Rev-erba and Rev-erbb are unique NRs that lack the activation function 2 domain required for Keywords apoCIII; Rev-erbb; transcriptional regulation; zinc finger HIT domain-containing pro
Trang 1interacts with orphan nuclear hormone receptor Rev-erbb and removes Rev-erbb-induced inhibition of apoCIII
transcription
Jiadong Wang1, Yang Li1, Min Zhang1, Zhongle Liu1, Cong Wu1, Hanying Yuan1, Yu-Yang Li1, Xin Zhao2and Hong Lu1
1 State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
2 Department of Animal Science, McGill University, Montreal, Canada
The nuclear receptors (NRs) are a family of
transcrip-tion factors which regulate a wide array of biological
processes, including those involved in diabetes, obesity,
cardiovascular disease and cancer [1] Many important
medicines, including 12 of the top 100 selling drugs,
target NRs [2] Generally, NRs regulate the activity of
transcription by binding to specific ligands and
coregu-lators [3] However, the regulation of transcriptional
activity by orphan nuclear hormone receptors, whose
ligands are not identified or are lost during evolution,
is less clear
The Rev-erb family is a subgroup of orphan recep-tors, two members of which have been isolated from mammalian genotypes: Rev-erba, also known as Ear-1
or NR1D1 (official nomenclature), and Rev-erbb, also known as RVR, BD73 or NR1D2 (official nomencla-ture) [4] Rev-erba and Rev-erbb are unique NRs that lack the activation function 2 domain required for
Keywords
apoCIII; Rev-erbb; transcriptional regulation;
zinc finger HIT domain-containing protein 1;
ZNHIT-1
Correspondence
H Lu, State Key Laboratory of Genetic
Engineering, School of Life Sciences, Fudan
University, Shanghai 200433, China
Fax: +86 21 65642505
Tel: +86 21 65642505
E-mail: honglu0211@yahoo.com
(Received 27 June 2007, revised 15 August
2007, accepted 22 August 2007)
doi:10.1111/j.1742-4658.2007.06062.x
The orphan receptors, Rev-erba and Rev-erbb, are members of the nuclear receptor superfamily and specifically repress apolipoprotein CIII (apoCIII) gene expression in rats and humans Moreover, Rev-erba null mutant mice have elevated very low density lipoprotein triacylglycerol and apoCIII levels However, ligands for Rev-erb are unknown and the regulatory mechanism of Rev-erb is poorly understood Conceivably, co-factors for Rev-erb may play an important role in the regulation of lipid metabolism In this study, a zinc finger HIT domain-containing protein, ZNHIT-1, interacted with Rev-erbb ZNHIT-1 was found to be a con-served protein in eukaryotes and was highly abundant in human liver Furthermore, ZNHIT-1 was identified as a nuclear protein Serial trun-cated fragments and substitution mutations established a putative nuclear localization signal at amino acids 38–47 of ZNHIT-1 A putative ligand-binding domain of Rev-erbb and the FxxLL motif of ZNHIT-1 were required for their interaction Finally, ZNHIT-1 was recruited by Rev-erbb to the apoCIII promoter and removed the Rev-Rev-erbb-induced inhibi-tion of apoCIII transcripinhibi-tion These findings demonstrate that ZNHIT-1 functions as a cofactor to regulate the activity of Rev-erbb, and may play
a role in lipid metabolism
Abbreviations
ChIP, chromatin immunoprecipitation; Co-IP, coimmunoprecipitation; GFP, green fluorescent protein; GST, glutathione S-transferase; LBD, ligand-binding domain; NCBI, National Center for Biotechnology Information; NLS, nuclear localization signal; NR, nuclear receptor; RFP, red fluorescent protein; SRCAP, SNF-2 related CBP activator protein; VLDL, very low density lipoprotein; ZNHIT-1, zinc finger HIT domain-containing protein 1.
