3 on chromosomes. Polytene chromosome sets stained with a mixture of antibodies to NH3-UpSET (D’–D’’, E’–E’’,
F–G; green) and Rpd3 (D, D,’ G; red) or Sin3 (E, E’’, F; red). DNA was counterstained with DAPI (blue). (F and G) Higher magnification views showing colocalization
of UpSET with Rpd3 (G) or Sin3 (F).
(H) UpSET interacts with Rpd3 and Sin3. Coimmunoprecipitation of UpSET with Rpd3 and Sin3 from Kc nuclear extracts. Arrows indicate the specified protein.
20% input is shown.
Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc. 1219
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Figure 4. UpSET Modulates Active Chromatin Features of Transcriptionally Active Genes
(A) RNAi-based knockdown of UpSET levels. Western blot for UpSET from nuclear extracts of Mock and UpSET-specific RNAi expressing cells same antibodies
as in Figure 1J. RNA Pol II was used as loading control.
(B) UpSET knockdown increases histone acetylation over UpSET bound promoters (TSS). Native ChIP of H3Ac, H3K9Ac and H4K16Ac from Mock and UpSET
knockdown cells as indicated. Data are represented as mean ± SEM. N: Notch; Rel; Relish; Neur: neuralized; Dl: Delta; Orb: oo18 RNA-binding protein; Su(H):
Suppressor of Hairle ss, and the PcG-silenced C15 gene, as negative control. (*p < 0.05; **p < 0.01; ***p < 0.001, nonsignificant p values are not marked).
(C and D) Cross-linked ChIP for Rpd3 and Sin3 from Mock and UpSET knockdown cells. Bars represent mean ± SEM.
(E and F) Histone acetylation increases around UpSET-depleted genes. Native ChIP of H3Ac, H3K9Ac and H4K16Ac from Mock and UpSET knockdown cells as
indicated. UpSET chromatin profiles show the location of the used primers. Repressive chromatin reported from (Bartkuhn et al., 2009); blue line). Bars represent
mean ± SEM.
(G and H) Native ChIP of H3K4m1, H3K4m2, H3K4m3 and H3K27m3 from Mock and UpSET knockdown cells. Primers are same as in Figures 4E and 4F. Bars
represent mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001).
1220 Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc.
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Interestingly, and regardless of its interaction with UpSET, SIN3
binding was not dramatically affected in UpSET knockdown
cells, suggesting that recruitment of Rpd3, but not Sin3, to active
promoters is UpSET-dependent.
It has been suggested that HDAC recruitment to the promoter
region functions to constrain active histone modifications to this
region (Buratowski and Kim, 2010). Thus, UpSET-dependent
histone deacetylation may be responsible for restricting active
marks to promoter regions. To test this hypothesis, we selected
two genes (CG13689 and CG4389) that are flanked by repressive
chromatin (H3K27m3) and reside in regions of low gene density
(Bartkuhn et al., 2009). We chose genes with these characteris-
tics to avoid the potentially confounding effect of active neigh-
boring genes. We performed ChIP-qPCR by using three sets of
primers per gene that target 2.5 kb upstream, the promoter
region, and 2.5 kb downstream of the gene. Upon UpSET
depletion, the flanking repressed regions exhibit higher levels
of histone acetylation, suggesting that histone acetylation is
relocalized to neighboring regions in the absence of UpSET
(Figures 4 E and 4F). To investigate whether other histone marks
associated with transcription also spread to flanking regions in
the absence of UpSET, we performed ChIP of H3K4me1,
H3K4me2, H3K4me3, and control H3K27m3 in UpSET knock-
down cells. In mock-treated cells, H3K4 methylation is reduced
in repressive regions containing H3K27m3 (Figures 4 G and 4H).
