Article

The enhancer of trithorax and polycomb gene Caf1/p55 is essential for cell survival and patterning in Drosophila development

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Development (Impact Factor: 6.46). 05/2011; 138(10):1957-66. DOI: 10.1242/dev.058461
Source: PubMed

ABSTRACT

In vitro data suggest that the human RbAp46 and RbAp48 genes encode proteins involved in multiple chromatin remodeling complexes and are likely to play important roles in development and tumor suppression. However, to date, our understanding of the role of RbAp46/RbAp48 and its homologs in metazoan development and disease has been hampered by a lack of insect and mammalian mutant models, as well as redundancy due to multiple orthologs in most organisms studied. Here, we report the first mutations in the single Drosophila RbAp46/RbAp48 homolog Caf1, identified as strong suppressors of a senseless overexpression phenotype. Reduced levels of Caf1 expression result in flies with phenotypes reminiscent of Hox gene misregulation. Additionally, analysis of Caf1 mutant tissue suggests that Caf1 plays important roles in cell survival and segment identity, and loss of Caf1 is associated with a reduction in the Polycomb Repressive Complex 2 (PRC2)-specific histone methylation mark H3K27me3. Taken together, our results suggest suppression of senseless overexpression by mutations in Caf1 is mediated by participation of Caf1 in PRC2-mediated silencing. More importantly, our mutant phenotypes confirm that Caf1-mediated silencing is vital to Drosophila development. These studies underscore the importance of Caf1 and its mammalian homologs in development and disease.

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Available from: Umesh C Karandikar, Mar 02, 2016
1957RESEARCH ARTICLE
INTRODUCTION
The development of complex multicellular organisms requires the
precise patterning of a wide range of appendages, organs, tissues
and cell types. To a large extent, this task is accomplished by the
reiterative use of a limited number of conserved pathways and
proteins. However, we are only beginning to understand how
pathways and proteins can be used repeatedly and still achieve
developmental diversity. Chromatin remodeling is an important
mechanism that allows cells to stably yet reversibly lock in
repressed or active chromatin states, thereby restricting the fate of
the cell during development (Kadonaga, 1998; Schulze and
Wallrath, 2007; Vermaak et al., 2003).
Drosophila Chromatin Assembly Factor 1 (Caf1, also known
as p55; RbAp48 – FlyBase) is a 55 kDa protein containing seven
WD repeats and -helical regions in the N and C termini, and
binds directly to histone H4 (Song et al., 2008; Tyler et al.,
1996). The Caf1 gene has homology to two human genes,
retinoblastoma associated protein 46 (RbAp46; RBBP7 – Human
Gene Nomenclature Database) and retinoblastoma associated
protein 48 (RbAp48; RBBP4 – Human Gene Nomenclature
Database) (Tyler et al., 1996; Verreault et al., 1996). Caf1 shares
87% and 84% amino acid identity with human RbAp48 and
RbAp46, respectively, and is therefore likely to prove an
excellent tool for studying the functional and developmental
roles of the human proteins (Tyler et al., 1996). Drosophila Caf1,
first identified as a component of the Chromatin Assembly
Factor 1 complex (CAF-1) which acts in nucleosome assembly
following DNA replication, is a component of many chromatin
remodeling complexes (Tyler et al., 1996). Caf1 is also found as
a component of the Nucleosome Remodeling Factor (NURF)
and, like its human homologs RbAp46 and RbAp48, is a
component of retinoblastoma (RB)-containing complexes
(Korenjak et al., 2004; Martinez-Balbas et al., 1998; Qian and
Lee, 1995; Qian et al., 1993; Taylor-Harding et al., 2004).
Caf1 is also a member of the Polycomb group (PcG) complex
Polycomb Repressive Complex 2 (PRC2), along with Extra Sex
Combs (ESC), Suppressor of Zeste 12 [SU(Z)12] and Enhancer of
Zeste [E(Z)] (Muller et al., 2002; Tie et al., 2001). The role of PcG
proteins has been best characterized in silencing of Hox genes,
although PcG silencing also occurs at many non-Hox loci (Bello et
al., 1998; Chen and Rasmuson-Lestander, 2009; Dura and Ingham,
1988; Netter et al., 1998; Pelegri and Lehmann, 1994;
Schuettengruber et al., 2007). PRC2 silences genes by effecting
histone modifications, particularly trimethylation of histone H3 at
lysine 27 (Czermin et al., 2002) (for a review, see Schuettengruber
et al., 2007). Beyond its important role in development, PRC2 has
also been implicated in cancer pathways. For example, in
mammals, aberrant epigenetic modification of PRC2 target genes
is associated with colorectal cancer (Widschwendter et al., 2007).
With participation in such diverse complexes, Drosophila Caf1
is likely to have important roles in multiple aspects of
development. Until now, mutant or reduced expression phenotypes
of Caf1 homologs have been reported in yeast, Arabidopsis and C.
elegans, but no mutations in Drosophila Caf1 have been identified
and genetically characterized (Bouveret et al., 2006; Guitton and
Development 138, 1957-1966 (2011) doi:10.1242/dev.058461
© 2011. Published by The Company of Biologists Ltd
1
Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA.
2
Department of Pathology, Baylor College of Medicine,
Houston, TX 77030, USA.
3
Department of Biochemistry and Molecular Biology,
M. D. Anderson Cancer Center, Houston, TX 77030, USA.
4
Program in
Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.
5
Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030,
USA.
6
Department of Ophthalmology, Baylor College of Medicine, Houston, TX
77030, USA.
*Author for correspondence (gmardon@bcm.tmc.edu)
Accepted 14 February 2011
SUMMARY
In vitro data suggest that the human RbAp46 and RbAp48 genes encode proteins involved in multiple chromatin remodeling
complexes and are likely to play important roles in development and tumor suppression. However, to date, our understanding of
the role of RbAp46/RbAp48 and its homologs in metazoan development and disease has been hampered by a lack of insect and
mammalian mutant models, as well as redundancy due to multiple orthologs in most organisms studied. Here, we report the first
mutations in the single Drosophila RbAp46/RbAp48 homolog Caf1, identified as strong suppressors of a senseless overexpression
phenotype. Reduced levels of Caf1 expression result in flies with phenotypes reminiscent of Hox gene misregulation. Additionally,
analysis of Caf1 mutant tissue suggests that Caf1 plays important roles in cell survival and segment identity, and loss of Caf1 is
associated with a reduction in the Polycomb Repressive Complex 2 (PRC2)-specific histone methylation mark H3K27me3. Taken
together, our results suggest suppression of senseless overexpression by mutations in Caf1 is mediated by participation of Caf1 in
PRC2-mediated silencing. More importantly, our mutant phenotypes confirm that Caf1-mediated silencing is vital to Drosophila
development. These studies underscore the importance of Caf1 and its mammalian homologs in development and disease.
