Science - USA (2018-12-21)

nuclear localization signal (NLS) at positions
488 to 492 (Lys-Arg-Pro-Arg-Pro) and a bipartite
NLS-like peptide (bNLS) at positions 345 to 359
(Lys-Arg-Arg-Val-Leu-Pro-Leu-Lys-Ser-Ser-Lys-
Val-Arg-Gln-Lys), adjacent to an alanine-rich
region (ARR; Ala^367 to Ala^402 ) (fig. S3A) that
shows characteristics of a helix-turn-helix struc-
ture [GYM 2.0 ( 37 )]. These domains are evolu-
tionarily conserved among species (fig. S3, B
and C). Fusion of a short peptide containing this
monopartite NLS to mCherry resulted in nu-
clear enrichment of the fusion proteins (fig.
S3D). Deletion of this sequence (JP2NTDNLS)
abolished nuclear localization of eGFP-JP2NT
in HEK293T cells (Fig. 2A, iii and iv) and cardio-

myocytes (fig. S3E), indicating that this NLS

is indispensable for nuclear localization of

JP2NT. A fusion protein containing mCherry

and the bNLS peptide together with the ARR

was imported into nuclei (fig. S3D). However,

deletion of the bNLS sequence from JP2NT

(eGFP-JP2NTDbNLS) did not prevent its nuclear

importation in HEK293T cells (Fig. 2A, v) and

cardiomyocytes (fig. S3E), indicating that this

region is not necessary for nuclear importation

of JP2NT. However, the subnuclear localiza-

tion of eGFP-JP2NTDbNLSwas mutually exclusive

from To-Pro-3 staining, which labels genomic

DNA (Fig. 2A, v), suggesting physical dissocia-

tion of eGFP-JP2NTDbNLSfrom genomic DNA.

Deletion of the adjacent ARR from JP2NT

(eGFP-JP2NTDARR) induced greater separation

of eGFP-JP2NT from DNA and was accompa-

nied by accumulation of DNA at the nuclear

periphery (Fig. 2A, vi). These data indicate that

bNLS and ARR are involved in DNA or chro-

matin binding.

To further confirm the association of JP2NT

with chromatin, we applied a biochemical frac-

tionation procedure ( 38 ) (Fig. 2B, i). JP2NT was

detected in both soluble (S3) and chromatin-

containing insoluble (P4) nuclear fractions (Fig. 2B,

ii, a). MNASE-mediated DNA digestion released

JP2NT from the insoluble chromatin fraction

(MNASE+/S4) (Fig. 2B, ii, a). Deletion of the eight

Guoet al.,Science 362 , eaan3303 (2018) 21 December 2018 4of9

-2.5Kb 2.5Kb

TSS TTS

-2.5Kb 2.5Kb

TSS TTS

-2.5Kb 2.5Kb

TSS TTS

Genomic binding profile of JP2NT

Promoter

Exon

Intron

Intergenic

TSS

Other

A

JP2NTΔMORNs/ΔARR

GGGATCCTGAGTCGCAGTATAAAAGAAGCTTTTCGGGCGTT GGGATCCTGAGTCGCAGGAAGCTTTTCGGGCGTTTATATTA

GGGATCCTGAGTCGCAGTATAAAAGAAGCTTTTCGGGCGTT GGGATCCTGAGTCGCAGTATATAAGAAGCTTTTCGGGCGTT GGGATCCTGAGTCGCAGTATAGAAGCTTTTCGGGCGTTTAA

Freeprobes

Freeprobes

Shiftedprobes

Shiftedprobes

GST-JP2NT

Blank

GST

200 ng 50 ng

100 ng

200 ng

GST-

JP2331-405

Blank

GST

50 ng100 ng200 ng

200 ng

GST-

JP2331-405

Blank

GST

50 ng100 ng200 ng

200 ng

Consensus TATA BOX

(promoter cMyc)

Mutant TATA BOX

(promoter cMyc)

WT TATA BOX

(promoter cMyc)

Mutant TATA BOX

(promoter cMyc)

WT TATA BOX (promoter cMyc)

D i ii

iv v

Blank

GST

200 ng100 ng50 ng200 ng GS Blank

T

200 ng

50 ng

100 ng

200 ng

GST- GST-

JP2NTΔMORNs

GST-JP2NT

Blank

GST

200 ng 50 ng

100 ng

200 ng

iii

TATA box core

sequence variation

TATAAA

TATAAT

TATATA

TATATT

TATTAA

TTTAAA

Binding

by JP2NT

yes

yes

yes

no

no

no

C

JP2NT

(p-value<10-10)

(p-value<10-10)

471 8943 22354

H3K9ac

JP2NT

2109 7305 19889

Pol II

0

1

2

3

4

5

6

7

8

9

10

ChIP-seq signal

ChIP-seq signal density

B Input_mm10 IP replicate1_mm10 IP replicate2_mm10

18

14

10

6

2

-2.5Kb 2.5Kb

TSS TTS

-2.5Kb 2.5Kb

TSS TTS

-2.5Kb 2.5Kb

TSS TTS

Fig. 3. JP2NT is a TATA-box binding
protein enriched at transcription start
sites and interacts with basic tran-
scription machinery.(A) Genomic DNA
binding profile of JP2NT in cardiomyocytes
as revealed by ChIP-seq. (B) JP2NT is
preferentially localized around transcrip-
tion start sites (TSSs), as revealed by two
replicates of ChIP-seq. (C) Overlap chart of
DNA binding peaks of indicated proteins.
(D)JP2NTbindstoTATAboxDNAsequences
in vitro. (i and ii) Gel shift assays of purified
GST-JP2NT binding to wild-type
(WT) (i) or mutant TATA box–containing
sequences (ii) derived from the cMyc
promoter. (iii) Summary of the results of
gel shift assays with various TATA box
variants or mutants. Mutation of the core
TATA sequences abolished the interaction
with GST-JP2NT. (iv) Deletion of the ARR,
but not the N-terminal MORN domains,
eliminated JP2NT binding to the TATA box
sequence. (v) The peptide JP2331-405
containing the ARR specifically binds to
the consensus TATA box sequence.

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