RNF8-dependent histone ubiquitination during DNA damage response
Teng Ma†, Jennifer A. Keller†, and Xiaochun Yu*
Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109,
†These authors contributed equally to this work.
*Correspondence address. Tel: þ1-734-615-4945; Fax: þ1-734-936-6684; Email: email@example.com
Histone ubiquitination regulates the chromatin structure
that is important for many biological processes. Recently,
ubiquitination of histones was observed during the DNA
damage response (DDR), and this modification is con-
trolled by really interesting new gene (RING) domain
E3 ligase, RNF8. Together with the E2 conjugating
enzyme UBC13, RNF8 catalyzes ubiquitination of the his-
tones H2A and H2AX during the DDR, thus facilitating
downstream recruitment of DDR factors, such as p53
binding protein 1 (53BP1) and breast cancer type 1 sus-
Accordingly, the RNF8 knockout mice display phenotypes
associated with failed DDR, including hypersensitivity to
ionizing radiation, V(D)J recombination deficiency, and a
predisposition to cancer. In addition to the DDR pheno-
types, RNF8 knockout mice fail to generate mature sperm
during spermatogenesis, resulting in male sterility. The
RNF8 knockout mice also have a drastic reduction in
histone ubiquitination in the testes. These findings indi-
cate that the role of histone ubiquitination during chro-
matin remodeling in two different biological events could
be linked by an RNF8-dependent mechanism. Here, we
review the molecular mechanism of RNF8-dependent
histone ubiquitination both in DDR and spermatogenesis.
Received: December 2, 2010Accepted: January 12, 2011
Chromatin fibers are composed of nucleosomes, in which
DNA is wrapped around a histone octamer core. The core his-
tones include H2A, H2B, H3, and H4 [1,2].These four cano-
nical histone proteins are composed of a structured central
(globular) domain that is in close contact with the DNA and
much more flexible N-terminal and C-terminal tails .Both
the globular domain and histone tails undergo post-
translational modifications, which can either directly change
the chromatin structure by affecting the accessibility of DNA
to other proteins or provide docking sites to recruit down-
stream chromatin remodeling factors [4,5]. These modifi-
cations, such as phosphorylation, acetylation, methylation,
and ubiquitination, combine to form the ‘histone code’ that is
associated with diverse cellular processes such as chromo-
some condensation, gene expression, and DNA damage
repair [6–14]. Histone modifications are not insulated from
each other. Instead, these modifications display cross-talks
and function together in biological events [15,16]. One
recently identified example is the RING domain E3 ligase
RNF8-dependent histone ubiquitination, which mediates
histone acetylation to promote histone eviction during both
spermatogenesis and DNA damage response (DDR) .
Protein ubiquitination is a chemical reaction with three
sequential steps in which ubiquitin, a 76 amino acid poly-
peptide, is covalently conjugated to the substrate in the
presence of ubiquitin E1, E2, and E3 enzymes . The
first-reported ubiquitination substrate was histone H2A,
identified in vivo by Goldknopf and Busch in 1977 .
Subsequently, histone H2B was found to be ubiquitinated
as well by West and Bonner . Like other protein ubi-
quitinations, histone ubiquitination is catalyzed by the for-
mation of an isopeptide bond between the carboxy-
terminal glycine of ubiquitin and lysine resides on H2A
and H2B . The ubiquitination sites have been mapped
to lysines 119 and 120 on the tails of H2A and H2B in
mammals, respectively . Considering that H2A and
H2B contain only 131 and 125 residues, respectively, the
large molecular size of ubiquitin relative to the histones
Acta Biochim Biophys Sin 2011, 43: 339–345 | ª The Author 2011. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmr016.