Trang 2ligand-dependent activation of transcription by other
members of the NR superfamily [5] Therefore,
Rev-erb receptors constitutively behave as unliganded
receptors and repress transcription by binding to
core-pressor molecules [6] Both Rev-erba and Rev-erbb are
widely expressed [4] Rev-erb represses transcription of
the Rev-erba gene itself [7], as well as N-myc [8], Bmal1
[9], enoyl-CoA hydratase⁄ 3-hydroxyacyl-CoA
dehydro-genase bifunctional enzyme [10], a-fetoprotein [11] and
rat apoAI [12] genes Rev-erba is also involved in
cir-cadian timing in brain and liver tissue, and regulates
Bmal1 which is the master regulator of circadian
rhythm in mammals [13–16] However, it is not known
whether Rev-erbb is involved in circadian regulation
Rev-erb also represses the expression of apoCIII
[17] ApoCIII is a 79-residue glycoprotein It is
synthe-sized in the liver as part of the very low density
lipo-protein (VLDL) It is well established that the plasma
concentration and synthesis rate of apoCIII are
posi-tively correlated with plasma triacylglycerols in both
normal and hypertriglyceridaemic subjects [18–20]
Large scale clinical trials have indicated that
hyper-triglyceridaemia is an independent risk factor for
coronary artery disease and atherosclerosis [21,22]
Therefore, a better understanding of the regulation of
the expression of the apoCIII gene is of major
impor-tance for the treatment of dyslipidaemia
In this study, a zinc finger HIT domain-containing
protein, ZNHIT-1 (NM_006349.2), was found to
interact with Rev-erbb by a yeast two-hybrid assay
The sequence of ZNHIT-1 was first identified by high
throughput genomic sequences in humans This
pro-tein contains a conserved zinc finger HIT domain
originally found in the yeast HIT-1 protein [23], and
so has been named ZNHIT-1 (‘zinc finger HIT
domain-containing protein 1’) Previous knowledge
about the function of ZNHIT-1 is limited One report
has described ZNHIT-1 as a subunit of the SNF-2
related CBP activator protein (SRCAP) complex,
which can remodel chromatin by incorporating the
histone variant H2A.Z into nucleosomes [24] The
interaction between ZNHIT-1 and Rev-erbb was
further confirmed using a glutathione S-transferase
(GST) pull-down assay and a coimmunoprecipitation
(Co-IP) assay In addition, the homologues and tissue
distribution of ZNHIT-1 were analysed Furthermore,
a nuclear localization signal (NLS) of ZNHIT-1 was
identified Using the chromatin immunoprecipitation
(ChIP) assay, we found that ZNHIT-1 was recruited
to the human apoCIII promoter by Rev-erbb, and
subsequently removed Rev-erbb-induced inhibition of
apoCIII transcription without changing the
DNA-binding activity of Rev-erbb
Results
Yeast two-hybrid screening
In order to identify Rev-erbb binding partners, a human fetal liver cDNA library was screened in a yeast two-hybrid assay using Rev-erbb as bait A screen of approximately 106 yeast transformants revealed that ZNHIT-1 interacted with Rev-erbb The zinc finger HIT domain is a motif found in many proteins, and plays an important role in gene regulation and chro-matin remodelling [25] So far, five human proteins containing the HIT domain have been identified A phylogenetic tree was constructed to show the relation-ship between ZNHIT-1 and other members of the zinc finger HIT domain proteins According to the evolu-tionary distance between the ZNHIT protein members, estimated from our phylogenetic analysis, it was concluded that a close relationship existed between ZNHIT-1 and ZNHIT-4, as well as between ZNHIT-3 and ZNHIT-5 (see Supplementary material Fig S1)
A liquid assay for b-galactosidase activity was further performed to establish the interaction between Rev-erbb and ZNHIT-1 As shown in Fig 1, the Rev-Rev-erbb homodimer was used as a positive control ZNHIT-1 and Rev-erbb cotransformants showed a stronger b-galactosidase activity than the Rev-erbb homodimer, whereas almost no b-galactosidase activity was detected
in ‘no insert’ or lamin-negative control vectors
Homologues and tissue distribution analysis of human ZNHIT-1
blastpof human ZNHIT-1 was performed at National Center for Biotechnology Information (NCBI) sites,
Fig 1 Evaluation of the interaction strength by a relative b-galacto-sidase activity assay The homodimer of Rev-erbb was used as a positive control No insert or lamin was used as a negative control Results are the means ± SD of three independent experiments performed in triplicate ‘BD’ and ‘AD’ represent the DNA-binding domain in pGBKT-7 and the DNA activation domain in pADKT-7, respectively These domains were fused with target proteins.
