Supplementary data (doc 172K)

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Supplementary data
PcG/ TrxG system
The PcG was originally defined as a set of genes whose individual mutation results in homeotic gene
overexpressed phenotypes similar to those Pc mutations, or which can enhance the phenotypes of Pc
mutant alleles. In contrast, TrxG genes are defined by their ability to counteract the activity of PcG genes in
homeotic gene regulation. There are at least 30 identified members of the PcG and TrxG family. Most of
these have mammalian homologs (Brock and van Lohuizen 2001, Dillon et al 2005, Francis and Kingston
2001, Kennison 1995, Ringrose and Paro 2004) (supplementary Table 1). PcG and TrxG genes encode
proteins that work in complex to modify chromatin. Two distinct PcG protein complexes have been
characterized to date: Polycomb repressive complex 1 (PRC1), and the E(z)-Esc complex (or PRC2). The
PRC1 complex contains Polycomb (Pc), Polyhomeotic (Ph), Posterior sex comb (Psc), zeste (z), dSbf1 and
Sex comb extra (Sce) (Brown et al 2005, Shao et al 1999) and five general transcription factors (Saurin et
al 2001). The E(z)-Esc complex contains Esc, E(z), Su(z)12, and histone binding NURF-55. The histone
deacetylase HDAC1 (Rpd3) may be present in some forms of the PRC2 complex (Czermin et al 2002,
Huang and Chang 2004). Biochemically purified PRC1 and PRC2 do not contain Pho or Phol (Muller et al
2002, Ng et al 2000, Saurin et al 2001). A new finding demonstrated that Pho exists in two distinct protein
assemblies: a Pho-dINO80 complex containing Drosophila INO80 nucleosome-remodeling complex, and a
Pho-repressive complex (PhoRC) containing the uncharacterized gene product dSfmbt (Klymenko et al
2006).
Similar PcG complexes are present in mammals. The first human PcG complex, termed the HPC-HPH PcG
complex, contains HPC, HPH, BMI1 and RING1. The second human PcG complex, termed the EED-EZH2
PcG complex, contains EED, EZH2, YY1, SUZ12, and RbAp46/48(Ng et al 2000, Satijn et al 1997, Satijn
et al 2001, Sewalt et al 1998, van Lohuizen et al 1991, van Lohuizen et al 1998). As EED has four isomers,
EED-EZH2 complex is further divided into PRC2 complex and PRC3 complex. The PRC2 complex
contains the longest EED form (form1) whereas PRC3 complex contains the two smallest EED forms
(form3 and form4). These EED isoforms selectively associate with distinct EED-EZH2 complexes with
resultant differential targeting of their associated histone lysine methyltransferase (HMTase)
activity
toward histone H3K27 or histone H1K26(Kuzmichev et al 2002, Kuzmichev et al 2004).
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To date, 4 TrxG protein complexes have been identified from Drosophila, all with different
chromatin-modifying properties (Cernilogar and Orlando 2005). The first complex contains BRM, MOR,
and OSA TrxG proteins, and at least 4 accessory proteins (Collins and Treisman 2000). The BRM proteins
functions as the ATPase subunit of this complex which is strongly related to the yeast SWI/SNF
nucleosome remodeling complex. The TrxG proteins Ash1 and TRX exist in the other two distinct
multiprotein complexes. Both are SET-domain proteins and function as histone methyltarnsferase. Ash1
selectively methylates H3K4, H3K9 and H4K20 (Beisel et al 2002, Byrd and Shearn 2003), whereas TRX
(trithorax) complex selectively methylates H3K4. The fourth TrxG complex, TAC1, contains the TrxG
protein TRX, the histone acetyltransferase CBP (CREB-binding protein), and the anti-phosphatase Sbf1
(Petruk et al 2001). A mammalian trithorax family histone methyltransferase complex has been extensively
investigated recently. This SET1-complex contains MLL1, MLL2, ASH2L, WDR5 and Rbp5. They have
been shown to be required for binding of the methyltransferase complex to the K4-dimethylated
H3(H3K4M2) tail as well as for global H3K4 trimethylation (H3K4M3) and Hox gene activation in human
cells (Hughes et al 2004, Wysocka et al 2005).
PcG and TrxG maintenance elements
Drosophila transgenic assays show that PcG and TrxG complexes are recruited to specific DNA elements
named as PcG and TrxG response elements (PREs and TREs, respectively). Both PREs and TREs are
complex elements, made up of binding sites for many proteins. The DNA binding proteins and the
consensus binding sites include Pho/phol (GCCAT), GAF/psg (GAGAG), zeste (BGAGTGV, YGAGYG),
Dsp1 (GAAAA) and Sp1/Klf (RRGGYG (Busturia et al 2001, Hodgson et al 2001, Mihaly et al 1998,
Mishra et al 2001, Schweinsberg et al 2004). Several of these proteins have been reported to have dual
roles in the recruitment of PcG and TrxG components. For example, Zeste binding sites appear to be
necessary for TrxG recruitment on PRE/TRE sequence (Dejardin and Cavalli 2004). At the same time,
Zeste binding sites are critical for PcG-dependent silencing at Ubx (Hur et al 2002). Similarly, GAF is a
member of the TrxG genes but may also play a role in PcG repression (Busturia et al 2001, Horard et al
2000, Mahmoudi et al 2003). Structural analysis of PREs and TREs shows that the elements required for
TrxG-dependent activation and PcG-dependent repression are separable but within 30-40 bp of each other.
