Introduction: X chromosome inactivation (XCI) is an essential mechanism for dosage compensation between females and males in mammals. In females, XCI is controlled by a complex, conserved locus termed the X inactivation center (Xic), in which the lncRNA Xist is the key regulator. However, little is known about the Xic in species with unusual sex chromosomes. The genus Tokudaia includes three rodent species endemic to Japan. Tokudaia osimensis and Tokudaia tokunoshimensis lost the Y chromosome (XO/XO), while Tokudaia muenninki (TMU) acquired a neo-X region by fusion of the X chromosome and an autosome (XX/XY). We compared the gene location and structure in the Xic among Tokudaia species. Methods: Gene structure of nine genes in Xic was predicted, and the gene location and genome sequences of Xic were compared between mouse and Tokudaia species. The expression level of the gene was confirmed by transcripts per million calculation using RNA-seq data. Results: Compared to mouse, the Xic gene order and location were conserved in Tokudaia species. However, remarkable structure changes were observed in lncRNA genes, Xist and Tsix, in the XO/XO species. In Xist, important functional repeats, B-, C-, D-, and E-repeats, were partially or completely lost due to deletions in these species. RNA-seq data showed that female-specific expression patterns of Xist and Tsix were confirmed in TMU, however, not in the XO/XO species. Additionally, three deletions and one inversion were confirmed in the intergenic region between Jpx and Ftx in the XO/XO species. Conclusion: Our findings indicate that even if the Xist and Tsix lncRNAs are expressed, they are incapable of producing a successful and lasting XCI in the XO/XO species. We hypothesized that the significant structure change in the intergenic region of Jpx-Ftx resulted in the inability to perform the XCI, and, as a result, a lack of Xist expression. Our results collectively suggest that structural changes in the Xic occurred in the ancestral lineage of XO/XO species, likely due to the loss of one X chromosome and the Y chromosome as a consequence of the degradation of the XCI system.

1.
Loda
A
,
Collombet
S
,
Heard
E
.
Gene regulation in time and space during X-chromosome inactivation
.
Nat Rev Mol Cell Biol
.
2022
;
23
(
4
):
231
49
.
2.
Payer
B
,
Lee
JT
.
X chromosome dosage compensation: how mammals keep the balance
.
Annu Rev Genet
.
2008
;
42
(
42
):
733
72
.
3.
Wutz
A
,
Gribnau
J
.
X inactivation Xplained
.
Curr Opin Genet Dev
.
2007
;
17
(
5
):
387
93
.
4.
Lyon
MF
.
Possible mechanisms of X chromosome inactivation
.
Nat New Biol
.
1971
;
232
(
34
):
229
32
.
5.
Monkhorst
K
,
de Hoon
B
,
Jonkers
I
,
Mulugeta Achame
E
,
Monkhorst
W
,
Hoogerbrugge
J
, et al
.
The probability to initiate X chromosome inactivation is determined by the X to autosomal ratio and X chromosome specific allelic properties
.
PLoS One
.
2009
;
4
(
5
):
e5616
.
6.
Yamada
F
,
Kawauchi
N
,
Nakata
K
,
Abe
S
,
Kotaka
N
,
Takashima
A
, et al
.
Rediscovery after thirty years since the last capture of the critically endangered Okinawa spiny rat Tokudaia muenninki in the northern part of Okinawa island
.
Mammal Study
.
2010
;
35
(
4
):
243
55
.
7.
Honda
T
,
Suzuki
H
,
Itoh
M
.
An unusual sex chromosome constitution found in the amami spinous country-rat, tokudaia osimensis osimensis
.
Jpn J Genet
.
1977
;
52
(
3
):
247
9
.
8.
Honda
T
,
Suzuki
H
,
Itoh
M
,
Hayashi
K
.
Karyotypical differences of the amami spinous country-rats, Tokudaia osimensis osimensis obtained from two neighbouring islands
.
Jpn J Genet
.
1978
;
53
(
4
):
297
9
.
9.
Murata
C
,
Yamada
F
,
Kawauchi
N
,
Matsuda
Y
,
Kuroiwa
A
.
Multiple copies of SRY on the large Y chromosome of the Okinawa spiny rat, Tokudaia muenninki
.
Chromosome Res
.
2010
;
18
(
6
):
623
34
.
10.