Advance Access Publication 28 March 2011
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 5 | Page 339
modifications. Although structural analysis indicates that
ubiquitin protrudes to the outside of the nucleosome, this
bulky modification existing in the nucleosome potentially
changes the chromatin structure. Thus, it is not surprising
that both H2A and H2B ubiquitination regulate chromatin
remodeling during gene transcription. Interestingly, the
roles of ubH2A and ubH2B are different in transcription. It
has been shown that ubH2A is enriched in gene loci with
low transcription activity and participate in gene silencing
with Polycomb repressive complex 1 [22–24]. In addition,
during the pachytene stage of meiotic prophase I, ubH2A
is highly enriched in the XY body where X and Y chromo-
somes are transcriptionally silenced . In contrast,
ubH2B marks highly transcribed gene loci and facilitates
transcription elongation [26–30]. Recently, both ubH2A
and ubH2B have been shown to be involved in DDR
RNF8 regulates histone ubiquitination
during DNA damage response
Genomic DNA that stores genetic information can easily
be damaged by numerous environmental and internal
hazards. The most deleterious damage is DNA double-
strand breaks (DSBs). In response to DSBs, a group of
PI3-like kinases, including Ataxia Telangiectasia Mutated
(ATM), Ataxia Telangiectasia and RAD3 related (ATR),
and DNA-dependent protein kinase catalytic subunit
(DNAPKc), are activated and transmit signals through
various mediators to arrest cell cycle progression and facili-
tate DNA damage repair [37,38]. One of those important
mediators during DDR is histone H2AX, a variant of H2A
with a C-terminal tail that can be phosphorylated by ATM
ATM-phosphorylated H2AX recruits mediator of DNA
damage checkpoint 1 (MDC1), which can also be phos-
phorylated by ATM at DNA damage sites. The H2AX and
MDC1 complex stabilizes a large group of DNA damage
repair factors, such as p53 binding protein 1 (53BP1) and
breast cancer type 1 susceptibility protein (BRCA1), at
DNA damage sites, which mediates cell cycle arrest and
DNA damage repair [39,40]. In addition to this, protein
MDC1 also regulate a unique ubiquitination cascade at
DNA damage sites through the E3 ligase RNF8 [31,33,34].
First reported in 1998, RNF8 is a 485-amino acid
nuclear polypeptide ubiquitously expressed in human
tissues . The RNF8 protein contains an N-terminal
forkhead-associated (FHA) domain and a C-terminal RING
domain . The FHA domain is a phospho-threonine
binding domain . Peptide library screening indicates
that the RNF8 FHA domain recognizes a pTXXF motif
. Following DNA damage, we and others have found
that the RNF8 FHA domain recognizes three different
pTXXF motifs in MDC1, and MDC1 targets RNF8 to
DNA damage sites through this phospho-dependent inter-
action [31,33,34]. The RING domain of RNF8 is an E3
ubiquitin ligase. It can interact with Ubc13 to catalyze
lysine-63 polyubiquitin chain formation as well as with
class III E2s (UBE2E2, UbcH6, and UBE2E3) for
Ubiquitination of H2A, H2AX, and H2B are known to be
regulated by RNF8 at DNA damage sites. H2A and H2AX
can be both mono- and poly-ubiquitinated, while H2B is
only mono-ubiquitinated. Although RNF8 is an E3 ligase
and does ubiquitinate histones in vitro , it is not clear
as to whether RNF8 or other E3 ligases directly ubiquiti-
nate histones in vivo.
Accumulating evidence suggests that histone ubiquitina-
tion could be recognized by ubiquitin-binding proteins .
For example, the ubiquitin interacting motif (UIM) domain
of receptor-associated protein 80 (RAP80) recognizes
ubH2A and ubH2B at DNA damage sites . RAP80
forms a complex with CCDC98 and BRCA1 [46–50]. The
UIM domain of RAP80 targets the whole complex to DNA
damage sites, which facilitates the DNA repair function of
BRCA1. Recently, we found that MRG15, a subunit of both
the histone acetyltransferase complex and deacetylase
complex [51–54], might also recognize ubH2B and induce
histone acetylation by two acetyltransferases, males-absent
on the first protein (MOF) and tat-interactive protein 60 kDa
(TIP60) (T. M., J. A. K., X. Y.). Since histone acetylation
brings negative charges onto the chromatin, it may poten-
tially change the topology of chromatin into a more relaxed
status, thus allowing other DNA damage repair factors to
access DNA damage sites.
