1. Developmental Biology
  2. Chromosomes and Gene Expression
Download icon

The Brm-HDAC3-Erm repressor complex suppresses dedifferentiation in Drosophila type II neuroblast lineages

  1. Chwee Tat Koe
  2. Song Li
  3. Fabrizio Rossi
  4. Jack Jing Lin Wong
  5. Yan Wang
  6. Zhizhuo Zhang
  7. Keng Chen
  8. Sherry Shiying Aw
  9. Helena E Richardson
  10. Paul Robson
  11. Wing-Kin Sung
  12. Fengwei Yu
  13. Cayetano Gonzalez
  14. Hongyan Wang  Is a corresponding author
  1. National University of Singapore, Singapore
  2. Duke-NUS Graduate Medical School Singapore, Singapore
  3. Institute for Research in Biomedicine, Spain
  4. Temasek Life Sciences Laboratory, Singapore
  5. Peter MacCallum Cancer Centre, Australia
  6. University of Melbourne, Australia
  7. University of Melbourne, Parkville, Australia
  8. Genome Institute of Singapore, Singapore
  9. Institució Catalana de Recerca i Estudis Avançats (ICREA), Spain
Research Article
Cite this article as: eLife 2014;3:e01906 doi: 10.7554/eLife.01906
5 figures and 2 tables

Figures

Figure 1 with 1 supplement
The Brm complex suppresses the formation of ectopic type II neuroblasts.

(AC) Type II MARCM clones of control (the MARCM driver; D), brm2 (B, B′) and brmT362 (C, C′) were labeled with Dpn (blue), Ase (red) and CD8::GFP (green). (D) Quantification of neuroblast number per type II MARCM clone for AC. (EH) Type II neuroblast lineage from control (‘the type II driver’: wor-Gal4 ase-Gal80; E), brm knockdown (F), snr1 knockdown (108599 KK; G), and bap60 knockdown (H) were labeled with Dpn (blue), Ase (red) and CD8 (green). (IJ′) type II MARCM clone of control (I) and bap55LL05955 (J, J′) were labeled with Dpn (blue), Ase (red) and CD8 (green). (K) Quantification of neuroblast number per type II lineage for EI. Arrows indicate neuroblasts. Clones are marked by CD8::GFP and indicated by white dotted line. Scale bars, 10 µm. *** indicates p<0.001.

https://doi.org/10.7554/eLife.01906.003
Figure 1—figure supplement 1
Analysis of chromatin remodelers in larval brains.

(A and B) Larval brains of control (elav-Gal4 driver) and brm knockdown under the control of elav-Gal4 driver were labeled with Insc and Mira (A) and dMyc (B). Central brain is to the left of white dotted line. (C) Type II MARCM clones of brmT362, brm2 with or without the expression of UAS-Brm were labeled with Dpn, Ase and CD8. (D) Type II driver control, snr1 TRiP RNAi (BDRC#32372), and snr1 VDRC RNAi (12645GD) under the type II driver were labeled with Dpn, Ase and CD8. (E) Control (MARCM driver) and snr1R3 type II MARCM clones were labeled with Dpn, Ase and CD8. (F) Control (MARCM driver) and bap55LL5955 type I MARCM clones were labeled with Dpn, Ase and CD8. (G) Type II neuroblast lineages of control (type II driver), iswi knockdown, nurf301 knockdown and acf1 knockdown were labeled with Dpn, Ase and CD8. Arrows, neuroblasts. Clone outline is indicated by white dotted line (CG). Scale bars, 10 μm.

https://doi.org/10.7554/eLife.01906.004
Figure 2 with 1 supplement
The Brm complex suppresses INP dedifferentiation into type II neuroblasts.