KEY WORDS: Caf1, Drosophila, Epigenetics, Polycomb, RbAp48, Senseless
The enhancer of trithorax and polycomb gene Caf1/p55 is
essential for cell survival and patterning in Drosophila
development
Aimée E. Anderson
1
, Umesh C. Karandikar
2
, Kathryn L. Pepple
1
, Zhihong Chen
3
, Andreas Bergmann
3,4
and
Graeme Mardon
1,2,4,5,6,
*
DEVELOPMENT
Page 1
1958
Berger, 2005; Hennig et al., 2003; Jullien et al., 2008; Lu and
Horvitz, 1998; Ruggieri et al., 1989). Furthermore, reduced
expression of the mammalian Caf1 homologs RbAp46 and RbAp48
is observed in human cancers, and in vitro studies suggest both
genes may act as tumor suppressors, but the lack of Caf1 mouse
models hinders our understanding of its potential role in human
disease (Guan et al., 2001; Guan et al., 1998; Ishimaru et al., 2006;
Kong et al., 2007; Li et al., 2003; Pacifico et al., 2007; Thakur et
al., 2007).
Chromatin remodeling adds a unique level of transcriptional
regulation to the signaling pathways that control development by
stably repressing genes in a heritable manner, and may therefore be
of particular importance to genes and pathways that are re-used in
development. The Drosophila senseless (sens) gene is one example
of a gene that is reiteratively employed. Specifically, Sens functions
in a number of processes in both the embryo and larval imaginal
tissues, including the compound eye. In the third instar eye disc,
Sens is necessary for differentiation of the R8 photoreceptor, the
founding cell of each ommatidium (Frankfort et al., 2001).
Continued expression of Sens is necessary for maintenance of R8
fate during pupal development (Xie et al., 2007). Sens is also
necessary for formation of interommatidial bristles (Domingos et
al., 2004; Frankfort et al., 2004; Morey et al., 2008).
Recently, we performed a screen in Drosophila for dominant
modifiers of a disorganized eye phenotype caused by
overexpression of senseless (Pepple et al., 2007). Here, we report
the identification of the complementation group S(ls)3, comprising
three loss-of-function alleles, as the first mutations in Drosophila
Caf1. In the current study, we describe functional and
developmental consequences of Caf1 gain- and loss-of-function in
Drosophila. Our observations indicate that severe loss of Caf1
expression results in cell death, consistent with key roles for Caf1
in chromatin remodeling complexes necessary for basic cellular
functions. However, at intermediate levels of wild-type Caf1
expression, we observe homeotic transformations and eye
phenotypes consistent with a major role for Caf1 in Polycomb
silencing. We present evidence suggesting that the genetic
interaction between Caf1 and sens is mediated by Polycomb
silencing. These results suggest that Caf1 is essential not only for
basic cellular functions and survival, but also for maintenance of
cellular identity and normal body plan.
MATERIALS AND METHODS
Fly stocks and mosaic analysis
Caf1 alleles generated in our laboratory were reported previously as S(ls)3
(Pepple et al., 2007). The Caf1 Genomic Rescue construct (Caf1GR) was
generated by recombineering a fragment from BACR32A03 into pACMAN
(Venken et al., 2006). pACMAN vector was a gift from Hugo Bellen (Jan
and Dan Duncan Neurological Research Institute, Houston, TX, USA). The
UAS-Caf1 construct was generated by insertion of the Caf1 cDNA
(LD33761, Drosophila Genomics Resource Center) into pUAST-attB
(Bischof et al., 2007). Transgenic flies were obtained by injection of
Caf1GR or UAS-Caf1 into the second chromosome insertion site of the
VK1 line (Venken et al., 2006). Other fly stocks used include w
11 8
, lz-GAL4,
UAS-GFP and UAS-sens/FM7 (ls). eyFLP; FRT82B p{w
+
} cl/TM6B,
Dp
49FK-1
c
1
/SM5, cn
1
, w; Dp
a1bw1
sp
1
/CyO, w; E2f2
76Q1
cn
1
bw
1
/CyO,
E2f
07172
/TM3, E(z)
731
, Rpd3
04556
ry
506
/TM3 ry
RK
Sb
1
Ser
1
, esc
1
, UAS-pb and
UAS-Antp were obtained from the Bloomington Drosophila Stock Center.
Caf1
short
, Caf1
med
and Caf1
long
alleles were recombined onto the FRT82B
chromosome. Mosaic heads were generated by crossing mutant alleles on
FRT chromosomes with eyFLP; FRT82B p{w
+
} cl/TM6B, obtained from
the Bloomington Drosophila Stock Center (Newsome et al., 2000).
Mapping and sequencing
Three rounds of P-element mapping were performed to localize S(ls)3
mutations to a 50 kb region as described (Zhai et al., 2003). Sequencing
was performed by the MD Anderson DNA Analysis Facility and
Macrogen. Sequence analysis was performed using Sequencher software
(Genecodes).
Light microscopy
Tangential sections of the adult retina were performed as described
(Tomlinson and Ready, 1987). Images were acquired with a Zeiss Axioplan
2 microscope, Zeiss Axiocam digital camera and Axiovision software.
Adobe Photoshop software was used to resize images and adjust brightness
and contrast.
Scanning electron microscopy (SEM)
Samples were prepared as described previously (Pepple et al., 2007).
Antibody staining and confocal microscopy
The following antibodies were used: rabbit anti-Caf1 (1:1000, AbCam);
guinea-pig anti-Sens (1:5000, a gift from Hugo Bellen); mouse anti-active-
Caspase 3 (1:1000, R&D Systems); rat anti-Elav (1:500, Developmental
Studies Hybridoma Bank); and rabbit anti-H3K27me3 (1:200, Lake
Placid). The following secondary antibodies were all used at 1:500: Alexa-
conjugated goat anti-rabbit secondary antibody (Molecular Probes), goat
anti-guinea pig Cy3 (Jackson ImmunoResearch), goat anti-rat Cy3
(Jackson ImmunoResearch) and goat anti-rat Cy5 (Jackson
ImmunoResearch). For histone methylation staining experiments, discs
were dissected and stained as described previously (Fan and Bergmann,
2010). For all other staining experiments, eye-antennal imaginal discs were
dissected and stained, and images acquired, as described previously (Pepple
et al., 2007). Adobe Photoshop and Gimp software were used to process
image brightness, color, contrast and noise, and to merge channels.