Trang 3and homologues were identified in dog, mouse, frog,
zebra fish, fruit fly and yeast As shown in Fig 2A,
human ZNHIT-1 encodes a full-length protein of 154
amino acids and shares high identity with its
homo-logues (100% with Canis familiaris, 97% with Mus
musculus, 87% with Xenopus tropicalis, 87% with
Danio rerio, 68% with Drosophila melanogaster and
33% with Schizosaccharomyces pombe), indicating that
ZNHIT-1 is a conservative protein in eukaryotes
To investigate the tissue distribution of ZNHIT-1, a
real-time quantitative PCR was performed with human
multiple tissue cDNA panels 1 and 2 from 16 human
tissues (Catalogue # 636742, 636752; Clontech, Tokyo,
Japan) as templates ZNHIT-1 was expressed in all
analysed tissues, abundantly in human liver, but weakly
in skeletal muscle, ovary and small intestine (Fig 2B)
Subcellular localization analysis of ZNHIT-1
Bioinformatic analyses by PSORT and other databases
failed to predict the potential localization of ZNHIT-1
To examine the subcellular localization of ZNHIT-1 proteins, Hela and HepG2 cells were transfected with expression vectors for pEGFPC1-ZNHIT-1 or pDS-RED1C1-ZNHIT-1 Their subcellular localization was visualized by laser microscopy Similar results were obtained from both types of cell As shown in Fig 3A,B for Hela cells, both red fluorescent protein (RFP)-ZNHIT-1 and green fluorescent protein (GFP)-ZNHIT-1 accumulated in the nucleus and exhibited a punctate distribution
To map the ZNHIT-1 NLS, serial deletion mutants
of ZNHIT-1 were constructed (Fig 3E) Like the full-sized protein, the ZNHIT-1 (1–54) mutant was local-ized exclusively in the nucleus of Hela cells In con-trast, the ZNHIT-1 (54–154) mutant, without the
NH2-terminus, was localized in both the nucleus and cytoplasm, similar to the empty GFP vector Further deletions in ZNHIT-1 (1–54) showed that amino acids 38–47 were sufficient for the nuclear localization
of ZNHIT-1 (Fig 3C) Alignment, even with yeast ZNHIT-1, revealed that four amino acids
A
B
Fig 2 Homologue and expression analysis of human ZNHIT-1 (A) Sequence comparison of human ZNHIT-1 and its homologues in Canis familiaris, Mus musculus, Xenopus tropicalis, Danio rerio, Drosophila melanogaster and Schizosaccharomyces pombe (GenBank accession nos XP_536855.2, Q8R331, NP_001017056.2, NP_001017401.1, NP_608895.1 and NP_595833.1) Residues identical in all compared sequences are presented on a black background, whereas residues similar in four or more compared sequences are presented on a grey background (B) The relative expression levels of ZNHIT-1 mRNA in 16 human tissues (human multiple tissue cDNA panels 1 and 2 from Clontech) determined by real-time quantitative PCR using b 2 -microglobulin as a reference standard.
Trang 4(38DNFxD42) of ZNHIT-1 were highly conserved.
Substitution mutations of these conserved amino acids
decreased the nuclear localization of ZNHIT-1
(Fig 3D) Collectively, the data in Fig 3 established
that the fragment 38DNFQDDPHAG47 of ZNHIT-1
contains a putative NLS, which is sufficient to target
ZNHIT-1 into the nucleus
Characterization of the interaction between
ZNHIT-1 and Rev-erb
The interaction between Rev-erbb and ZNHIT-1 was
further verified by a GST pull-down assay and a
Co-IP assay As shown in Fig 4A, bacterially expressed GST-ZNHIT-1, when conjugated to gluta-thione–Sepharose beads, efficiently and specifically pulled down 6His-tagged Rev-erbb, which was about
66 kDa Conversely, GST alone did not In the Co-IP assay, anti-Myc serum and protein A⁄ G agarose were added to the HepG2 cell lysates containing overexpressed Myc-ZNHIT-1 so as to precipitate the Myc-ZNHIT-1⁄ Rev-erbb complex, and anti-Rev-erbb serum was used in western blotting to detect Rev-erbb
in the precipitate As shown in Fig 4B, endogenous Rev-erbb interacted with ZNHIT-1 in mammalian cells
It is well known that Rev-erba is closely related to Rev-erbb, especially in the DNA-binding domain and putative ligand-binding domain (LBD) In order to
A
B
C
D
E
Fig 3 Subcellular localization analysis of ZNHIT-1 in Hela cells.
(A) Nuclear localization of RFP-ZNHIT-1 (B) Nuclear localization of
GFP-ZNHIT-1 (C) Nuclear localization of GFP-ZNHIT-1 (38–47).
(D) Cytoplasmic and nuclear localization of GFP-ZNHIT-1 NLS
mutant (E) Subcellular localization of various ZNHIT-1 deletion
mutants.
A
B
C
Fig 4 Validation of the interaction between ZNHIT-1 and Rev-erbb or Rev-erba (A) The GST pull-down assay in vitro GST-ZNHIT-1 or GST immobilized on the beads was incubated with 6His-Rev-erbb Interacting proteins were immunoblotted with anti-6His serum Lane 2 is the input, which acts as a positive control (B) The interaction between ZNHIT-1 and Rev-erbb vali-dated by a Co-IP assay HepG2 cells were transfected with pCMV-Myc-ZNHIT-1 Expression of Myc-ZNHIT-1 in transfected cells was analysed by western blotting Cell lysates were then precipitated by anti-Myc serum The precipitated proteins were eluted from the protein A ⁄ G PLUS agarose and analysed by western blotting using anti-Rev-erbb serum HC represents the heavy chain of mouse IgG (C) The interaction between ZNHIT-1 and Rev-erba validated by a Co-IP assay HepG2 cells were cotransfected with Myc-ZNHIT-1 and pCMV-HA-Rev-erba LC represents the light chain of mouse IgG.
