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HIC1 epigenetically represses CIITA transcription in B lymphocytes
HIC1 epigenetically represses CIITA transcription in B lymphocytes Sheng Zeng, Yuyu Yang, Xian Cheng, Bisheng Zhou, Ping Li, Yuhao Zhao, Xiaocen Kong, Yong Xu PII: DOI: Reference:
S1874-9399(16)30199-7 doi:10.1016/j.bbagrm.2016.10.003 BBAGRM 1089
To appear in:
BBA - Gene Regulatory Mechanisms
Received date: Revised date: Accepted date:
26 August 2016 5 October 2016 5 October 2016
Please cite this article as: Sheng Zeng, Yuyu Yang, Xian Cheng, Bisheng Zhou, Ping Li, Yuhao Zhao, Xiaocen Kong, Yong Xu, HIC1 epigenetically represses CIITA transcription in B lymphocytes, BBA - Gene Regulatory Mechanisms (2016), doi:10.1016/j.bbagrm.2016.10.003
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ACCEPTED MANUSCRIPT HIC1 epigenetically represses CIITA transcription in B lymphocytes Sheng Zeng,1,6 Yuyu Yang,1,2,6 Xian Cheng,1,3,6 Bisheng Zhou,1,6 Ping Li,1,4 Yuhao Zhao,1
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Xiaocen Kong,1,5 Yong Xu1* 1
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Department of Pathophysiology, Key Laboratory of Cardiovascular Disease and Molecular
Intervention, Nanjing Medical University, Nanjing, China; 2
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing,
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China; 3
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Jiangsu Institute of Nuclear Medicine, Wuxi, China;
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Department of Gastroenterology, Second Hospital Affiliated to Nanjing Medical University,
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Nanjing, China; 5
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Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, Nanjing,
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China
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*Correspondence to:
Yong Xu, PhD, Nanjing Medical University, 101 Longmian Ave, Nanjing, Jiangsu 211166, China Email: [email protected], Tel/Fax: 86-25-86869371; or Xiaocen Kong, MD/PhD, Nanjing First Hospital, 68 Changle Rd, Nanjing, Jiangsu 210006, China Email: [email protected]
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These authors contributed equally to this work.
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ACCEPTED MANUSCRIPT Abstract Differentiation of B lymphocytes into isotope-specific plasma cells represents a hallmark
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event in adaptive immunity. During B cell maturation, expression of class II transactivator
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(CIITA) gene is down-regulated although the underlying epigenetic mechanism is not completely defined. Here we report that hypermethylated in cancer 1 (HIC1) was up-regulated in differentiating B lymphocytes paralleling CIITA repression. Over-expression
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of HIC1 directly repressed endogenous CIITA transcription in B cells. Reporter assay and
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chromatin immunoprecipitation (ChIP) assay confirmed that HIC1 bound to the proximal CIITA type III promoter (-545/-113); mutation of a conserved HIC1 site within this region
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abrogated CIITA trans-repression. More important, depletion of HIC1 with small interfering
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RNA (siRNA) restored CIITA expression in differentiating B cells. Mechanistically, HIC1
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preferentially interacted with and recruited DNMT1 and DNMT3b to the CIITA promoter to synergistically repress CIITA transcription. On the contrary, silencing of DNMT1/DNMT3b or
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inhibition of DNMT activity with 5-aza-dC attenuated CIITA trans-repression. Therefore, our data identify HIC1 as a novel factor involved in B cell differentiation acting as an epigenetic repressor of CIITA transcription.
Key words: transcriptional regulation; epigenetics; HIC1; CIITA; DNMT
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ACCEPTED MANUSCRIPT Introduction The bifurcation of the immune system in higher eukaryotes marks an evolutionarily
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significant step in host defense. Unlike the native immune system which usually launches
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fast and non-specific attacks against invading pathogens, the adaptive immune system relies on highly specialized lymphocytes to initiate a more delicate but slower response to combat aliens [1]. Differentiation of B lymphocytes represents a paradigm for the mode of action of
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the adaptive immune system: in response to a range of activating signals (e.g., LPS or helper
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T cells), mature B cells transition into antibody-secreting plasma cells and participate in the elimination of a specific set of pathogens [2]. A complex network of transcription factors,
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including PAX5, BCL6, IRF4, and BLIMP-1, cumulatively define the differentiation process of B
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lymphocytes and hence contribute to adaptive immunity [3].
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Class II transactivator, or CIITA, is the master regulator of major histocompatibility complex II molecules required for activation of CD4+ T lymphocytes [4]. CIITA expression
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defines the identity of mature B cells because during B cell differentiation CIITA is silenced at the transcriptional levels. CIITA transcription is driven by four distinct promoters in different immune cells: type I and type III promoters are responsible for constitutive CIITA expression in dendritic cells and B cells respectively while type IV promoter mediates IFN- induced CIITA expression in macrophages and non-myeloid cells [5, 6]. Previously, BLIMP-1 has been shown to bind to the proximal CIITA type III promoter (-180/-171) and represses CIITA transcription during B cell differentiation [7]. In addition, it has been documented that BLIMP-1 recruits a histone H3K9 dimethyltransferase (G9a) rendering a repressive chromatin structure to facilitate trans-repression of the CIITA promoter in differentiating B cells [8]. On 3
ACCEPTED MANUSCRIPT the other hand, it has been noted that the formation of a repressive CIITA promoter, as defined by the loss of active histone marks, following B cell differentiation precedes the full
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activation of BLIMP-1 [9, 10], suggesting that transcription factors other than BLIMP-1 also
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contribute to CIITA trans-repression.
Hypermethylated in cancer 1 (HIC1) is a transcriptional factor initially identified as a target of p53 activation [11]. HIC1 contains five Kruppel-like C2H2 zinc fingers that mediate
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its binding to target promoters and a BTB/POZ domain that is required for transcriptional
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repression [12]. Previous investigations have primarily implicated HIC1 as a tumor repressor by down-regulating the transcription of a host of genes including SIRT1 [13], CXCR7 [14], p21
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[15], and EphA2 [16]. Here we report that HIC1 expression is up-regulated during B cell
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differentiation paralleling CIITA down-regulation and that HIC1 recruits DNMT1 and
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DNMT3b to repress CIITA repression. Therefore, our data identify HIC1 as a novel factor
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involved in B cell differentiation acting as an epigenetic repressor of CIITA transcription.
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ACCEPTED MANUSCRIPT Methods and materials Cell culture
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HEK293 cells, U2OS cells, and Raji cells were maintained in DMEM supplemented with
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10% fetal bovine serum (Gibco). Primary B lymphocytes were isolated and differentiated as previously described [17].
Plasmids, transient transfection, and luciferase assay
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FLAG-HIC1, GFP-HIC1, FLAG-DNMT1, FLAG-DNMT3a, FLAG-DNMT3b, and CIITA type III
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promoter luciferase plasmids have been described previously [6, 18, 19]. Small interfering RNAs were purchased from Dharmacon. Transient transfections in HEK293 cells or U2OS cells
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were performed with Lipofectamine 2000 (Invitrogen). Transient transfections in Raji cells or
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primary B cells were performed with an electroporator (NEON, Life Technologies). To
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construct stable Raji cells expressing GFP-HIC1 or GFP, puromycin (200g/ml) was added to the media 48 hours after transfection; the cells were selected for 2 weeks with selection
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media changed every other day. Protein extraction, immunoprecipitation and Western blot Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche). Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysate overnight before being absorbed by Protein A/G-plus Agarose beads. Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2, Sigma) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blot 5
ACCEPTED MANUSCRIPT analyses were performed with anti-FLAG, anti-GFP, anti--actin (Sigma), anti-CIITA, anti-HIC1, (Santa Cruz), anti-DNMT1, anti-DNMT3a, and anti-DNMT3b (Proteintech) antibodies. All
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experiments were repeated at least three times.
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RNA Isolation and Real-time PCR
RNA was extracted with the RNeasy RNA isolation kit (Qiagen). Reverse transcriptase reactions were performed using a SuperScript First-strand Synthesis System (Invitrogen).
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Real-time PCR reactions were performed on an ABI Prism 7500 system. Primers and Taqman
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probes used for real-time reactions were purchased from Applied Biosystems. All experiments were repeated at least three times.
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Chromatin Immunoprecipitation (ChIP)
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Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described
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before [20]. In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1%
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Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼500 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-HIC1 (Santa Cruz), anti-DNMT1, anti-DNMT3a, anti-DNMT3b (Proteintech), or pre-immune IgG. Precipitated genomic DNA was amplified by real-time PCR with the following primers: for human CIITA promoter, GAGTAGGCATGGTAGAGGAGAGC and TACCACACTCCCTTAAGC; for human GAPDH promoter, TACTAGCGGTTTTACGGGCG and TCGAACAGGAGGAGCAGAGAGCGA; for mouse Ciita promoter, CATGGGCAAATTAGAGGGTATCC and
GCAAAGAAAGCGAGCAAGGG;
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mouse
Gapdh
promoter,
ACCEPTED MANUSCRIPT ATCACTGCCACCCAGAAGACTGTGGA
and
CTCATACCAGGAAATGAGCTTGACAAA.
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experiments were repeated at least three times.
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CIITA promoter DNA methylation analysis
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CIITA promoter methylation status was analyzed with bisulfite conversion followed by sequencing essentially as described previously [21]. Briefly, genomic DNA isolated from Raji cells primary HSCs was subjected to bisulfite treatment using the EZDNA Methylation Gold
AAACACAAACTCCTATTCCCATCCTCAC;
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TTAAGGGAGTGTGGTAAAATTAGAGGGTG;
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Kit (ZymoResearch). The modified DNA was amplified using the following primers: Forward, Reverse
and
Reverse
Nested, Outer,
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AAACAACTCTTTCACATCTTCCAATAACC TAC. The PCR products were cloned into pCRII-TA
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Statistical Analysis
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vectors (Invitrogen) and sequenced using the Sanger sequencing.