RESULTS
Mutations in the gene Chromatin Assembly Factor
1 (Caf1) interact with senseless
Overexpression of UAS-sens under control of lozenge-GAL4
(hereafter abbreviated ls) results in eyes with a disruption of the
regular hexagonal array of ommatidia in the adult retina. This
disruption is caused, in part, by formation of extra interommatidial
bristles (Pepple et al., 2007). Previously, we have reported the
isolation of a three-member lethal complementation group, S(ls)3,
that was discovered in a screen for mutations that dominantly
modify ls (Pepple et al., 2007). Each of the three alleles of S(ls)3
dominantly suppresses the extra bristles and disrupted ommatidial
array of ls to a similar degree (Fig. 1B-I), indicating a role in the
Sens pathway. We performed P-element recombination mapping
(Zhai et al., 2003) to narrow the lethal mutations to a 50 kb region
containing 15 genes. Sequencing of all exons from this region
revealed mutations in the gene Caf1 (CG4236) in all three alleles
(Fig. 1A). Caf1 encodes a 430 amino acid protein with seven WD
repeats (Song et al., 2008; Tyler et al., 1996). Caf1
long
contains a
G-to-A transition that changes a conserved amino acid from glycine
to asparagine. The mutation in Caf1
med
truncates the protein in its
third WD repeat. The Caf1
short
mutation, which would truncate the
protein in the first WD repeat, occurs in the first exon and probably
results in a null allele due to nonsense-mediated decay (Valencia-
Sanchez and Maquat, 2004).
To test whether the lethality of the three mutants comprising
S(ls)3 is due to mutations in Caf1, we generated flies containing a
12 kb genomic rescue construct, designated Caf1GR, designed to
ensure all regulatory regions of Caf1 are included (not shown).
This resulted in the inclusion of several other genes: Rpb7,
CG31344, Art3, mRPS10 and the 3 end of CG12241. The Caf1GR
construct rescues the lethality of the most severe truncation allele,
RESEARCH ARTICLE Development 138 (10)
DEVELOPMENT
Page 2
Caf1
short
, in combination with either Caf1
med
or Df(3R)ED5664, a
deficiency that uncovers Caf1. Rescued flies are viable and appear
phenotypically normal, indicating that the lethality of Caf1
short
is
due to mutations within the 12 kb region.
Reduced Caf1 activity disrupts segment identity
We attempted to rescue flies homozygous or trans-heterozygous for
Caf1 alleles with expression of a UAS-Caf1 construct under the
control of Ubiquitin-GAL4 (Ubi-GAL4). In a wild-type
background, Ubi-GAL4/UAS-Caf1 flies are viable and
phenotypically normal. Ubi-GAL4/UAS-Caf1; Caf1
short
/Caf1
long
flies are also viable and are phenotypically normal, suggesting that
lethality of the mutant alleles is due to mutations in Caf1 (data not
shown). Both Ubi-GAL4/UAS-Caf1; Caf1
short
/Caf1
med
and Ubi-
GAL4/UAS-Caf1; Caf1
short
/Caf1
short
flies raised at 25°C survive to
become pharates, though few adults eclose. Pharates dissected live
from pupae and the few adults that do eclose appear phenotypically
normal (data not shown). Given the differential ability of UAS-Caf1
to rescue different allelic combinations at 25°C, we reasoned that
raising Ubi-GAL4/UAS-Caf1; Caf1
short
/Caf1
med
flies at 18°C would
allow us to analyze the effects of reduced levels of Caf1. All flies
of this genotype raised at 18°C have disorganized kidney-shaped
eyes, as well as homeotic transformations of the aristae to leg-like
structures that occasionally terminate in claws. Other defects,
including duplications of scutellar bristles and wing defects, are
sometimes seen.
We performed a series of temperature shift experiments and
determined that the eye and antennal phenotypes are dependent on
the temperature during the third larval instar (data not shown). Ubi-
GAL4/UAS-Caf1; Caf1
short
/Caf1
med
flies raised at 25°C during third
instar have normal eyes and antennae (Fig. 2A,B,E). By contrast,
flies raised at 18°C have small, disorganized eyes (Fig. 2C,D,F). In
Fig. 2D, the transformed arista terminates in a claw (arrow).
Tangential sections of adult eyes from animals raised at 18°C
display abnormalities in the arrangement of rhabdomeres, including
loss of small central rhabdomeres (Fig. 2G, arrowheads). These
defects are probably due to insufficient levels of Caf1 given the
temperature-sensitive nature of the GAL4-UAS system (for a
review, see Duffy, 2002) and are reminiscent of phenotypes caused
by overexpression of Hox genes (Bello et al., 1998). Hox genes are
major targets of the PcG complexes, which are responsible for
establishing and maintaining silencing of Hox genes in regions
where their expression would interfere with proper anterior-
posterior patterning (Schuettengruber et al., 2007). In addition to
eye and antennal defects, wing defects are seen in many animals
1959RESEARCH ARTICLEDrosophila Caf1 and development
Fig. 1. Loss of Caf1 function suppresses an ectopic senseless
phenotype. (A)
Three mutations in Caf1 resulting in amino acid
substitution or truncation of the Caf1 protein are shown. Purple boxes
indicate WD repeats. The positions of mutations are indicated by yellow
vertical bars. (B,F)
lz-GAL4, UAS-sens/+ (ls) flies have disorganized eyes
characterized by extra bristles. Three lethal mutations in Caf1 can
dominantly suppress the disorganized eyes and extra bristles of the ls
phenotype. (C,G)
ls/+; Caf1
long
/+. (D,H)
ls/+; Caf1
med
/+. (E,I)
ls/+;
Caf1
short
/+. F-I are magnified regions of the eyes shown in B-E,
respectively. Scale bars: 100
m.
Fig. 2. Reduced levels of Caf1 expression result in homeotic
transformations and loss of neuronal structures in the eye. Caf1
mutant flies expressing UAS-Caf1 under the control of Ubi-GAL4 were
raised at 25°C or 18°C during larval development to modulate the level
of Caf1 expression. (A,B)
Flies raised at 25°C during larval development
have normal head morphology (A; eye and arista magnified in B).
(C,D)
A fly raised at 18°C during larval development has disorganized
eye structure (C; eye and arista magnified in D) and altered arista
morphology, resembling transformation to tarsus. The arrow in D
indicates a claw-like structure. The eye of a fly raised at 18°C during
larval development is disorganized; many interommatidial bristles are
missing (F) in contrast to the eye of a fly raised at 25°C during larval
development (E). (G)
A section through the eye of a fly raised at 18°C
during larval development reveals several ommatidia missing small
rhabdomeres (two examples are indicated by red arrowheads). A
normally constructed ommatidium is indicated by the green arrow. The
genotype of all flies in this figure is ubi-GAL4/UAS-
Caf1;Caf1
short
/Caf1
med
. Scale bars: 100
m.
DEVELOPMENT
Page 3
1960
raised at 18°C during larval development; an example of a fly with
reduced wings and scutellar bristle duplication is shown in Fig. S1
in the supplementary material.
Caf1 is required for cell survival
Larvae homozygous for Caf1
med
or Caf1
short
appear grossly
normal but mature slowly and survive to first or second instar.