Strikingly, UpSET knockdown increases the levels of H3K4m2
and H3K4m3 in the flanking regions (Figures 4G and 4H). This
increase in active marks in the absence of UpSET is not due to
a global increase on histone acetylation and H3K4 methylation,
as the levels of these modifications are not affected in knock-
down Kc cells or in upSET mutant ovaries, as determined by
western blot and polytene chromosome staining (Figures S3E–
S3L). Interestingly, H3K27m3 levels are also increased, perhaps
as a compensatory response to the presence of active marks
(Figures 4G and 4H). Together, these results reveal that UpSET
is required to restrict active chromatin marks to promoter/gene
In mammals, the only currently known target of MLL5 is
the CCNA2 gene, whose expression is regulated in C2C12 cells
by binding of MLL5 to the cell-cycle regulatory element located
in the first exon, resembling the positioning of UpSET binding
in Drosophila (Figures 2 and S4A) (Seba stian et al., 2009). To
determine whether the modulation of histone acetylation is a
conserved feature of MLL5, we knocked down MLL5 in C2C12
cells. Interestingly, we find that histone acetylation, in particular
H4K16Ac, increases at the MLL5 binding sites of CCNA2, as well
as on neighboring regions located 1–2 kb from the binding site
(Figures S4B and S4C). Regions further away were not affected.
Thus, our results suggest that the ability to modulate histone
acetylation is conserved among these proteins.
UpSET Modulates Chromatin Accessibility of Active
Promoter Regions
The UpSET-dependent targeting of HDAC machinery to active
genes is required to maintain proper control of active his-
tone marks. As histone acetylation usually correlates with a
more open chromatin configuration, we hypothesized that the
absence of UpSET must alter chromatin accessibility of active
promoter regions. Using MNaseI digestion and nucleosome ex-
tractions at low salt concentrations (Figure S5A) (Henikoff et al.,
2009), we assessed chromatin accessibility in UpSET knock-
down and mock RNAi-expressing control cells. As shown in Fig-
ures 4I and 4J, compared to 6,000 random sequences, UpSET
depletion increases chromatin accessibility of the 6,000
regions usually bound by this factor. To determine whether this
increase in accessibility correlates with gene expression levels,
we performed end analysis of genes clustered into quintiles by
their expression levels. We find that the three main clusters (quin-
tiles 1–3; genes with highest expression) of UpSET-bound genes
are more accessible over 1.5 kb upstream of the TSS (Figure 4K).
Interestingly, this accessibility is higher over the first 1.5 kb
downstream of the TSS, as well as the 3
end of the genes
than over promoter regions (Figure 4K). Nucleosomes located
just downstream of the TSS are more affected, which correlates
with regions preferentially enriched for UpSET (Figure 2E). Con-
sistent with this, genes that are hyperacetylated in UpSET KD
show different degrees of increased chromatin accessibility (Fig-
ure S5B). Our results indicate that UpSET is required to control
chromatin accessibility around the promoter and transcribed
upSET mutants are sterile suggesting a key role for this protein
in oogenesis. To establish whether mutant ovary tissues possess
a more open chromatin, we adapted a DNA-methylation-based
chromatin accessibility protocol for Drosophila tissues by using
the M.SssI methyltransferase (Figures 4L and S5C–S5G). The
Drosophila genome has minimal cytosine methylation, thereby
allowing efficient M.SssI modification of mCpG dinucleotides
based on their chromatin accessibility. This methylation can sub-
sequently be identified with a 5mC monoclonal antibody (Bell
et al., 2010). 5mC methylation cannot be detected in genomic
DNA from untreated wild-type or upSET mutant ovaries (Figures
4M, 4N, and S5C), whereas extended incubation of genomic
DNA with M.SssI reveals all CpG availability for methylation
(Figures S5 H and S5I). upSET mutant and wild-type ovaries
(I) UpSET depletion increases the accessibility of UpSET bound regions. Chromatin accessibility ratio in KD:mock-treated cells from a 150 kb region of chro-
mosome 2L.
(J) UpSET associated regions show increased accessibility. Chromatin accessibility signal distributions over UpSET bound regions or random sites.p<
2.2 3 10
(K) UpSET modulates chromatin accessibility of transcriptionally active genes. End analysis of the chromatin accessibility signal in UpSET knockdown cells over
the 5
and 3
ends of genes clustered by expression quintiles from high (Q1) to low (Q5) gene expression as indicated.
(L–P) Chromatin accessibility in situ. Schematic of M.SssI DNA methyltransferase accessibility assay in ovaries. Low CpG methylation as detected by 5-mC
immunofluorescence in untreated Drosophila germarium from wild-type (M) and upSET
mutants (N). Higher chromatin accessibility in M.SssI-treated
mutant ovaries (P) compared to wild-type (O).