Although RNF8 can trigger DSB-associated ubiquitina-
tions, it might not be sufficient to sustain conjugated ubi-
quitin at DNA damage sites due to the weak E3 ligase
activity of RNF8 in vitro  and competition with strong
deubiquitinase activity in vivo [55–57]. The persistence of
ubiquitinated histones at DNA lesions was unexplained
until the discovery of another E3 ligase, RNF168.
Performing a meticulous monitoring of the DSB-associated
ubiquitinations during the first 10 min after DNA damage,
researchers found that the temporal accumulation of conju-
gated ubiquitin at DSBs tightly correlated with the reten-
tion of RNF168 in this compartment, and that no increase
in local ubiquitin concentration was observed in cells with
depleted RNF168, even at the earliest time points. RNF168
contains ubiquitin-binding domains (MIU1 and MIU2) that
allow interaction with ubH2A [32,35]. Like RNF8,
RNF168 interacts with UBC13 to ubiquitinate histones
adjacent to DSBs [32,35]. RNF168 ubiquitination is RNF8
dependent, and, by targeting H2A and H2AX, amplifies
the local concentration of ubiquitin conjugates to the
Histone ubiquitination during DNA damage response and spermatogenesis
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 5 | Page 340
threshold required for retention of 53BP1 and BRCA1
[32,35]. These data indicate that the ubiquitin conjugates
generated by RNF8 are transient and/or unstable and
require amplification and/or stabilization by RNF168 to
achieve the threshold needed for the completion of the
DSB-induced chromatin response. Interestingly, it was
found that overexpression of RNF8 rescues cellular pheno-
types in cells with moderate, but not strong, down-
regulation of RNF168 , indicating that high activity of
RNF8 can maintain unstable ubiquitin conjugates to com-
pensate for a weaker, but not absent, RNF168 response.
Additionally, recent work has shown that the silencing of
genes near sites of DNA damage (DISC, Double-strand
break-Induced Silencing in Cis) is dependent on H2A ubi-
quitination, and that DISC is only lost when both RNF8
and RNF168 are inactivated . Thus, the functional
interaction between RNF8 and RNF168 needs to be further
In addition to RNF168, it was also reported that another
factor, HERC2, forms a complex with RNF8 in response to
ionizing radiation and is involved in the DDR .
HERC2 is an HECT-type E3 ubiquitin ligase. The
inducible phosphorylation of HERC2 at Thr 4827, which
is recognized by the FHA domain of RNF8. HERC2 facili-
tates assembly of the ubiquitin-conjugating enzyme Ubc13
with RNF8, thereby promoting DNA damage-induced for-
mation of poly-ubiquitin chains. It has also been shown
that HERC2 interacts with and maintains the levels of
RNF168, implicating HERC2 in maintenance of both com-
ponents of the histone ubiquitination pathway.
Taken together, RNF8 plays a central role in DDR.
RNF8 recognizes phosphorylated MDC1 in order to relo-
cate to DSBs and ubiquitinate histones at DNA lesions.
RNF8 acts upstream of a number of repair factors including
RNF168, HECT domain and RCC-like domain-containing
protein 2 (Herc2), 53BP1 and BRCA1, and its activity
tethers these proteins to the damaged chromatin to trans-
duce the repair signal for DNA damage in the cell. To
examine the function of RNF8 in vivo, we and others have
generated RNF8-deficient mice [17,60,61]. To our surprise,
the phenotype of RNF8 null mice is very mild. Although
RNF8 null mice are sensitive to ionizing radiation and
have subtle defects in V(D)J recombination during T-cell
development and immunoglobulin class-switching during
B-cell differentiation, the mice are viable and seldom
develop T-cell or B-cell lymphomas [17,60,61]. These mild
phenotypes lead us to search for other similar proteins that
could play a redundant functional role with RNF8.