Many Caf1
long
larvae survive to third instar, but die shortly after
forming a disorganized pupa. Homozygous Caf1 germline
mutant clones are not recovered. To assess the role of Caf1 in
Drosophila development and overcome the problem of larval
lethality, we attempted to make clones using heat shock-Flippase
(hs-FLP). Caf1
long
clones survive well in the Drosophila eye
(data not shown). However, hs-FLP clones of Caf1
short
and
Caf1
med
do not survive in adults, suggesting cell death or
proliferation defects. To recover clones of Caf1
short
and Caf1
med
,
we used the ey-FLP;FRT cell-lethal system, which generates
eyes composed mostly of homozygous mutant tissue and has the
advantage of marking any remaining heterozygous ommatidia
with w
+
(Newsome et al., 2000). We used two approaches to
analyze mutant eyes: light microscopy (LM), which reveals the
gross structure of the eye and can differentiate between
homozygous and heterozygous tissue but does not reveal the fine
details of ommatidial architecture; and scanning electron
microscopy (SEM), which shows the detailed structure of the eye
but does not allow identification of clones.
Eyes mutant for Caf1
long
have large clones of mutant tissue with
only a subtle disruption in the ommatidial array, leading to a
slightly disorganized appearance (Fig. 3B,F,J). Misplaced bristles
are occasionally seen in these eyes and many bristles are shorter
than wild type. By contrast, Caf1
short
eyes contain very little mutant
tissue (white tissue in Fig. 3K). The remaining eye, composed
mostly of heterozygous tissue, is small, has irregular ommatidial
structure, and is almost completely devoid of bristles (Fig.
3C,G,K). This suggests impaired cell viability or proliferation in
Caf1
short
homozygous tissue. Consistent with these results, no adult
Caf1
short
clones are recovered in the thorax using Ubx-FLP; FRT
82B Sb
63
P{w
+
y
+
}, whereas large wild-type or Caf1
long
clones are
recovered in most animals of the correct genotype with this
technique (data not shown). Approximately 20% of flies with ey-
FLP-induced Caf1
short
or Caf1
med
clones have extra or missing
appendages on the head, including the ocelli, antennae and
maxillary palps. In some cases, structures are only partially
duplicated, leading to a branched appearance. An example of a fly
with a duplicated antenna is shown in Fig. 3D and enlarged in Fig.
3H. Although the original publication of the eyFLP; FRT p{w
+
} cl
chromosome described the use of an eye-specific version of ey-
GAL4, with our stock, we have observed clones outside of the eye
proper, including the peripodial epithelium and the antennal disc
(Newsome et al., 2000). Therefore, although adult clones outside
the eye field are not marked, these duplicated structures are
probably due to Caf1 clones outside the eye field.
Overexpression of Caf1 resembles the Caf1 loss-
of-function phenotype
To assess the effects of varying levels of Caf1 expression, we
generated an ey-GAL4,UAS-Caf1 recombinant chromosome to
overexpress Caf1 in the eye antennal disc. These flies are viable
RESEARCH ARTICLE Development 138 (10)
Fig. 3. Altered expression of Caf1 disrupts eye and head
development.. (B-D,F-H,J,K) The ey-FLP; cl technique was
used to generate eyes composed mostly of Caf1 mutant
tissue. (A,B,E,F) Eyes mostly homozygous for Caf1
long
(B,F) are
slightly disorganized compared with wild type (A,E). Some
interommatidial bristles (IOBs) are missing (circle in F) or
occasionally occur at adjacent vertices (bracket in F).
(I-K)
Homozygous Caf1
long
clones produced with the ey-FLP; cl
technique are large (white tissue in J), similar to wild-type (I),
while Caf1
short
clones are small compared with remaining
heterozygous tissue (K). (C,G,K) Eyes generated with the ey-
FLP; cl technique and the Caf1
short
allele are small, highly
disorganized and almost completely devoid of bristles (C,G),
and are composed mostly of heterozygous tissue (K). (D,H)
An
example of a fly of this genotype with a duplicated antenna is
shown in D, with area in red rectangle enlarged in H. (L)
The
head of a wild-type fly. (M,N)
The eyes of ey-GAL4 +/+ UAS-
Caf1 flies raised at 25°C are large and disrupted, with reduced
numbers of IOBs (M; magnified in N). Increased dose of Caf1
in ey-GAL4, UAS-Caf1 homozygous flies results in smaller,
more highly disorganized eyes. (O,P)
An example with an
ectopic macrochaete is shown in O and magnified in P. Scale
bars: 100
m.
DEVELOPMENT
Page 4
when raised at 18°C, 25°C and even 30°C. The eyes of ey-
GAL4/UAS-Caf1 flies raised at 25°C are disorganized and slightly
smaller than wild type (Fig. 3M), and resemble the Caf1
long
phenotype (Fig. 3J). Additionally, the eyes of these flies have
reduced numbers of interommatidial bristles (Fig. 3N). When
raised at 30°C, the eyes are smaller and more disorganized,
probably owing to the increased activity of GAL4 at higher
temperatures in Drosophila (Duffy, 2002). Eyes and heads of ey-
GAL4, UAS-Caf1 homozygotes raised at 25°C have an even more
severe phenotype reminiscent of Caf1 complete loss-of-function:
the eyes are extremely small and ommatidial structure is highly
disrupted. Occasionally, ectopic structures are seen in these eyes
(Fig. 3O,P). Outside the eye, duplications and deletions of other
head structures are seen frequently in ey-GAL4, UAS-Caf1
homozygotes, similar to Caf1
short
clones (data not shown).
Eyes with Caf1
short
clones have excess apoptosis
and disrupted early development
Even when using the ey-FLP; FRT cl technique, Caf1
short
clones
are small during third instar, and the spacing of developing R8
photoreceptors is disrupted (see Fig. S2 in the supplementary
material). Caf1
short
clones generated with this technique are rare at
6 hours of pupal development and almost completely absent by 24
hours (data not shown). To determine whether the small size, loss
of mutant tissue and disorganization of Caf1
short
eyes is due in part
to apoptosis, we stained third instar eye discs with an antibody
against active Caspase 3 (Fan and Bergmann, 2010). Normally,
programmed cell death occurs in the Drosophila eye disc during
pupal development to remove excess cells during differentiation of
secondary and tertiary pigment cells, and formation of the
hexagonal lattice (Frohlich, 2001). At late third instar, control eye
discs using the eyFLP; FRT 82B P{w+} cl chromosome show very
little Caspase 3 staining (Fig. 4A,C). Consistent with their large
adult size, third instar ey-FLP; FRT P{w
+
} cl/FRT Caf1
long
eye
discs also show very low levels of active Caspase 3 (Fig. 4D,F).