One-way ANOVA with post-hoc Scheffe analyses were performed using an SPSS package.
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P values smaller than .05 were considered statistically significant (*).
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ACCEPTED MANUSCRIPT Results HIC1 expression is up-regulated during B cell differentiation
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We started off by examining the expression of HIC1 in a human B lymphocyte line (Raji)
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before and after differentiation. Stimulation with IL-2/IL-5/LPS led to significant down-regulation of CIITA message and protein indicative of plasma cell differentiation. Paralleling CIITA repression, an increase in HIC1 expression was observed (Fig.1A and Fig.1B).
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Likewise, HIC1 expression was up-regulated during the differentiation of primary mouse B
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lymphocytes (Fig.1C and Fig.1D). Of note, the changes in HIC1 expression followed a different kinetics compared to those of BLIMP-1, a well characterized plasma cell marker and
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CIITA repressor [7]. While BLIMP-1 expression was continuously elevated up to 24h following
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IL-2/IL-5/LPS treatment, the window for HIC1 induction was narrower: HIC1 expression
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started to increase at 6h, peaked at 12h but declined at 24h. Collectively, these data suggest that HIC1 might be an early response protein during B cell differentiation contributing to
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CIITA repression.
HIC1 binds to CIITA type III promoter and represses CIITA transcription To directly assess whether HIC1 is a de novo repressor of CIITA transcription, we performed the following experiments. As shown in Fig.2A, co-transfection of HIC1 expression construct with CIITA type III promoter-luciferase constructs of different lengths into HIC1-negative U2OS cells resulted in robust repression of luciferase activities of two long CIITA promoter constructs (-1007 and -545) but not that of the shortest one (-113). Similar observation was made in HEK293 cells (Fig.2A), suggesting that there might be a HIC1-response element with the CIITA proximal promoter between -545 and -113 with 8
ACCEPTED MANUSCRIPT respect to the transcription start site. Close examination of this region revealed a putative HIC1 motif between -298 and -288 (GCTGGCACCAG). Deletion of this potential HIC1 site
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augmented basal CIITA promoter activity but also abrogated HIC1-mediated repression
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(Fig.2B). To examine whether HIC1 could down-regulate endogenous CIITA messages, we constructed a stable Raji cell line in which GFP-tagged HIC1 expression construct was incorporated into the genome (GFP-HIC1). Compared to the control stable cell line in which
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an empty vector (GFP) was transfected, HIC1 expression was increased by more than 2 fold in
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two different GFP-HIC1 clones as measured by qPCR (Fig.2C). Importantly, there was a modest (~40%) but significant down-regulation of CIITA messages in GFP-HIC1 cells as
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opposed to GFP cells. In comparison, BLIMP1 levels were not significantly altered by HIC1
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GFP-HIC1 cells (Fig.2D).
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over-expression. Western blotting showed that CIITA protein levels were also decreased in
Next, we evaluated direct binding of HIC1 to the CIITA gene in cells. Chromatin
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immunoprecipitation (ChIP) assay performed with an anti-GFP antibody showed significantly higher levels of binding of GFP-HIC1 to the CIITA type III promoter than GFP whereas ChIP assay performed with a pre-immune IgG detected no such enrichment of GFP-HIC1 over GFP (Fig.2E). On the contrary, we did not find any significant binding of HIC1 on the GAPDH promoter (Fig.2E). We also examined the binding of endogenous HIC1 to the CIITA promoter in B cells. As shown in Fig.2F, IL-2/IL-5/LPS stimulation resulted in augmented binding of HIC1 to the CIITA promoter but not the GAPDH promoter. In addition, ChIP assays performed in primary mouse B lymphocytes showed that occupancies of HIC1 on the CIITA promoter started to increase at 12h following IL-2/IL-5/LPS stimulation and continued to increase at 24h 9
ACCEPTED MANUSCRIPT following a kinetics slightly lagging behind that of its expression (Fig.2G). Taken together, we conclude that HIC1 could bind to CIITA type III promoter and repress CIITA transcription.
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HIC1 is essential for CIITA down-regulation during B cell differentiation
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Having determined that HIC1 over-expression is sufficient to drive CIITA trans-repression, we asked whether HIC1 is indispensable for CIITA down-regulation during B cell differentiation. Knockdown of HIC1 with two different siRNAs in Raji cells completely blunted
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the induction of HIC1 expression by IL-2/IL-5/LPS treatment. More important, CIITA
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repression was significantly alleviated (Fig.3A and Fig.3B). Similarly, siRNA targeting HIC1 partially rescued CIITA expression in differentiating primary B lymphocytes (Fig.3C and
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cell differentiation.
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Fig.3D). Therefore, HIC1 is both adequate and necessary for CIITA trans-repression during B
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HIC1 interacts with DNMT during B cell differentiation HIC1 is known to engage the epigenetic machinery, histone deacetylase for instance, to
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repress gene transcription [18, 22-24]. Previously, it has been shown that DNA methyltransferases (DNMTs) play a role in CIITA silencing in cancer cells [25]. We tested the hypothesis that HIC1 interacts with DNMT to repress CIITA transcription in B cells. To this end, we transfected GFP-tagged HIC1 into HEK293 along with FLAG-tagged DNMT expression constructs. Immunoprecipitation with an anti-FLAG antibody pulled down HIC1 along with DNMT1, DNMT3a, and DNMT3b, suggesting that HIC1 might be in a complex with DNMTs (Fig.4A). More important, an anti-HIC1 antibody precipitated all three DNMT isoforms along with HIC1 in Raji cells (Fig.4B). To further examine the functional consequence of this DNMT-HIC1 interaction, we performed Re-ChIP assay in Raji cells and primary B lymphocytes. 10
ACCEPTED MANUSCRIPT Of interest, both a HIC1-DNMT1 complex and a HIC1-DNMT3b complex but not a HIC1-DNMT3a were detected on the CIITA promoter following IL-2/IL-5/LPS induction
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indicating that the interaction between HIC1 and different DNMT isoforms is locus-specific
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(Fig.4C and Fig.4D); HIC1 did not interact with any of the three DNMT isoforms on the GAPDH promoter either with or without IL-2/IL-5/LPS treatment. Thus it appears that HIC1 may recruit DNMT1/DNMT3b to repress CIITA transcription.
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We performed a series of experiments to further clarify the functional relevance of the
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HIC1-DNMT complex. In response to IL-2/IL-5/LPS treatment, there were enhanced occupancies of DNMT1 and DNMT3b but not DNMT3a on the CIITA promoter in Raji cells;
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over-expression of HIC1 further augmented the binding of DNMT1 and DNMT3b (Fig.5A).
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Bisulfite modification followed by sequencing revealed that following IL-2/IL-5/LPS treatment
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there was a significant increase in CpG methylation surrounding the CIITA promoter, which was further up-regulated by HIC1 over-expression (Fig.5B). In reporter assays, co-transfection
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of DNMT1 and DNMT3b, but not DNMT3a, enhanced the repression of CIITA promoter activity by HIC1, confirming that HIC1 only interacts with DNMT1 and DNMT3b instead of DNMT3a on the CIITA promoter (Fig.5C). In addition, depletion of endogenous HIC1 completely blocked the recruitment of DNMT1 and DNMT3b to the CIITA promoter in both Raji cells (Fig.5D) and primary B lymphocytes (Fig.5E). Accordingly, CpG methylation surrounding the CIITA promoter was attenuated (Fig.5F). Together, these data support a role for the HIC1-DNMT complex in repressing CIITA transcription during B cell differentiation. DNMT depletion or inhibition attenuates CIITA repression during B cell differentiation
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ACCEPTED MANUSCRIPT Finally, we tackled the question whether DNMT inhibition/depletion would achieve similar effects as HIC1 silencing with regard to the recovery of CIITA expression. Indeed,
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exposure to a DNMT inhibitor 5-azacytidine (5-Aza-dc) dose-dependently relieved the
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repression of CIITA promoter activity by HIC1 in both HEK293 cells and U2OS cells (Fig.6A). Furthermore, 5-Aza-dc treatment also partially normalized endogenous CIITA expression in differentiating B cells (Fig.6B and Fig.6C). Finally, siRNA targeting DNMT1 and DNMT3b, but
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not DNMT3a, antagonized the repression of CIITA in primary B lymphocytes exposed to
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IL-2/IL-5/LPS. Therefore, HIC1 recruitment of DNMT1 and DNMT3b may play an essential role
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in CIITA trans-repression during B cell differentiation.
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ACCEPTED MANUSCRIPT Discussion Silencing of CIITA by transcriptional repression is a hallmark event during B cell
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differentiation. We present evidence that HIC1 is activated during B cell differentiation and
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directly binds to the CIITA promoter to repress CIITA transcription. Previous investigations have portrayed BLIMP-1, a zinc-finger transcription factor, as the major regulator of CIITA expression during B cell differentiation [8, 10, 26, 27]. At the same time evidence is
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accumulating that BLIMP-1 full activation and its binding to the CIITA promoter are preceded
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by the loss of active histone marks from the same region and the initiation of CIITA repression suggesting that BLIMP-1 is the sole transcription factor responsible for CIITA
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repression [9, 10]. Boss and colleagues have recently identified a zinc-finger containing
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transcription factor ZBTB32 as an early repressor of CIITA transcription during B cell
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differentiation [28]. ZBTB32 binds to the proximal CIITA promoter at a site adjacent to the BLIMP-1 site (separated by ~50bp) and can interact with BLIMP-1 in plasma cells although it
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is unclear whether there is any functional interplay/inter-dependence between BLIMP-1 and ZBTB32 [28]. By comparison, HIC1 binds to a more distal site of the CIITA promoter (~100bp from the BLIMP-1 site). BLIMP-1, HIC1, and ZBTB32 all belong to zinc-finger family of transcription factors that are known to form homodimers or heterodimers when binding to DNA [29]. It is likely that BLIMP1, HIC1, ZBTB32, and other transcription factors form a large repressor complex cooperatively regulate CIITA expression during B cell differentiation. In support of this model, we found that depletion of HIC1 attenuated but not completely blocked CIITA repression in B cells (Figure 3). Coincidently, ZBTB32 deficiency delayed instead of abrogating CIITA repression [28]. Clearly, more work is warranted to clarify the 13
ACCEPTED MANUSCRIPT functional reliance/redundancy of different transcription factors in regulating the kinetics of CIITA repression during B cell differentiation.