Trang 5determine whether Rev-erba could also interact with
ZNHIT-1, Rev-erba was amplified from a human fetal
liver cDNA library, and a Co-IP assay between
Rev-erba and ZNHIT-1 was performed As shown in
Fig 4C, Rev-erba was also capable of binding
ZNHIT-1
Mapping the regions of interaction between ZNHIT-1 and Rev-erbb
Rev-erbb consists of five domains: A⁄ B, C, D and E [5] The highly conserved region C is responsible for DNA binding, and region E contains a putative LBD and mediates the recruitment of cofactors [5] To iden-tify the region of Rev-erbb essential for the interaction with ZNHIT-1, serial deletion assays were performed using the yeast two-hybrid method N- and C-terminal deletion constructs of Rev-erbb (Fig 5A) were fused in-frame to the Gal4-binding domain and tested for their abilities to bind ZNHIT-1 by checking the activa-tion of the reporter genes Ade2, His3 and LacZ It was shown that ZNHIT-1 binds specifically to the LBD of Rev-erbb The interaction between ZNHIT-1 and all the serial truncated fragments of Rev-erbb was further confirmed by the GST pull-down in vitro assay (Fig 5B) Similarly, the serial deletion constructs of ZNHIT-1 were fused to the Gal4 activation domain and tested for their abilities to bind Rev-erbb As shown in Fig 5C, ZNHIT-1D2 (amino acids 72–110) was required for the interaction with Rev-erbb This fragment contains an FxxLL motif (x denotes any amino acid), which has been reported to function in the same manner as an LxxLL motif, and mediates transcriptional coactivator binding to NRs [26–28] To further investigate whether the ZNHIT-1 FxxLL motif was required for the interaction with Rev-erbb, a dou-ble L87A⁄ L88A mutation in the context of full-length ZNHIT-1 was introduced As a result, the mutant of the FxxLL motif failed to interact with Rev-erbb The results from the GST pull-down assay (Fig 5D) were consistent with those from the yeast two-hybrid assay
A
B
C
D
E
Fig 5 Mapping the regions for the interaction between Rev-erbb and ZNHIT-1 (A) The Rev-erbb region required for interaction with ZNHIT-1 was revealed by a yeast two-hybrid assay The serial trun-cated fragments of Rev-erbb were separately coexpressed with full-length ZNHIT-1 in AH109 (B) Serial truncated fragments of Rev-erbb interacting with ZNHIT-1 were confirmed by a pull-down assay (C) The ZNHIT-1 region required for the interaction with Rev-erbb was mapped by a yeast two-hybrid assay The serial truncated fragments of ZNHIT-1 were individually coexpressed with full-length Rev-erbb in AH109 The activation of the reporters was analysed (D) All serial truncated fragments of ZNHIT-1 interacting with Rev-erbb were confirmed by a pull-down assay (E) The interaction between Rev-erbb, ZNHIT-1 and N-CoR GST-ZNHIT-1 was first immobilized on glutathione–Sepharose and was then incubated with 6His-Rev-erbb together with a myc-tagged N-CoR fragment (amino acids 2053–2453 containing IDI + IDII domains) The mix-ture was incubated at 4 C for 6 h After three washes with NaCl ⁄ P i buffer, the sample was boiled and analysed by western blotting using anti-6His or anti-myc.
Trang 6The transcriptional repressor, N-CoR, has been
reported to interact with Rev-erbb through the E
region [5,29] Deletion of one of two fragments (amino
acids 394–416 and 561–576) in the E region of
Rev-erbb ablates N-CoR⁄ Rev-erbb interaction [29] In
order to identify whether the region mediating the
Rev-erbb⁄ N-CoR interaction is the same as that
medi-ating the Rev-erbb⁄ ZNHIT-1 interaction, the amino
acid fragment 416–561 (Rev-erbb D6) was tested for its
ability to bind to ZNHIT-1 It was shown that
Rev-erbb D6 interacted with ZNHIT-1 (lane 2 in the right
panel of Fig 5E), suggesting that the regions
mediat-ing the Rev-erbb⁄ N-CoR and Rev-erbb ⁄ ZNHIT-1
interactions are different As ZNHIT-1 and N-CoR
bind to different regions of the E domain of Rev-erbb,
the possibility that these three proteins could form a
ternary complex exists To answer this question,
GST-ZNHIT-1 was affinity immobilized on glutathione–
Sepharose and incubated with 6His-Rev-erbb and a
myc-tagged N-CoR fragment (amino acids 2053–2453
containing IDI + IDII domains) As shown in
Fig 5E, ZNHIT-1 could pull-down 6His-Rev-erbb
directly, but not the myc-tagged N-CoR fragment, in
the presence of Rev-erbb These results suggest that
these three proteins could not form a ternary complex
in vitro
Colocalization of ZNHIT-1 and Rev-erbb in the
nucleus
To determine whether the interaction between ZNHIT-1
and Rev-erbb might be of physiological relevance in
mammalian cells, the subcellular localization and
dis-tribution of ZNHIT-1 and Rev-erbb in Hela and
HepG2 cells were assessed using laser microscopy
Similar results were obtained from both types of cell
GFP-Rev-erbb accumulated in the nucleus and
exhib-ited a diffuse distribution (Fig 6A) As shown in
Fig 6B, confocal images of cells expressing wild-type
RFP-ZNHIT-1 and GFP-Rev-erbb indicated that both
proteins were nuclear and that the majority of these
