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Cell Reports, Volume 14
Supplemental Information
Alternative Splicing of G9a
Regulates Neuronal Differentiation
Ana Fiszbein, Luciana E. Giono, Ana Quaglino, Bruno G. Berardino, LorenaSigaut, Catalina von Bilderling, Ignacio E. Schor, Juliana H. Enriqué Steinberg, MarioRossi, Lía I. Pietrasanta, Julio J. Caramelo, Anabella Srebrow, and Alberto R. Kornblihtt
Figure S1
N2a Cells qPCR B
siLUC siE10 siG9a A
D
Figure S2
C
B
A
RA 2 days
RA 2 days +
control 2 days RA 2 days +
BIX 2 days
Figure S3
A
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
1,1
A B C D E F G H I J K
Dif
Undif
0
0,2
0,4
0,6
0,8
1
1 2
nC
hIP
: IP
H3
K9
me
2/H
3to
tal
Promoter Ex 4 Ex10 Ex 7 Ex 14 Ex 22
Ex 27
A B C D E F G
H I J K
Intergenic
nC
hIP
: IP
H3
K9
me
2/H
3to
tal
B C
CpG Island
- DMSO
D
RA
-
Figure S4
- DMSO
N2a cells
E9 E10 E11
Globin
Promoter
I9 I10 I globin
E globin
B
A
Figure S5
Figure S6 A B
Figure S7
- DMSO
ED1+
ED1-
Exon 25
Intergenic region
Promoter
Exon 1 Exon 20 Exon 33 (ED1)
Exon 47
0,
4,
8,
12,
16,
Promoter Exon 10 Exon 20 Exon 28 Exon 35 Exon 45
(H3K
9m
e2
-IgG
)/In
put
rel to
HP
RT
E2
FN Differ
Undif -
DMSO
1
Supplemental Information
Supplemental Figure Legends
Figure S1. Selectively down regulation of G9a E10+ isoform does not affect G9a levels. Related to
Figure 3
N2a cells were differentiated by treatment with 1 µM RA (RA) or maintained undifferentiated (-) and
transfected with siLUC as a control, siE10 or siG9a previously to differentiation induction. (A) mRNA
levels of total G9a were assessed by RT-qPCR and relativized to HSPCB. (B) Pictures are shown and
levels of total G9a fluorescence intensity were measure after immunofluorescence.
Figure S2. Neuronal differentiation triggers a nuclear accumulation of G9a protein. Related to Figure 5
N2a cells were differentiated with 2% (v/v) DMSO or 1 µM RA for 6 or 3 days, respectively. Cells
were then fixed and G9a protein localization was assessed by indirect immunofluorescence. (A and B)
Plots show total fluorescence intensity of G9a in the nuclear and cytoplasmic compartments for N2a
cells differentiated with DMSO (A) or RA (B). (C) HaCaT, HeLa, MCF7 and T47D cells were fixed
and G9a protein localization was assessed by indirect immunofluorescence. Alternative splicing of the
G9a gene was assessed by radioactive RT-PCR.
Figure S3. BIX does not inhibit differentiation when cells were treated after the differentiation program
was already established. Related to Figure 7
N2a cells were differentiated by treatment with 1 µM RA for 2 days and then 3 µM BIX or vehicle was
added. The percentage of cells displaying neurite outgrowth (differentiated cells) was determined and
representative pictures are shown. Scale bars represent 10 µm.
Figure S4. Chromatin structure regulates G9a alternative splicing. Related to Figure 7
(A) Schematic diagram of G9a showing the exon-intron organization and the distribution of qPCR
amplicons (top). N2a cells were differentiated with RA for 3 days and H3K9me2 levels at the indicated
regions were determined by nChIP assay followed by qPCR (bottom). Values of two independent
immunoprecipitations, relativized to the mean value for total H3, are shown for each region. An
amplicon located at an intergenic region (left) was used as a control. (B-C) N2a cells were
2
differentiated with 2% DMSO for 6 days and treated with 5 µM 5-azacytidine (B) or 2 ng/µl
trichostatin A (C) for 48 and 16 h, respectively. Cells were then harvested, RNA was extracted and
alternative splicing of the G9a gene was assessed by radioactive RT-PCR. Statistical analysis
correspond to two-way ANOVA, Bonferroni post hoc test (*) p<0.05, (***) p<0.0001. (D) N2a cells
were differentiated with DMSO for 6 days and DNA methylation of a CpG island close to the
alternative exon 10 (red box) was determined by sodium bisulfite modification followed by DNA
sequencing. Black circles represent methylated CpG dinucleotides.
Figure S5. Alternative splicing elicited by a G9a minigene is modulated during neuron differentiation.
Related to Figure 7
(A) Schematic diagram of the G9a minigene. (B) N2a cells were transfected with the minigene vector
and differentiated with 2% DMSO for 6 days. Cells were harvested, RNA was extracted and alternative
splicing was assessed by radioactive RT-PCR as described.
Figure S6. Position of G9a (EHMT2) and SUV39H1 in a functional interactions splicing network
(Papasaikas et al., 2015). Related to Discussion
Connections between factors indicate consistent effects of their knockdowns on an extensive set of
alternative splicing events, suggesting mechanistic associations in splicing regulation. Positive or
negative functional interactions are represented by green or red edges, respectively. Edge thickness
indicates the strength of the functional interaction, while node size is proportional to the overall impact
of a given knockdown in the regulation of alternative splicing. Node coloring corresponds to the
natural separation of the network in distinct, coherent modules. Black dotted lines represent
known physical interactions as reported in the STRING database (Franceschini et al., 2013). Factors
implicated in chromatin structure modification are shown with red labeling.
Figure S7. Chromatin structure and alternative splicing of Fibronectic gene during neuron
differentiation. Related to Discussion
Schematic diagram of Fibronectin showing the exon-intron organization (top). N2a cells were
differentiated with DMSO for 6 days. Cells were harvested, RNA was extracted, and alternative
splicing of the Fibronectin gene was assessed by radioactive RT-PCR. H3K9me2 levels at the
3
indicated regions of Fibronectin gene were determined by nChIP assay followed by qPCR. Values of
two independent immunoprecipitations are shown for each region.
Supplemental Experimental Procedures
Bisulfite sequencing
Unmethylated cytosine was converted to uracil by sodium bisulfite using the EpiTect Bisulfite Kit
(Quiagen). Two µl of the converted samples were used as PCR template for amplification with primers
designed with the online tool Bisearch. PCR products were sequenced using a BI 3130xl Genetic
Analyzer (Applied Biosystems).
nChIP
Native chromatin immunoprecipitation was performed as previously described (Schor et al., 2009). For
each immunoprecipitation we used 10 µg of H3K9me2 (07-441) antibody from Millipore or 5 µg of
H3total (07-690) antibody from Millipore. Protein A agarose beads, pre-blocked with salmon sperm
(Millipore), were used to recover the immuno-complexes. DNA was purified using QIAquick
purification kit (QIAGEN) and quantitative PCR analysis was performed using SYBR green.
Immunoprecipitated chromatin was normalized to input chromatin, and H3 total amount. All primer
sequences and real time PCR conditions are available upon request.
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