ZBTB32 Is an Early Repressor of the CIITA and MHC Class II Expression during B Cell Differentiation to Plasma Cells

This information is current as Hye Suk Yoon, Christopher D. Scharer, Parimal Majumder, of October 1, 2021. Carl W. Davis, Royce Butler, Wendy Zinzow-Kramer, Ioanna Skountzou, Dimitrios G. Koutsonanos, Rafi Ahmed and Jeremy M. Boss J Immunol published online 30 July 2012 http://www.jimmunol.org/content/early/2012/07/29/jimmun ol.1103371 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 30, 2012, doi:10.4049/jimmunol.1103371 The Journal of Immunology

ZBTB32 Is an Early Repressor of the CIITA and MHC Class II during B Cell Differentiation to Plasma Cells

Hye Suk Yoon,*,1 Christopher D. Scharer,*,1 Parimal Majumder,* Carl W. Davis,† Royce Butler,* Wendy Zinzow-Kramer,* Ioanna Skountzou,* Dimitrios G. Koutsonanos,* Rafi Ahmed,*,† and Jeremy M. Boss*,†

CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation -1 (Blimp-1), the regulator of plasma cell fate and suppressor Downloaded from of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and http://www.jimmunol.org/ a potential regulatory partner of Blimp-1 in repressing gene expression. The Journal of Immunology, 2012, 189: 000–000.

o initiate and regulate the adaptive and humoral immune mature into Ab-secreting plasma cells (10). As B cells transition to responses, MHC class II (MHC-II) encode hetero- plasma cells, the expression of CIITA is silenced, resulting in the T dimeric that present antigenic peptides to CD4 subsequent loss of MHC-II expression (11, 12). T lymphocytes (1, 2). MHC-II genes are transcriptionally regu- Plasma cell differentiation requires extensive epigenetic re- lated by CIITA. CIITA is expressed in a limited number of cell programming and morphological changes that allow these cells to types, including dendritic cells, macrophages, and B lymphocytes secrete high levels of serum Abs (13, 14). This process represents by guest on October 1, 2021 (3, 4). CIITA functions as a transcriptional coactivator, connecting a critical event in the development and control of the humoral the DNA-binding transcription factors at MHC-II promoters with immune response. The B lymphocyte-induced maturation protein- chromatin-modifying complexes and the RNA polymerase ma- 1 (Blimp-1) encoded by the Pdrm1 gene is an important regulator chinery (5–7). Depending on the species, the expression of CIITA of this transition. Ectopic expression of Blimp-1 in some B cell is controlled by three or four tissue-specific promoters, with lines induces plasma cell differentiation (15, 16) and the silencing promoter III (pIII) principally used by B cells (8, 9). Following of CIITA and MHC-II gene expression (17). Moreover, B cells immune stimulation, activated B lymphocytes differentiate or carrying a B cell-specific, conditional knockout allele of Blimp-1 fail to form plasma cells, leading to a decrease of serum Ig levels, even though B cell development to this point was normal (18, 19). *Department of Microbiology and Immunology, Emory University School of Med- icine, Atlanta, GA 30322; and †Emory Vaccine Center, Emory University School of Biochemically, Blimp-1 functions as a transcriptional repressor of Medicine, Atlanta, GA 30322 gene activity (18, 20). However, Blimp-1 is only part of this cell 1H.S.Y. and C.D.S. contributed equally to this work. fate decision. A complex network of activators and repressors Received for publication November 23, 2011. Accepted for publication June 29, controls B cell fate and development. For example, whereas B cell 2012. lymphoma-6 (BCL6) maintains the B cell program and represses This work was supported by National Institutes of Health Grants R56AI3400 and Blimp-1 expression, Blimp-1 represses the B cell program by si- R01AI34000 (to J.M.B.), HHSN266 200700006C (to I.S. and D.G.K.), and R01A1030048 and U19A1091493 (to R.A.). lencing BCL6, Pax5, and SpiB, critical modulators of B cell fate (20, 21). Blimp-1 also serves to induce the plasma cell program by The microarray data presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus database (http:// contributing to the regulation of XBP-1 and syndecan-1 (22). www.ncbi.nlm.nih.gov/gds) under accession number GSE17999. Although this seems complete, additional data indicate that other Address correspondence and reprint requests to Dr. Jeremy M. Boss, Emory Univer- factors may act independently of Blimp-1 to initiate this differ- sity, 1510 Clifton Road, Room 3001, Atlanta, GA 30322. E-mail address: jmboss@emory. entiation program (23). With respect to CIITA silencing in plasma edu cells, Blimp-1 was found to bind directly to sequences upstream of The online version of this article contains supplemental material. three tissue-specific promoters of the CIITA gene (promoters I, III, Abbreviations used in this article: BCL6, B cell lymphoma-6; Blimp-1, B lymphocyte-induced maturation protein-1; ChIP, chromatin immunoprecipitation; and IV), suggesting that it directly repressed CIITA expression HDAC, histone deacetylase; LCMV, lymphocytic choriomeningitis virus; MHC-II, (17, 24, 25). The molecular mechanism(s) by which Blimp-1 MHC class II; pIII, promoter III; ROG, repressor of GATA3; shRNA, short hairpin mediates repression is still not well defined. Blimp-1 was found RNA. to interact with histone deacetylase (HDAC)-1 and HDAC-2 in Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 repressing the c- gene during plasma cell differentiation (26).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103371 2 ZBTB32 REPRESSES CIITA EXPRESSION