Elav staining in these eye discs shows a nearly regular array of
developing photoreceptors, similar to that of control discs (compare
Fig. 4E with 4B). By contrast, eye discs with homozygous clones
for Caf1
short
are small and have extensive Caspase 3 staining (Fig.
4G,I). Notably, the majority of Caspase 3 staining in discs
harboring Caf1
short
clones is observed anterior to Elav staining,
implying that most cell death occurs anterior to the furrow in cells
that would normally be proliferating. The arrangement of Elav-
positive photoreceptor clusters is also irregular (Fig. 4H). Similar
to ey-FLP; FRT82B Caf1
short
/FRT82B P{w
+
} cl eye discs,
significant apoptosis marked by Caspase 3 staining occurs in the
anterior region of the eye disc in ey-GAL4, UAS-Caf1 third instar
eye discs (Fig. 4J-L). Increased apoptosis is also observed in the
Ubx-FLP; FRT82B Caf1
short
/FRT82B ubi-GFP wing disc (see Fig.
S3 in the supplementary material).
The ls eye phenotype can be rescued by
overexpression of Hox genes
The homeotic transformations observed in Caf1 mutant flies
partially rescued by Caf1 cDNA expression (Fig. 2) suggest that
PRC2 function may be impaired when levels of Caf1 are low,
resulting in ectopic expression of one or more Hox genes. To test
whether overexpression of Hox genes could account for dominant
suppression of the ls phenotype by mutations in Caf1, we crossed
ls flies to UAS lines for several Hox genes, including proboscipedia
(pb), abdominal-A (abd-A), Deformed (Dfd), Ultrabithorax (Ubx),
Sex combs reduced (Scr), Antennapedia (Antp) and labial (lab).
Overexpression of Hox genes alone in the eye also leads to a
disorganized eye phenotype; each of the above UAS lines, when
crossed to lz-GAL4 alone, also generates disorganized eyes (data
not shown). Therefore, suppression of ls can only occur if levels of
Hox and Sens activity functionally cancel each other out.
Consistent with our hypothesis, ectopic expression from two lines,
UAS-Antp and UAS-pb, results in strong suppression of the ls
disorganized eye phenotype (Fig. 5B,C,F,G) compared with ls
crossed to UAS-GFP (Fig. 5A,E).
Mutations in Polycomb Group genes suppress the
ls phenotype
As Caf1 is a component of PRC2, we hypothesize that suppression
of ls by Caf1 mutations is due to loss of PRC2 function and predict
that mutations in other PcG genes should also dominantly suppress
the ls phenotype. To test this prediction, we crossed ls flies to
mutations in PRC1 and PRC2 genes. The PRC2 complex mutations
1961RESEARCH ARTICLEDrosophila Caf1 and development
Fig. 4. Neural differentiation is disrupted and programmed cell
death is increased in Caf1
short
mutant eye discs. (A-I)
Control eye
discs (ey-FLP; FRT82B/FRT82B p{w
+
} CL; A-C) and Caf1
long
eye discs (ey-
FLP; FRT82B Caf1
long
/FRT82B p{w
+
} CL; D-F) stained for active Caspase 3
(A,D, and green in C,F) show only diffuse and sporadic staining, while
Caf1
short
eye discs (ey-FLP; FRT82B Caf1
short
/FRT82B p{w
+
} CL; G-I)
display bright patches of Caspase 3 staining. (J-L)
Similar to Caf1
short
discs, Caspase 3 staining is increased in ey-GAL4, UAS-Caf1 discs. Elav
staining (B,E,H,K; magenta in C,F,I,L) marks the position of
differentiating photoreceptors, which is highly disrupted in Caf1 mutant
discs. Scale bars: 100
m.
DEVELOPMENT
Page 5
1962
Su(z)12
2
, Su(z)12
3
, Su(z)12
4
, Rpd3
303
, Rpd3
04556
, E(z)
731
, esc
1
and
esc
2
each suppressed the ls phenotype, as did PRC1 mutations
Polycomb
3
(Pc
3
) and Pc
15
(Fig. 5D,H; data not shown). We also
tested mutations in genes encoding factors from other Caf1-
containing complexes. Mutations in three members of the dREAM
complex were suppressors of ls (see Fig. S4 in the supplementary
material), consistent with previous observations of cooperation
between PcG and dREAM complex members (Dahiya et al., 2001;
Kotake et al., 2007; Tonini et al., 2004).
Caf1 interacts genetically with a Polycomb Group
gene
We tested for a genetic interaction with the PRC1 gene Pc (Fig.
5I). Males heterozygous for the Pc
15
allele display ectopic sex
combs on the second and sometimes third legs, consistent with
homeotic transformation of second or third legs to first leg.
Crossed to wild type, we observe that an average of 82% of male
Pc
15
/+ flies have an ectopic sex comb on at least one posterior
leg. By contrast, only 12.3% of Pc
15
/Caf1
short
and 30% of
Pc
15
/Caf1
long
double heterozygote male flies have one or more
ectopic sex combs. Thus, Caf1 is a strong dominant suppressor
of the Pc
15
phenotype. Although this result seems consistent with
a Trithorax Group-like activity for Caf1, mutations in other bona
fide PcG members sometimes interact genetically in a similar
manner; these data suggest that like these PcG members, Caf1
can be placed in the Enhancer of Trithorax and Polycomb (ETP)
group (for a review, see Fedorova et al., 2009). Furthermore,
ETP-like activity of Caf1 is not surprising, given its
aforementioned participation in multiple chromatin remodeling
complexes. To confirm that the interaction is specific to Pc, we
repeated this analysis with the Pc
3
allele, which also results in
ectopic sex combs on second and sometimes third legs. Eighty-
five percent of Pc
3
/+ males exhibit ectopic sex combs, compared
with 64% of Pc
3
/Caf1
long
flies (P<0.06; 170 Pc
3
/+ and 88
Pc
3
/Caf1
P9
flies counted over six replicates). There was no
significant difference between Pc
3
/+ and Pc
3
/Caf1
long
. Thus,
although mutations in Caf1 suppress Pc
3
less strongly than Pc
15
,
the genetic interaction is consistent between the two alleles.
