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Figure legends

Fig. 1. Identification of a gene locus enriched with genes that are upregulated during neuronal

maturation. (A) Gene clustering analysis of genes transcribed in hippocampal neurons cultured

for 1, 4, and 10 days. (B) Gene density of all genes and maturation-dependent genes (>twofold increase from day 1 to day 10 of culture) in each chromosome. (C) Genomic structure of

the 14qD2 locus. Genes found to be upregulated during maturation by microarray are depicted

in red circles and others are shown in gray circles. BAC clones used for DNA FISH are

indicated in the lower bars. (D) RT-qPCR analysis of mRNA expression of maturationdependent genes (red) and genes without changes during maturation (gray). Data were

normalized to mRNA levels of each gene at day 1 of in-vitro culture (1 DIV).

Fig. 2. Subnuclear positioning of 14qD2L in hippocampal neurons and brain sections. (A)

Representative images of DNA FISH for 14qD2L (red) and lamin B1 immunostaining (green)

at 1 DIV and 21 DIV of hippocampal neurons. Nuclei were counterstained with DAPI. Scale

bars: 5 μm. (B) Percentage alleles of 14qD2L at the nuclear periphery in hippocampal neurons

cultured at the indicated DIV. Error bars indicate the mean ± SD of 4–6 biological replicates (n

= 42–68 alleles). Statistical analysis was conducted with Kruskal–Wallis with post-hoc Steel’s

test. *p < 0.05 (C) Representative images of immunostaining for DAPI (blue), Tuj1 (red), GFP

(green), and GFAP (cyan) at 1, 4, and 10 DIV of culture of hippocampal neurons prepared from

GAD67-GFP knock-in mice. Scale bar: 5 μm. (D) Percentage of GFP-positive and -negative

neurons, and glia in hippocampal neuron culture prepared from GAD67-GFP knock-in mice.

(E) Representative images of DNA FISH for 14qD2L (red) and lamin B1 immunostaining

(blue) and GFP immunostaining (green) in GAD67-negative and -positive hippocampal

neurons cultured for 10 days prepared from GAD67-GFP knock-in mice. Scale bars: 5 μm. (F)

Percentage 14qD2 alleles at the nuclear periphery in GAD67-negative and -positive

31

hippocampal neurons cultured for 10 DIV prepared from GAD67-GFP knock-in mice. Error

bars indicate the mean ± SD of three biological replicates (n = 134–164). Statistical analysis

was conducted with Mann-Whitney U test. (G) Images of DNA FISH for 14qD2L in frozen

sections of the hippocampus from mice at postnatal day 1 (P1) and in adult mice. (H)

Percentage 14qD2 alleles at the nuclear periphery of P1 and adult hippocampi. Data are the

mean ± SD of 5–6 biological replicates (n = 42–68) and were analyzed with Mann-Whitney U

test. **p < 0.01

Fig. 3. Subnuclear positioning of 14qD2L in non-neuronal cells and human cell line. (A)

Representative images of DNA FISH for 14qD2L (red) and lamin B1 immunostaining (green)

in murine primary cardiomyocytes at 2, 3, 6, and 8 DIV. Nuclei were counterstained with DAPI.

Scale bar: 5 μm. (B) Percentage 14qD2 alleles at the nuclear periphery in primary

cardiomyocytes cultured for the indicated DIV. Data are presented as the mean ± SD from three

experiments (n = 98–176 alleles). (C) Egr3 expression in brains and hearts as assessed by metamicroarray analysis. In total, 429 and 222 microarray datasets for mouse brain and heart were

obtained from Gene Expression Omnibus (GEO), respectively, and were classified as early

embryo (18 brain, 17 heart), mid embryo (38 brain, 39 heart), late embryo (12 brain, 29 heart),

neonate (32 brain, 16 heart), and adult (329 brain, 121 heart). Data are shown as log signal

intensity. (D) Representative images of DNA FISH for 14qD2L (red) and lamin B1

immunostaining (green) in neurons differentiated from a human iPS cell line for 7 or 14 days.

Nuclei were counterstained with DAPI. (E) Percentage 14qD2 alleles at the nuclear periphery

in human iPS cell line-derived neurons after induction for the indicated periods. Data are

presented as the mean ± SD of six experiments (n = 42–84 alleles). Statistical analysis was

conducted with Mann-Whitney U test. *p < 0.05

32

Fig. 4. Expression of lamin B1 in hippocampal neurons and the brain. (A) RT-qPCR analysis

of nuclear lamins and Lbr in hippocampal neurons. (B) Western blot analysis of lamin B1

expression with antibody targeting the C-terminus or N-terminus of lamin B1 in cultured

hippocampal neurons. The arrowheads and arrow indicate intact and cleaved lamin B1,

respectively. (C, D) Western blot analysis of lamin B1 in the brains of mice at embryonic day

17 (E17) and adult mice using antibody targeting the C terminus (C) or N terminus (D) of lamin

B1.

Fig. 5. Lentiviral expression of lamin B1 in hippocampal neurons. (A, C) Representative

images of DNA FISH for 14qD2L (A) or Bdnf (B) (in red) and immunostaining of lamin B1

(blue) and GFP (green) in GFP-lamin B1 or GFP control cells. (B, D) Percentage alleles at the

nuclear periphery. Error bars indicate means ± SDs of 5–6 biological replicates (n = 55–72).

Data were analyzed with a Mann-Whitney U test. *p < 0.05 (E) Representative images of DNA

FISH for 14qD2L (red) and immunostaining of lamin B1 (blue) and GFP (green) in cells

infected with pCSII mock, flag-lamin B1, flag-ΔN-lamin B1, or flag-ΔC-lamin B1. (F)

Percentage 14qD2 alleles at the nuclear periphery of CSII mock, flag-lamin B1, flag-ΔN-lamin

B1, and flag-ΔC-lamin B. Error bars indicate means ± SDs of 3–6 biological replicates (n =

61–78). Data were analyzed by Kruskal-Wallis test with post-hoc Steel’s test. *p < 0.05 (G)

mRNA expression levels of Egr3 and Bdnf after depolarization in GFP control or GFP-lamin

B1 cells stimulated with 5 or 25 μM bicuculline for 1 h. Data are presented as fold increase

compared to GFP control without bicuculline. Error bars indicate means ± SDs of 6 replicates.

Data were analyzed with ANOVA and post-hoc Tukey’s test. **p < 0.01 (H) Percentage alleles

of Egr3 or Bdnf at the nuclear periphery before and after depolarization with 25 μM bicuculline

for 1 h. Error bars indicate means ± SDs of 6 biological replicates (n = 42–48). Statistical

analysis was conducted with Mann-Whitney U test.

33

Table 1. Loci enriched with maturation-dependent genes

Chromosome Size (Mbp) All genes Maturation-dependent genes %

0.9

35

10

29

1.9

127

13

10

1.2

56

10

18

123

15

12

11

1.6

102

12

12

14

1.4

33

11

33

16

1.5

46

10

22

34

35

36

37

38

39

...