During dendritic cell maturation, Blimp-1 bound CIITA promoter I Primary murine splenic B cells were purified from 5- to 8-wk-old and recruited G9a and HDAC2, resulting in the acquisition of C57BL/6 mice, using magnetic separation columns (MACS; Miltenyi repressive histone marks (25). In other systems, Blimp-1 formed Biotec), as previously described (35). Ex vivo differentiation of B cells to plasma-like cells (35) was performed by incubating murine primary a complex with histone methyltransferases G9a (27) and PRMT5 splenic B cells for the indicated time in the above culture medium sup- (28), suggesting a role in modulating the remodeling of chro- plemented with LPS (20 mg/ml; Sigma-Aldrich), IL-2 (20 ng/ml; Sigma- matin. However, the participating factors and the mechanism of Aldrich), and IL-5 (5 ng/ml; Sigma-Aldrich). In addition, each 24 h the m Blimp-1 repression at CIITA pIII in plasma cells are largely un- culture medium was supplemented with LPS (10 g/ml), IL-2 (10 ng/ml), and IL-5 (2.5 ng/ml) for the duration of the time course. The Burkitt’s known. lymphoma B cell line Raji was grown in RPMI 1640 medium supple- ZBTB32 encodes a transcriptional repressor and member of the mented with 5% FBS (HyClone Laboratories) and 5% bovine calf serum zinc finger, broad complex, tram track, bric-a-brac (BTB) family (HyClone Laboratories). The plasmacytoma cell line H929 was cultured in of proteins (29). ZBTB32, which is also known as repressor of RPMI 1640 medium with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, GATA3 (ROG), PLZF-like zinc finger protein (PLZP), testis zinc 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 0.05 mM 2-ME. HEK293 and HEK293FT cells were cultured in DMEM with 10% finger protein (TZFP), and Fanconi anemia zinc finger (FAZF), heat-inactivated FBS. was identified in multiple screens involving either immune cell tumorigenesis or development (30–32). In T cells, ZBTB32 ac- Primary plasmablast purification tivity inhibits the development of Th2 cells by interfering with the To obtain primary plasmablasts from the spleen, BALB/c mice (six per activity of GATA3, a factor required for Th2 development and experiment) were infected by i.p. injection with 2 3 105 PFU lymphocytic IL-4 expression (33, 34). ZBTB32 repressed IL-13 expression choriomeningitis virus (LCMV), Armstrong strain (37). At 8 d postinfection, through direct binding and recruitment of HDAC1 and HDAC2 mice were euthanized and their spleens harvested. After homogenization and RBC lysis with ACK buffer (Life Technologies), splenocytes were Downloaded from to deacetylate the (34). ZBTB32 also plays a role in suspended in Dulbecco’s PBS with 2% FBS and 2 mM EDTA and stained T lymphocyte homeostasis (31, 34), but a function for ZBTB32 in with PE- or APC-labeled anti-mouse CD138, PerCP-labeled anti-mouse terminal differentiation events, such as B cell to plasma cell, has B220, and APC- or PE-labeled anti-mouse CD44. For flow cytometry, not been previously described. Abs were purchased from BD Pharmingen. CD138-positive cells were enriched by magnetic bead-positive selection using LS columns and anti-PE Although Blimp-1 is highly expressed in plasma cells, it is not hi hi or anti-APC microbeads (Miltenyi Biotec). CD138 CD44 cells were then fully expressed at early time points in the in vivo or ex vivo dif- sorted by flow cytometry. Postsort purity was ∼90–95%, and ∼5–10 million ferentiation models when CIITA is repressed and the CIITA locus sorted cells were obtained per experiment. To assess Ab-secreting plasma- http://www.jimmunol.org/ has lost the binding of positive regulators of transcription and its blasts, ELISPOT assays, performed essentially as described (37), were used. active chromatin marks (23, 35). This suggests that other factors Multiscreen HA plates (Millipore) were coated with goat anti-mouse IgG/ IgA/IgM capture Ab (0.25 mg per well) (Caltag) and blocked with 10% FCS may initiate or contribute to CIITA silencing. To examine early in PBS. Then 3-fold serial dilutions of sorted plasma cells were seeded into events, cDNA microarrays were used to analyze the gene ex- each well in growth medium (RPMI 1640 with 10% FCS, 10 mM HEPES, pression profiles of ex vivo differentiated murine B cells; a set of 5.5 mM 2-ME, and 100 U/ml penicillin–streptomycin). Plates were incu- transcriptional and chromatin regulators during this transition was bated for 6–8 h at 37˚C in a 5% CO2 atmosphere. After washing, foci of Ab identified. Among these, ZBTB32 was highly induced, and further secretion were detected with anti–IgG-biotin or anti–IgM-biotin (Southern Biotech) and Avidin-HRP (Vector Laboratories), using chromogen 3-amino- analysis demonstrated that ZBTB32 could directly suppress CIITA 9-ethylcarbazole substrate (Sigma-Aldrich). Spots were counted manually by guest on October 1, 2021 gene expression. In comparison with wild-type and Blimp-1–de- and divided by the number of cells seeded to determine the proportion of ficient B cells, B cells from ZBTB32 knockout animals displayed Ab-secreting cells in the sorted population. delayed downregulation of CIITA gene expression, suggesting a Flow cytometry role for ZBTB32 early in the differentiation process. The results suggest that ZBTB32 may function by direct binding of ZBTB32 Flow cytometry was performed following standard protocols using 3 6 to the proximal promoter region of CIITA and by formation of a FACSCalibur. Briefly, 1 10 control or LPS-, IL-2–, IL-5–stimulated primary B cells were stained with either FITC- or PE-conjugated I-A/E a complex with Blimp-1. Thus, these data describe ZBTB32 as and PE-conjugated CD138 (syndecan-1) Abs (BD Pharmingen). For a novel repressor of CIITA gene expression and suggest a role for assessing splenic B cell populations, 1 3 106 total splenocytes were ZBTB32 in the regulation of CIITA and the silencing of MHC-II stained with the following fluorophore-Ab conjugations: FITC-CD19, PE- gene expression during the formation of plasma cells. I-A, APC-B220, Cy5.5PerCP-IgM, and V450-CD5 (BD Pharmingen). Cells were washed with PBS containing 0.1% BSA, incubated for 30 min on ice, and washed again in PBS containing 0.1% BSA. Flow cytometry Materials and Methods data were analyzed using CellQuest software. All experiments were per- Mice, cells, and cell culture formed at least three times from independently isolated and purified cells.