See also Figures S3, S4, and S5.
Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc. 1221
Page 8
Figure 5. UpSET Modulates Off-Target Gene and Transposon Expression
(A and B) UpSET knockdown upr egulates silent genes. Gene expression analysis of knockdown Kc cells with primers (W-Z) targeting the genomic region from
Figure 4F, as well as the coding sequence of the UpSET target gene CG4389. (*p < 0.05; **p < 0.01; not significant p values are not marked). Data are represented
as mean ± SEM.
1222 Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc.
Page 9
were treated in situ with the M.SssI methyltransferase in non-
saturating conditions. Detection of 5mC in mutant ovaries by
immunofluorescence with the 5mC antibody shows higher chro-
matin accessibility in mutant ovaries (2-fold increase; p < 0.01)
(Figures 4O, 4P, and S5J). Interestingly, this function does not
seem to be conserved with its murine ortholog by assessing
chromatin accessibility at the only described MLL5 binding site
in the CCNA2 gene (Figures S4D and S4E). We do not know
whether this is a general property of MLL5 targets or whether it
is specific to this MLL5 target, as the region surrounding the
CCNA2 gene is already highly accessible. Altogether, our results
show that UpSET plays a key role in modulating chromatin
accessibility, preferentially at actively transcribed genes.
UpSET Modulates Gene Expression
The spreading of histone acetylation and H3K4 methylation in
the absence of UpSET suggests that UpSET-containing com-
plexes may control intergenic transcription, as well as modulate
gene expression levels of UpSET-regulated genes. To test this
hypothesis, we analyzed the expression levels of coding and
noncoding regions of the genes CG13689 and CG4389 by RT-
qPCR in UpSET KD cells. Although some UpSET target genes
display a subtle upregulation in their levels, intergenic transcrip-
tion remains the same (Figures 5A, 5B, S6 A, and S6B). Interest-
ingly, silent genes neighboring UpSET target genes become
upregulated (Figures 5A and 5B). To establish whether this was
a global feature due to UpSET, we performed a transcriptome
analysis of KD cells and evaluated which genes are affected
based on the presence or not of RNA polymerase II (Gilchrist
et al., 2010). In Kc cells, UpSET KD affected approximately 438
genes, 80% of which are not transcribed in Kc cells (Figures
5C and S6C). We also analyzed upSET mutant and wild-type
ovaries to determine whether upSET mutants have a similar tran-
scriptional phenotype. These analyses revealed that 859 genes
are differentially regulated (Figures 5D and 5E). By gene ontology
analysis, the affected genes are preferentially involved in signal
transduction pathways, as well as processes generally silent
during oogenesis but expressed during postembryonic, larval,
and pupal development (Figure 5E). In addition, we categorized
the modENCODE RNA-seq data for ovaries into expression
quintiles (Q1, high to Q5, low) and evaluated the percentage of
affected genes from each quintile. We found that low expressing
or silent genes (Q4–5) are the most affected genes in the
absence of UpSET ( Figure 5F). These results were confirmed
by RT-qPCR, with specific primers for genes from Q4–Q5 (Fig-
ure 5G). We also examined whether repeat elements are up-
regulated in UpSET mutant ovaries and KD cells. In higher
eukaryotes, repetitive elements including transposons and retro-
elements are the main component of heterochromatin, but they
are also interspersed in euchromatic regions, including introns
and intergenic regions (Kaminker et al., 2002). We found that
transposable elements located near UpSET binding sites are
consistently upregulated (Figures 5H–5K). Thus UpSET function
is required to modulate gene expression of nonexpressing genes
and repeat elements.