(AC′) Type II MARCM clones of control (the MARCM driver; A, A′), brm2 (B, B′) and brmT362 (C, C′) were labeled with Dpn (blue), Ase (red) and CD8::GFP (green). (D) Quantifications of INP number per type II clone for AC′. *** indicates p<0.001. (EG) Type II MARCM clones of control (E), brm2 (F) and brmT362 (G) were labeled with Dpn (blue), PntP1 (red) and CD8::GFP (green). (HI′) INP clones of a control (driver: erm-Gal4 [II]; erm-Gal4 [III]; (H) and brm RNAi under erm-Gal4 (II); erm-Gal4 (III) with UAS-Dcr2 UAS-CD8-GFP (I, I′) were labeled with Dpn (blue), Ase (red) and CD8 (green). White arrows indicate neuroblasts, yellow arrows indicate Dpn+ Ase+ mature INPs and yellow arrowheads indicate Dpn PntP1+ INPs. Clones are marked by CD8::GFP and indicated by white dotted line. Scale bars, 10 µm (AG) and 5 µm (HI′).

https://doi.org/10.7554/eLife.01906.005
Figure 2—figure supplement 1
Partial knock down of brm in INP clones.

Left panels, INP clones of a control (driver: erm-Gal4 [II]; erm-Gal4 [III]) and brm RNAi under erm-Gal4 (II); erm-Gal4 (III) with UAS-Dcr2 UAS-CD8-GFP were labeled with Brm (red) and CD8 (green). Right panels, Brm is absent in type II neuroblast clones under the type II driver. Clones are indicated by white dotted line. Scale bars, 10 µm.

https://doi.org/10.7554/eLife.01906.006
Figure 3 with 1 supplement
HDAC3 acts cooperatively with the Brm complex to suppress the formation of ectopic type II neuroblasts.

(AD) The driver control (A), brm RNAi (B), hdac3 RNAi (C), brm hdac3 double knockdown (D) under the type II driver were labeled with Dpn, Ase, and CD8. (E) Quantification of neuroblast number per type II MARCM clone in AD. (FG) Type II MARCM clones from the driver control (F) and snr16c hdac36c (G) homozygous MARCM clones were labeled with Dpn, Ase and CD8. Arrows indicate neuroblasts. (H) Quantification of neuroblast number per type II MARCM clone in FG. *** indicates p<0.001. (IJ) Clones are marked by CD8::GFP and indicated by white dotted line. Larval brain tissues from the wild-type MARCM clones (I) and snr16c hdac36c MARCM clones (J) were implanted into the abdomen of wild-type hosts. Scale bar, 10 µm.

https://doi.org/10.7554/eLife.01906.008
Figure 3—figure supplement 1
Brm is not important for the apical-basal polarity of neuroblasts.

(AC) Neuroblast of control MARCM clones and brm2 MARCM clones were co-labeled with aPKC (white), CD8 (green) and Phospho-Histone H3 (PH3; green) (A) or Numb, GFP and DNA (B) or Brat, GFP and DNA (C). Lower panels are enlarged images of the boxed region. (D) Type I MARCM clones from control (MARCM driver) and snr16c hdac36c were labeled with Dpn, Ase and CD8. (E) Type II MARCM clones from control (driver) and hdac3N were labeled with Dpn, Ase and CD8. Arrows, neuroblasts. Scale bars, 5 µm (AC), 10 μm (DE).

https://doi.org/10.7554/eLife.01906.009
Figure 4 with 1 supplement
The Brm remodeling complex physically associates with Erm and HDAC3.

(A) Co-immunoprecipitation (Co-IP) between Flag-Brm and Myc-Erm. (B) An illustration of Erm domains and truncated constructs. (C) Co-IP between Myc-Brm and Flag-Erm-N or Flag-Erm-C. (D) Co-IP between Flag-Bap60 and Myc-Erm-N or Myc-Erm-C. (E) Co-IP between Myc-Snr1 and Flag-Erm-N or Flag-Erm-C. (F) Co-IP was Flag-HDAC3 and Myc-Brm. (G) Co-IP between Flag-HDAC3 and Myc-Erm-N or Myc-Erm-C. IP was performed using anti-Flag or anti-Myc antibodies. Western blot was performed using anti-Flag and anti-Myc antibodies. (H) Protein pull-down assay. MBP, MBP-Erm-N and MBP-ErmC bound beads were incubated with protein extracts from S2 cells expressing Myc-Brm, Myc-Snr1 or Myc-HDAC3. Western blot was performed using an anti-Myc antibody. Coomassie blue (CB) staining showed 10% input of various purified MBP or MBP fusion proteins.