C57BL/6 and BALB/c mice were purchased from The Jackson Laboratory. cDNA microarray analysis ZBTB32/ROG mice were described previously (36) and were generously provided by Dr. I. Cheng Ho (Harvard University, Cambridge, MA) RNA was prepared from purified murine primary B cells that were either through Dr. Jianlin Gang (Boston University, Boston, MA). Blimp-1fl/fl, untreated or treated as described above with IL-2, IL-5, and LPS for 24 h. CD19-Cre transgenic mice (19) were generously provided by Dr. Kathryn RNA was submitted to the Emory Winship Cancer Institute Cancer Calame (Columbia University, New York, NY) through Dr. S. Speck Genomics core facility for whole-genome expression profiling using Ap- (Emory University, Atlanta, GA). Double knockout mice were generated plied Biosystems Mouse Genome Survey Microarray chips. Single-color by crossing and backcrossing the ZBTB32 and Blimp-1/CD19-Cre mice microarray technology was used to measure the absolute level of RNA. and monitoring for the respective alleles. Mice were housed in the Emory Two biological replicate experiments were performed. The data were an- University School of Medicine Facilities. All animal protocols were ap- alyzed using significant analysis of microarray with a 2-fold cutoff and false proved by the Emory University Institutional Animal Care and Use discovery rate of ,0.05 (38). Genes with 2-fold or more change in ex- Committee. pression are presented in Supplemental Table II. The full dataset has been CW13.20-3B3 (CRL-1669), referred to in this article as BCL1 cells, Raji deposited into National Center for Biotechnology Information Gene Ex- (CCL-86), P3X63Ag8 (CRL-1580), and NCI-H929 (CRL-9068) cell lines pression Omnibus database (http://www.ncbi.nlm.nih.gov/gds), accession were purchased from the American Type Tissue Collection. Murine BCL1 number GSE17999. cells, P3X63Ag8 plasma cells, and murine splenic B cells were grown in RPMI 1640 medium (Mediatech) supplemented with 10% heat-inactivated RNA isolation and real-time RT-PCR FBS (HyClone Laboratories), 10 mM HEPES (HyClone Laboratories), 1 mM sodium pyruvate (HyClone Laboratories), 1X non-essential amino Total RNA was isolated using an RNeasy mini prep (Qiagen). RNA, 2 acids (HyClone Laboratories), and 0.05 mM 2-ME (Sigma-Aldrich). mg, was used to generate cDNA with reverse transcriptase (Invitrogen) and The Journal of Immunology 3 random hexamers and oligo dT (Applied Biosystems). Approximately 1/ H929 cell immunoprecipitation experiments, nuclear extracts (250 mg) 100 of the reverse transcription-generated cDNA was analyzed by real- were incubated with specific Abs for 1 h on ice, followed by the addition of time PCR using an iCycler (Bio-Rad) to measure SYBR Green incorpo- 40 ml Protein A Sepharose and an additional 3 h incubation at 4˚C. Fol- ration of the PCR product. Primers used in real-time RT-PCR are provided lowing incubation, the beads were washed in 50 mM Tris (pH 8), 150 mM in Supplemental Table I. The 18S rRNA measurements by real-time PCR NaCl, and 1% Nonidet P-40. Precipitates were separated by SDS-PAGE, were used to normalize between samples. Normalized data from at least and immunoblotting was conducted with the indicated Abs. Abs used in three independent cell preparations were averaged and plotted as fold over these experiments include the following: monoclonal anti-GST (sc-138; control treated samples. Student t tests were used to determine if signifi- Santa Cruz Biotech); anti-Myc (16-213; Millipore); anti-Flag (F4042; cant differences between control and experimental samples were observed. Sigma-Aldrich); anti-ZBTB32 and anti–Blimp-1, as described above; anti- SEM was used to represent experimental variation. RFX5 (42); secondary anti-mouse IgG (A6782; Sigma-Aldrich); and anti- rabbit IgG (A0545; Sigma-Aldrich). Plasmids and transfection Listeria monocytogenes and influenza infection Expression plasmids used in transient transfection experiments, purchased from OriGene Technologies, included those for CBX2 (MC204062), L. monocytogenes, strain 10403S, grown in Difco Brain Heart Infusion HMGN3 (SC110044), Rad54l (MC205395), SATB1 (MC200989), and (BHI; BD Biosciences) to an OD of ∼0.2, was concentrated, and 9.8 3 106 TfDP1 (SC127935). These vectors were transfected transiently into 2 3 CFU were used to infect wild-type C57BL/6 and ZBTB32ko mice. 106 log phase growing Raji cells, using Amaxa Nucleofector Kit V Cohorts of wild-type and ZBTB32ko mice were also infected with 0.1 3 according to the manufacturer’s protocol. Myc-tagged ZBTB32 and Flag- LD50 A/PR/8/34 mouse-adapted virus. Sera were individually collected on tagged Blimp-1 cDNAs were cloned into the pcDNA 3.1(2) expression the indicated days, and anti-influenza–specific Ab levels were determined vector directly from PCR-amplified cDNA from P3X63Ag8 cells. Flag- quantitatively by hemagglutination inhibition assays, as described previ- tagged GFP was cloned from the pEGFP expression construct. A total of ously (43). Briefly, MaxiSorp Nunc 96-well plates (Southern Biotechnology 1 3 107 log phase growing Raji cells were electroporated as described Associates) were coated with 4 mg/ml inactivated A/PR/8/34 egg-grown m m previously (39) with 50 g of the indicated expression vector. HEK293 inactivated virus and sera incubated with 4 HA units/50 l A/PR/8/34 virus Downloaded from cells were transfected with Lipofectamine 2000 (Invitrogen) in 10-cm for 30 min. Infected mice were monitored daily for signs of morbidity dishes with 10 mg plasmid DNA encoding Myc-ZBTB32 or Flag–Blimp-1. (body weight changes, fever, and hunched posture) and mortality. Lentivirus generation and infection LPS injection Flag-tagged ZBTB32 cDNA was cloned into the YFP-expressing lentivirus Mice were injected retro-orbitally with 50 mg LPS (Enzo Life Sciences) vector (pHR-UBQ-IRES-eYFP). These lentivirus constructs were cotrans- diluted into 100 ml PBS. At 3 d postinjection, splenocytes were harvested, fected with the pseudo envelope protein VSV-G gene into HEK293FT RBCs were lysed in ACK lysis buffer (0.15M NH Cl, 10mM KHCO ,