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Another major finding of this current report is that HIC1 recruits DNMT1 and DNMT3b
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to repress CIITA transcription. Although immunoprecipitation experiments showed that DNMT1, DNMT3a, and DNMT3b all bound to HIC1 with comparable affinity, DNMT1 and DNMT3b were preferentially recruited by HIC1 to the CIITA promoter to repress
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transcription. These data suggest that although the physical interactions between HIC1 and
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DNMTs do not rely on DNA, recruitment of DNMTs to HIC1 target genes are locus and context-dependent and may be dependent on specific chromatin structure. Alternatively,
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HIC1 may rely on differential recruitment of DNMTs to repress the transcription of different
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genes. Of note, DNMT depletion or inhibition only partially rescued CIITA repression. HIC1 is
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known to interact with multiple epigenetic factors including histone methyltransferases (HMTs) [30] and histone deacetylases (HDACs) [22]. DNMTs have also been found to be in
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large complexes with HMTs and HDACs [31-33]. Therefore, DNMTs may constitute only part of the epigenetic mechanism whereby HIC1 contributes to CIITA trans-repression. Indeed, CIITA silencing in cancer cells can be reversed by treatment with either DNMT inhibitors (e.g., 5-Aza-dc) or HDAC inhibitors (e.g., TSA) [25, 34]. In addition, Londhe et al have reported that simultaneous treatment with TSA and 5-Aza-dc resulted in a more robust restitution of CIITA expression in rhabdomyosarcoma cells in individual treatment [35]. These data allude to a model wherein B cell activation signals stimulate coordinated recruitment of different epigenetic factors including DNMTs, some of which may be independent of HIC1, to the CIITA promoter, to repress CIITA transcription. 14
ACCEPTED MANUSCRIPT A lingering issue that remains unanswered is whether HIC1, in addition to contributing to CIITA repression, plays a role in B cell differentiation per se. HIC1 was initially identified as
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a tumor repressor gene that can be activated by p53 and has been implicated in the
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pathogenesis of a range of human cancers [36]. Recently, it has been shown that BLIMP-1 plays a major role protecting against B cell lymphoma by promoting B cell differentiation [37]. If future studies can expand the role of HIC1 in B cells beyond a mere repressor of CIITA
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transcription, then it would not only reinforce the characterization of HIC1 as a
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wide-spectrum tumor repressor, but provide strong rationale for screening small-molecule
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compounds that can boost HIC1 activity as novel therapeutic solutions to treat cancer.
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ACCEPTED MANUSCRIPT Acknowledgements This work was supported, in part, by the National Natural Science Foundation of China
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(31270805, 81503067, 81602352, 81402550, 81400840, 81500426). YX is a Fellow at the
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Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.
Disclosure
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None.
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[23] N. Stankovic-Valentin, S. Deltour, J. Seeler, S. Pinte, G. Vergoten, C. Guerardel, A. Dejean, D. Leprince, An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcriptional repression activity, Molecular
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and cellular biology, 27 (2007) 2661-2675. [24] C. Van Rechem, G. Boulay, S. Pinte, N. Stankovic-Valentin, C. Guerardel, D. Leprince, Differential regulation of HIC1 target genes by CtBP and NuRD, via an acetylation/SUMOylation switch, in quiescent versus proliferating cells, Molecular and cellular biology, 30 (2010) 4045-4059.
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[25] A. Satoh, M. Toyota, H. Ikeda, Y. Morimoto, K. Akino, H. Mita, H. Suzuki, Y. Sasaki, T. Kanaseki, Y. Takamura, H. Soejima, T. Urano, K. Yanagihara, T. Endo, Y. Hinoda, M. Fujita, M. Hosokawa, N. Sato, T. Tokino, K. Imai, Epigenetic inactivation of class II transactivator (CIITA) is associated with the absence of interferon-gamma-induced HLA-DR expression in colorectal and gastric cancer cells, Oncogene, 23 (2004) 8876-8886. [26] M. Shapiro-Shelef, K.I. Lin, L.J. McHeyzer-Williams, J. Liao, M.G. McHeyzer-Williams, K. Calame, Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells, Immunity, 19 (2003) 607-620. [27] M.A. Smith, G. Wright, J. Wu, P. Tailor, K. Ozato, X. Chen, S. Wei, J.F. Piskurich, J.P. Ting, K.L. Wright, Positive regulatory domain I (PRDM1) and IRF8/PU.1 counter-regulate MHC class II transactivator (CIITA) expression during dendritic cell maturation, The Journal of biological chemistry, 286 (2011) 7893-7904. [28] H.S. Yoon, C.D. Scharer, P. Majumder, C.W. Davis, R. Butler, W. Zinzow-Kramer, I. Skountzou, D.G. Koutsonanos, R. Ahmed, J.M. Boss, ZBTB32 is an early repressor of the CIITA and MHC class II gene expression during B cell differentiation to plasma cells, J Immunol, 189 (2012) 2393-2403. [29] S.S. Krishna, I. Majumdar, N.V. Grishin, Structural classification of zinc fingers: survey and summary, Nucleic acids research, 31 (2003) 532-550. 18
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ACCEPTED MANUSCRIPT Figure legends Figure 1: HIC1 expression is up-regulated during B cell differentiation. (A, B) Raji cells were treated with IL-2/IL-5/LPS or vehicle for 24 hours. Gene expression levels were examined by
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qPCR (A) and Western (B). (C, D) Primary B lymphocytes were induced to differentiate by IL-2/IL-5/LPS. Cells were harvested at indicated time points and gene expression levels were
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examined by qPCR (C) and Western (D).
Figure 2: HIC1 binds to CIITA type III promoter and represses CIITA transcription. (A) CIITA promoter constructs of different lengths were transfected into U2OS or HEK293 cells with or
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without HIC1. Luciferase activities were normalized by protein concentration and GFP fluorescence. (B) Wild type CIITA promoter construct (WT) or CIITA promoter construct
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harboring the HIC1 site mutation (MT) was transfected into HEK293 cells with or without HIC1. Luciferase activities were normalized by protein concentration and GFP fluorescence.
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(C-E) Raji cells were stably transfected with GFP-HIC1 or GFP. Gene expression levels were
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examined by qPCR (C) and Western (D). Binding of HIC1 to the CIITA promoter was evaluated by ChIP with anti-GFP or IgG (E). (F) Raji cells were treated with IL-2/IL-5/LPS or vehicle for 24
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hours. ChIP assays were performed with anti-HIC1 or IgG. (G) Primary B lymphocytes were induced to differentiate by IL-2/IL-5/LPS. Cells were harvested at indicated time points and ChIP assays were performed with anti-HIC1 or IgG.
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Figure 3: HIC1 is essential for CIITA down-regulation during B cell differentiation. (A, B) Raji cells were transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with IL-2/IL-5/LPS for 24 hours. Gene expression levels were examined by qPCR (A) and Western (B). (C, D) Primary B lymphocytes were transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with IL-2/IL-5/LPS. Cells were harvested at indicated time points and gene expression levels were examined by qPCR (C) and Western (D). Figure 4: HIC1 interacts with DNMT during B cell differentiation. (A) HEK293 cells were transfected with GFP-HIC1 and FLAG-tagged DNMT expression constructs as indicated. Immunoprecipitation was performed with anti-FLAG. (B) Whole cell lysates extracted from Raji cells were immunoprecipitated with indicated antibodies. (C) Raji cells were treated with IL-2/IL-5/LPS or vehicle for 24 hours. Re-ChIP assays were performed with indicated 20
ACCEPTED MANUSCRIPT antibodies. (D) Primary B lymphocytes were induced to differentiate by IL-2/IL-5/LPS. Re-ChIP assays were performed with indicated antibodies. Figure 5: HIC1 and DNMT cooperate to repress CIITA differentiation. (A) Raji cells were
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stably transfected with GFP-HIC1 or GFP. ChIP assays were performed with indicated antibodies. (B) Bisulfite conversion coupled with sequencing was performed as described
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under Materials and methods using genomic DNA isolated from Raji cells. (C) A CIITA promoter-luciferase construct was transfected into HEK293 or U2OS cells with indicated expression constructs. Luciferase activities were normalized by protein concentration and
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GFP fluorescence. (D) Raji cells were transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with IL-2/IL-5/LPS for 24 hours. ChIP assays were performed with
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indicated antibodies. (E) Primary B lymphocytes were transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with IL-2/IL-5/LPS. (F) Raji cells were
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transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with
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IL-2/IL-5/LPS for 24 hours. Bisulfite conversion coupled with sequencing was performed as described under Materials and methods.
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Figure 6: DNMT depletion or inhibition attenuates CIITA repression during B cell differentiation. (A) A CIITA promoter-luciferase construct was transfected into HEK293 or U2OS cells with HIC1 followed by exposure to 5-Aza-dC (0.1 M and 0.5M ) for 24 hours.
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Luciferase activities were normalized by protein concentration and GFP fluorescence. (B, C) Primary B lymphocytes were induced to differentiate by IL-2/IL-5/LPS in the presence or absence of 5-Aza-dC (0.5M) for 24 hours. CIITA expression was examined by qPCR (B) and Western (C). (D, E) Primary B lymphocytes were transfected with siRNAs targeting specific DNMT and induced to differentiate by IL-2/IL-5/LPS in the presence or absence of 5-Aza-dC (0.5M). Cells were harvested at 12h and 24h post-treatment and CIITA expression was examined by qPCR (D) and Western (E).