upSET Mutants Enhance Pc Homeotic Transformation
Our results show that UpSET modulates gene expression by
controlling histone acetylation and chromatin accessibility. As
silent genes are upregulated upon UpSET depletion, we hypoth-
esized that lack of UpSET could enhance phenotypes associ-
ated with repressive complexes. Polycomb group (PcG) proteins
are required for silencing homeotic and many other genes and
are key players of transcriptional memory (Ringrose and Paro,
2004). As the loss of PcG genes causes homeotic transforma-
tion due the ectopic expression of homeotic genes in tissues
where their expression is repressed, we performed a genetic
analysis to determine whether UpSET increases PcG-associ-
ated mutant phenotypes. Pc
and Pc
alleles show homeotic
transformations of the second and third legs into the first leg in
33%–40% of male Pc flies (Table 1). Strikingly, when these Pc
mutants are also heterozygous for upSET
, the frequency
of this transformation increased to 75%–89% (Table 1). We
confirmed this striking genetic interaction by using a second
upSET allele, upSET
, a hypomorphic allele resulting
from the insertion of a Minos transposon after amino acid
T2599 (Figures S7A–S7C’). upSET
also enhances the
homeotic transformation associated with the Pc alleles to
75% (Table 1). These genetic interactions are not due to a direct
protein-protein interaction because polytene chromosomes cos-
tained with Pc and UpSET antibodies show no overlap (Fig-
ure S7D), UpSET is not enriched at regions bound by PcG
proteins (Figure 2D, S7E, and S7F), and does not affect PcG-
associated H3K27me3 mark (Figures S7G and S7H) (Schwartz
et al., 2006; Ne
gre et al., 2010). These results genetically confirm
that upregulated transcription in upSET mutants enhances PcG
associated phenotypes.
(C) Nonexpressed genes are more affected upon UpSET knockdown. Genes differentially expressed in UpSET knockdo wn cells were clustered by their
association with RNA polymerase II.
(D and E) Transcriptome analysis of upSET
mutant ovaries. Heat maps show the summary of three biological replicates of independent upSET
ovaries compared to wild-type (D). Upregulated (red) and downregulated (green) genes are shown. Gene ontology analysis of the differentially expressed genes
(E) Twenty-two categories were enriched in the data set (p < 0.01).
(F) RNA-seq from wild-type ovaries (modENCO DE) was clustered in gene expression quintiles from high (Q1) to low (Q5) and upSET
-affected gene
percentage for each quintile is shown.
(G) Low/silent genes are upregulated in upSET
mutants. RT-qPCR of low expressing genes (Q4–5) was compared to highly expressed genes (Q1). (*p < 0.05;
**p < 0.01; not significant p values are not marked). Bars represent mean ± SEM.
(H–K) Loss of UpSET increases expression of transposons. RT-qPCR of transposons from nine representative repetitive element families found near UpSET
binding sites in upSET
mutant ovaries (H) or UpSET knockdown Kc cells (K). cDNA tiling analysis of transposon expression in upSET
ovaries with
DamID chromatin profile for a single peak (I) or region (J) of UpSET binding (see also Figure S2A). The middle track shows mRNA expression in ovaries. Repeat
element locations are shown in lower panel. Bars represent mean ± SEM (*p < 0.05; **p < 0.01; not significant p values are not marked).
See also Figure S6.
Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc. 1223
Page 10
UpSET Controls Cell Specification by Modulating Notch
mutant egg chambers exhibit several distinct phe-
notypes: anterior-posterior axis asymmetry (13%; n = 164),
reduced numbers of nurse cells (25%; n = 164), and egg
chamber fusions (16%; n = 164) (Figures 6A, 6B, 6D, and 6E).
When egg chambers mature beyond stage 9 (S9) (15%; n =
215), 80% of these display abnormally large nucleoli (Figures
6C and 6F). Staining with Orb, a marker for oocyte specifica-
tion, confirmed that although upSET mutants properly specify
the oocyte (Figures 6G–6I), egg chambers are not pinched off
properly, resulting in a variable number of nurse cells (Figure 6I).
UpSET is expressed in somatic and germ cell lineages
and exhibits a dynamic temporal expression pattern in the
germarium and during egg chamber development (Figures
6J–6K’). Together, these phenotypes suggest that UpSET is
required to establish proper signaling pathways within egg
Figure 6. UpSET Modulates Developmentally Regulated Gene Silencing
(A–F) upSET mutants show incomplete or fused egg chambers (arrows) in comparison to wild-type. Wild-type (A–C) and upSET
mutant (D–F) ovarioles
stained with DAPI (blue; to visualize DNA) and phalloidin (white). Confocal projections of 5 serial 1 mm optical sections are shown.
(G–I) UpSET is required for the establishment of anterior-posterior axis asymmetry and proper egg chamber formation. Wild-type (G) or upSET
(H and I) ovarioles stained with the oocyte-specific marker Orb showing alterations on the oocyte position and improper pinching off of egg chambers (arrowhead)
from the germarium. Confocal projections of eight serial 1 mm optical sections are shown.