4 3 http://www.jimmunol.org/ packaging cells using FuGENE 6 (Roche), according to the manufacturer’s 0.1M EDTA, pH 7.2), and total splenocytes were stained with PE-conjugated protocols. At 48 and 72 h posttransfection, virus was harvested and was anti-CD138 and APC-conjugated anti-B220 (BD Biosciences) for 30 min on infected into purified primary murine B cells in the presence of 8 mg/ml ice. Samples were washed once in cold PBS and resuspended in cold PBS polybrene (Sigma-Aldrich). Infection efficiency was determined by YFP containing 0.1% BSA. Flow cytometry data were analyzed with CellQuest expression and Western blot using Flag Ab. Lentivirus constructs (pLKO.1) software. harboring short hairpin RNA (shRNA) to specific ZBTB32 (cat. RMM4534- NM_021397; clone number, TRCN0000096484, TRCN0000096485, TRCN0000096486, TRCN0000096487, TRCN0000096488) were pur- Results chased from Open Biosystems (now Thermo Scientific). For primary B cell A large number of transcription factors and chromatin experiments, five different lentiviruses that express shRNA specific to remodeling proteins are differentially regulated during ex vivo ZBTB32 were infected into purified murine primary B cells. Cells were B cell differentiation by guest on October 1, 2021 then treated as above with LPS, IL-2, and IL-5 to induce plasma cell differentiation for 5 d. To deplete ZBTB32 in the plasma cell line Ex vivo differentiation of murine B cells to plasma cells, using LPS, P3X63Ag8, lentivirus expressing the indicated shRNA to ZBTB32 was IL-2, and IL-5 (35), was used as a model system to dissect the used, and cells were selected on puromycin (encoded in pLKO.1) for 6 d, mechanisms that silence CIITA and ultimately MHC-II gene ex- prior to analysis. pression. This model results in the silencing of CIITA and MHC-II, Chromatin immunoprecipitation as well as the acquisition of canonical plasma cell markers, such Chromatin immunoprecipitation (ChIP) assays were performed as described as syndecan-1 (CD138) (Fig. 1A, Ref. 35). Although plasma cell previously (35, 40). A total of 1 3 107 P3X63Ag8 or A20 were crosslinked phenotypic changes develop over at least a 5-d period using this with 1% formaldehyde for 15 min in culture media. Chromatin was iso- protocol, a significant reduction in the steady-state levels of CIITA lated and sonicated to generate an average 600-bp size of DNA. Chromatin mRNA was observed within the first 24 h of culture (35). At this m (30 g) was precleared and immunoprecipitated with the indicated Abs early time point, the expression of Blimp-1, a transcriptional re- overnight at 4˚C. DNA was purified from the reverse crosslinked chro- matin, and 1/10 of each sample was analyzed by quantitative real-time pressor of CIITA (17) and master regulator of plasma cell fate PCR using SYBR Green incorporation for the CIITA DNA sequence of (20), is not fully expressed (Fig. 1B), suggesting that other factors interest. Primers for CIITA pIII and a control region are provided in may also play a role in CIITA silencing. To identify potential Supplemental Table I. Quantitation for each real-time PCR assay was candidate genes that were differentially expressed between B cells carried out by comparison with a standard curve generated with genomic and plasma cells at this 24-h time point, the above ex vivo dif- DNA. The average of at least three experiments was normalized to an 2 irrelevant Ab control for the locus and plotted with respect to the amount ferentiation model was used. Freshly isolated and purified CD43 of input chromatin. Anti-ZBTB32 (sc-25358) and anti-PU.1 (sc-352) Abs splenic B cells were cultured in media or with LPS, IL-2, and IL-5 were purchased from Santa Cruz Biotechnology. The anti–Blimp-1 anti- for 24 h, and RNA transcripts were compared using cDNA serum was developed in collaboration with Rockland Immunochemicals. microarrays. Of the 32,970 murine targets queried, 2370 genes This antiserum recognizes both murine and human Blimp-1 (data not . , shown). Nonimmune control IgG antiserum was purchased from Millipore. were differentially expressed 2-fold (false discovery rate 0.05), with 1169 upregulated and 1201 downmodulated in the Coimmunoprecipitation and immunoblotting cells treated for 24 h (Fig. 1C, Supplemental Table II). A heat map Myc-ZBTB32 and Flag–Blimp-1 expression vectors were transiently of the 2370 modulated genes, sorted by fold change, from two transfected into HEK293 cells for 48 h. Nuclear lysates were prepared as biological replicates showed clear segregation of the modulated described previously (41). Abs were preincubated with magnetic beads genes (Fig. 1C). In addition to the MHC-II genes, some of the (Invitrogen) overnight at 4˚C. Nuclear extract, 500 mg, was immunopre- many highly differentially expressed genes were transcription cipitated with Ab-prebound beads overnight at 4˚C. After incubation, the beads were washed in 50 mM Tris (pH 8.0), 100 mM NaCl, and 0.05% factors or modifiers of chromatin structure (Table I). For example, Nonidet P-40, and eluted with SDS-PAGE loading buffer. Immunoblotting key B cell transcription factors, such as BCL6, EBF1, CD19, and using the indicated antisera was carried out under standard conditions. For SpiC, were significantly repressed during the differentiation process. 4 ZBTB32 REPRESSES CIITA EXPRESSION Downloaded from http://www.jimmunol.org/

FIGURE 1. Ex vivo differentiation of murine B cells to plasma-like cells results in the induction and repression of 2370 genes. Primary mouse B cells purified from splenocytes of C57BL/6 mice were either untreated (Con) or differentiated ex vivo using LPS, IL-2, and IL-5 for the various times as in- by guest on October 1, 2021 dicated. (A) Flow cytometry using FITC-conjugated I-A and PE-conjugated CD138 (syndecan-1) Abs was carried out after 5 d of the above treatment to assess the loss of MHC-II surface expression and acquisition of a key plasma cell marker. Histograms are representative of three experiments with similar results. (B) Quantitative RT-PCR was used to determine the steady-state mRNA levels of representative genes encoding transcription factors and chromatin remodeling proteins identified from the cDNA microarrays over a 5-d time course of the ex vivo differentiation protocol. The data from three independent biological replicates were analyzed and normalized to the expression level of 18S rRNA and plotted with respect to the day 0 time point. SEM was used to represent experimental variability. (C) To assess events that occurred early in the differentiation process, control and 24-h–treated cultures were examined by RNA profiling using cDNA microarrays (See Materials and Methods). A heat map, representing 2370 Z-score normalized genes displaying a change in expression .2-fold, was plotted with respect to fold change. The relative locations of significantly changed genes discussed in this article are indicated. Two biological replicates are shown.

Enhancer of zest 2 (EZH2) and chromobox homology 2 (CBX2), and LPS. For the repressed genes, two overall kinetic profiles were genes that encode members of the chromatin remodeling polycomb observed. Profile 1 showed a nearly continual decline in gene group (PcG) repressor complexes PRC2 and PRC1, respectively, expression over the time course (Fig. 1B). BCL6, SpiC, and CIITA were induced, whereas EZH1, a component of the PRC2 complex, fell into this group. Profile 2 transcripts (EBF1, PAX5, and SpiB) was repressed. The expression of other chromatin assembly/ were initially induced up to a few-fold before being repressed remodeling factor genes, Chalf, TfDP1, HMGN3, and Smyd2,was around day 2. Differences in these initial expression profiles from also increased. In addition, ZBTB32, which encodes a zinc finger the 24-h microarray results may reflect the overall sensitivity of repressor protein previously characterized in the regulation of Th the cDNA microarrays. Two profiles were also observed for the cell differentiation as a repressor of GATA3, was increased 102-fold induced genes. The profile exhibited by Blimp-1 and XBP-1 during the differentiation process and was the most significantly showed an increase and stabilization of mRNA expression over induced transcript. These results raised the possibility that the above the time course (Fig. 1B). The other induced gene profile, which genes may function to regulate CIITA expression and/or B cell included ZBTB32, Tfdp1, Smyd2, and EZH2, displayed a transient, differentiation. high burst of induction over the first 2 d and a slow decline in expression over the rest of the time course, with the final levels B cell-specific genes show diverse kinetics of regulation still significantly above the starting steady-state levels in resting To verify the cDNA microarray data and to provide a kinetic view B cells. of the expression of some of the differentially regulated genes, real- To determine whether a similar set of genes were differentially time RT-PCR was carried out on six repressed genes and six in- regulated in primary plasma cells, plasmablasts (CD138+CD44+) duced genes over a 5-d period following treatment with IL-2, IL-5, were purified from the spleens of mice 8 d following LCMV in- The Journal of Immunology 5

Table I. Differential expression of selected genes during ex vivo plasma cell differentiation