expressed proteins were colocalized in Hela cells To
extend this observation, coexpression studies with
Rev-erbb and the ZNHIT-1 NLS mutant were performed
As illustrated in Fig 3D, the ZNHIT-1 NLS mutant
was expressed in both the cytoplasm and nucleus in
Hela cells without transfection of Rev-erbb
Surpris-ingly, in cells with coexpressed ZNHIT-1 mutant and
RFP-Rev-erbb, the ZNHIT-1 NLS mutant adopted a
predominantly nuclear expression profile and
colocal-ized with Rev-erbb (Fig 6C) Thus, these data provide
additional evidence that Rev-erbb interacts with
ZNHIT-1 in the nucleus, and implies that this
interac-tion with Rev-erbb recruits the cytoplasmic ZNHIT-1 NLS mutant into the nuclear compartment
Recruitment of ZNHIT-1 by Rev-erbb to the apoCIII promoter and its effect on the apoCIII promoter
Rev-erbb has been reported to repress the expression
of apoCIII [17] To extend this observation, Rev-erbb was found to bind to the promoter of apoCIII in vivo (Fig 7A) and to function as a transcriptional silencer (Fig 7B) To study the effect of the interaction between ZNHIT-1 and Rev-erbb on the Rev-erbb-mediated transcription of apoCIII, recruitment of ZNHIT-1 to the apoCIII promoter was first studied
No ZNHIT-1 was detectable in association with the apoCIII promoter in the absence of transfected Rev-erbb However, a relatively strong association of ZNHIT-1 with the apoCIII promoter was detected after transfection of Rev-erbb These results suggest that Rev-erbb is essential to recruit ZNHIT-1 to the apoCIII promoter (Fig 7A) In order to further address the functional significance of ZNHIT-1-medi-ated regulation on Rev-erbb, HepG2 cells expressing endogenous ApoCIII were transiently transfected with HA-Rev-erbb and either Myc-ZNHIT-1 or empty vector, together with the pGL3-apoCIII-pro reporter, which contains the promoter of the human apoCIII gene (nucleotides )1408 to +24) and pRL As
illus-A
B
C
Fig 6 Colocalization of ZNHIT-1 and Rev-erbb in Hela cells (A) Nuclear localization of GFP-Rev-erbb (B) Colocalization of RFP-ZNHIT-1 and GFP-Rev-erbb (C) Effect of Rev-erbb on the localiza-tion of the ZNHIT-1 NLS mutant Hela cells were cotransfected with RFP-Rev-erbb and GFP-ZNHIT-1 NLS mutant.
Trang 7trated in Fig 7B, overexpression of Rev-erbb repressed
the expression of apoCIII by approximately 60%, and
the repression was totally removed by coexpressed
ZNHIT-1 In contrast, the L87A⁄ L88A mutant of
ZNHIT-1 did not relieve the repression, suggesting
that ZNHIT-1-mediated regulation is dependent on
the binding of Rev-erbb Furthermore, ZNHIT-1 D4, without the zinc finger HIT domain, did not remove the inhibition of apoCIII (Fig 7B), indicative of the requirement of the HIT domain in ZNHIT-1-mediated coregulation
To further evaluate the effect of ZNHIT-1 on apoC-III expression in vivo, HepG2 cell lines with stable ZNHIT-1 siRNA expression were established These HepG2 RNAi cell lines showed a significant decrease
in mRNA expression of the endogenous ZNHIT-1 genes, as measured by quantitative real-time PCR The apoCIII level was also measured by quantitative real-time PCR As shown in Fig 7C, the endogenous apoCIIIlevel was reduced to about 70% in the HepG2 cell line with stable ZNHIT-1 siRNA expression, sug-gesting that endogenous ZNHIT-1 affects apoCIII transcription
No change in Rev-erbb DNA-binding activity to the apoCIII promoter by ZNHIT-1
Next, the possible effect of ZNHIT-1 on the binding capacity of Rev-erbb to the apoCIII promoter was investigated ChIP analysis using anti-Rev-erbb serum was performed in HepG2 cells transfected with Rev-erbb alone or cotransfected with Rev-erbb and ZNHIT-1 The binding capacity of Rev-erbb to the apoCIII promoter was assessed by quantitative PCR for ChIP DNA As shown in Fig 8, ZNHIT-1 did not affect Rev-erbb binding to the apoCIII promoter These results indicate that Rev-erbb remains on the apoCIIIpromoter after recruitment of ZNHIT-1
Discussion
It is well known that Rev-erb is involved in lipid metabolism by the regulation of apoCIII transcription
A
B
C
Fig 7 Recruitment of ZNHIT-1 by Rev-erbb to the apoCIII promoter and its effect on the apoCIII promoter (A) Recruitment of ZNHIT-1
by Rev-erbb to the apoCIII promoter A ChIP assay of the apoCIII promoter was performed in human HepG2 cells with the indicated antibodies Promoter-specific primers are described in ‘Experimen-tal procedures’ (B) The effect of ZNHIT-1 on Rev-erbb activity was assessed by a luciferase assay HepG2 cells were cotransfected with pGL3-apoCIII-pro and pRL, as well as the plasmid or plasmids
as indicated Plasmid pRL was used to normalize the transfection efficiencies, and the empty vector pCMV-HA was used as a nega-tive control All luciferase activities are the means ± SD of three independent experiments performed in triplicate (C) The effect of ZNHIT-1 on endogenous apoCIII expression was analysed by real-time PCR with total RNA from the stable knock-down of ZNHIT-1 cells The mRNA levels of ZNHIT-1 and apoCIII were normalized to the endogenous b2-microglobulin mRNA level.