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ACCEPTED MANUSCRIPT Figure 1 A
B 5
CIITA (135kd)
*
4.5
Vehicle IL-2/IL-5/LPS
HIC1 (78kd)
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Relative mRNA levels
4 3.5
IL-2/IL-5/LPS
*
1 0.5 0
HIC1
Hic1
Blimp1
5
20 18
2
1
14 12 10 8 6
D
3
1.2
1.2
1
1
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Relative mRNA levels
Relative mRNA levels
4
Ciita
NU
C
Relative mRNA levels
CIITA
4
0.8 0.6 0.4 0.2
Relative mRNA levels
1.5
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2
IP
-actin (42kd)
3 2.5
dc4
0.8 0.6 0.4 0.2
AC
D
0
6 12 24
0
0
6 12 24
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0 IL-2/IL-5/LPS (h) 0
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IL-2/IL-5/LPS (h)
0 0 6 12 24
Hic1 (78kd) Ciita (135kd) -actin (42kd)
0
6
12
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0 6 12 24
ACCEPTED MANUSCRIPT Figure 2
HIC1
CIITA 1.2 *
0
E
0.8 0.6 0.4 0.2 0
CIITA type III ChIP
7
T
IP CIITA (135kd) GFP-HIC1 (96kd)
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0.5
Relative mRNA levels
1
GFP-HIC1#2
GFP-HIC1#1
1.5
GFP
*
2
*
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
MT
D
BLIMP1
1
GFP-HIC1#1
Relative mRNA levels
*
2.5
GFP
Relative mRNA levels
3
0.5
-113
-545
GFP-HIC1#2
C
*
1
WT
-1007
-113
-545
1.5
0
0 -1007
HIC1 HIC1
-actin (42kd)
GAPDH ChIP
1.4
5
Relative enrichment
Relative enrichment
1.2
GFP IgG
3 2
P GF
0.4 0.2
#2 #1 C1 C1 HI HI P P GF GF
F 7 6 5 4 3 2 1 0
*
AC
Relative enrichment
0.6
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1 0
1 0.8
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*
4
D
*
6
0
P GF
#1 #2 C1 C1 HI HI P P GF GF
Vehicle IL-2/IL-5/LPS
HIC1 IgG CIITA type III ChIP
HIC1 IgG GAPDH ChIP
G CIITA type III ChIP
GAPDH ChIP
*
0
6
12
24
1.4
Relative enrichment
Hic1 IgG
*
Relative enrichment
4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 IL-2/IL-5/LPS (h)
1.2 1 0.8 0.6 0.4 0.2
0 IL-2/IL-5/LPS (h)
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0
GFP-HIC1#2
0.5
2
WT MT
GFP-HIC1#1
*
1
-288
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0.2
1.5
GCTGGCACCAG GCTGGCACCAG
GFP
0.4
*
Relative luciferase activity
0.6
2
2.5
NU
0.8
-298
HIC1 HIC1
GFP-HIC1#2
Relative luciferase activity
Relative luciferase activity
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0
B
HEK293
2.5
*
*
GFP-HIC1#1
U2OS
1.2
GFP
A
6
12
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ACCEPTED MANUSCRIPT Figure 3 A
B HIC1
1
*
CIITA (135kd)
0.5
*
T
0.6 0.4
SCR siHIC1#1 siHIC1#2
0.2
0 IL-2/IL-5/LPS SCR siHIC1#1 siHIC1#2
HIC1 (78kd)
0.8
Hic1
1 0.5
0 IL-2/IL-5/LPS (h)
12
24
NU *
0.6
*
0.4 0.2
0 IL-2/IL-5/LPS (h)
0
12
24
D
0
1 0.8
MA
1.5
Relative mRNA levels
Relative mRNA levels
SCR siHic1
*
2
Ciita
1.2 *
2.5
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D
SCR siHic1 IL-2/IL-5/LPS
Ciita (135kd) Hic1 (78kd) -actin (42kd)
24h
AC
12h
IL-2/IL-5/LPS
C 3
IP
1
CIITA
*
1.5
0 IL-2/IL-5/LPS SCR siHIC1#1 siHIC1#2
1.2
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*
2
Relative mRNA levels
Relative mRNA levels
2.5
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-actin (42kd)
ACCEPTED MANUSCRIPT Figure 4 A
GFP-HIC1
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HIC1 (78kd)
←FLAG-DNMT1 ←FLAG-DNMT3a ←FLAG-DNMT3b
DNMT1 (180kd)
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Input
DNMT3a (100kd)
GFP-HIC1
DNMT3b (100kd)
C CIITA type III ChIP
GAPDH ChIP
1.4
* *
4
Vehicle IL-2/IL-5/LPS
3
1
D
Ciita type III ChIP 6 *
5
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Vehicle IL-2/IL-5/LPS
4 3 2
CE P
Relative enrichment
*
D
0 1st IP HIC1 HIC1 HIC1 IgG IgG IgG 2nd IP DNMT1 DNMT3a DNMT3b DNMT1 DNMT3a DNMT3b
1
1.2
1
0.8 0.6 0.4
MA
2
Relative enrichment
5
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6
Gapdh ChIP 1.4 1.2 1 0.8 0.6 0.4 0.2
0 1st IP Hic1 Hic1 Hic1 IgG IgG IgG 2nd IP Dnmt1 Dnmt3a Dnmt3b Dnmt1 Dnmt3a Dnmt3b
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0 1st IP Hic1 Hic1 Hic1 IgG IgG IgG 2nd IP Dnmt1 Dnmt3a Dnmt3b Dnmt1 Dnmt3a Dnmt3b
0.2
0 1st IP HIC1 HIC1 HIC1 IgG IgG IgG 2nd IP DNMT1 DNMT3a DNMT3b DNMT1 DNMT3a DNMT3b
Relative enrichment
Relative enrichment
7
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GFP-HIC1 FLAG-DNMT1 FLAG-DNMT3a FLAG-DNMT3b
-HIC1
Input
←FLAG-DNMT1 ←FLAG-DNMT3a ←FLAG-DNMT3b
Eluate
IgG
B IP: FLAG
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ACCEPTED MANUSCRIPT Figure 5 A GAPDH ChIP
*
DNMT1
GFP GFP IL-2/IL-5/LPS GFP-HIC1 IL-2/IL-5/LPS
1.4 1.2 1 0.8 0.6 0.4 0.2 0
T
*
DNMT3a DNMT3b
DNMT1
IP
Relative enrichment
Relative enrichment
CIITA type III ChIP 7 6 5 4 3 2 1 0
DNMT3a DNMT3b
P
S B
hI
C
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B rw Fo
d
-288
d
ar
ar
rw
Fo -298 HIC1
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% Methylation
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Relative luciferase activity
U2OS CIITA promoter (-545)
1
* *
D
*
0.8
0.4 0.2
D
AC
4 3 2 1 0
DNMT1
DNMT3a
DNMT3b
0.4 0.2 0
GAPDH ChIP
1.4
Relative enrichment
*
*
5
0.6
HIC1 DNMT1 DNMT3a DNMT3b
CIITA type III ChIP
6
0.8
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0.6
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Relative luciferase activity
*
HIC1 DNMT1 DNMT3a DNMT3b
er ut
d te
1.2
HEK293 CIITA promoter (-545)
1
0
O
es N
20
0
Relative enrichment
se er ev
R
se er ev
R
se er ev
R *
30
10
1.2
S
S
P
hI GFP GFP IL-2/IL-5/LPS GFP-HIC1 IL-2/IL-5/LPS
40
C
B
B
C
50
SCR SCR IL-2/IL-5/LPS SCR siHIC1 IL-2/IL-5/LPS
1.2 1
SCR IL-2/IL-5/LPS siHIC1 IL-2/IL-5/LPS
0.8 0.6 0.4 0.2 0
DNMT1
DNMT3a
DNMT3b
E *
*
6 4 2
0 LPS/IL-2/IL-5 (h) 0
12 24 12 24
SCR siHic1
8
Ciita ChIP-Dnmt3a
6 4 2
0 LPS/IL-2/IL-5 (h) 0
12 24 12 24
SCR siHic1
F 30
SCR SCR IL-2/IL-5/LPS siHIC1 IL-2/IL-5/LPS
*
% Methylation
8
Relative enrichment
Ciita ChIP-Dnmt1
Relative enrichment
Relative enrichment
8
20
10
0
26
Ciita ChIP-Dnmt3b
6
*
*
4 2
0 LPS/IL-2/IL-5 (h) 0
12 24 12 24
SCR siHic1
ACCEPTED MANUSCRIPT Figure 6 B
0.4 0.2
0 HIC1 5-Aza-dc
*
0.6
*
0.4 0.2
12
24
24
Ciita mRNA
Relative mRNA levels
*
0.6
12
24
0.2
0
12
24
1.2 SCR siDnmt3b
1 0.8 0.6
*
*
0.4 0.2
0 IL-2/IL-5/LPS (h)
IL-2/IL5/LPS
SCR
siDnmt1
0.4
0 IL-2/IL-5/LPS (h)
SCR
0
AC
SCR
0.6
0
12
24
IL-2/IL5/LPS
SCR
0.2
Ciita mRNA
1.4
SCR siDnmt3a
0.8
siDnmt3a
0.4
IL-2/IL5/LPS
SCR
1
D
*
CE P
E
SCR siDnmt1
0.8
c -d
Ciita mRNA
1.2
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Relative mRNA levels
1
0 IL-2/IL-5/LPS (h)
MA
D 1.2
5
za -A
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12
0 12 24 12 24
icl
-actin (42kd) 0
0.2
h Ve
Ciita(135kd)
0.4
e
C
IL-2/IL-5/LPS (h) 5-Aza-dc
*
0.6
0 IL-2/IL-5/LPS (h)
0 HIC1 5-Aza-dc
*
T
0.6
0.8
IP
*
1 0.8
siDnmt3b
0.8
1
Relative mRNA levels
*
Ciita mRNA
1.2
U2OS
SCR
1
1.2
Relative mRNA levels
HEK293
Relative luciferase activity
Relative luciferase activity
CIITA promoter (-545) 1.2
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A
Ciita (135kd)
Ciita (135kd)
Ciita (135kd)
Dnmt1 (180kd)
Dnmt3a (100kd)
Dnmt3b (100kd)
-actin (42kd)
-actin (42kd)
-actin (42kd)
27
ACCEPTED MANUSCRIPT
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Highlights HIC1 expression is up-regulated during B cell differentiation. HIC1 is essential for CIITA trans-repression during B cell differentiation. HIC1 interacts with and recruits DNMT1/3b to repress CIITA transcription in B cells. Depletion or inhibition of DNMT1/3b alleviates CIITA trans-repression.