Terao
M
,
Ogawa
Y
,
Takada
S
,
Kajitani
R
,
Okuno
M
,
Mochimaru
Y
, et al
.
Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9
.
Proc Natl Acad Sci USA
.
2022
;
119
(
49
):
e2211574119
.
11.
Tsuchiya
K
,
Wakana
S
,
Suzuki
H
,
Hattori
S
,
Hayashi
Y
.
Taxonomic study of Tokudaia (Rodentia: muridae): I.genetic differentiation
.
Mem Natl Sci Mus
.
1989
;
22
:
227
34
.
12.
Murata
C
,
Yamada
F
,
Kawauchi
N
,
Matsuda
Y
,
Kuroiwa
A
.
The Y chromosome of the Okinawa spiny rat, Tokudaia muenninki, was rescued through fusion with an autosome
.
Chromosome Res
.
2012
;
20
(
1
):
111
25
.
13.
Matveevsky
S
,
Kolomiets
O
,
Bogdanov
A
,
Hakhverdyan
M
,
Bakloushinskaya
I
.
Chromosomal evolution in mole voles Ellobius (cricetidae, Rodentia): bizarre sex chromosomes, variable autosomes and meiosis
.
Genes
.
2017
;
8
(
11
):
306
.
14.
Just
W
,
Baumstark
A
,
Süss
A
,
Graphodatsky
A
,
Rens
W
,
Schäfer
N
, et al
.
Ellobius lutescens: sex determination and sex chromosome
.
Sex Dev
.
2007
;
1
(
4
):
211
21
.
15.
Mulugeta
E
,
Wassenaar
E
,
Sleddens-Linkels
E
,
van IJcken
W
,
Heard
E
,
Grootegoed
JA
, et al
.
Genomes of Ellobius species provide insight into the evolutionary dynamics of mammalian sex chromosomes
.
Genome Res
.
2016
;
26
(
9
):
1202
10
.
16.
Chureau
C
,
Prissette
M
,
Bourdet
A
,
Barbe
V
,
Cattolico
L
,
Jones
L
, et al
.
Comparative sequence analysis of the X-inactivation center region in mouse, human, and bovine
.
Genome Res
.
2002
;
12
(
6
):
894
908
.
17.
Augui
S
,
Nora
EP
,
Heard
E
.
Regulation of X-chromosome inactivation by the X-inactivation centre
.
Nat Rev Genet
.
2011
;
12
(
6
):
429
42
.
18.
Anguera
MC
,
Ma
W
,
Clift
D
,
Namekawa
S
,
Kelleher
RJ
3rd
,
Lee
JT
.
Tsx produces a long noncoding RNA and has general functions in the germline, stem cells, and brain
.
PLoS Genet
.
2011
;
7
(
9
):
e1002248
.
19.
Hierholzer
A
,
Chureau
C
,
Liverziani
A
,
Ruiz
NB
,
Cattanach
BM
,
Young
AN
, et al
.
A long noncoding RNA influences the choice of the X chromosome to be inactivated
.
Proc Natl Acad Sci U S A
.
2022
;
119
(
28
):
e2118182119
.
20.
Lee
J
,
Davidow
LS
,
Warshawsky
D
.
Tsix, a gene antisense to Xist at the X-inactivation centre
.
Nat Genet
.
1999
;
21
(
4
):
400
4
.
21.
Brown
CJ
,
Hendrich
BD
,
Rupert
JL
,
Lafrenière
RG
,
Xing
Y
,
Lawrence
J
, et al
.
The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus
.
Cell
.
1992
;
71
(
3
):
527
42
.
22.
Nesterova
TB
,
Slobodyanyuk
SY
,
Elisaphenko
EA
,
Shevchenko
AI
,
Johnston
C
,
Pavlova
M
, et al
.
Characterization of the genomic Xist locus in rodents reveals conservation of overall gene structure and tandem repeats but rapid evolution of unique sequence
.
Genome Res
.
2001
;
11
(
5
):
833
49
.
23.
Navarro
P
,
Page
DR
,
Avner
P
,
Rougeulle
C
.
Tsix-mediated epigenetic switch of a CTCF-flanked region of the Xist promoter determines the Xist transcription program
.
Genes Dev
.
2006
;
20
(
20
):
2787
92
.
24.
Pugacheva
EM
,
Tiwari
VK
,
Abdullaev
Z
,
Vostrov
AA
,
Flanagan
PT
,
Quitschke
WW
, et al
.