Induced Genes Repressed Genes

Symbola Foldb Symbol Fold Symbol Fold Symbol Fold ZBTB32 102 Nek2 15 Pax5 0.479 Fyb 0.163 Ptgir 68 Kif2c 15 Elf2 0.479 Emr1 0.162 Cdc6 30 Pycr1 14 Pou2af1 0.460 Acas2l 0.154 Il2ra 29 Cdc25c 14 Klf7 0.419 Metrnl 0.154 Il6 27 Bcat1 14 Jarid2 0.399 Zfp318 0.153 Bub1 24 Ckap2 13 Arid1a 0.385 Btg1 0.072 Melk 24 Mybl2 13 Vezf1 0.384 Zfp36 0.071 Ccna2 23 Sytl3 12 EBF1 0.373 C1qb 0.016 Rad51 23 Idi1 12 CD19 0.300 Slpi 0.013 Plk1 22 Pole2 11 Zfp353 0.292 Tgfbi 0.012 Pbk 19 Sgol2 11 Foxo1 0.287 SpiC 0.012 Rrm2 19 Cenph 11 Hhex 0.254 Sgk 0.009 Tpx2 19 Aurka 11 Nfatc1 0.243 Axl 0.009 Chek1 18 Nusap1 11 Ifngr1 0.228 Fcna 0.009 Shcbp1 18 Prc1 11 Il17r 0.222 C1qg 0.004 Gcat 18 Bcl2l1 11 Btla 0.222 C1qa 0.002 Pask 17 Cdc20 10 BCL6 0.218 MHC Genes Clspn 17 Smyd2 9.3 Pink1 0.213 Symbol Fold Downloaded from Birc5 16 HMGN3 7.8 Satb1 0.182 H2-Ab1 0.20 Rad54l 16 TfDP1 7.5 EZH1 0.170 H2-DMb 0.18 Batf 16 EZH2 6.5 Add3 0.170 H2-DMa 0.16 Chaf1a 16 Bach 6.4 Apc 0.166 CIITA 0.14 Fignl1 15 CBX2 6.0 Hbp1 0.166 H2-Oa 0.14 Kif11 15 Acat2 6.0 Sectm1 0.163 H2-Ob 0.07

aGenes in bold are referred to in the article. http://www.jimmunol.org/ bFold reflects the level in stimulated cells divided by control untreated cells. fection (Fig. 2A), and the RNA profiles of 18 genes were ana- the induction/repression of genes was similar to that observed in lyzed. Enrichment of plasmablasts led to a population of pre- the ex vivo system (Figs. 1B, 2C). Induction of Blimp-1, XBP-1, dominantly IgG-secreting cells that were 94% CD138+CD44+ and syndecan-1 was more pronounced. The steady-state level of (Fig. 2A, 2B). Although differences in magnitude were present, ZBTB32 mRNA, at 13-fold over naive B cells, was less than the by guest on October 1, 2021

FIGURE 2. Primary murine plasmablasts repress B cell genes while inducing plasma cell genes. Primary murine splenic plasmablasts were collected and analyzed for expression of select transcription factors identified in the microarray screen above. (A) At 8 d following infection with LCMV, splenocytes were isolated and plasmablasts were sorted based on their expression of high levels of CD138 and CD44. (B) Purified cells from two independent isolations were tested for Ab secretion by ELISPOT. Between 70 and 100% of the sorted cells represented Ab-secreting cells (ASC). (C) Steady-state mRNA levels of representative genes identified in the microarray screen were analyzed by quantitative RT-PCR. Samples were analyzed as above, using three independent RNA samples and plotted relative to fold change over B cells. SEM was used to represent experimental variability. 6 ZBTB32 REPRESSES CIITA EXPRESSION peak but similar to the day 5 levels observed in the ex vivo system. As expected (44), CIITA, I-Ab, CD19, and c-Myc genes were re- pressed in the plasmablasts (Fig. 2C). Overall, these results verify the ex vivo cDNA microarray data obtained in the in vivo model and provide a series of candidates that may regulate CIITA and potentially other genes involved at the early stages of B cell dif- ferentiation.

ZBTB32 regulates CIITA and MHC-II gene expression To determine if some of the induced plasma cell transcription factors could repress CIITA expression, cDNA expression vectors encoding seven of the genes were obtained and transiently trans- fected into the MHC-II– and CIITA-expressing human B lym- phoblast cell-line Raji (Fig. 3A). Selection of this set of candidates was based on the overall induction strength and known function such that transcriptional repressors and chromatin assembly/ remodeling proteins were chosen. The DNA-binding protein Satb1 was repressed following B cell differentiation and was chosen as a control gene that was not expected to repress CIITA Downloaded from expression. The transient transfection efficiency by nucleofection of Raji cells ranged between 35 and 60% (H.S. Yoon, unpublished observations). Of the seven genes transfected, only three had a statistically significant effect on the repression of CIITA. Ectopic expression of CBX2 and EZH2 in Raji cells, which are members of the polycomb PRC1 and PRC2 complexes, respectively, re- http://www.jimmunol.org/ sulted in small but statistically significant decreases in CIITA expression. By contrast, ectopic expression of ZBTB32 resulted in a 65% reduction in steady-state mRNA levels of CIITA. Thus, ZBTB32 could potentially represent a novel regulator of CIITA expression and, as described below, was further characterized. Because the available vectors did not contain an epitope tag, ex- pression of the transfected genes in this system could not be easily