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S1874-9399(16)30199-7 doi:10.1016/j.bbagrm.2016.10.003 BBAGRM 1089
To appear in:
BBA - Gene Regulatory Mechanisms
Received date: Revised date: Accepted date:
26 August 2016 5 October 2016 5 October 2016
Please cite this article as: Sheng Zeng, Yuyu Yang, Xian Cheng, Bisheng Zhou, Ping Li, Yuhao Zhao, Xiaocen Kong, Yong Xu, HIC1 epigenetically represses CIITA transcription in B lymphocytes, BBA - Gene Regulatory Mechanisms (2016), doi:10.1016/j.bbagrm.2016.10.003
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ACCEPTED MANUSCRIPT HIC1 epigenetically represses CIITA transcription in B lymphocytes Sheng Zeng,1,6 Yuyu Yang,1,2,6 Xian Cheng,1,3,6 Bisheng Zhou,1,6 Ping Li,1,4 Yuhao Zhao,1
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Xiaocen Kong,1,5 Yong Xu1* 1
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Department of Pathophysiology, Key Laboratory of Cardiovascular Disease and Molecular
Intervention, Nanjing Medical University, Nanjing, China; 2
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing,
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China; 3
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Jiangsu Institute of Nuclear Medicine, Wuxi, China;
4
Department of Gastroenterology, Second Hospital Affiliated to Nanjing Medical University,
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Nanjing, China; 5
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Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, Nanjing,
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China
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*Correspondence to:
Yong Xu, PhD, Nanjing Medical University, 101 Longmian Ave, Nanjing, Jiangsu 211166, China Email: [email protected], Tel/Fax: 86-25-86869371; or Xiaocen Kong, MD/PhD, Nanjing First Hospital, 68 Changle Rd, Nanjing, Jiangsu 210006, China Email: [email protected]
6
These authors contributed equally to this work.
1
ACCEPTED MANUSCRIPT Abstract Differentiation of B lymphocytes into isotope-specific plasma cells represents a hallmark
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event in adaptive immunity. During B cell maturation, expression of class II transactivator
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(CIITA) gene is down-regulated although the underlying epigenetic mechanism is not completely defined. Here we report that hypermethylated in cancer 1 (HIC1) was up-regulated in differentiating B lymphocytes paralleling CIITA repression. Over-expression
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of HIC1 directly repressed endogenous CIITA transcription in B cells. Reporter assay and
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chromatin immunoprecipitation (ChIP) assay confirmed that HIC1 bound to the proximal CIITA type III promoter (-545/-113); mutation of a conserved HIC1 site within this region
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abrogated CIITA trans-repression. More important, depletion of HIC1 with small interfering
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RNA (siRNA) restored CIITA expression in differentiating B cells. Mechanistically, HIC1
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preferentially interacted with and recruited DNMT1 and DNMT3b to the CIITA promoter to synergistically repress CIITA transcription. On the contrary, silencing of DNMT1/DNMT3b or
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inhibition of DNMT activity with 5-aza-dC attenuated CIITA trans-repression. Therefore, our data identify HIC1 as a novel factor involved in B cell differentiation acting as an epigenetic repressor of CIITA transcription.
Key words: transcriptional regulation; epigenetics; HIC1; CIITA; DNMT
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ACCEPTED MANUSCRIPT Introduction The bifurcation of the immune system in higher eukaryotes marks an evolutionarily
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significant step in host defense. Unlike the native immune system which usually launches
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fast and non-specific attacks against invading pathogens, the adaptive immune system relies on highly specialized lymphocytes to initiate a more delicate but slower response to combat aliens [1]. Differentiation of B lymphocytes represents a paradigm for the mode of action of
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the adaptive immune system: in response to a range of activating signals (e.g., LPS or helper
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T cells), mature B cells transition into antibody-secreting plasma cells and participate in the elimination of a specific set of pathogens [2]. A complex network of transcription factors,
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including PAX5, BCL6, IRF4, and BLIMP-1, cumulatively define the differentiation process of B
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lymphocytes and hence contribute to adaptive immunity [3].
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Class II transactivator, or CIITA, is the master regulator of major histocompatibility complex II molecules required for activation of CD4+ T lymphocytes [4]. CIITA expression
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defines the identity of mature B cells because during B cell differentiation CIITA is silenced at the transcriptional levels. CIITA transcription is driven by four distinct promoters in different immune cells: type I and type III promoters are responsible for constitutive CIITA expression in dendritic cells and B cells respectively while type IV promoter mediates IFN- induced CIITA expression in macrophages and non-myeloid cells [5, 6]. Previously, BLIMP-1 has been shown to bind to the proximal CIITA type III promoter (-180/-171) and represses CIITA transcription during B cell differentiation [7]. In addition, it has been documented that BLIMP-1 recruits a histone H3K9 dimethyltransferase (G9a) rendering a repressive chromatin structure to facilitate trans-repression of the CIITA promoter in differentiating B cells [8]. On 3
ACCEPTED MANUSCRIPT the other hand, it has been noted that the formation of a repressive CIITA promoter, as defined by the loss of active histone marks, following B cell differentiation precedes the full
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activation of BLIMP-1 [9, 10], suggesting that transcription factors other than BLIMP-1 also
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contribute to CIITA trans-repression.
Hypermethylated in cancer 1 (HIC1) is a transcriptional factor initially identified as a target of p53 activation [11]. HIC1 contains five Kruppel-like C2H2 zinc fingers that mediate
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its binding to target promoters and a BTB/POZ domain that is required for transcriptional
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repression [12]. Previous investigations have primarily implicated HIC1 as a tumor repressor by down-regulating the transcription of a host of genes including SIRT1 [13], CXCR7 [14], p21
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[15], and EphA2 [16]. Here we report that HIC1 expression is up-regulated during B cell
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differentiation paralleling CIITA down-regulation and that HIC1 recruits DNMT1 and
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DNMT3b to repress CIITA repression. Therefore, our data identify HIC1 as a novel factor
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involved in B cell differentiation acting as an epigenetic repressor of CIITA transcription.
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ACCEPTED MANUSCRIPT Methods and materials Cell culture
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HEK293 cells, U2OS cells, and Raji cells were maintained in DMEM supplemented with
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10% fetal bovine serum (Gibco). Primary B lymphocytes were isolated and differentiated as previously described [17].
Plasmids, transient transfection, and luciferase assay
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FLAG-HIC1, GFP-HIC1, FLAG-DNMT1, FLAG-DNMT3a, FLAG-DNMT3b, and CIITA type III
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promoter luciferase plasmids have been described previously [6, 18, 19]. Small interfering RNAs were purchased from Dharmacon. Transient transfections in HEK293 cells or U2OS cells
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were performed with Lipofectamine 2000 (Invitrogen). Transient transfections in Raji cells or
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primary B cells were performed with an electroporator (NEON, Life Technologies). To
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construct stable Raji cells expressing GFP-HIC1 or GFP, puromycin (200g/ml) was added to the media 48 hours after transfection; the cells were selected for 2 weeks with selection
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media changed every other day. Protein extraction, immunoprecipitation and Western blot Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche). Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysate overnight before being absorbed by Protein A/G-plus Agarose beads. Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2, Sigma) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blot 5
ACCEPTED MANUSCRIPT analyses were performed with anti-FLAG, anti-GFP, anti--actin (Sigma), anti-CIITA, anti-HIC1, (Santa Cruz), anti-DNMT1, anti-DNMT3a, and anti-DNMT3b (Proteintech) antibodies. All
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experiments were repeated at least three times.
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RNA Isolation and Real-time PCR
RNA was extracted with the RNeasy RNA isolation kit (Qiagen). Reverse transcriptase reactions were performed using a SuperScript First-strand Synthesis System (Invitrogen).
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Real-time PCR reactions were performed on an ABI Prism 7500 system. Primers and Taqman
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probes used for real-time reactions were purchased from Applied Biosystems. All experiments were repeated at least three times.
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Chromatin Immunoprecipitation (ChIP)
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Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described
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before [20]. In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1%
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Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼500 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-HIC1 (Santa Cruz), anti-DNMT1, anti-DNMT3a, anti-DNMT3b (Proteintech), or pre-immune IgG. Precipitated genomic DNA was amplified by real-time PCR with the following primers: for human CIITA promoter, GAGTAGGCATGGTAGAGGAGAGC and TACCACACTCCCTTAAGC; for human GAPDH promoter, TACTAGCGGTTTTACGGGCG and TCGAACAGGAGGAGCAGAGAGCGA; for mouse Ciita promoter, CATGGGCAAATTAGAGGGTATCC and
GCAAAGAAAGCGAGCAAGGG;
for 6
mouse
Gapdh
promoter,
ACCEPTED MANUSCRIPT ATCACTGCCACCCAGAAGACTGTGGA
and
CTCATACCAGGAAATGAGCTTGACAAA.