Caf1 mutant tissue is deficient in a PRC2-
dependent histone methylation mark
The PRC2 complex is associated with trimethylation of histone 3
at lysine 27 (H3K27me3) (Czermin et al., 2002) due to the
methyltransferase activity of the E(z) protein. The H3K27me3
mark is associated with repression of gene transcription. Our
previous results suggest that loss of Caf1 results in a reduction in
PRC2 activity, and therefore predicts a reduction in global levels
of H3K27me3. To test this, we generated Caf1
short
and Caf1
long
clones in the eye disc using ey-FLP and stained third instar discs
with an antibody against H3K27me3 (Fig. 6). The H3K27me3
mark is reduced in clones of both Caf1 alleles compared with
surrounding heterozygous and wild-type tissue. A similar reduction
of H3K27me3 in Caf1 clones is seen in the wing disc (see Fig. S5
in the supplementary material). Taken together, these results
RESEARCH ARTICLE Development 138 (10)
Fig. 5. The ls phenotype is suppressed by ectopic expression of
Hox genes. Genetic interactions between sens, Hox, Caf1 and PcG are
shown. (A,E)
Similar to ls, the eyes of lz-GAL4, UAS-sens/w; UAS-GFP/+
flies are disorganized and have numerous ectopic bristles (A; magnified
in E). (B,C,F,G) The eyes of lz-GAL4, UAS-sens/w; UAS-pb/+ (B;
magnified in F) and lz-GAL4, UAS-sens/w; UAS-Antp/+ (C; magnified in
G) flies have a more wild-type appearance, with reduced numbers of
interommatidial bristles and a more regular pattern of hexagonal
ommatidia. (D,H,I) ls is also dominantly modified by mutations in PcG
members, including E(z) (D; magnified in H). In addition to its
interaction with sens, Caf1 interacts genetically with Pc (I). Eighty-two
percent of Pc
15
/+ animals have at least one ectopic sex comb. By
contrast, only 12.3% of Pc
15
/Caf1
short
animals have ectopic sex combs
(P<0.0000001) and 30.0% of Pc
15
/Caf1
long
animals (P<0.000001). For
each genotype, six separate crosses were scored. A total of 297 Pc
13
/+,
212 Pc
13
/Caf1
short
and 156 Pc
13
/Caf1
long
animals were counted. Scale
bars: 100
m.
Fig. 6. Trimethylation at lysine 27 of histone 3 is reduced in Caf1
mutant clones. (A-C)
Genotype of eye discs shown is ey-FLP; FRT82B
Caf1
long
/FRT82B ubi-GFP. (D-F)
Genotype of eye disc in ey-FLP; FRT82B
Caf1
short
/FRT82B ubi-GFP. Clones are marked by absence of GFP
staining (A,D; green in C,F). Reduced staining of H3K27me3 (B;
magenta in C) is observed in Caf1
long
clones (A; green in C). H3K27me3
(E; magenta in F) is also reduced in Caf1
short
clones (D; green in F).
Examples of clones are marked by arrows. Scale bars: 100
m.
DEVELOPMENT
Page 6
suggest that at least some of the phenotypes and genetic
interactions we observe with partial or complete loss of Caf1 are
due to loss of PRC2 activity and H3K27 trimethylation.
DISCUSSION
Loss or reduction of Caf1 disrupts development
via altered PRC2 activity
Several lines of evidence suggest that the participation of Caf1 in
PcG complexes may account for many of the phenotypes we
observe in flies with altered expression of Caf1. First, Caf1 loss-
of-function clones in the eye have phenotypes ranging from slight
disorganization and bristle defects (Fig. 3B,F,J) to almost complete
loss of homozygous tissue in adults (Fig. 3C,G,K), and incomplete
rescue of Caf1 results in adult eyes that are small and disorganized
(Fig. 2C,F). Clones of many PcG genes have similar phenotypes in
the eye. Loss of E(z) or Pc causes mild defects in differentiation in
the third instar disc, but clones fail to survive in adults (Brook et
al., 1996; Janody et al., 2004). An analogous situation occurs in
Caf1
short
clones, where expression of Elav, which marks
differentiating neurons, is present in Caf1 clones at third instar but
Caf1
short
tissue is largely missing in the adult. Derepression of Hox
genes could account for these phenotypes, as ectopic expression of
many Hox genes in the eye field causes small disorganized eyes in
adults (Plaza et al., 2001).
Second, flies with incomplete rescue of Caf1 display a range of
homeotic phenotypes, notably transformation of arista to leg (Fig.
2C,D). Similar homeotic transformations, including antenna-to-leg
transformations, are a hallmark of mutations in PcG genes (Denell,
1973; Lewis, 1978; Wang et al., 2006). We also observe a genetic
interaction between Caf1 and the PRC1 gene Pc, as mutations in
Caf1 are able to dominantly suppress the homeotic transformation
of second or third leg to first leg in Pc
15
/+ males (Fig. 5).
Third, the disrupted patterning of Caf1
short
mutant heads may
also result from PcG dysfunction (Fig. 3). It is possible that these
patterning defects are non-cell autonomous and may be an indirect
result of widespread apoptosis in the eye disc (Fig. 4). Normally,
when an imaginal disc is injured, remaining cells proliferate and
assume correct identities, leading to a perfectly patterned adult
structure (McClure and Schubiger, 2007) (for a review, see
Bergmann and Steller, 2010). This type of regeneration requires
that some determined cells must change their fates and involves
substantial chromatin remodeling. Under specific circumstances,
the disc can regenerate with incorrect patterning, leading to
duplication, deletion or transformation of structures, a phenomenon
referred to as transdetermination (McClure and Schubiger, 2007).
Levels of many PcG transcripts are increased in transdetermining
imaginal discs, and heterozygous mutations in PcG genes can
enhance transdetermination in regenerating imaginal discs (Klebes
et al., 2005). Therefore, one interpretation of the patterning defects
in Caf1
short
mutant discs is that under the stress of widespread
apoptosis, the remaining heterozygous tissue is haploinsufficient
for the chromatin remodeling activity required to properly
regenerate and pattern the injured disc. Consistent with this
interpretation, no extra or missing appendages are observed in flies
with Caf1
long
clones, which show less active Caspase 3 staining at
third instar.
Finally, in Caf1 mutant tissue, we observe a reduction in levels
of the H3K27me3 mark, which is associated with inactive
chromatin and PRC2 activity (Fig. 6). These data are consistent
with a disruption of PRC2 function as a result of loss of Caf1 and
represent the first in vivo evidence that Caf1 is an essential member
of this chromatin remodeling complex in an animal model.
Sens and Caf1 may act in parallel competing
pathways
One obvious question arises from the current study: why were
multiple Caf1 alleles identified in a screen for modifiers of the sens
overexpression phenotype? Moreover, it is surprising that
mutations in Caf1 were not identified in previous Drosophila
modifier screens involving PcG or Rb pathway members (Ambrus
et al., 2009; Janody et al., 2004; Steele et al., 2009). We propose
that the link between sens and Caf1 is due to the role of Caf1 in
PcG-mediated silencing.
Recent evidence suggests that Sens and Hox proteins can
compete for binding at overlapping sites at an enhancer of the
rhomboid (rho) locus (Li-Kroeger et al., 2008). When the Hox
protein Abdominal-A (Abd-A) binds, transcription of rho is
activated, whereas binding by Sens leads to repression of rho. In
the embryo, this mechanism acts as a molecular switch to allow
differentiation of either chordotonal organs (under control of Sens)
or hepatocyte-like cells called oenocytes (by the action of Abd-A).