Familial cases of point mutations in the XIST promoter reveal a correlation between CTCF binding and pre-emptive choices of X chromosome inactivation
.
Hum Mol Genet
.
2005
;
14
(
7
):
953
65
.
25.
Raposo
AC
,
Casanova
M
,
Gendrel
AV
,
da Rocha
ST
.
The tandem repeat modules of Xist lncRNA: a swiss army knife for the control of X-chromosome inactivation
.
Biochem Soc Trans
.
2021
;
49
(
6
):
2549
60
.
26.
Sheardown
SA
,
Duthie
SM
,
Johnston
M
,
Newall
AET
,
Formstone
EJ
,
Arkell
RM
, et al
.
Stabilization of Xist RNA mediates initiation of X chromosome inactivation
.
Cell
.
1997
;
91
(
1
):
99
107
.
27.
Maenner
S
,
Blaud
M
,
Fouillen
L
,
Savoye
A
,
Marchand
V
,
Dubois
A
, et al
.
2-D Structure of the A region of Xist RNA and its implication for PRC2 association
.
PLoS Biol
.
2010
;
8
(
1
):
e1000276
.
28.
Schoeftner
S
,
Sengupta
AK
,
Kubicek
S
,
Mechtler
K
,
Spahn
L
,
Koseki
H
, et al
.
Recruitment of PRC1 function at the initiation of X inactivation independent of PRC2 and silencing
.
EMBO J
.
2006
;
25
(
13
):
3110
22
.
29.
Duszczyk
MM
,
Wutz
A
,
Rybin
V
,
Sattler
M
.
The Xist RNA A-repeat comprises a novel AUCG tetraloop fold and a platform for multimerization
.
RNA
.
2011
;
17
(
11
):
1973
82
.
30.
Jeon
Y
,
Lee
JT
.
YY1 tethers Xist RNA to the inactive X nucleation center
.
Cell
.
2011
;
146
(
1
):
119
33
.
31.
Sarma
K
,
Levasseur
P
,
Aristarkhov
A
,
Lee
JT
.
Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome
.
Proc Natl Acad Sci U S A
.
2010
;
107
(
51
):
22196
201
.
32.
Zushi
H
,
Murata
C
,
Mizushima
S
,
Nishida
C
,
Kuroiwa
A
.
Unique XCI evolution in Tokudaia: initial XCI of the neo-X chromosome in Tokudaia muenninki and function loss of XIST in Tokudaia osimensis
.
Chromosoma
.
2017
;
126
(
6
):
741
51
.
33.
Kudo
R
,
Yoshida
I
,
Matiz Ceron
L
,
Mizushima
S
,
Kuroki
Y
,
Jogahara
T
, et al
.
The neo-X does not form a Barr body but shows a slightly condensed structure in the Okinawa spiny rat (Tokudaia muenninki)
.
Cytogenet Genome Res
.
2023
;
162
(
11–12
):
632
43
.
34.
Okuno
M
,
Mochimaru
Y
,
Matsuoka
K
,
Yamabe
T
,
Matiz-Ceron
L
,
Jogahara
T
, et al
.
Chromosomal-level assembly of Tokudaia osimensis, Tokudaia tokunoshimensis, and Tokudaia muenninki genomes
.
Sci Data
.
2023
;
10
(
1
):
927
.
35.
Kim
D
,
Langmead
B
,
Salzberg
SL
.
HISAT: a fast spliced aligner with low memory requirements
.
Nat Methods
.
2015
;
12
(
4
):
357
60
.
36.
Shumate
A
,
Wong
B
,
Pertea
G
,
Pertea
M
.
Improved transcriptome assembly using a hybrid of long and short reads with StringTie
.
PLoS Comput Biol
.
2022
;
18
(
6
):
e1009730
.
37.
Grabherr
MG
,
Haas
BJ
,
Yassour
M
,
Levin
JZ
,
Thompson
DA
,
Amit
I
, et al
.
Full-length transcriptome assembly from RNA-Seq data without a reference genome
.
Nat Biotechnol
.
2011
;
29
(
7
):
644
52
.
38.
Li
W
,
Godzik
A
.
Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences
.
Bioinformatics
.
2006
;
22
(
13
):
1658
9
.
39.
Wu
TD
,
Watanabe
CK
.