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experiments were repeated at least three times.
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CIITA promoter DNA methylation analysis
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CIITA promoter methylation status was analyzed with bisulfite conversion followed by sequencing essentially as described previously [21]. Briefly, genomic DNA isolated from Raji cells primary HSCs was subjected to bisulfite treatment using the EZDNA Methylation Gold
AAACACAAACTCCTATTCCCATCCTCAC;
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TTAAGGGAGTGTGGTAAAATTAGAGGGTG;
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Kit (ZymoResearch). The modified DNA was amplified using the following primers: Forward, Reverse
and
Reverse
Nested, Outer,
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AAACAACTCTTTCACATCTTCCAATAACC TAC. The PCR products were cloned into pCRII-TA
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Statistical Analysis
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vectors (Invitrogen) and sequenced using the Sanger sequencing.
One-way ANOVA with post-hoc Scheffe analyses were performed using an SPSS package.
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P values smaller than .05 were considered statistically significant (*).
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ACCEPTED MANUSCRIPT Results HIC1 expression is up-regulated during B cell differentiation
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We started off by examining the expression of HIC1 in a human B lymphocyte line (Raji)
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before and after differentiation. Stimulation with IL-2/IL-5/LPS led to significant down-regulation of CIITA message and protein indicative of plasma cell differentiation. Paralleling CIITA repression, an increase in HIC1 expression was observed (Fig.1A and Fig.1B).
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Likewise, HIC1 expression was up-regulated during the differentiation of primary mouse B
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lymphocytes (Fig.1C and Fig.1D). Of note, the changes in HIC1 expression followed a different kinetics compared to those of BLIMP-1, a well characterized plasma cell marker and
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CIITA repressor [7]. While BLIMP-1 expression was continuously elevated up to 24h following
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IL-2/IL-5/LPS treatment, the window for HIC1 induction was narrower: HIC1 expression
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started to increase at 6h, peaked at 12h but declined at 24h. Collectively, these data suggest that HIC1 might be an early response protein during B cell differentiation contributing to
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CIITA repression.
HIC1 binds to CIITA type III promoter and represses CIITA transcription To directly assess whether HIC1 is a de novo repressor of CIITA transcription, we performed the following experiments. As shown in Fig.2A, co-transfection of HIC1 expression construct with CIITA type III promoter-luciferase constructs of different lengths into HIC1-negative U2OS cells resulted in robust repression of luciferase activities of two long CIITA promoter constructs (-1007 and -545) but not that of the shortest one (-113). Similar observation was made in HEK293 cells (Fig.2A), suggesting that there might be a HIC1-response element with the CIITA proximal promoter between -545 and -113 with 8
ACCEPTED MANUSCRIPT respect to the transcription start site. Close examination of this region revealed a putative HIC1 motif between -298 and -288 (GCTGGCACCAG). Deletion of this potential HIC1 site
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augmented basal CIITA promoter activity but also abrogated HIC1-mediated repression
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(Fig.2B). To examine whether HIC1 could down-regulate endogenous CIITA messages, we constructed a stable Raji cell line in which GFP-tagged HIC1 expression construct was incorporated into the genome (GFP-HIC1). Compared to the control stable cell line in which
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an empty vector (GFP) was transfected, HIC1 expression was increased by more than 2 fold in
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two different GFP-HIC1 clones as measured by qPCR (Fig.2C). Importantly, there was a modest (~40%) but significant down-regulation of CIITA messages in GFP-HIC1 cells as
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opposed to GFP cells. In comparison, BLIMP1 levels were not significantly altered by HIC1
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GFP-HIC1 cells (Fig.2D).
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over-expression. Western blotting showed that CIITA protein levels were also decreased in
Next, we evaluated direct binding of HIC1 to the CIITA gene in cells. Chromatin
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immunoprecipitation (ChIP) assay performed with an anti-GFP antibody showed significantly higher levels of binding of GFP-HIC1 to the CIITA type III promoter than GFP whereas ChIP assay performed with a pre-immune IgG detected no such enrichment of GFP-HIC1 over GFP (Fig.2E). On the contrary, we did not find any significant binding of HIC1 on the GAPDH promoter (Fig.2E). We also examined the binding of endogenous HIC1 to the CIITA promoter in B cells. As shown in Fig.2F, IL-2/IL-5/LPS stimulation resulted in augmented binding of HIC1 to the CIITA promoter but not the GAPDH promoter. In addition, ChIP assays performed in primary mouse B lymphocytes showed that occupancies of HIC1 on the CIITA promoter started to increase at 12h following IL-2/IL-5/LPS stimulation and continued to increase at 24h 9
ACCEPTED MANUSCRIPT following a kinetics slightly lagging behind that of its expression (Fig.2G). Taken together, we conclude that HIC1 could bind to CIITA type III promoter and repress CIITA transcription.
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HIC1 is essential for CIITA down-regulation during B cell differentiation
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Having determined that HIC1 over-expression is sufficient to drive CIITA trans-repression, we asked whether HIC1 is indispensable for CIITA down-regulation during B cell differentiation. Knockdown of HIC1 with two different siRNAs in Raji cells completely blunted
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the induction of HIC1 expression by IL-2/IL-5/LPS treatment. More important, CIITA
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repression was significantly alleviated (Fig.3A and Fig.3B). Similarly, siRNA targeting HIC1 partially rescued CIITA expression in differentiating primary B lymphocytes (Fig.3C and
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cell differentiation.
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Fig.3D). Therefore, HIC1 is both adequate and necessary for CIITA trans-repression during B
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HIC1 interacts with DNMT during B cell differentiation HIC1 is known to engage the epigenetic machinery, histone deacetylase for instance, to
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repress gene transcription [18, 22-24]. Previously, it has been shown that DNA methyltransferases (DNMTs) play a role in CIITA silencing in cancer cells [25]. We tested the hypothesis that HIC1 interacts with DNMT to repress CIITA transcription in B cells. To this end, we transfected GFP-tagged HIC1 into HEK293 along with FLAG-tagged DNMT expression constructs. Immunoprecipitation with an anti-FLAG antibody pulled down HIC1 along with DNMT1, DNMT3a, and DNMT3b, suggesting that HIC1 might be in a complex with DNMTs (Fig.4A). More important, an anti-HIC1 antibody precipitated all three DNMT isoforms along with HIC1 in Raji cells (Fig.4B). To further examine the functional consequence of this DNMT-HIC1 interaction, we performed Re-ChIP assay in Raji cells and primary B lymphocytes. 10
ACCEPTED MANUSCRIPT Of interest, both a HIC1-DNMT1 complex and a HIC1-DNMT3b complex but not a HIC1-DNMT3a were detected on the CIITA promoter following IL-2/IL-5/LPS induction
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indicating that the interaction between HIC1 and different DNMT isoforms is locus-specific
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(Fig.4C and Fig.4D); HIC1 did not interact with any of the three DNMT isoforms on the GAPDH promoter either with or without IL-2/IL-5/LPS treatment. Thus it appears that HIC1 may recruit DNMT1/DNMT3b to repress CIITA transcription.
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We performed a series of experiments to further clarify the functional relevance of the
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HIC1-DNMT complex. In response to IL-2/IL-5/LPS treatment, there were enhanced occupancies of DNMT1 and DNMT3b but not DNMT3a on the CIITA promoter in Raji cells;
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over-expression of HIC1 further augmented the binding of DNMT1 and DNMT3b (Fig.5A).
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Bisulfite modification followed by sequencing revealed that following IL-2/IL-5/LPS treatment
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there was a significant increase in CpG methylation surrounding the CIITA promoter, which was further up-regulated by HIC1 over-expression (Fig.5B). In reporter assays, co-transfection
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of DNMT1 and DNMT3b, but not DNMT3a, enhanced the repression of CIITA promoter activity by HIC1, confirming that HIC1 only interacts with DNMT1 and DNMT3b instead of DNMT3a on the CIITA promoter (Fig.5C). In addition, depletion of endogenous HIC1 completely blocked the recruitment of DNMT1 and DNMT3b to the CIITA promoter in both Raji cells (Fig.5D) and primary B lymphocytes (Fig.5E). Accordingly, CpG methylation surrounding the CIITA promoter was attenuated (Fig.5F). Together, these data support a role for the HIC1-DNMT complex in repressing CIITA transcription during B cell differentiation. DNMT depletion or inhibition attenuates CIITA repression during B cell differentiation
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ACCEPTED MANUSCRIPT Finally, we tackled the question whether DNMT inhibition/depletion would achieve similar effects as HIC1 silencing with regard to the recovery of CIITA expression. Indeed,
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exposure to a DNMT inhibitor 5-azacytidine (5-Aza-dc) dose-dependently relieved the
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repression of CIITA promoter activity by HIC1 in both HEK293 cells and U2OS cells (Fig.6A). Furthermore, 5-Aza-dc treatment also partially normalized endogenous CIITA expression in differentiating B cells (Fig.6B and Fig.6C). Finally, siRNA targeting DNMT1 and DNMT3b, but
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not DNMT3a, antagonized the repression of CIITA in primary B lymphocytes exposed to
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IL-2/IL-5/LPS. Therefore, HIC1 recruitment of DNMT1 and DNMT3b may play an essential role
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in CIITA trans-repression during B cell differentiation.