We propose that a similar mechanism underlies the suppression of
the Sens overexpression phenotype (Fig. 7). We hypothesize that
one or more targets of Sens in the eye contain similar overlapping
sites that can be bound by either Sens or a Hox protein. During
normal development, these loci are bound by neither Sens nor Hox
in undifferentiated cells posterior to the furrow, as no Hox genes
are known to be widely expressed in the eye field (Hueber and
Lohmann, 2008). In the absence of both types of factors, these loci
are transcriptionally active, and are necessary to ultimately attain
the proper fates of the cells in which they are expressed. When
Sens is overexpressed, as in ls, Sens binds to its recognition site in
the downstream loci, repressing transcription. Repression of these
genes initiates a cascade leading to a change in cell fate; for
example, some of the cells that would normally become secondary
or tertiary pigment cells now become bristle precursors, giving rise
to the extra bristles of ls. However, when one copy of Caf1 is lost,
a slight derepression of the Hox genes occurs due to loss of PcG
activity. Hox proteins are now able to compete with Sens for the
overlapping binding sites, tipping the balance towards activation of
downstream genes and attainment of normal cell fate – effectively
suppressing ls. The ability of ectopic expression of pb and Antp in
the eye to suppress ls is consistent with this hypothesis.
Suppression of ls by Hox proteins is particularly significant given
that ectopic expression of Antp alone in the eye field leads to a
small and disorganized eye (Bello et al., 1998; Plaza et al., 2008;
Plaza et al., 2001). As Sens activity is exquisitely sensitive to Hox
proteins, especially in the eye, our screen for modifiers of a sens
overexpression phenotype was therefore ideal for identifying
mutations in Caf1.
Previous studies have explored pro-apoptotic roles of Hox
proteins and anti-apoptotic roles of Sens. It is therefore possible
that one effect of Hox gene derepression in ls eyes suppressed by
Caf1 may be restoration of an apoptotic fate in cells that would
otherwise form bristle precursors due to ectopic Sens. Abd-A
expression in the abdomen during normal third instar larval
development leads to apoptosis of proliferating neuroblasts of the
central nervous system, and ectopic expression of other Hox genes
can also cause neuroblast apoptosis (Bello et al., 2003).
Accordingly, survival of neuroblasts is dependent on PcG activity
to repress Hox gene expression (Bello et al., 2003). Furthermore,
expression of Sens is necessary in the Drosophila embryonic
salivary gland to prevent apoptosis (Chandrasekaran and
Beckendorf, 2003). Thus, one possible mechanism for suppression
of ls by Caf1 mutations is that in the ls eye, Sens may promote the
1963RESEARCH ARTICLEDrosophila Caf1 and development
DEVELOPMENT
Page 7
1964
ectopic bristle fate partly by repressing apoptotic genes in cells
normally fated to die, whereas in the ls eye suppressed by
mutations in Caf1, ectopic Hox proteins may promote apoptosis
and prevent bristle formation. We were unable to detect increased
Ubx expression by antibody staining; however, a very small
increase in one or more Hox proteins may be all that is necessary
to change the transcriptional state of downstream loci and prevent
the ectopic bristles and other defects in the highly sensitized ls eye
– especially in the eye field, where no Hox genes are known to be
highly expressed. Furthermore, the fact that multiple Hox proteins
can recognize the same DNA binding site offers the possibility that
the competitive effect of each Hox protein type on genes with
overlapping Sens/Hox binding sites would be additive (Hueber and
Lohmann, 2008). Therefore, although loss of one copy of Caf1
may only cause a small derepression of any one Hox gene, mild
derepression of many Hox genes collectively can lead to strong
repression of the ls phenotype.
Caf1 has multiple roles in Drosophila
development
Biochemical evidence suggests that Caf1 is a member of multiple
complexes that effect gene regulation through chromatin
remodeling, suggesting that it is a vital component of the cell’s
arsenal of chromatin modifying factors (Henikoff, 2003). Although
our results suggest that disruption of PRC2 function may be the
most important consequence of Caf1 gain- or loss-of-function,
many phenotypes we observed in Caf1 mutant tissue are also
reminiscent of mutations in members of other complexes
previously shown to contain Caf1. It is not surprising that all three
alleles of Caf1 in the current study are homozygous lethal, and that
Caf1
short
cells have poor viability, considering that Caf1 has been
found in the NURF and CAF-1 complexes, which have
fundamental roles in nucleosome assembly and spacing (Bulger et
al., 1995; Kamakaka et al., 1996; Martinez-Balbas et al., 1998;
Tyler et al., 1996; Verreault et al., 1996). The apoptosis we observe
in eyes with Caf1
short
clones is also consistent with a role for Caf1
in the dREAM (Drosophila Rbf, E2F2, and Myb-interacting
proteins) complex. Members of the E2f family of transcription
factors can complex with Dp proteins and bind short recognition
sites to activate transcription (Brehm and Kouzarides, 1999;
Classon and Harlow, 2002; Korenjak and Brehm, 2006; Macaluso
et al., 2006). When Rb binds the E2f-Dp complex, transcription is
repressed. Like Caf1 homozygotes, homozygous rbf1 null flies die
in early larval development (Du and Dyson, 1999). Fully rbf1-
deficient embryos display increased apoptosis, a phenotype
reminiscent of the increased active Caspase-3 staining seen anterior
to the morphogenetic furrow in eye discs with Caf1
short
clones (Fig.
4).
The mammalian homologs of sens, Growth Factor
Independence 1 (Gfi1) and Gfi1b are essential to the development
of multiple cell types and have been implicated as oncogenes
(Duan et al., 2005; Gilks et al., 1993; Gilks et al., 1995; Grimes et
al., 1996; Hochberg et al., 2008; Karsunky et al., 2002; Kazanjian
et al., 2004; Liao et al., 1995; Person et al., 2003; Schmidt et al.,
1998; Wallis et al., 2003; Yucel et al., 2003) (for reviews, see Duan
and Horwitz, 2003; Hock and Orkin, 2006). Therefore, the
possibility that Caf1 links Sens with the activity of PcG complexes
through parallel, competing pathways has implications for both
Drosophila development and the activity of Gfi1 family members
in human development and disease, and warrants additional study
beyond the scope of the present work. Our results underscore the
importance of Caf1 to diverse processes, including cell survival and
tissue identity, and highlight the participation of Caf1 in multiple
chromatin remodeling complexes. Further studies are needed to
fully assess the importance of Caf1 in Drosophila development, as
well as its developmental role in other chromatin remodeling
complexes.