Trang 8The ApoCIII protein is a major component of VLDL
and plays a key role in hypertriglyceridaemia [18–20]
Human Rev-erba and Rev-erbb specifically repress
apoCIII gene expression in rats and humans [17,30]
Rev-erba null mutant mice have elevated VLDL
tri-acylglycerol and ApoCIII [31] Cofactors that interact
with Rev-erb and modify its effect on transcription will
no doubt be involved in the regulation of its target
genes One corepressor, N-CoR, has been reported to
interact with Rev-erba and Rev-erbb and,
conse-quently, to intensify the transcriptional repression
[6,9] However, no study has reported on how the
tran-scriptional repression mediated by erba or
Rev-erbb can be removed In this study, it has been shown
that ZNHIT-1 can interact with Rev-erbb and relieve
its inhibitory effect on the transcription of apoCIII
Interestingly, ZNHIT-1 is highly abundant in the liver
(Fig 2B) where ApoCIII is synthesized Thus, it is
plausible that ZNHIT-1 can affect lipid metabolism
However, further experimentation will be needed to
confirm this assumption
ZNHIT-1 was identified as a nuclear protein Serial
truncated fragments and substitution mutations
estab-lished a putative NLS in amino acids 38–47 of
ZNHIT-1 Furthermore, the ZNHIT-1 NLS mutant
was expressed in both the cytoplasm and nucleus Interestingly, in cells that coexpressed the ZNHIT-1 NLS mutant together with Rev-erbb, the ZNHIT-1 NLS mutant adopted a predominantly nuclear expres-sion profile This shuttling of the ZNHIT-1 NLS mutant to the nucleus was selective for Rev-erbb expression, as the expression of another nuclear pro-tein, such as p53, did not alter the cytoplasmic locali-zation of the ZNHIT-1 NLS mutant (data not shown) Thus, these data provide evidence that the interaction with Rev-erbb recruits the cytoplasmic ZNHIT-1 NLS mutant to the nuclear compartment
Many cofactors bind to a common LBD region of NRs to regulate transcription These cofactors usually contain a short conserved LxxLL motif, which was originally identified in the NR-interacting domain of transcriptional intermediary factor 1a [26], and subse-quently found in other putative NR coactivators [27,28] Furthermore, LxxLL appears to be both necessary and sufficient for such interactions [26] However, some NR-binding proteins, such as NR-binding SET domain-containing protein, also contain a variant motif FxxLL that is responsible for ligand-dependent binding of NRs [32] In ZNHIT-1, a segment extending from residue 72
to residue 110 is required for interaction with Rev-erbb, and is predicted to form an a-helix This segment con-tains the FxxLL motif Our data show that the FxxLL motif of ZNHIT-1 is required for the binding of ZNHIT-1 to Rev-erbb.The analysis of homologues showed that ZNHIT-1 is conserved in eukaryotes In particular, its six cysteines are absolutely conserved from humans to yeast Interestingly, ZNHIT-1 D4, with-out the zinc finger HIT domain, did not remove the inhibitory effect of Rev-erbb on the transcription of apoCIII(Fig 7B) This suggests that the HIT domain is
a functional activity domain for ZNHIT-1
Rev-erbb is a transcriptional silencer and does not possess an activation domain; however, it does contain
a transcriptional silencing domain in the C-terminal putative LBD [5] ChIP assay showed that Rev-erbb could recruit ZNHIT-1 to the apoCIII promoter (Fig 7A), and this recruitment did not modulate Rev-erbb DNA-binding activity (Fig 8) Furthermore, ZNHIT-1 removed the repression of apoCIII induced
by Rev-erbb (Fig 7B) The function of ZNHIT-1 was dependent on the presence of the FxxLL motif for binding to Rev-erbb through its interaction with LBD (Fig 5), and the HIT domain for its activity (Fig 7B) Like Rev-erbb, Rev-erba was also capable of bind-ing ZNHIT-1 (Fig 4C) It is well known that Rev-erba
is a major player in the circadian rhythm However, ZNHIT-1 expression in the liver and heart was not cir-cadian in an extensive and divergent circir-cadian gene
Fig 8 No effect of ZNHIT-1 on Rev-erbb DNA-binding activity to
the apoCIII promoter Quantitative PCR for ChIP products
precipi-tated with anti-Rev-erbb serum was performed using
promoter-specific primers The relative ChIP DNA level was normalized
against the input DNA.