https://doi.org/10.7554/eLife.01906.010
Figure 4—figure supplement 1
Brm and Erm may regulate gene expression of some common downstream targets.

(A) Co-IP was performed using S2 cells expressing Flag-PntP1 and Myc-Brm. IP was performed using anti-Flag or anti-Myc antibodies. Western blot was performed using anti-Flag and anti-Myc antibodies. (B) The DNA binding preferences of the first zinc-finger ‘GTAG’ and the fourth zinc-finger ‘RAAA’. They are observed to be enriched in 270 Brm binding sites. (C) The distant distribution between the ChIP–chip peak and the occurrences of the motif in (D). (D) The de novo Erm-binding motif learned by SEME based on the 270 Brm binding sites.

https://doi.org/10.7554/eLife.01906.011
Figure 5 with 1 supplement
Brm genetically interacts with Erm to prevent dedifferentiation of INPs to neuroblasts.

(AD) Type II clones of control (the type II driver; A), brm knockdown (B), erm knockdown (C) and brm erm double knockdown (D) were labeled with Dpn (blue), Ase (red) and CD8 (green). (E) Quantifications of neuroblast number per type II neuroblast lineage for AD. (FI) Type II MARCM clones of brm2 (F, F′), UAS-Erm (G) and UAS-Erm, brm2 (HI) were labeled with Dpn (blue), Ase (red) and CD8 (green). (JM) Larval brains of control (J, elav-Gal4 driver), erm knockdown (K), snr1 knockdown (L) and erm snr1 double knockdown (M) were labeled with Dpn (blue), Ase (red) and Mira (green). (N) Quantifications of the number of type II neuroblasts per brain hemisphere in various genotypes in JM. Control (elav-Gal4), 7 ± 0; erm RNAi, 76.6 ± 14.2; snr1 RNAi, 219.5 ± 52.2; erm snr1 double knockdown (KD), 862.0 ± 106.7. (OQ) Larval brains of control (driver; O), erm knockdown (P) and erm hdac3 double knockdown (Q) under the type II driver were labeled with Dpn (blue), Ase (red) and CD8 (green). (R) Quantifications of neuroblast number per brain hemisphere in OQ. Central brain is to the left of white dotted lines. Arrows indicate neuroblasts. Clones were indicated by white dotted lines. Scale bars, 10 µm. *** indicates p<0.001.

https://doi.org/10.7554/eLife.01906.012
Figure 5—figure supplement 1
Knocking down of erm enhanced the neuroblast overgrowth observed in brm mutants.

(A) Type II MARCM clones of brmT362 and UAS-Dcr2; erm RNAi, brmT362 were labeled with Dpn, Ase and CD8. Arrows, neuroblasts. Clone outline is indicated by white dotted line. Scale bar, 10 µm. (B) Quantification of neuroblast number per type II MARCM clone. MARCM Control, 1.0 ± 0; brmT362, 4.1 ± 2.4; erm RNAi, brmT362, 9.9 ± 5.5. (C) Simultaneous knockdown of brm and notch in type II neuroblast lineages partially suppressed the ectopic neuroblast phenotype, compared with brm knockdown alone. Type II neuroblast lineages are labeled with Dpn, Ase and CD8. (D) Quantification of genotypes in C. brm notch double knockdown, 6.0 ± 4.7 neuroblasts/lineage, n = 76; brm knockdown, 10.8 ± 4 neuroblasts/lineage, n = 39. *** indicates p<0.001.