(J–K’) UpSET protein levels are differentially regulated during oogenesis. Wild-type ovarioles were stained with a mix of antibodies against UpSET NH3- and
COOH- termini and costained with DAPI (blue). Confocal projections of five serial 1 mm optical sections are shown. Arrowhead indicates regions with differential
UpSET levels.
(L and M) Wild-type (L) and upSET
mutant (M) ovaries stained with antibodies against the Notch intracellular domain (green) and DAPI (blue).
(N and O) Costaining of Socs36E (green) and Eya (red) in wild-type (N) and upSET
mutant (O) ovarioles. DAPI (blue) was used to visualize DNA. Overlapping
expression is indicated with an arrowhead in upSET mutant ovary.
(P) UpSET restricts histone acetylation and chromatin accessibility around promoter/g enic regions via interaction and stabilization of Rpd3/Sin3-containing
HDAC machinery. Lack of UpSET increases chromatin accessibility that correlates with a higher histone acetylation level due to the loss of Rpd3 from tran-
scriptionally active genes. Changes in the chromatin landscape increase the probability of activating transposon expression and off-target genes.
Posterior is oriented to the right in all images. Scale bars, 50 mm.
See also Figure S7.
1224 Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc.
Page 11
Drosophila oogenesis is regulated by a fine interplay of sig-
naling pathways that control asymmetric organization and cell
specification. Our results show that UpSET functions are re-
quired to restrict activation of silent genes; thus we predicted
that components of developmental pathways would be affected.
The Notch pathway plays a key role in the specification of cells
that control the asymmetry of the Drosophila ovary (Bastock
and St Johnston, 2008, Assa-Kunik et al., 2007). Notch staining
in upSET mutant ovarioles shows proper activation in the germa-
rium, but Notch protein levels remain high in the maturing egg
chambers compared to wild-type, indicating a role for UpSET
in maintaining developmentally controlled Notch expression
(Figures 6L and 6M). Consistent with this, we find that Notch
target genes are likewise upregulated (Figures 6N, 6O, S7 I,
and S7J). upSET mutant flies exhibit higher levels of Socs36E
in polar cells compared to the rest of the follicle cells in early
oogenesis (Figure 6O), and S5 egg chambers show high and
heterogeneous accumulation of Socs36E in follicle cells regard-
less of the presence of Eya. upSET S8 mutant egg chambers
exhibit high levels of Soc36E and, unexpectedly, border cells
retain higher levels of this protein compared to wild-type (Figures
S7I and S7J). Upregulation of these genes was confirmed by
western blot (Figure S7K).
Notch signaling is also involved in a complicated interplay with
other developmental signaling pathways, including the JAK/
STAT pathway (Assa-Kunik et al., 2007). Consistent with upregu-
lation of the Notch pathway in upSET mutants, UpSET also
antagonizes JAK/STAT signaling (Figures S7L–S7O). Together,
our results suggest that uncontrolled Notch signaling, caused
by the lack of UpSET, impairs polar cell establishment early in
oogenesis and confirms that UpSET plays a key role in regulating
gene expression in vivo.
upSET encodes an inactive SET domain most closely related to
the mammalian protein MLL5. UpSET interacts with HDAC com-
plexes to restrict active histone marks to promoter and genic
regions. upSET mutant flies are female sterile due to uncon-
trolled upregulation of developmentally important signaling path-
ways such as that mediated by Notch in oogenesis. Thus, UpSET
defines a metazoan SET-containing epigenetic regulator: it func-
tions to recruit the HDAC machinery to transcriptionally active
regions to maintain proper chromatin opening and regulation of
gene expression (Figure 6P).