Trang 9expression study [33] In order to further verify this,
mRNA of mouse liver was extracted every 6 h for
30 h, and real-time quantitative PCR showed that
ZNHIT-1 expression was not circadian (data not
shown)
In addition to ZNHIT-1, we recently found that
Rev-erbb could interact with different cofactors,
includ-ing coactivators such as histone acetyl-transferase and
corepressors such as histone deacetylase (J Wang et al.,
School of Life Sciences, Fundan University, Shanghai,
China, unpublished results) In the case of ‘classical’
NRs, NRs recruit corepressors to repress transcription
in the absence of ligands In contrast, ligand binding
induces a conformational change of the receptor,
excludes corepressors from the complex and leads to
the binding of coactivators instead However, Rev-erbb
is an orphan NR, whose ligands, if any, have not been
identified Reinking et al [34] have shown that the
ligand-binding pocket of E75, a Drosophila orthologue
of human Rev-erb, is tightly bound by haem Haem
binding of E75 can be disrupted by mutating the two
most highly conserved histidine residues in the LBD,
and these two residues are also present in the vertebrate
orthologue of E75, Rev-erba and Rev-erbb
Interest-ingly, they also identified E75 as a potential gas sensor,
and showed that CO or NO binds to E75 to interfere
with E75-mediated repression All of the above findings
suggest that there may be certain unidentified ligands
which may regulate the activity of Rev-erbb and
con-trol the recruitment of different cofactors
The mechanism by which ZNHIT-1 removes the
Rev-erbb-induced inhibition of transcription remains
unclear Most cofactors affect transcription through
direct regulation of chromatin remodelling or
recruit-ment of other chromatin remodelling proteins Human
ZNHIT-1 shares 30% identity with Vps71 (P46973)
from Saccharomyces cerevisiae and 67% identity with
its HIT domains Vps71 is a subunit of the SWR1
chromatin remodelling complex that incorporates the
histone variant H2A.Z into nucleosomes [35] The
histone variant H2A.Z is implicated in transcription
activation and the prevention of ectopic spread of
heterochromatin [36] Cai et al [24] reported that the
ZNHIT-1 protein was a subunit of the SRCAP
com-plex, which is the closest mammalian homologue of
SWR1 Recent research has found that the human
SRCAP complex can remodel chromatin and activate
gene transcription [37] The above findings imply that
ZNHIT-1 may affect transcriptional regulation
through the recruitment of a chromatin remodelling
complex Further studies are needed to clarify the
molecular mechanism underlying the activation of
apoCIIIby ZNHIT-1
In summary, a zinc finger HIT domain-containing protein, ZNHIT-1, interacted with Rev-erbb The FxxLL motif of ZNHIT-1 and the putative LBD of Rev-erbb were required for this interaction Further-more, ZNHIT-1 was recruited to the apoCIII promoter
by Rev-erbb and relieved the transcriptional repression
of Rev-erbb These findings indicate that ZNHIT-1 functions as a cofactor to regulate the activity of Rev-erbb, possibly by chromatin remodelling
Experimental procedures
Plasmid construction and protein expression
The coding sequence (CDS) of human Rev-erbb was ampli-fied from the human marathon liver cDNA library (Clon-tech, Tokyo, Japan) by PCR and subcloned into pET28a (catalogue # 69864-3; Novagen, Darmstadt, Germany) and pCMV-HA (Clontech) vectors The CDS of human ZNHIT-1 was amplified from the human liver cDNA library (Clontech) by PCR and subcloned into pGEX4T-3 (catalogue # U13855; Pharmacia Biotech, Piscataway, NJ, USA) and pCMV-Myc (Clontech) vectors ZNHIT-1 L87A⁄ L88A was generated by PCR, and subsequently cloned into pEGX4T-3 and pCMV-Myc vectors pET28a-Rev-erbb, pGEX4T-3-ZNHIT-1 and plasmids containing serial truncated fragments were expressed in Escherichia coli strain BL21 DE3 The purification of protein was performed according to the manufacturer’s instructions (Novagen)
Yeast two-hybrid screening
Yeast two-hybrid screening was performed as described previously [37] Briefly, the yeast two-hybrid screen was car-ried out using the Matchmaker Two-hybrid system 3 (Clon-tech) Rev-erbb was used as bait to screen the human fetal liver cDNA library (Clontech) The mating test was used to pick out the specific interaction between bait and prey
GST pull-down assay
GST-ZNHIT-1 and GST-ZNHIT-1 fragment fusion proteins were expressed in bacteria and purified on glutathione– Sepharose (catalogue # 17-0757-01; Pharmacia) as specified