https://doi.org/10.7554/eLife.01906.013

Tables

Table 1

Histone modifiers and their RNAi lines

https://doi.org/10.7554/eLife.01906.007
S/No.Gene nameFull nameCG #Main functionVDRC RNAi lines
1enokEnoki mmushroomCG11290HATKK108400, GD37527
2nejNejire/CBPCG15319HATKK105115
3CG1894CG1894HATGD41575, GD41574
4CG2051CG2051HATGD33458
5MofMales absent on the firstCG3025HATKK105370
6Rpb4Rpb4CG33520HATGD21985, GD23308
7PcafGcnCG4107HATKK108943, GD21786
8YL-1YL-1CG4621HATGD21903
9ChmChameauCG5229HATKK105542
10DikDisketteCG7098HATGD46320
11lidLittle imaginal discsCG9088HATGD42203, KK103830
12Ada2bCG9638HATGD24076
13Sirt7CG11305HDACGD18043, GD18045
14HDAC4CG1770HDACGD20522
15HDAC3CG2128HDACKK107073
16HDACXCG31119HDACKK108098
17Sirt4CG3187HDACGD40295, KK110639
18Sirt2CG5085HDACKK103790
19Sir2CG5216HDACGD23199, KK108241, KK105502
20Bin1Bicoid interacting proteinCG6046HDACKK105352, GD15710
21Sirt6CG6284HDACGD22483
22GugGrungeCG6964HDACGD13687
23Rpd3HDAC1CG7471HDACGD46929, GD30600, GD46929
24Sin3aCG8815HDACKK105852
25Rtf1CG10955Methyl transferaseKK110392
26Vig2CG11844Methyl transferaseKK107081, GD17245
27eggegglessCG12196Methyl transferaseKK101677, GD33730
28escextra sexcombsCG14941Methyl transferaseGD5690, GD5692
29set2CG1716Methyl transferaseGD30707
30g9aCG2995Methyl transferaseGD25474
31pr-set7CG3307Methyl transferaseKK105422
32trrtrithorax-relatedCG3848Methyl transferaseGD10749, KK110276
33CG40351CG40351Methyl transferaseGD40683, GD10833, GD45267
34CG4565CG4565Methyl transferaseGD5665
35mes-4CG4976Methyl transferaseGD10836
36Art4Arginine methyl transferase 4CG5358Methyl transferaseKK107009
37Su(var)3–9auppressor of variegation 3–9CG6476Methyl transferaseGD39377
38Art1Arginine methyl transferase 11CG6554Methyl transferaseGD40388, KK110391
39ash2absent, small or homeotic discs 2CG6677Methyl transferaseKK100718
40LKRLysine ketoglutarate reductaseCG7144Methyl transferaseGD51346
41Su(z)12Suppressor of Zeste 205CG8013Methyl transferaseGD42422, GD42423
42Su(var)205Suppressor of variegation 205CG8409Methyl transferaseKK107477
43Ash1Absent, small or homeotic discs 1CG8887Methyl transferaseGD28928
Table 2