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ACCEPTED MANUSCRIPT Discussion Silencing of CIITA by transcriptional repression is a hallmark event during B cell
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differentiation. We present evidence that HIC1 is activated during B cell differentiation and
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directly binds to the CIITA promoter to repress CIITA transcription. Previous investigations have portrayed BLIMP-1, a zinc-finger transcription factor, as the major regulator of CIITA expression during B cell differentiation [8, 10, 26, 27]. At the same time evidence is
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accumulating that BLIMP-1 full activation and its binding to the CIITA promoter are preceded
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by the loss of active histone marks from the same region and the initiation of CIITA repression suggesting that BLIMP-1 is the sole transcription factor responsible for CIITA
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repression [9, 10]. Boss and colleagues have recently identified a zinc-finger containing
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transcription factor ZBTB32 as an early repressor of CIITA transcription during B cell
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differentiation [28]. ZBTB32 binds to the proximal CIITA promoter at a site adjacent to the BLIMP-1 site (separated by ~50bp) and can interact with BLIMP-1 in plasma cells although it
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is unclear whether there is any functional interplay/inter-dependence between BLIMP-1 and ZBTB32 [28]. By comparison, HIC1 binds to a more distal site of the CIITA promoter (~100bp from the BLIMP-1 site). BLIMP-1, HIC1, and ZBTB32 all belong to zinc-finger family of transcription factors that are known to form homodimers or heterodimers when binding to DNA [29]. It is likely that BLIMP1, HIC1, ZBTB32, and other transcription factors form a large repressor complex cooperatively regulate CIITA expression during B cell differentiation. In support of this model, we found that depletion of HIC1 attenuated but not completely blocked CIITA repression in B cells (Figure 3). Coincidently, ZBTB32 deficiency delayed instead of abrogating CIITA repression [28]. Clearly, more work is warranted to clarify the 13
ACCEPTED MANUSCRIPT functional reliance/redundancy of different transcription factors in regulating the kinetics of CIITA repression during B cell differentiation.
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Another major finding of this current report is that HIC1 recruits DNMT1 and DNMT3b
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to repress CIITA transcription. Although immunoprecipitation experiments showed that DNMT1, DNMT3a, and DNMT3b all bound to HIC1 with comparable affinity, DNMT1 and DNMT3b were preferentially recruited by HIC1 to the CIITA promoter to repress
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transcription. These data suggest that although the physical interactions between HIC1 and
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DNMTs do not rely on DNA, recruitment of DNMTs to HIC1 target genes are locus and context-dependent and may be dependent on specific chromatin structure. Alternatively,
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HIC1 may rely on differential recruitment of DNMTs to repress the transcription of different
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genes. Of note, DNMT depletion or inhibition only partially rescued CIITA repression. HIC1 is
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known to interact with multiple epigenetic factors including histone methyltransferases (HMTs) [30] and histone deacetylases (HDACs) [22]. DNMTs have also been found to be in
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large complexes with HMTs and HDACs [31-33]. Therefore, DNMTs may constitute only part of the epigenetic mechanism whereby HIC1 contributes to CIITA trans-repression. Indeed, CIITA silencing in cancer cells can be reversed by treatment with either DNMT inhibitors (e.g., 5-Aza-dc) or HDAC inhibitors (e.g., TSA) [25, 34]. In addition, Londhe et al have reported that simultaneous treatment with TSA and 5-Aza-dc resulted in a more robust restitution of CIITA expression in rhabdomyosarcoma cells in individual treatment [35]. These data allude to a model wherein B cell activation signals stimulate coordinated recruitment of different epigenetic factors including DNMTs, some of which may be independent of HIC1, to the CIITA promoter, to repress CIITA transcription. 14
ACCEPTED MANUSCRIPT A lingering issue that remains unanswered is whether HIC1, in addition to contributing to CIITA repression, plays a role in B cell differentiation per se. HIC1 was initially identified as
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a tumor repressor gene that can be activated by p53 and has been implicated in the
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pathogenesis of a range of human cancers [36]. Recently, it has been shown that BLIMP-1 plays a major role protecting against B cell lymphoma by promoting B cell differentiation [37]. If future studies can expand the role of HIC1 in B cells beyond a mere repressor of CIITA
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transcription, then it would not only reinforce the characterization of HIC1 as a
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wide-spectrum tumor repressor, but provide strong rationale for screening small-molecule
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compounds that can boost HIC1 activity as novel therapeutic solutions to treat cancer.
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ACCEPTED MANUSCRIPT Acknowledgements This work was supported, in part, by the National Natural Science Foundation of China
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(31270805, 81503067, 81602352, 81402550, 81400840, 81500426). YX is a Fellow at the
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Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.
Disclosure
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None.
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tumour cells, British journal of cancer, 90 (2004) 844-852.
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ACCEPTED MANUSCRIPT Figure legends Figure 1: HIC1 expression is up-regulated during B cell differentiation. (A, B) Raji cells were treated with IL-2/IL-5/LPS or vehicle for 24 hours. Gene expression levels were examined by
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qPCR (A) and Western (B). (C, D) Primary B lymphocytes were induced to differentiate by IL-2/IL-5/LPS. Cells were harvested at indicated time points and gene expression levels were
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examined by qPCR (C) and Western (D).
Figure 2: HIC1 binds to CIITA type III promoter and represses CIITA transcription. (A) CIITA promoter constructs of different lengths were transfected into U2OS or HEK293 cells with or
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without HIC1. Luciferase activities were normalized by protein concentration and GFP fluorescence. (B) Wild type CIITA promoter construct (WT) or CIITA promoter construct
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harboring the HIC1 site mutation (MT) was transfected into HEK293 cells with or without HIC1. Luciferase activities were normalized by protein concentration and GFP fluorescence.
D
(C-E) Raji cells were stably transfected with GFP-HIC1 or GFP. Gene expression levels were
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examined by qPCR (C) and Western (D). Binding of HIC1 to the CIITA promoter was evaluated by ChIP with anti-GFP or IgG (E). (F) Raji cells were treated with IL-2/IL-5/LPS or vehicle for 24
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hours. ChIP assays were performed with anti-HIC1 or IgG. (G) Primary B lymphocytes were induced to differentiate by IL-2/IL-5/LPS. Cells were harvested at indicated time points and ChIP assays were performed with anti-HIC1 or IgG.
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Figure 3: HIC1 is essential for CIITA down-regulation during B cell differentiation. (A, B) Raji cells were transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with IL-2/IL-5/LPS for 24 hours. Gene expression levels were examined by qPCR (A) and Western (B). (C, D) Primary B lymphocytes were transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with IL-2/IL-5/LPS. Cells were harvested at indicated time points and gene expression levels were examined by qPCR (C) and Western (D). Figure 4: HIC1 interacts with DNMT during B cell differentiation. (A) HEK293 cells were transfected with GFP-HIC1 and FLAG-tagged DNMT expression constructs as indicated. Immunoprecipitation was performed with anti-FLAG. (B) Whole cell lysates extracted from Raji cells were immunoprecipitated with indicated antibodies. (C) Raji cells were treated with IL-2/IL-5/LPS or vehicle for 24 hours. Re-ChIP assays were performed with indicated 20
ACCEPTED MANUSCRIPT antibodies. (D) Primary B lymphocytes were induced to differentiate by IL-2/IL-5/LPS. Re-ChIP assays were performed with indicated antibodies. Figure 5: HIC1 and DNMT cooperate to repress CIITA differentiation. (A) Raji cells were
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stably transfected with GFP-HIC1 or GFP. ChIP assays were performed with indicated antibodies. (B) Bisulfite conversion coupled with sequencing was performed as described
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under Materials and methods using genomic DNA isolated from Raji cells. (C) A CIITA promoter-luciferase construct was transfected into HEK293 or U2OS cells with indicated expression constructs. Luciferase activities were normalized by protein concentration and
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GFP fluorescence. (D) Raji cells were transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with IL-2/IL-5/LPS for 24 hours. ChIP assays were performed with
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indicated antibodies. (E) Primary B lymphocytes were transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with IL-2/IL-5/LPS. (F) Raji cells were
D
transfected with siRNA targeting HIC1 or random siRNA (SCR) followed by treatment with
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IL-2/IL-5/LPS for 24 hours. Bisulfite conversion coupled with sequencing was performed as described under Materials and methods.
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Figure 6: DNMT depletion or inhibition attenuates CIITA repression during B cell differentiation. (A) A CIITA promoter-luciferase construct was transfected into HEK293 or U2OS cells with HIC1 followed by exposure to 5-Aza-dC (0.1 M and 0.5M ) for 24 hours.
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Luciferase activities were normalized by protein concentration and GFP fluorescence. (B, C) Primary B lymphocytes were induced to differentiate by IL-2/IL-5/LPS in the presence or absence of 5-Aza-dC (0.5M) for 24 hours. CIITA expression was examined by qPCR (B) and Western (C). (D, E) Primary B lymphocytes were transfected with siRNAs targeting specific DNMT and induced to differentiate by IL-2/IL-5/LPS in the presence or absence of 5-Aza-dC (0.5M). Cells were harvested at 12h and 24h post-treatment and CIITA expression was examined by qPCR (D) and Western (E).