UpSET Modulates Chromatin Opening
In silenced loci, HDACs are recruited to chromatin via transcrip-
tional repressors/corepressors. However, recent genome-wide
analyses of HDACs and associated proteins uncovered a com-
plex binding pattern that also includes their recruitment to tran-
scriptionally active loci in metazoans (Filion et al., 2010; Wang
et al., 2009). In yeast, Set3 and Set4 recruit HDACs to the 5
regions of active genes, resulting in the recruitment of Rpd3,
which suppresses spurious transcription within the transcribing
gene (Kim and Buratowski, 2009; Govind et al., 2010; Carrozza
et al., 2005). Like Set3, UpSET is recruited to coding regions
of transcriptionally active genes and interacts with HDAC
machinery, and its depletion increases histone acetylation levels
over transcribed regions. However, UpSET is also required to
restrict active marks to promoters and transcribed regions via
its direct interaction with an Rpd3/Sin3-containing complex,
as well as to regulate chromatin accessibility. Our results also
suggest that although UpSET binding peaks at the 5
end of
active genes, UpSET also targets the 3
ends of active genes,
albeit to a lower level. UpSET binding correlates with that
of H2A.Z(His2Av)-containing nucleosomes, which are enriched
mainly over coding sequences in metazoan and generally more
accessible (Mavrich et al., 2008). In yeast, H2A.Z is enriched
mainly at the 5
regions of active genes, correlating with the
preferential recruitment of Set3 to the same region (Raisner
et al., 2005). Therefore, it is possible that UpSET-like machinery
is required to modulate chromatin accessibility of H2A.Z en-
riched regions.
In the absence of UpSET, promoter-associated active marks,
including H3K9Ac, H4K16, and H3K4m3, spread away from the
gene into neighboring flanking regions, which may increase the
probability of spurious transcription from cryptic TSSs or repet-
itive elements in the flanking regions (Carrozza et al., 2005). We
have shown previously that mammalian MLL2, which contains
an active SET domain, can spread along the chromatin fiber until
reaching an open promoter where it trimethylates H3K4 and acti-
vates gene expression ( Demers et al., 2007). We speculate that
further spreading of this active mark is restricted by the recruit-
ment of UpSET and its associated HDAC.
An Unusual SET Domain Containing Protein
The UpSET SET domain is a well conserved domain (Emerling
et al., 2002) and appears to be the Drosophila ortholog of yeast
Set3 and Set4 and mammalian MLL5 (Eissenberg and Shilati-
fard, 2010; Buratowski and Kim, 2010). None of these SET
domains encode H3K4 methyltransferase activity, other than
a small MLL5 isoform whose histone methyltransferase activity
may be activated by T440 glycosylation (Fujiki et al., 2009).
Although UpSET contains this residue, it does not exhibit meth-
yltransferase activity in vitro, and H3K4 methylation is unaffected
in upSET mutant flies and knockdown cells. One possibility is
that histones are not the main UpSET substrate because the
functions of different proteins including RNA pol II and PC2 are
methylation-dependent (Sims et al., 2011; Yang et al., 2011).
However, this seems unlikely as UpSET’s SET domain lacks
several key amino acids required for methyltransferase activity.
A second possibility is that the SET domain is used to identify
H3K4 methylation as a docking site (Emerling et al., 2002; Madan
et al., 2009). However, recent studies of the yeast ortholog Set3
suggest that the PHD domain performs this function (Kim and
Buratowski, 2009). A third possibility is that the SET domain is
required for protein-protein interactions or protein-RNA interac-
tions. Consistent with this, PcG proteins require stable interac-
tion with specific noncoding RNAs to promote silencing (Zhao
et al., 2008).
Biologic Implications of UpSET Function
Dysregulation of active chromatin marks could have broad impli-
cations for gene regulation and cell biology. We find that lack of
UpSET has severe consequences in gametogenesis.
Cell 151, 1214–1228, December 7, 2012 ª2012 Elsevier Inc. 1225
Page 12
Interestingly, deletion of set3 and MLL5 also affect gametogen-
esis in yeast and mouse, respectively. mll5 knockout mice are
viable, yet show aberrant terminal differentiation during sper-
matogenesis (Yap et al., 2011). Similarly, in yeast, deletion of
set3 results in normal growth, but deregulation of sporulation
genes during meiosis results in reduced ascus formation (Pijnap-
pel et al., 2001). Our results suggest that UpSET is required to
silence developmentally regulated and inducible genes. The
continuous presence of HDAC machinery near transcriptionally
active genes would make gene silencing a more efficient pro-
cess. In this regard, lack of UpSET impairs shut down of the
Notch pathway during oogenesis, resulting in a cascade of
noncontrolled expression of Notch target genes that ultimately
affects cell fate specification in the ovary.