Acknowledgements
We thank Mardelle Atkins for helpful discussions and reading of the
manuscript, Georg Halder for expertise with sequencing, Richard Atkinson and
Jodie Polan for advice on confocal microscopy, and Kenneth Dunner (Cancer
Center Core Grant CA16672) for assistance with scanning electron
microscopy. This work was supported by the Retina Research Foundation
RESEARCH ARTICLE Development 138 (10)
Fig. 7. A model for suppression of ls by mutations in Caf1. We
propose that overlapping Sens- and Hox-binding sites occur in one or
more Sens targets that are necessary to suppress bristle fate. (A)
In the
absence of Sens, as occurs in non-neuronal eye cells, the normal
transcriptional state is on. Activity of this gene suppresses bristle fate
and/or promotes other cell fates, such as pigment cell (oblong cell
marked P) or apoptosis (starburst labeled A). Hox genes are not
transcribed due to repressive chromatin state induced and maintained
by PcG complexes with the participation of Caf1. (B)
When excess Sens
is introduced into the cell by ls, Sens occupies its binding site in
downstream targets and transcription is halted. Downstream, cells that
would normally achieve fates such as pigment cells or apoptosis instead
become bristle precursors (cluster of grey cells labeled B). (C)
Cells
heterozygous for Caf1 mutations have fewer functional PcG complexes
and therefore some level of Hox gene derepression due to
haploinsufficiency of Caf1. Some Hox protein (H) is able to bind its site
in Sens target genes, which remain on and no fate change occurs.
(D)
In the sensitized ls background, haploinsufficiency of Caf1 also leads
to derepression of Hox genes. Ectopic Hox proteins compete with
excess Sens produced by lz-GAL4,UAS-sens for binding sites, and ens is
unable to repress transcription of its target genes, thus preventing fate
change and production of ectopic interommatidial bristles.
DEVELOPMENT
Page 8
(G.M.), and National Institutes of Health grants R01 EY011232 (G.M.), R01
GM068016 (A.B.), R01 GM074977 (A.B.), R01 GM081543 (A.B.), T32
CA009299 (A.E.A.) and T32 EY007102 (K.L.P.). Deposited in PMC for release
after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058461/-/DC1
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RESEARCH ARTICLE Development 138 (10)
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    • "(A) A schematic representation of a PRC2 component, CAF1p55, containing seven WD40 domains, is shown. The Caf1p55short allele carries a point mutation that changes Gly to a stop codon at AA position 79, whereas the Caf1p55long alleles carries a point mutation that changes Gly to Asp at AA position 375 (Anderson et al. 2011). (B) To determine the effect of altering Caf1p55 activity in Psc-induced rough eye phenotype, a single copy of either Caf1p55short mutant or Caf1p55long mutant chromosome was introduced in GMR-Gal4;f00391 flies. "
    [Show abstract] [Hide abstract] ABSTRACT: A Polycomb group protein, Posterior sex combs (Psc), was identified in a genetic screen designed to find factors that can specifically induce morphological defects in rbf1 mutant eyes. We discovered that rbf1 mutations enhance developmental phenotypes caused by Psc overexpression such as ectopic cell death and disorganized ommatidia. Our genetic analysis revealed that Psc-induced developmental defects are strongly influenced by Caf1p55, which is a shared component of several chromatin-associated complexes including a histone chaperone complex dCAF-1. Interestingly, the expression levels of dCAF-1 components, CAF1p105 and CAF1p180, are increased in rbf1 mutants, while the expression level of CAF1p55 itself remains relatively unchanged. We demonstrated that the increased levels of CAF1p105 and CAF1p180 are required for the hypersensitivity of rbf1 mutant cells to Psc-induced cell death and for the developmentally regulated cell death normally observed in rbf1 mutant eyes. We propose that Caf1p105 and Caf1p180 are important determinants of cell death sensitivity in rbf1 mutant cells and contribute to the genetic interaction between Psc and rbf1.
    Full-text · Article · Jul 2013 · G3-Genes Genomes Genetics
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    • "In Drosophila larvae, Pcl facilitates the recruitment of PRC2 to chromosomes (Savla, Benes, Zhang, & Jones, 2008). Classical Polycomb phenotypes related to the misregulation of Hox genes have been described for all PRC2 members and with the exception of esc and escl, all homozygous null alleles show larval lethality (Anderson et al., 2011; Birve et al., 2001; Duncan, 1982; Phillips & Shearn, 1990; Struhl & Brower, 1982). According to the Drosophila developmental transcriptome project (Gelbart & Emmert, 2011; McQuilton, St Pierre, & Thurmond, 2012), mRNA levels for all PRC2 genes peak in early embryos, then decline at larval stages and increase in female but not male adults. "
    [Show abstract] [Hide abstract] ABSTRACT: Proper development of an embryo requires tightly controlled expression of specific sets of genes. In order to generate all the lineages of the adult, populations of pluripotent embryonic stem cells differentiate and activate specific transcriptional programs whereas others are shutdown. The role of transcription factors is obvious in promoting expression of such developmental genes; however maintenance of specific states throughout cell division needs additional mechanisms. Indeed, the nucleoprotein complex of DNA and histones, the chromatin, can act as a facilitator or barrier to transcription depending on its configuration. Chromatin-modifying enzymes regulate accessibility of DNA by establishing specific sets of chromatin, which will be either permissive or repressive to transcription. In this review, we will describe the H3K9/HP1 and Polycomb pathways, which mediate transcriptional repression by modifying chromatin. We discuss how these two major epigenetic silencing modes are dynamically regulated and how they contribute to the early steps of embryo development.
    Full-text · Article · Apr 2013 · Current Topics in Developmental Biology
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    • "SUZ12 directly binds to the promoters of the PRC2 target genes probably through its Zinc-finger domains, and Suz12 expression is increased in human colon tumors [22]. In the mammalian PRC2 complex, there is a pair of highly homologous WD40 proteins RbAP46/48, which is shown to be essential for cell survival and patterning in Drosophila development, and knockdown of the Drosophila RbAP46/48 P55 causes severe reduction in histone H3K27me3 methylation level [23]. Although AEBP2 is not essential for PRC2 activity, it is required for optimal enzymatic activity [21]. "
    [Show abstract] [Hide abstract] ABSTRACT: PRC2 is the major H3K27 methyltransferase and is responsible for maintaining repressed gene expression patterns throughout development. It contains four core components: EZH2, EED, SUZ12 and RbAp46/48 and some cell-type specific components. In this study, we focused on characterizing the histone binding domains of PHF1 and PHF19, and found that the Tudor domain of PHF1 and PHF19 selectively binds to histone H3K36me3. Structural analysis of these Tudor domains also shed light on how these Tudor domains selectively binds to H3K36me3. The Tudor domain binding of H3K36me3 of PHF1, PHF19 and likely MTF2 provide another recruitment and regulatory mechanism for the PRC2 complex. The first PHD domains of PHF1 and PHF19 do not exhibit histone H3K4 binding ability, nor do they affect the Tudor domain binding to histones.
    Full-text · Article · Dec 2012 · Biochemical and Biophysical Research Communications
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