Predicted common target genes of Brm and Erm

https://doi.org/10.7554/eLife.01906.014
S/No.CG nameGene name
1CG10033for
2CG10117ttv
3CG10137CG10137
4CG10159BEAF-32
5CG10388Ubx
6CG10610ECSIT
7CG1071E2f2
8CG10844RyR
9CG1100Rpn5
10CG11228hpo
11CG11309CG11309
12CG11589VhaM9.7-c
13CG12165Incenp
14CG12321CG12321
15CG12333CG12333
16CG12387zetaTry
17CG12797Ciao1
18CG12818CG12818
19CG12819sle
20CG12855HPS1
21CG12994CG12994
22CG13004CG13004
23CG13016CG13016
24CG13018CG13018
25CG13117CG13117
26CG1322zfh1
27CG13316Mnt
28CG13350Ctf4
29CG13366CG13366
30CG13432qsm
31CG13472CG13472
32CG13688Ipk2
33CG13900CG13900
34CG13919CG13919
35CG14291CG14291
36CG14463CG14463
37CG1453Klp10A
38CG14813deltaCOP
39CG14814CG14814
40CG14938crol
41CG14939CycY
42CG15010ago
43CG15027CG15027
44CG15120CG15120
45CG15387CG15387
46CG15701CG15701
47CG15706CG15706
48CG15845Adf1
49CG1600Drat
50CG1616dpa
51CG17033elgi
52CG17035GXIVsPLA2
53CG17052obst-A
54CG17233CG17233
55CG17249CG17249
56CG17259CG17259
57CG17260CG17260
58CG1765EcR
59CG17803CG17803
60CG1785CG1785
61CG1817Ptp10D
62CG18292CG18292
63CG1845Br140
64CG18660Nckx30C
65CG18675CG18675
66CG2004CG2004
67CG2019disp
68CG2051CG2051
69CG2146didum
70CG2189Dfd
71CG2446Amun
72CG2698CG2698
73CG2720Hop
74CG2813cold
75CG2977Inx7
76CG3059NTPase
77CG3127Pgk
78CG31481pb
79CG3157gammaTub23C
80CG3165CG3165
81CG3166aop
82CG31712CG31712
83CG31713Apf
84CG3178Rrp1
85CG31794Pax
86CG31852Tap42
87CG31855CG31855
88CG31911Ent2
89CG32022CG32022
90CG32556chas
91CG32592hiw
92CG33116CG33116
93CG33162SrpRbeta
94CG3587CG3587
95CG3666Tsf3
96CG3842CG3842
97CG3857CG3857
98CG3920Reph
99CG42254CG42254
100CG42311grh
101CG42334comm3
102CG42362CG42362
103CG42363CG42363
104CG42365CG42365
105CG42379CG42379
106CG42380CG42380
107CG42381CG42381
108CG4400CG4400
109CG4590Inx2
110CG4619CG4619
111CG4645CG4645
112CG4798l(2)k01209
113CG4996CG4996
114CG5229chm
115CG5393apt
116CG5505scny
117CG5548CG5548
118CG5588Mtl
119CG5599CG5599
120CG5611CG5611
121CG5613CG5613
122CG5824l(3)07882
123CG5836SF1
124CG6022Cchl
125CG6202Surf4
126CG6218CG6218
127CG6235tws
128CG6241CG6241
129CG6272CG6272
130CG6322U4-U6-60K
131CG6343ND42
132CG6401CG6401
133CG6511CG6511
134CG6556cnk
135CG6565CG6565
136CG6604H15
137CG6634mid
138CG6829Ark
139CG6948Clc
140CG6951CG6951
141CG6983CG6983
142CG7082papi
143CG7085l(2)s5379
144CG7186SAK
145CG7191CG7191
146CG7372CG7372
147CG7379CG7379
148CG7564CG7564
149CG7597Cdk12
150CG7632CG7632
151CG7685CG7685
152CG7734shn
153CG7771sim
154CG7828APP-BP1
155CG7845CG7845
156CG7849CG7849
157CG7957MED17
158CG7961alphaCop
159CG8067CG8067
160CG8241pea
161CG8287Rab8
162CG8360CG8360
163CG8372CG8372
164CG8396Ssb-c31a
165CG8409Su(var)205
166CG8481CG8481
167CG8790Dic1
168CG8798Lon
169CG8817lilli
170CG9042Gpdh
171CG9054Ddx1
172CG9063Rich
173CG9065CG9065
174CG9243CG43345
175CG9243CG43346
176CG9244Acon
177CG9249CG9249
178CG9250Mpp6
179CG9305CG9305
180CG9376CG9376
181CG9473MED6
182CG9596CG9596
183CG9635RhoGEF2
184CG9641CG9641
185CG9730mRpL21
186CG9750rept
187CG9829poly
188CG9865CG9865

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)