Repeat elements, including transposons, are often expressed
at high levels during gametogenesis (Castan
eda et al., 2011).
Thus, a further increase in expression of repeat elements result-
ing from deregulation of chromatin architecture in the absence of
UpSET may have severe consequences on genome stability, as
well as on gene expression. In mammals, repeat elements are
regulated by DNA methylation, as well as by corepressors,
including PcG proteins (Leeb et al., 2010). Based on our results,
we hypothesize that deletion of UpSET ortholog proteins,
including MLL5 or its sister gene SETD5, would increase histone
acetylation and/or active methylation marks. Interestingly, loss
of the human chromosome segment 7q22 encoding the MLL5
gene is among the most common recurring cytogenetic aberra-
tions detected in myeloid malignancies with poor prognosis
(Emerling et al., 2002; Zhang et al., 2009; Madan et al., 2009;
Heuser et al., 2009). We suggest that the alterations in chromatin
structure associated with MLL5 deletions would act syner-
gistically with DNA demethylating agents often used to treat
leukemias to generate a highly unstable chromatin structure.
Consistent with this notion, hematopoietic cells from MLL5
knockout mice exhibit hypersensitivity to DNA demethylating
agents (Heuser et al., 2009).
Taken together, our results suggest that UpSET and its ortho-
logs represent a class of epigenetic regulators that restrict the
location of active chromatin marks to control promoter architec-
ture and may provide a new therapeutic target in cancer. In
addition, UpSET contributes to the stability of the genome by
preventing the spread of active marks to flanking regions and
the subsequent activation of cryptic promoters and repetitive
Fly Stocks
All fly stocks are maintained and crossed on yeast-cornme al-molasses-
malt extract medium at 25
C. The upSET alleles used in this study are: w;
(Exelixis Collection at Harvard), and w
; Mi{ET1}
(Bloomington Stock Center). See Extended Experimental
Procedures for additional alleles used in this study.
UpSET Antibody Production and Characterization
Polyclonal mouse antiserum against the N-end and C-end of UpSET was
generated by immunizing BALB/c BYJ Rb(8.12) 5BNR/J mice (Jackson
Labs) with a GST- UpSET N-end (1–319 aa) or UpSET C-end (2740–3155
aa) fusion protein. See Extended Experimental Procedures for antibody
Chromatin Accessibility Assay
Kc cells were harvested and nuclei were prepared as described (Henik-
off et al., 2009). Nuclei were treated with MNase I (1.6 U/ml, Sigma) for 10 min
at 37
C and the reaction stopped by adding EDTA to a final 20 mM concentra-
tion. Nuclei were nutated for 2 hr in 80 mM NaCl at 4
C in order to extract
hypersensitive nucleosomes. Nuclei were spun at 1,500 3 g for 10 min and
the supernatant was recovered and treate d with 100 mg/ml proteinase K for
6 hr. Nucleosomal DNA was purified with a QIAGEN PCR purification kit and
labeled for hybridization according to Nimblegen’s protocol.
NCBI Gene Expression Omnibus accession numbers for the data sets re-
ported in this article are GSE34720.
Supplemental Information includes Extended Experimental Procedures and
seven figures and can be found with this article online at
We thank Jorja Henikoff, Steve Henikoff, and Galina Filippova for their interest,
advice and comments on the manuscript. We thank Jorja Henikoff, Ryan Ba-
som and Janet Young for bioinformatic help. We also thank Erika Bach, Al
Courey, Barry Honda, Steven Hou, Lori Pile, Liz Wayner, the Harvard Exelixis
Collection, the Bloomington/Kyoto Stock Centers, and the Developmental
Studies Hybridoma Bank for antibodies, flies and other reagents used in this
study. This work was supported by NIH grants GM073021 and GM097083
(to SMP) and DK44746 and HL65440 (to MG).