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ACCEPTED MANUSCRIPT Figure 1 A
B 5
CIITA (135kd)
*
4.5
Vehicle IL-2/IL-5/LPS
HIC1 (78kd)
T
Relative mRNA levels
4 3.5
IL-2/IL-5/LPS
*
1 0.5 0
HIC1
Hic1
Blimp1
5
20 18
2
1
14 12 10 8 6
D
3
1.2
1.2
1
1
MA
16
Relative mRNA levels
Relative mRNA levels
4
Ciita
NU
C
Relative mRNA levels
CIITA
4
0.8 0.6 0.4 0.2
Relative mRNA levels
1.5
SC R
2
IP
-actin (42kd)
3 2.5
dc4
0.8 0.6 0.4 0.2
AC
D
0
6 12 24
0
0
6 12 24
CE P
0 IL-2/IL-5/LPS (h) 0
TE
2
IL-2/IL-5/LPS (h)
0 0 6 12 24
Hic1 (78kd) Ciita (135kd) -actin (42kd)
0
6
12
24
22
0 6 12 24
ACCEPTED MANUSCRIPT Figure 2
HIC1
CIITA 1.2 *
0
E
0.8 0.6 0.4 0.2 0
CIITA type III ChIP
7
T
IP CIITA (135kd) GFP-HIC1 (96kd)
MA
0.5
Relative mRNA levels
1
GFP-HIC1#2
GFP-HIC1#1
1.5
GFP
*
2
*
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
MT
D
BLIMP1
1
GFP-HIC1#1
Relative mRNA levels
*
2.5
GFP
Relative mRNA levels
3
0.5
-113
-545
GFP-HIC1#2
C
*
1
WT
-1007
-113
-545
1.5
0
0 -1007
HIC1 HIC1
-actin (42kd)
GAPDH ChIP
1.4
5
Relative enrichment
Relative enrichment
1.2
GFP IgG
3 2
P GF
0.4 0.2
#2 #1 C1 C1 HI HI P P GF GF
F 7 6 5 4 3 2 1 0
*
AC
Relative enrichment
0.6
CE P
1 0
1 0.8
TE
*
4
D
*
6
0
P GF
#1 #2 C1 C1 HI HI P P GF GF
Vehicle IL-2/IL-5/LPS
HIC1 IgG CIITA type III ChIP
HIC1 IgG GAPDH ChIP
G CIITA type III ChIP
GAPDH ChIP
*
0
6
12
24
1.4
Relative enrichment
Hic1 IgG
*
Relative enrichment
4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 IL-2/IL-5/LPS (h)
1.2 1 0.8 0.6 0.4 0.2
0 IL-2/IL-5/LPS (h)
23
0
GFP-HIC1#2
0.5
2
WT MT
GFP-HIC1#1
*
1
-288
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0.2
1.5
GCTGGCACCAG GCTGGCACCAG
GFP
0.4
*
Relative luciferase activity
0.6
2
2.5
NU
0.8
-298
HIC1 HIC1
GFP-HIC1#2
Relative luciferase activity
Relative luciferase activity
1
0
B
HEK293
2.5
*
*
GFP-HIC1#1
U2OS
1.2
GFP
A
6
12
24
ACCEPTED MANUSCRIPT Figure 3 A
B HIC1
1
*
CIITA (135kd)
0.5
*
T
0.6 0.4
SCR siHIC1#1 siHIC1#2
0.2
0 IL-2/IL-5/LPS SCR siHIC1#1 siHIC1#2
HIC1 (78kd)
0.8
Hic1
1 0.5
0 IL-2/IL-5/LPS (h)
12
24
NU *
0.6
*
0.4 0.2
0 IL-2/IL-5/LPS (h)
0
12
24
D
0
1 0.8
MA
1.5
Relative mRNA levels
Relative mRNA levels
SCR siHic1
*
2
Ciita
1.2 *
2.5
CE P
TE
D
SCR siHic1 IL-2/IL-5/LPS
Ciita (135kd) Hic1 (78kd) -actin (42kd)
24h
AC
12h
IL-2/IL-5/LPS
C 3
IP
1
CIITA
*
1.5
0 IL-2/IL-5/LPS SCR siHIC1#1 siHIC1#2
1.2
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*
2
Relative mRNA levels
Relative mRNA levels
2.5
24
-actin (42kd)
ACCEPTED MANUSCRIPT Figure 4 A
GFP-HIC1
T
HIC1 (78kd)
←FLAG-DNMT1 ←FLAG-DNMT3a ←FLAG-DNMT3b
DNMT1 (180kd)
IP
Input
DNMT3a (100kd)
GFP-HIC1
DNMT3b (100kd)
C CIITA type III ChIP
GAPDH ChIP
1.4
* *
4
Vehicle IL-2/IL-5/LPS
3
1
D
Ciita type III ChIP 6 *
5
TE
Vehicle IL-2/IL-5/LPS
4 3 2
CE P
Relative enrichment
*
D
0 1st IP HIC1 HIC1 HIC1 IgG IgG IgG 2nd IP DNMT1 DNMT3a DNMT3b DNMT1 DNMT3a DNMT3b
1
1.2
1
0.8 0.6 0.4
MA
2
Relative enrichment
5
NU
6
Gapdh ChIP 1.4 1.2 1 0.8 0.6 0.4 0.2
0 1st IP Hic1 Hic1 Hic1 IgG IgG IgG 2nd IP Dnmt1 Dnmt3a Dnmt3b Dnmt1 Dnmt3a Dnmt3b
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0 1st IP Hic1 Hic1 Hic1 IgG IgG IgG 2nd IP Dnmt1 Dnmt3a Dnmt3b Dnmt1 Dnmt3a Dnmt3b
0.2
0 1st IP HIC1 HIC1 HIC1 IgG IgG IgG 2nd IP DNMT1 DNMT3a DNMT3b DNMT1 DNMT3a DNMT3b
Relative enrichment
Relative enrichment
7
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GFP-HIC1 FLAG-DNMT1 FLAG-DNMT3a FLAG-DNMT3b
-HIC1
Input
←FLAG-DNMT1 ←FLAG-DNMT3a ←FLAG-DNMT3b
Eluate
IgG
B IP: FLAG
25
ACCEPTED MANUSCRIPT Figure 5 A GAPDH ChIP
*
DNMT1
GFP GFP IL-2/IL-5/LPS GFP-HIC1 IL-2/IL-5/LPS
1.4 1.2 1 0.8 0.6 0.4 0.2 0
T
*
DNMT3a DNMT3b
DNMT1
IP
Relative enrichment
Relative enrichment
CIITA type III ChIP 7 6 5 4 3 2 1 0
DNMT3a DNMT3b
P
S B
hI
C
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B rw Fo
d
-288
d
ar
ar
rw
Fo -298 HIC1
NU
% Methylation
MA
Relative luciferase activity
U2OS CIITA promoter (-545)
1
* *
D
*
0.8
0.4 0.2
D
AC
4 3 2 1 0
DNMT1
DNMT3a
DNMT3b
0.4 0.2 0
GAPDH ChIP
1.4
Relative enrichment
*
*
5
0.6
HIC1 DNMT1 DNMT3a DNMT3b
CIITA type III ChIP
6
0.8
TE
0.6
CE P
Relative luciferase activity
*
HIC1 DNMT1 DNMT3a DNMT3b
er ut
d te
1.2
HEK293 CIITA promoter (-545)
1
0
O
es N
20
0
Relative enrichment
se er ev
R
se er ev
R
se er ev
R *
30
10
1.2
S
S
P
hI GFP GFP IL-2/IL-5/LPS GFP-HIC1 IL-2/IL-5/LPS
40
C
B
B
C
50
SCR SCR IL-2/IL-5/LPS SCR siHIC1 IL-2/IL-5/LPS
1.2 1
SCR IL-2/IL-5/LPS siHIC1 IL-2/IL-5/LPS
0.8 0.6 0.4 0.2 0
DNMT1
DNMT3a
DNMT3b
E *
*
6 4 2
0 LPS/IL-2/IL-5 (h) 0
12 24 12 24
SCR siHic1
8
Ciita ChIP-Dnmt3a
6 4 2
0 LPS/IL-2/IL-5 (h) 0
12 24 12 24
SCR siHic1
F 30
SCR SCR IL-2/IL-5/LPS siHIC1 IL-2/IL-5/LPS
*
% Methylation
8
Relative enrichment
Ciita ChIP-Dnmt1
Relative enrichment
Relative enrichment
8
20
10
0
26
Ciita ChIP-Dnmt3b
6
*
*
4 2
0 LPS/IL-2/IL-5 (h) 0
12 24 12 24
SCR siHic1
ACCEPTED MANUSCRIPT Figure 6 B
0.4 0.2
0 HIC1 5-Aza-dc
*
0.6
*
0.4 0.2
12
24
24
Ciita mRNA
Relative mRNA levels
*
0.6
12
24
0.2
0
12
24
1.2 SCR siDnmt3b
1 0.8 0.6
*
*
0.4 0.2
0 IL-2/IL-5/LPS (h)
IL-2/IL5/LPS
SCR
siDnmt1
0.4
0 IL-2/IL-5/LPS (h)
SCR
0
AC
SCR
0.6
0
12
24
IL-2/IL5/LPS
SCR
0.2
Ciita mRNA
1.4
SCR siDnmt3a
0.8
siDnmt3a
0.4
IL-2/IL5/LPS
SCR
1
D
*
CE P
E
SCR siDnmt1
0.8
c -d
Ciita mRNA
1.2
TE
Relative mRNA levels
1
0 IL-2/IL-5/LPS (h)
MA
D 1.2
5
za -A
NU
12
0 12 24 12 24
icl
-actin (42kd) 0
0.2
h Ve
Ciita(135kd)
0.4
e
C
IL-2/IL-5/LPS (h) 5-Aza-dc
*
0.6
0 IL-2/IL-5/LPS (h)
0 HIC1 5-Aza-dc
*
T
0.6
0.8
IP
*
1 0.8
siDnmt3b
0.8
1
Relative mRNA levels
*
Ciita mRNA
1.2
U2OS
SCR
1
1.2
Relative mRNA levels
HEK293
Relative luciferase activity
Relative luciferase activity
CIITA promoter (-545) 1.2
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A
Ciita (135kd)
Ciita (135kd)
Ciita (135kd)
Dnmt1 (180kd)
Dnmt3a (100kd)
Dnmt3b (100kd)
-actin (42kd)
-actin (42kd)
-actin (42kd)
27
ACCEPTED MANUSCRIPT
T IP SC R NU MA D TE CE P AC
Highlights HIC1 expression is up-regulated during B cell differentiation. HIC1 is essential for CIITA trans-repression during B cell differentiation. HIC1 interacts with and recruits DNMT1/3b to repress CIITA transcription in B cells. Depletion or inhibition of DNMT1/3b alleviates CIITA trans-repression.
28