GMAP: a genomic mapping and alignment program for mRNA and EST sequences
.
Bioinformatics
.
2005
;
21
(
9
):
1859
75
.
40.
Kent
WJ
.
BLAT: the BLAST-like alignment tool
.
Genome Res
.
2002
;
12
(
4
):
656
64
.
41.
Altschul
SF
,
Gish
W
,
Miller
W
,
Myers
EW
,
Lipman
DJ
.
Basic local alignment search tool
.
J Mol Biol
.
1990
;
215
(
3
):
403
10
.
42.
Delcher
AL
,
Phillippy
A
,
Carlton
J
,
Salzberg
SL
.
Fast algorithms for large-scale genome alignment and comparison
.
Nucleic Acids Res
.
2002
;
30
(
11
):
2478
83
.
43.
Vaught
A
.
Graphing with Gnuplot and Xmgr: two graphing packages available under linux
.
Linux J
;
1996
.
44.
Dobin
A
,
Davis
CA
,
Schlesinger
F
,
Drenkow
J
,
Zaleski
C
,
Jha
S
, et al
.
STAR: ultrafast universal RNA-seq aligner
.
Bioinformatics
.
2013
;
29
(
1
):
15
21
.
45.
Li
B
,
Dewey
CN
.
RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome
.
BMC Bioinformatics
.
2011
;
12
:
323
.
46.
RStudio: Integrated development environment for R. Available from: http://www.posit.co/ (
2006
). Accessed Nov 10, 2023.
47.
Mise
N
,
Goto
Y
,
Nakajima
N
,
Takagi
N
.
Molecular cloning of antisense transcripts of the MouseXistGene
.
Biochem Biophys Res Commun
.
1999
;
258
(
3
):
537
41
.
48.
Bousard
A
,
Raposo
AC
,
Żylicz
JJ
,
Picard
C
,
Pires
VB
,
Qi
Y
, et al
.
The role of Xist-mediated Polycomb recruitment in the initiation of X-chromosome inactivation
.
EMBO Rep
.
2019
;
20
(
10
):
e48019
.
49.
Ridings-Figueroa
R
,
Stewart
ER
,
Nesterova
TB
,
Coker
H
,
Pintacuda
G
,
Godwin
J
, et al
.
The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory
.
Genes Dev
.
2017
;
31
(
9
):
876
88
.
50.
Van Bemmel
JG
,
Galupa
R
,
Gard
C
,
Servant
N
,
Picard
C
,
Davies
J
, et al
.
The bipartite TAD organization of the X-inactivation center ensures opposing developmental regulation of Tsix and Xist
.
Nat Genet
.
2019
;
51
(
6
):
1024
34
.
51.
Chureau
C
,
Chantalat
S
,
Romito
A
,
Galvani
A
,
Duret
L
,
Avner
P
, et al
.
Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region
.
Hum Mol Genet
.
2011
;
20
(
4
):
705
18
.
52.
Tian
D
,
Sun
S
,
Lee
JT
.
The long noncoding RNA, Jpx, is a molecular switch for X-chromosome inactivation
.
Cell
.
2010
;
143
(
3
):
390
403
.
53.
Barakat
TS
,
Gunhanlar
N
,
Pardo
CG
,
Achame
EM
,
Ghazvini
M
,
Boers
R
, et al
.
RNF12 activates Xist and is essential for X chromosome inactivation
.
PLOS Genet
.
2011
;
7
(
1
):
e1002001
.
54.
Lee
JT
.
Disruption of imprinted X inactivation by parent-of-origin effects at Tsix
.
Cell
.
2000
;
103
(
1
):
17
27
.
55.
Hwang
JY
,
Choi
KH
,
Lee
DK
,
Kim
SH
,
Kim
EB
,
Hyun
SH
, et al
.
Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts
.
J Reprod Dev
.
2015
;
61
(
6
):
533
40
.
56.
Fang
H
,
Disteche
CM
,
Berletch
JB
.
X inactivation and escape: epigenetic and structural features
.
Front Cell Dev Biol
.
2019
;
7
:
219
.
57.
Burgoyne
PS
,
Mahadevaiah
SK
,
Turner
JMA
.
The consequences of asynapsis for mammalian meiosis
.
Nat Rev Genet
.
2009
;
10
(
3
):
207
16
.
You do not currently have access to this content.