by the manufacturer The 6His-tagged erbb and Rev-erbb fragments were expressed in bacteria and purified on nickel nitrilotriacetic acid agarose (catalogue # 30210; Qiagen, Venlo, the Netherlands) GST pull-down assays were performed as described previously [37]
Co-IP assay
For the determination of the interaction between ZNHIT-1 and Rev-erbb, HepG2 cells were transfected with
Trang 10Myc-ZNHIT-1 The lysate was first precipitated with anti-Myc
and then detected with anti-Rev-erbb The interaction
between ZNHIT-1 and Rev-erba was assessed using HepG2
cells cotransfected with Myc-ZNHIT-1 and
pCMV-HA-Rev-erba Anti-HA and anti-Myc were used for
precip-itation and detection, respectively The Co-IP assay was
carried out as described previously [38]
Subcellular localization analysis
The complete open reading frames of Rev-erbb and
ZNHIT-1 were constructed in-frame in the plasmids
pEG-FP and pDsRed, respectively Hela and HepG2 cells grown
on coverslips were transiently transfected with plasmids
pEGFP-Rev-erbb and pDsRed-ZNHIT-1, or cotransfected
with both plasmids After 36 h of transfection, the cells
were fixed with 4% formaldehyde and stained with
1 lgÆmL)1of 4¢,6¢-diamidino-2-phenylindole to visualize the
nuclei with an Olympus IX71 laser microscope
Luciferase reporter gene assay
The promoter of the human apoCIII gene (nucleotides
)1408 to +24) was obtained from human genomic DNA
by PCR and subcloned into the promoter-less luciferase
reporter plasmid pGL3-basic (catalogue # E1751; Promega,
Madison, WI, USA), generating pGL3-apoCIII-pro HepG2
cells were cotransfected using FuGENE 6 reagent
(catalo-gue # 11814443001; Roche, Basel, Switzerland) with 100 ng
of apoCIII promoter-driven luciferase expression plasmid
pGL3-apoCIII-pro and the indicated amount of human
Rev-erbb-expressing plasmid pCMV-Rev-erbb, as well as
10 ng of pRL (sea pansy) as an internal control for
trans-fection efficiency The dosage of transfected plasmids in
one well was kept constant by the addition of appropriate
amounts of the empty vector pCMV After 36 h, the cells
were lysed by 200 lL of Promega lysis buffer for 10 min at
room temperature Firefly and Renilla luciferase activities
were measured using a Dual-Luciferase Reporter Assay
Kit (catalogue # 192445; Promega) on a Lumistar
lumi-nometer (BMG Laboratory Technologies, Offenburg,
Germany) Firefly luciferase activity values were divided by
Renilla luciferase activity values to obtain normalized
lucif-erase activities To facilitate comparisons within a given
experiment, the activity data were presented as relative
luciferase activities The final relative activity was calculated
from three independent experiments performed in triplicate
ChIP assay
HepG2 cells were cotransfected with 1 lg of
pCMV-Rev-erbb and either pCMV-ZNHIT-1 or pCMV vector After
incubation, ChIP assay and PCR were performed as
described previously [39] The primers for PCR were
designed to ensure specific amplification of a 230-bp frag-ment of the apoCIII promoter, with the forward primer (TCTCCTAGGGATTTCCCAACTCTCC) and the reverse primer (CTGCCTCTAGGGATGAACTGAGCAG) Quan-titative real-time PCR was performed as described previ-ously [40] Quantitative PCR for ChIP products precipitated with anti-Rev-erbb serum was performed using promoter-specific primers The relative ChIP DNA level was normalized against the input DNA
Stable knock-down of ZNHIT-1
The oligonucleotides encoding the ZNHIT-1 siRNA were 5¢-GATCCGAGACTGCCTCAGTTTGATTCAAGAGATCA AACTGAGGCAGTCTCTTTTTT-3¢ and 5¢-AGCTTAAA AAAGAGACTGCCTCAGTTTGATCTCTTGAATCAAA CTGAGGCAGTCTCG-3¢ These oligonucleotides were annealed and subcloned to downstream of the U6 promoter
in pGCsi-U6⁄ Neo ⁄ GFP (Genechem, Shanghai, China) using HindIII and BamHI The empty plasmid or RNAi plasmid was transfected into HepG2 cells using Fugene-HD transfect reagent (catalogue # 93539521; Roche) After 1 day of incu-bation, media from all plates were replaced with selective medium containing 300 lgÆmL)1of Geneticin (Gibco⁄ BRL, Grand Island, NY, USA) Cells were grown in selective media for 2 weeks, and G418-resistant colonies were estab-lished in six-well plates, expanded and cloned independently
Acknowledgements
This work was supported by grants from the National Nature Science Foundation of China (NSFC 30671175 and 30370752) and from the Specialized Research Fund for the Doctoral Program of High Education (SRFDP 20060246017)
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