Chromatin immunoprecipitation (ChIP) experiments demonstrated that the TrxG and PcG proteins bind to
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largely overlapping regions(Orlando et al 1998). Therefore, these DNA sequences required for maintenance
of gene regulation are generally called ‘maintenance elements’ or ‘cellular memory module’ (CMM) in
recognition of their dual function (Dejardin and Cavalli 2004).
The intermingling of PREs and TREs required for activation and repression raises the possibility that PRE
and TRE sequences may act in a concerted manner. Both of them might simultaneously be needed for
changing and maintaining opposite transcriptional states. In TrxG (ash1 and trx) mutants, expression of
Hox genes is lost within their normal expression domains, but the expression is restored in the TrxG and
PcG (E(z) double mutants (Klymenko and Muller 2004). Noticeably, a combination of zeste, Dsp1,
GAF/psg and pho /phol binding sites in the same number, orientation and spacing as in the native PRE is
insufficient to restore full PRE activity (Dejardin and Cavalli 2005). Thus, the PcG and TrxG proteins
recognizing the targeting chromatin is not only sequence-specific but also structure-dependent, where
additional interactions may be required to usher PcG and TrxG complexes to their DNA target. Current
work has suggested possible additional roles for other proteins as cofactors, such as the corepressor CtBP,
the DNA binding factor Grainhead (GRH) and members of the Sp1/KLF family (Blastyak et al 2006,
Brown et al 2005, Srinivasan and Atchison 2004, Srinivasan et al 2005). Most recently it has been found
that the Jumonji- and ARID-domain-containing protein, JARID2, is sufficient to recruit PcG proteins to a
heterologous promoter and form stable complex with PRC2(Pasini et al 2010). We have found the
coexistence of active and repressive histone modifications along the Hoxa9 regulatory region (Yan et al
2006). Ablation of menin abolishes both active and repressive histone modifications, including H3K4M3,
H3K4M2, H3K4M1, H3K9M2 and H3K27M2). These data suggest that co-localization of PcG-repressors
and TrxG-activators is able to guarantee regulated levels of transcription of active genes..
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Supplementary Table 1. PCG and TrxG proteins and their homologues
Drosophila proteins
Gene symbol
Human
homologues
Mouse
homologues
Protein domains
PcG
Extra sex combs
Multi sex combs
Pleiohomeotic
Pho-like
Polycomb
esc
mxc
pho
phol
Pc
eed
YY1
YY1
M33, mPc2
WD40 repeats
Polycomb-like
Polyhomeotic
Sex comb extra
Additional sex combs
Enhancer of zeste
Enhacer of Polycomb
Posterior sex combs
Pcl
ph
Sce
Asx
E(z)
E(Pc)
Psc
EED
YY1
YY1
M33 (HPC1), HPC2,
HPC3
PHF1
HPH1, HPH2
RING
ASXL1, ASXL2
EZH1, EZH2
EPC1, EPC2
BMI1, MEL18
Mph1, rae28, Mph2
Asxl1, Asxl2
Enx2, Enx2
Epc1, Epc2
Bmi1, mel18
PHD fingers
SAM
RING
Sex combs on midleg
Scm
Suppressor of zeste 2
Supressor of zest 12
Corto
Lola like (batman)
Pipsqueak
Super sex comb
Su(z)2
Su(z) 12
corto
lolal
psq
sxc
SCML1, SCML2,
SCMH1
BMI1, MEL18
SUZ12
Scml1, Scml2,
Scmh1
Bmi1, mel18
Chromodomain
SET
RING (zinc
finger)+helix-turn-helix
SAM
RING finger
Chromodomain
BTB/POZ domain
GA(n), DNA binding
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TrxG
Trithorax
Trithorax-like
trx
trxl
MLL/ALL1
MLL1
Absent, small, or
homeotic discs2
Absent, small, or
homeotic disc1
Brahma
Ash-2
hASH2
ASH2L
SET
Single zinc-finger
DNA-binding motif
PHD, SPRY domain
Ash-1
hASH1
ASH1L
SET, PHD
brm
HBRM, BRG1
Osa
Moria
osa
mor
ELD/OSA1
BAF170/BAF155
Zeste
GAF(GAGA factor)
Kismet
Tonalli
Devenir (breathless)
Kohtalo
Skuld
Little imaginal discs
z
Trl
kis
tna
Dev (btl)
kto
skd
lid
Urdur
Leg arista wing
complex
urd
lawc
SNF2/SWI2 ATPase
domain; bromodomain
ARID
SANT domain, leucine
zipper
GA(n), DNA binding
SNF2/SWI2 ATPase
SP-RING zinc finger
Fgfr, tyrosine kinase
ARID; RING/PHD;
Leucin zipper; PHD(2)
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