examined. Thus, this assay could not rule out a potential role for by guest on October 1, 2021 candidate genes that displayed no effect on CIITA expression. The repressive ability of ZBTB32 was compared with the known FIGURE 3. ZBTB32 and the polycomb complex proteins EZH2 and regulator of CIITA, Blimp-1. Epitope-tagged vectors expressing CBX2 repress CIITA expression. (A) cDNA expression vectors encoding Flag-GFP, Myc-ZBTB32, or Flag–Blimp-1 were electroporated seven transcription factors/chromatin assembly proteins identified from the into Raji cells, and the ability of each construct to regulate CIITA, cDNA microarrays were transiently expressed in the human CIITA-posi- HLA-DRA, BCL6, and a control gene, MTA3, was measured 72 h tive B cell line Raji. At 3 d posttransfection, RNA was extracted, and the level of CIITA mRNA was evaluated by quantitative RT-PCR. The origin of later (Fig. 3B). As expected, owing to a reduction in CIITA, HLA- the cDNAs is indicated by the prefix (m for mouse and h for human). The DRA mRNA expression was also significantly reduced by control lane represents a GFP expression vector. (B) The expression of ZBTB32, compared with a control GFP electroporation. GFP CIITA, HLA-DRA, BCL6, and MTA3 mRNA steady-state levels was eval- vector control transfection did not affect the level of HLA-DRA uated by quantitative RT-PCR following ectopic expression of Flag-GFP, mRNA. Importantly, MTA3 and BCL6 mRNA expression levels Myc-ZBTB32, or Flag–Blimp-1 in Raji cells. (C) Expression vectors were not affected, suggesting that ZBTB32’s role as a repressor containing Flag-GFP, Myc-ZBTB32, or Flag–Blimp-1 were electroporated was specific to CIITA. On its own, Blimp-1 significantly repressed into the human Raji B cell line. Expression levels of each tagged protein CIITA levels, but this repression was very modest. As such, HLA- were determined by Western blot with anti-Flag and anti-Myc Abs. The actin Western blot is provided as a loading control. The positions (arrows) DRA mRNA levels were not significantly affected. Consistent with of Blimp-1 and GFP are indicated in the middle anti-Flag epitope blot. The previous data (20), Blimp-1 repressed BCL6 levels but not a con- positions of relevant m.w. standards are shown. (D) Purified primary mu- trol gene, MTA3. Western blotting confirmed that each construct rine B cell cultures were infected with a lentivirus vector expressing either was expressed to significant levels (Fig. 3C). YFP or ZBTB32. RNA was prepared and analyzed for CIITA mRNA levels To determine whether ZBTB32 could repress CIITA in primary as above. Data from three independent experiments were averaged, normal- murine B cells, a YFP lentiviral expression vector coexpressing ized to the expression of 18S rRNA, and presented with respect to the levels ZBTB32 was used to infect freshly purified murine B cells. The of a control gene, which was arbitrarily set at 1. SEM was used to represent levels of CIITA mRNA were assessed after 4 d. In these cultures, experimental variability. *Differences between control and experiments that show significance, with p , 0.05 as determined by the Student t test. a low level of LPS (5 mg/ml) was provided to maintain cell via- bility over the course of the experiment. The level of CIITA mRNA in the YFP lentiviral control cultures was similar to that in uninfected cultures (data not shown), whereas the ectopic ex- ZBTB32-depleted plasma cells reactivate expression of CIITA pression of ZBTB32 in primary murine B cells repressed CIITA and I-A expression (Fig. 3D). These results indicate that ZBTB32 can The murine plasmacytoma cell line P3X63Ag8 represents a fully repress CIITA expression. differentiated murine plasma cell and expresses ZBTB32. To de- The Journal of Immunology 7 termine the effects of depletion of ZBTB32 in plasma cells, Consistent with CIITA expression, I-Ab expression was dere- P3X63Ag8 was infected with lentiviral vectors expressing a series pressed (70- to 300-fold) using the same vectors. The levels of of ZBTB32 shRNAs, and the effect on expression of CIITA, I-A, BCL6 mRNA were slightly induced at levels between 1.5- and 3- and other B cell- and plasma cell-specific genes was assessed. fold, depending on the shRNA. Although no significant difference Three of the five lentiviral shRNA vectors were able to reduce the was observed for syndecan-1 or XBP-1 mRNAs, a 20–50% re- ZBTB32 protein levels to varying degrees (Fig. 4A). The levels of duction in Blimp-1 mRNA levels was noted, suggesting that actin protein did not change with any of the shRNA vectors (Fig. ZBTB32 may affect the expression of other genes in this cell line 4A). ZBTB32 shRNA vector 3 demonstrated the greatest effect on that are required to maintain the plasma cell phenotype. CIITA derepression, resulting in an ∼2000-fold increase (Fig. 4B). Because ZBTB32 depletion in P3X63Ag8 cells resulted in the The other vectors (2, 4) showed an ∼250-fold increase (Fig. 4B). derepression of CIITA and I-Ab mRNA, it was of interest to de- termine if ZBTB32 depletion would also result in an increase in MHC-II surface expression. Flow cytometry of the P3X63Ag8 cells infected with a ZBTB32 shRNA lentivirus was compared with that of control vector-infected cells. In this case, ∼15% of the cells exhibited strong I-A surface expression (Fig. 4C), indicating that this subpopulation of cells had been derepressed for CIITA and MHC-II expression. This small proportion of cells may reflect an epigenetic pathway that must be overcome to allow derepres- sion of a terminally differentiated cell, such as that described with Downloaded from dedifferentiation of plasma cells transfected with vectors express- ing MTA3 and Bcl6 (41).

ZBTB32-deficient B cells fail to efficiently repress CIITA The ZBTB32/ROG-knockout mouse (referred to in this article

as ZBTB32ko) was created previously and contains a deletion http://www.jimmunol.org/ spanning exons 2–6 of the ZBTB32 gene (36). The ZBTB32ko mouse displays minor defects in Th2 cell development and homoeostasis (32, 33), but B cells appear to develop normally, as they express wild-type levels of typical B cell phenotypic markers, such as B220, CD5, CD19, MHC-II, and IgM (Supplemental Fig. 1A). B cell differentiation to plasma cells or the ability of these mice to repress CIITA transcription was not previously investi- gated in these mice. To determine if CIITA expression was altered in ZBTB32ko mice, splenic B cells from ZBTB32ko and wild- by guest on October 1, 2021 type mice were isolated and differentiated ex vivo in LPS, IL-2, and IL-5, and mRNA levels of CIITA were examined by real-time RT-PCR. In this experiment, a 3-d time course was used that corresponded to the peak and early induction of ZBTB32 mRNA. In wild-type cells, ZBTB32 mRNA levels were increased ∼50-fold and CIITA mRNA levels were reduced to ∼30% of their B cell levels at 4 h, the earliest time point examined (Fig. 5A). As a point of reference, Blimp-1 mRNA has not been induced at this time point in the wild-type differentiated cells (Fig. 5G). CIITA mRNA levels continued to decrease during the time course, and this oc- curred in a manner that was concordant with the increase in Blimp-1 mRNA levels after 48 h. Comparison of the kinetics of FIGURE 4. ZBTB32 is required for CIITA silencing in a plasma cell ZBTB32ko and wild-type B cells in this differentiation protocol A line. ( ) Lentiviruses containing five different shRNAs specific to ZBTB32 showed that ZBTB32ko cells were significantly delayed in their were produced and used to infect the murine plasma cell line P3X63Ag8. ability to silence CIITA (Fig. 5A), indicating that ZBTB32 plays The efficiency of ZBTB32 knockdown compared with the control empty vector pLKO.1 was evaluated by Western blot. The actin loading control a role early in the process. showed that none of the shRNAs affect expression of the actin gene. To examine the role of Blimp-1 and compare it with ZBTB32, Lentivirus with shRNAs 2, 3, and 4 reduced ZBTB32 expression, whereas the CD19-Cre, Blimp-1 conditional knockout mouse (Blimp- constructs 1 and 5 did not. (B) Quantitative RT-PCR was performed on 1cko), which deletes Blimp-1 from 85–90% of B cells (19), was RNA isolated 6 d after infection of P3X63Ag8 cells with lentivirus investigated in this ex vivo system. In addition, the influence of expressing shRNAs 2, 3, or 4. The RNA levels of CIITA and other B cell- both genes in this system was assessed using a ZBTB32ko/Blimp- specific genes, I-Ab and BCL6, as well as plasma cell-specific genes, 1cko double knockout (dko) mouse line. Ex vivo differentiation of Blimp-1, XBP-1, and syndecan-1, were examined and compared with the Blimp-1cko B cells resulted in a repression of CIITA mRNA control (pLKO.1) samples. The results were plotted with respect to the through the first 24 h that mirrored wild-type kinetics (Fig. 5B), control infection for each of the genes. (C) P3X63Ag8 cells, infected for suggesting that Blimp-1 does not play a role in the early sup- 6 d with control pLKO.1 or ZBTB32 shRNA number 3, were evaluated for I-A surface expression by flow cytometry and are shown with forward side pression of CIITA.At48h,CIITA transcript levels increased scatter (FSC). All experiments were performed at least three times from slightly, suggesting that repression was not fully maintained in the independent cultures. SEM was used to represent experimental variability. absence of Blimp-1. Ex vivo differentiation of B cells from the *p , 0.05, Student t test values. dko mouse displayed a delayed decrease in CIITA mRNA paral- 8 ZBTB32 REPRESSES CIITA EXPRESSION