From a similar domain architecture search, Chfr could
be a paralog of RNF8 . RNF8 and Chfr are the only
two human E3s that contain both the FHA domain and
RING domain. Like RNF8, the RING domain of Chfr is
also an E3 ligase and can interact with Ubc13, the key E2
enzyme to catalyze histone ubiquitination at DNA damage
sites . More interestingly, Chfr is down-regulated in
20%–40% of primary tumors and tumor cell lines, mainly
due to promoter hypermethylation-induced Chfr gene silen-
cing, suggesting that Chfr may play a role in tumor sup-
pression [64–71]. Since RNF8 and Chfr share similar
functional domains and interact with the same E2 ubiquitin
RNF8-deficient mice, Chfr-deficient mice are also viable
and have a mild phenotype. However, after we crossed
RNF8-deficient mice and Chfr-deficient mice to generate
double-knock-out mice (DKO), we found that DKO mice
were not only hypersensitive to ionizing radiation, but also
have significant V(D)J recombination defects during T-cell
development and develop T-cell lymphomas (unpublished
data). These phenotypes of DKO mice are very similar to
ATM-deficient mice . In the mouse embryonic fibro-
blasts (MEFs) extracted from DKO mice, the basal level of
histone ubiquitination is significantly abrogated, indicating
that RNF8 and Chfr may regulate not only DNA
damage-induced histone ubiquitination but also the basal
level of histone ubiquitination. As acetylation and destabili-
zation of the nucleosome have been linked to histone ubi-
quitination [17,73], RNF8 and Chfr-dependent histone
ubiquitination indirectly modulate chromatin structure and
condensation. In response to DNA damage, RNF8 could
be recruited to DNA damage site [31,33,34]. Its ubiquitina-
tion activity could relax the chromatin adjacent to DNA
lesions and allow DDR factors to access DNA damage
sites for proper repair. In the absence of RNF8 and Chfr,
DSBs, particularly generated during V(D)J recombination,
could not be correctly repaired, inducing genomic instabil-
ity and ultimately causing T-cell lymphoma.
RNF8 in spermatogenesis
In addition to playing important roles in DDR, histone ubi-
quitination is critical for spermatogenesis. Correspondingly,
loss of RNF8-dependent histone ubiquitination suppresses
spermatogenesis [17,61]. During spermatogenesis, progeni-
tor cells undergo successive mitotic and meiotic divisions
(spermatocytogenesis) and a metamorphic change (sper-
miogenesis) to produce spermatozoa. During the pachytene
stage of meiotic prophase I, ubH2A is highly enriched in
the XY body , where X and Y chromosomes become
partially synapsed through pseudo-autosomal regions and
are transcriptionally silenced. This phenomenon is known
as meiotic sex chromosome inactivation (MSCI) .
Consistent with its transcriptionally silenced status, the XY
body contains a unique combination of histone modifi-
cation marks associated with gene silencing including
dimethylation of histone H3 on lysine 9 (H3K9) and
Histone ubiquitination during DNA damage response and spermatogenesis
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 5 | Page 341
deacetylation of histone H3 and H4 . MSCI is impor-
tant for proper meiosis, and is controlled by H2AX.
Disruption of MSCI leads to the arrest of spermatocytes at
H2AX-deficient mice . The role of ubH2A in the XY
body is not clear, but it is thought that these modifications
may mediate MSCI .In RNF8 knockout spermatocytes,
ubiquitinated conjugates on the XY body in pachytene-
stage cells are strikingly lost, which is correlated with
RNF8’sroleas the E3
[31,33,34]. However, although ubH2A enrichment at the
XY body is abolished, both XY body formation and
meiosis are unaffected in RNF8-deficient testes as marked
by normal gH2AX . The transcription and replication
machinery are inactivated in the RNF8-deficient mice as in
the wild-type mice, as shown by the exclusion of RNA
polymerase II from the XY body and the low-expression
pattern of X chromosome genes . Thus, these findings
indicate that RNF8-dependent histone ubiquitination is not
required for MSCI and meiosis .
