Ubiquitination Associates with
Inactive X Chromosomes and
Is Involved in Initiation of
Received for publication, October 18, 2004
Published, JBC Papers in Press, October 26, 2004,
Jia Fang‡, Taiping Chen§, Brian Chadwick¶,
En Li§, and Yi Zhang‡?
From the ‡Department of Biochemistry and Biophysics,
Lineberger Comprehensive Cancer Center, University of
North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599-7295, the §Novartis Institute for
Biomedical Research, Cambridge, Massachusetts 02139,
and the ¶Department of Molecular Genetics &
Microbiology, Institute for Genome Sciences and Policy,
Duke University Medical Center, Durham, North
Histone modifications are thought to serve as epige-
netic markers that mediate dynamic changes in chroma-
tin structure and regulation of gene expression. As a
model system for understanding epigenetic silencing, X
chromosome inactivation has been previously linked to
a number of histone modifications including methyla-
tion and hypoacetylation. In this study, we provide evi-
dence that supports H2A ubiquitination as a novel epi-
genetic marker for the inactive X chromosome (Xi) and
links H2A ubiquitination to initiation of X inactivation.
We found that the H2A-K119 ubiquitin E3 ligase Ring1b,
a Polycomb group protein, is enriched on Xi in female
trophoblast stem (TS) cells as well as differentiating
embryonic stem (ES) cells. Consistent with Ring1b me-
diating H2A ubiquitination, ubiquitinated H2A (ubH2A)
is also enriched on the Xi of both TS and ES cells. We
demonstrate that the enrichment of Ring1b and ubH2A
on Xi is transient during TS and ES cell differentiation,
suggesting that the Ring1b and ubH2A are involved in
the initiation of both imprinted and random X inactiva-
tion. Furthermore, we showed that the association of
Ring1b and ubH2A with Xi is mitotically stable in non-
differentiated TS cells.
Dynamic changes in chromatin structure play important
roles in regulating DNA-templated processes such as transcrip-
tion. Regulation of chromatin dynamics can be achieved
through ATP-dependent chromatin remodeling and covalent
modifications of histones, such as acetylation, methylation,
phosphorylation, and ubiquitination (1–4). Studies in the past
several years have established a fundamental role for histone
acetylation and methylation in regulating gene activity (4–6).
However, the role of histone ubiquitination is just beginning to
be revealed (7).
Both histones H2A and H2B are subject to modification by
ubiquitin. The identification of E2 and E3 involved in H2B
ubiquitination (8–10) and the discovery of the “cross-talk” be-
tween histone methylation and ubiquitination (11, 12) have set
the stage for functional analysis of histone H2B ubiquitination.
Recent studies indicate that, unlike other reversible histone
modifications in which addition or removal of a group from a
histone molecule results in opposing transcriptional effects,
sequential ubiquitination and deubiquitination are both in-
volved in transcriptional activation (13, 14). Although histone
H2A was the first protein identified to be ubiquitinated (15), its
functional significance has been elusive until very recently
when we biochemically purified the H2A-K119 ubiquitin E3
ligase and linked its function to Polycomb silencing (16).
Dosage compensation in mammals is achieved by transcrip-
tional silencing of one of the two X chromosomes in female cells
(17). X inactivation occurs during early embryonic development
and is controlled by a large non-coding RNA, Xist (18, 19). After
coating the inactive X chromosome (Xi), Xist RNA is believed to
recruit silencing factors to mediate a series of epigenetic
changes to the chromosome. The epigenetic changes include
methylation and hypoacetylation of histones, incorporation of
macroH2A, and CpG methylation (20).
Genetic and biochemical studies have revealed that Poly-
comb group (PcG)1proteins function in at least two distinct
protein complexes including the Eed-Ezh2 complex and the
PRC1 complex (21, 22). Previously, we as well as others (23–
25), have demonstrated that the Eed-Ezh2 complex and its
associated H3-K27 methyltransferase activity are recruited to
Xi and participate in X inactivation. However, no link between
components of the PRC1 complex and X inactivation has been
established. Our recent finding that H2A ubiquitination is
mediated by a Ring1b-containing PcG complex (16) prompted
us to investigate a potential role of H2A ubiquitination in X
inactivation. Our study revealed that both Ring1b and ubiq-
uitinated H2A (ubH2A) are specifically enriched on the Xi of
trophoblast stem (TS) cells as well as differentiating embryonic
stem (ES) cells. In addition, analysis of the dynamic nature of
the Xi enrichment suggests that Ring1b and ubH2A are likely
involved in the initiation stage of X inactivation. Taken to-
gether, our study reveals a new epigenetic feature of Xi and
suggests a role for Ring1b and ubH2A in the initiation stage of
X inactivation in both extraembryonic and embryonic cell
Cell Culture and Differentiation—TS cells were kindly provided by
Gary Uy (26) and cultured as described (27). To induce TS cell differen-
tiation, human fibroblast growth factor-4 (Sigma), heparin (Sigma), and
embryonic mouse fibroblast-conditioned medium were removed from the
culture medium. The female mouse ES cell line 129/cas16, derived from a
female 129Sv/Jae-Castaneous hybrid blastocyst, was maintained and
differentiated following a previously described procedure (28).
* This work was supported by National Institutes of Health Grants
GM63076 and GM68804 (to Y. Z.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
S The on-line version of this article (available at http://www.jbc.org)
contains supplemental Figs. S1–S3.
? To whom correspondence should be addressed. Tel.: 919-843-8225;
Fax: 919-966-9673; E-mail: firstname.lastname@example.org.
1The abbreviations used are: PcG, Polycomb group; ubH2A, ubiquiti-
nated H2A; TS, trophoblast stem; ES, embryonic stem; PBS, phosphate-
buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; DAPI, 4,6-
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 279, No. 51, Issue of December 17, pp. 52812–52815, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
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