FIGURE 5. ZBTB32-deficient B cells fail to acutely repress CIITA during ex vivo dif- ferentiation. Splenic B cells from wild-type (n = 6), ZBTB32ko (n = 10), Blimp-1cko (n = 3), and ZBTB32/Blimp-1dko (n = 9) mice were ex vivo differentiated for 3 d. RNA was isolated at indicated time points and CIITA [top row,(A–C)], ZBTB32 [middle row,(D– F)], and Blimp-1 [bottom row,(G–I)] mRNA levels were quantitated by qRT-PCR, nor- malized to 18S rRNA, and plotted with re- spect to the day 0 time point for the gene analyzed. Data for the wild-type animals are replotted in each row so that a visual com- parison with each knockout can be made. SEM was used to represent experimental variability. Student t test differences from wild type, *p , 0.05, **p , 0.01. Downloaded from leling that in ZBTB32ko cells for the first 24 h and, as in the ZBTB32 associates with Blimp-1 Blimp-1cko, CIITA mRNA levels increased after 48 h (Fig. 5C). The binding of both ZBTB32 and Blimp-1 to the same pIII up- These data suggest that ZBTB32 initially functions to repress stream promoter region in plasma cells suggested the possibility CIITA and that Blimp-1 is required to maintain the repression. that the two proteins may interact. To determine if that may be the Analysis of ZBTB32 and Blimp-1 mRNAs over the same time case, Flag-tagged Blimp-1 was coexpressed with Myc-tagged http://www.jimmunol.org/ course verified each genetic deletion (Fig. 5D–I). In the Blimp- ZBTB32 in HEK293 cells, and the ability of the proteins to as- 1cko, slight increases in Blimp-1 mRNA were observed at the 48- sociate was tested by immunoprecipitation. Anti-Flag and anti-Myc and 72-h time points (Fig. 5H, 5I), confirming previous observa- Abs were each able to coimmunoprecipitate both Flag–Blimp-1 tions about the efficiency of the CD19-Cre system (19). Blimp-1– and Myc-ZBTB32 proteins (Fig. 6B). A control Ab to GST failed deficient B cells were found to express less ZBTB32 mRNA than to immunoprecipitate either protein (Fig. 6B). A similar set of ∼ did wild-type cells (Fig. 5E); however, this level was 25-fold immunoprecipitation assays was carried out on the endogenous above that in resting B cells and was likely sufficient to repress proteins using human H929 plasma cells. The results showed that CIITA at early time points. ZBTB32 immunoprecipitated with Blimp-1 but not a control by guest on October 1, 2021 protein RFX5 (Fig. 6C). Control antisera did not pull down either ZBTB32 directly binds CIITA pIII protein. These results therefore suggest that ZBTB32 can interact The above data suggest that ZBTB32 plays an important role in with Blimp-1 and that these two proteins may function together or CIITA silencing during plasma cell differentiation, but do not in a complex to silence gene expression. distinguish between a direct or indirect role, nor do the experi- ments provide mechanistic insight into how ZBTB32 may func- Discussion tion. ZBTB32 contains a Kru¨ppel-type zinc finger DNA binding The transition from B cell to plasma cell represents a critical fate domain and was previously reported to bind to the Aurora kinase decision leading to effective humoral immune responses. The si- C (Aie1) promoter (45) and IL-13 gene (34), suggesting that it may lencing of CIITA and MHC-II gene expression effectively removes also bind to the CIITA gene and directly silence expression. CIITA the ability of these cells to receive TCR–MHC-II–mediated sig- is expressed from three promoters in mouse cells, with pIII being nals. During this transition, transcriptionally active chromatin principally used in B cells (4, 8, 46). A sequence similar to the marks are removed and replaced by repressive chromatin mod- ZBTB32 binding site in the Aie1 gene was identified and lies ifications (35). Blimp-1 was identified previously as a key regu- within 50 bp of a known Blimp-1 binding site (17) upstream of lator of this transition and as a repressor of CIITA expression (15, CIITA pIII. ChIP assays were performed in P3X63Ag8 plasma 17, 49). However, using an ex vivo differentiation model to mimic cells, which express high levels of ZBTB32, and amplicons were early events, it was observed that silencing of CIITA begins at designed around the predicted binding site at pIII. A20 B cells, a time before Blimp-1 reached a high level of expression. Al- which do not express ZBTB32 and do express high levels of though it could be that the CIITA gene is sensitive to very low CIITA, were used as a control cell line. ZBTB32 was found to bind levels of Blimp-1 expression, this inconsistency prompted a search specifically to CIITA pIII, but not to a control amplicon from the for factors expressed early in this transition that could mediate CIITA locus or in A20 cells (Fig. 6A). Blimp-1 binding was also CIITA silencing. As shown, ZBTB32 was identified as such a tested and found to occur at the same amplicon, in agreement with factor and had the highest level of induction in this system. previous reports (17, 47). The known B cell activator PU.1 was An ex vivo system to globally stimulate and differentiate naive found to bind to a site upstream of pIII in A20 but not P3X63Ag8 B cells was chosen such that gene expression and biochemical cells, in accordance with previous reports (48), providing a posi- analyses could be achieved during early time points in the process. tive control for these chromatin lysates. Control IgG antisera did Intriguingly, the cDNA microarrays identified 2370 genes whose not precipitate either of the specific or nonspecific DNAs. These expression had already changed at 24 h, with several genes showing data support the hypothesis that ZBTB32 directly binds and log differences in their expression patterns. Verification of the represses the CIITA gene. microarray results, using primary plasmablasts isolated 8 d after The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ FIGURE 6. ZBTB32 binds CIITA pIII in differentiated plasma cells and can interact with Blimp-1. (A) ChIP assays from murine A20 B cells or P3X63Ag8 plasma cells were performed using Abs specific to ZBTB32, Blimp-1, PU.1, or nonimmune IgG control antisera. The occupancy of these factors at CIITA pIII and a negative control site was measured by quantitative PCR, compared with a standard curve generated for these regions. The positions of these ChIP PCR amplicons are indicated in the schematic, as are the positions of the putative binding sites for ZBTB32, Blimp-1, and PU.1. Data from three independent chromatin preparations were expressed with respect to the input chromatin. *Student t test differences from both the control site and the control cell line, p , 0.05. SEM was used to represent experimental variability. (B) Flag–Blimp-1 and Myc-ZBTB32 were coexpressed in HEK293 cells. Nuclear extracts were prepared and subjected to immunoprecipitation using anti-GST, anti-Myc, and anti-Flag Abs. The precipitates were analyzed by Western blot with the indicated antisera. Input lane represents 10% of the extract. The data are representative of three independent experiments. (C) Coimmunopre-