Ubiquitinated histones occur in other stages of spermato-
genesis beyond meiosis. For example, ubiquitinated H2A
and H2B are also enriched in elongating spermatids
[23,78]. During spermiogenesis, sperm DNA is highly con-
densed and tightly wrapped around histone-like protamines
instead of histone octamers . The transition from
nucleosomes to protamines occurs in round haploid sper-
matids that elongate and transform into mature sperm.
During this process, most nucleosomal histones are initially
replaced by two transition proteins, transition protein 1 and
2, and subsequently by two protamines, protamine 1 and 2
[80,81]. Both histone ubiquitination and hyper-acetylation
are implicated in nucleosome removal at post-meiotic
stages . Although the biological function of these
massive chromatin remodeling events is not clear, it is
hypothesized that the protamines promote increased DNA
condensation to facilitate the packaging of DNA into the
sperm heads. Failure to accomplish this global chromatin
restructuring causes male sterility [82–84]. In fact, the
male infertility in RNF8-deficient mice occurs during this
post-meiotic stage. Histological analysis of the testes
revealed that RNF8-deficient testes contained fewer con-
densing spermatids and drastically fewer condensed mature
spermatids. Further investigation indicated that chromatin-
bound transition proteins and protamines were reduced in
the testes of RNF8-deficient mice. During histone replace-
ment, it has been suggested that the N-terminal tail of
histone H4 is highly acetylated . Since acetylation adds
negative charges to nucleosomes, it has been hypothesized
that acetylation of H4 could loosen chromatin fibers to
enhance histone replacement [86–88]. Interestingly, the H4
acetylation level is also significantly reduced in testes from
RNF8-deficient mice, whereas other histone markers like
H3 methylation showed no change . Similarly, the
chromatin-associated histone acetyltransferase MOF, which
accounts for the majority of H4K16Ac, is also decreased in
the RNF8-deficient testes .
Collectively, these findings pose a trans-histone modifi-
cation model in which RNF8-dependent histone H2A/H2B
ubiquitination induces the H4 acetylation by MOF. In
support of this model, the N-terminal tail of H4 has been
shown to make an inter-particle contact with the H2A/H2B
heterodimer of adjacent nucleosomes . H4 acetylation
could be an essential step for histone removal in elongating
spermatids. A defect in H4 acetylation could significantly
suppress histone removal and histone-like protein incorpor-
ubH2A/ubH2B induces H4 acetylation in adjacent nucleo-
somes and promotes removal of histones from the chromo-
somes of elongating spermatids.
Concluding remarks and perspectives
The role of RNF8 in DDR and in spermatogenesis under-
scores the similarities between these diverse cellular events.
RNF8-dependent histone ubiquitination is required for both
biological processes, which are linked by the necessity for
loosening histone–DNA interactions (Fig. 1). The only
difference is that during DDR, RNF8-dependent histone
ubiquitination regulates histone acetylation at DNA damage
sites to induce local chromatin relaxation and potential
local histone eviction; whereas during spermiogenesis, it is
Figure 1 A model of RNF8-dependent histone eviction
regulates histone acetylation and facilitates histone eviction.
During DNA damage response or spermiogenesis, RNF8-dependent histone ubiquitination
Histone ubiquitination during DNA damage response and spermatogenesis
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 5 | Page 342
global histone acetylation, global chromatin relaxation, and
global histone eviction. However, the molecular mechan-
isms underlying these two biological events are almost
identical. It is possible that other histone ubiquitination-
dependent biological processes, such as gene transcription,
adopt a similar mechanism for chromatin remodeling.
Certainly the importance of RNF8 in vivo is broader than
originally expected, and suggests that many other factors
may have more extensive roles than are currently known.
This work was supported by American Cancer Society
(RSG-08–125-01-CCG to X.Y.), National Institute of
Health (CA132755 and CA130899 to X.Y.), the University
of Michigan Cancer Center and GI Peptide Research
Center. X.Y. is a recipient of the Era of Hope Scholar
Award from the Department of Defense.
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