Received: December 28, 2011
Revised: August 5, 2012
Accepted: September 26, 2012
Published online: November 21, 2012
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  • Source
    • "However, binding of the PHD-containing protein ING2 to H3K4me3 recruits HDAC complexes that promote the acquisition of an inactive chromatin conformation and repression of transcription (Becker, 2006). The Drosophila PHD-containing protein UpSET is also required to restrict chromatin accessibility by directly binding an Rpd3 HDAC complex (Rincon-Arano et al., 2012). In plants, the PHD-containing protein ORC1 has been shown to bind H3K4me3 and activate transcription of several target genes (de la Paz Sanchez and Gutierrez, 2009 ). "
    [Show abstract] [Hide abstract] ABSTRACT: The interplay among histone modifications modulates the expression of master regulatory genes in development. Chromatin effector proteins bind histone modifications and translate the epigenetic status into gene expression patterns that control development. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific proteins with a plant homeodomain (PHD) motif, SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS), function in the chromatin-mediated repression of floral initiation and play independent roles in the control of genes regulating flowering. Previous results showed that repression of the floral integrator FLOWERING LOCUS T (FT) requires EBS. We establish that SHL is necessary to negatively regulate the expression of SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), another floral integrator. SHL and EBS recognize di- and trimethylated histone H3 at lysine 4 and bind regulatory regions of SOC1 and FT, respectively. These PHD proteins maintain an inactive chromatin conformation in SOC1 and FT by preventing high levels of H3 acetylation, bind HISTONE DEACETYLASE6, and play a central role in regulating flowering time. SHL and EBS are widely conserved in plants but are absent in other eukaryotes, suggesting that the regulatory module mediated by these proteins could represent a distinct mechanism for gene expression control in plants.
    Preview · Article · Oct 2014 · The Plant Cell
  • Source
    • "Interestingly, UpSET, the MLL5 homologue of Drosophila, assembles with Rpd/Sin3-repressor complexes and thereby regulates promoter-associated histone acetylation and H3K4 tri-and di-methylation. Strikingly, loss or decrease of UpSET alters chromatin architectures of promoters, including broadening of H3K4me3 peaks [32]. Whether or not MLL5 plays a role in the observed H3K4me3 peak broadening that affects neuronal chromatin of some subjects with autism [11] will await further investigations (figure 2c ). "
    [Show abstract] [Hide abstract] ABSTRACT: The growing list of mutations implicated in monogenic disorders of the developing brain includes at least seven genes (ARX, CUL4B, KDM5A, KDM5C, KMT2A, KMT2C, KMT2D) with loss-of-function mutations affecting proper regulation of histone H3 lysine 4 methylation, a chromatin mark which on a genome-wide scale is broadly associated with active gene expression, with its mono-, di- and trimethylated forms differentially enriched at promoter and enhancer and other regulatory sequences. In addition to these rare genetic syndromes, dysregulated H3K4 methylation could also play a role in the pathophysiology of some cases diagnosed with autism or schizophrenia, two conditions which on a genome-wide scale are associated with H3K4 methylation changes at hundreds of loci in a subject-specific manner. Importantly, the reported alterations for some of the diseased brain specimens included a widespread broadening of H3K4 methylation profiles at gene promoters, a process that could be regulated by the UpSET(KMT2E/MLL5)-histone deacetylase complex. Furthermore, preclinical studies identified maternal immune activation, parental care and monoaminergic drugs as environmental determinants for brain-specific H3K4 methylation. These novel insights into the epigenetic risk architectures of neurodevelopmental disease will be highly relevant for efforts aimed at improved prevention and treatment of autism and psychosis spectrum disorders.
    Preview · Article · Sep 2014 · Philosophical Transactions of The Royal Society B Biological Sciences
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    [Show abstract] [Hide abstract] ABSTRACT: Appropriate gene expression relies on the sophisticated interplay between genetic and epigenetic factors. Histone acetylation and an open chromatin configuration are key features of transcribed regions and are mainly present around active promoters. Our recent identification of the SET-domain containing protein UpSET established a new functional link between the modulation of open chromatin features and active recruitment of well-known co-repressors in metazoans. Structurally, the SET domain of UpSET resembles H3K4 and H3K36 methyltransferases; however, it is does not confer histone methyltransferase activity. Rather than methylating histones to regulate gene expression like other SET domain-containing proteins, UpSET fine-tunes transcription by modulating the chromatin structure around active promoters resulting in suppression of expression of off-target genes or nearby repetitive elements. Chromatin modulation by UpSET occurs in part through its interaction with histone deacetylases. Here, we discuss the different scenarios in which UpSET could play key roles in modulating gene expression.
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