cipitation of Blimp-1 and ZBTB32 was performed with nuclear extracts from H929 human plasma cells, using control (anti-TCR), anti–Blimp-1, anti- by guest on October 1, 2021 ZBTB32, or anti-RFX5 antisera. The input represents 16% of extract. Data are representative of two independent experiments. virus infection, verified that the ex vivo system mimicked a plasma pIII provided evidence for direct binding of ZBTB32 to the up- cell fate differentiation process. Previously, a number of gene ex- stream promoter region of the CIITA gene. pression profile comparisons between B cells and in vivo generated Early kinetic analysis from wild-type mice suggests a model for plasma cells were performed to identify genes associated with this the regulation of CIITA during plasma cell differentiation in which genetic program (50, 51). Although the data generated here were ZBTB32 is induced and functions during the very early stages of at an earlier time point during the differentiation process, a num- the ex vivo plasma cell differentiation system, when CIITA si- ber of hallmark genes were differentially regulated in both studies, lencing occurred within 4 h. At this time point, Blimp-1 mRNA including the upregulation of cell cycle/proliferation-associated was not detectable. Cells from ZBTB32ko animals showed delayed genes and IL6, IRF4, IRF6,andTfDP1. Likewise, as in this study, kinetics of CIITA silencing and were consistent with a model in Pax5, CD19, BCL6, SpiB, and others were among some of the key which only Blimp-1 was controlling CIITA silencing. Likewise, at early time points, the cells from a Blimp-1cko mouse displayed downmodulated genes observed previously. Many of these genes kinetics more similar to those of the wild-type mouse, as ZBTB32 were found previously to be direct targets of Blimp-1, including (although reduced in this background) was fully functional at this Pax5, CIITA,andSpiB (20). Surprisingly, ZBTB32 was not identified stage. At later time points, the levels of CIITA were derepressed in in either of the two previous B cell–plasma cell microarray screens, the Blimp-1cko mouse, suggesting a role for Blimp-1 in main- perhaps because those studies were performed at later time points in taining or establishing a fully repressed state. These data are . the differentiation of plasma cells ( 6 d), a significant time after the consistent with the requirement for Blimp-1 in establishing plasma peak of ZBTB32 expression. cell fate. CIITA silencing in the dko mouse followed a combined ZBTB32 is a member of the BTB-containing zinc finger protein pattern of the two single knockouts with delayed silencing kinetics family that recruits corepressors, such as N-CoR and HDACs, to its and a late derepression of CIITA. The combined pattern was likely target genes (29, 34). ZBTB32 overexpression and shRNA anal- due to the loss of function of ZBTB32 and Blimp-1, respectively. It ysis revealed that ZBTB32 expression was able to repress CIITA should be emphasized that in the absence of Blimp-1, plasma cells and suggested that it could play a part in this differentiation pro- do not form either in vivo or through the ex vivo differentiation cess. Using homology searches from the Aie1 (45) gene, a potential protocol used in this study. Thus, at extended time points (after BTB-ZF family member binding site upstream of pIII of the CIITA 24 h; i.e., when Blimp-1 is expressed), it is likely that the cell gene was identified that was in close proximity to the Blimp-1 differentiation program in Blimp-1cko is altered and does not binding site (17). A positive ChIP assay result for ZBTB32 at follow a normal path. As such, it was not possible to separate 10 ZBTB32 REPRESSES CIITA EXPRESSION

Blimp-1 and ZBTB32, using the genetic models. The silencing of (59–62) and perhaps other cell types. The finding of ZBTB32 as CIITA by ZBTB32 early in the differentiation time course sug- a gene that is induced early upon the plasma cell differentiation gested that surface MHC-II expression could be reduced. This pathway may suggest that in some cases, this induction may be reduced expression was not observed (data not shown) and is likely important, but not necessary, to begin plasma cell differentiation due to the stability of CIITA bound to MHC-II promoters (52), as transition. Considering the stability of the CIITA enhanceosome well as the long half-life of MHC-II proteins on the cell surfaces complex (52), a rapid loss of CIITA expression through ZBTB32 (53). may allow for a more efficient loss of MHC-II during this pro- At the CIITA locus, activation-associated histone modifications cess. Decreases and ultimately loss of MHC-II would prevent are replaced with repressive modifications as cells transition from these cells from receiving further stimulation or help from Ag- B cells to plasma cells (35). One set of repressive marks, H3K27me3, specific T cells and lock the cell fate pathway that these cells is produced by the polycomb complexes. Consistent with the ap- undertake. Irrespective of its other potential functions during pearance of H3K27me3 in plasma cells was the fact that components terminal differentiation events in lymphocytes, the data reported of the polycomb repressor complex EZH2 and CBX2 were identified in this article introduce ZBTB32 as a novel regulator of CIITA in the microarray screen. In addition, when overexpressed, CBX2 gene expression. and EZH2 had small but significant repressive effects on CIITA ex- pression. Consistent with this finding is that EZH2 has been shown to function in regulating CIITA in other systems (54, 55). Although not Acknowledgments investigated in this study, the role of these proteins is likely pertinent We thank members of the laboratory for comments on this work, as well as to many of the genes repressed during this transition. the Emory Flow Cytometry Core and Emory Winship Cancer Genomics Kallies et al. (23) provided evidence that the plasma cell pro- Core; and Drs. Jianlin Gang, I. Cheng Ho, Kathryn Calame, and S. Speck Downloaded from for generously providing the mice. gram was initiated through the loss of Pax5, and that this occurred at a time prior to Blimp-1 expression. They suggested that an unknown factor could be the mediator of this process. Whether Disclosures ZBTB32 is this factor is not clear, but the study did raise the issue The authors have no financial conflicts of interest. of the breadth of the role that ZBTB32 may play in plasma cell differentiation and B cell-mediated immune responses. To exam- http://www.jimmunol.org/ ine the part that ZBTB32 may have in ultimately augmenting References humoral immune responses, ZBTB32ko mice were compared with 1. Saito, T., and R. N. Germain. 1988. The molecular basis of MHC-restricted antigen recognition by T cells. Int. Rev. Immunol. 3: 147–174. wild-type mice in two infection models. In the first, ZBTB32ko 2. Saito, T., and R. N. Germain. 1988. The generation and selection of the T cell were infected with a mouse-adapted influenza strain, which elicits repertoire: insights from studies of the molecular basis of T cell recognition. Immunol. Rev. 101: 81–113. a strong humoral immune response in wild-type mice. No differ- 3. Steimle, V., L. A. Otten, M. Zufferey, and B. Mach. 1993. Complementation ences in mortality or morbidity were observed, indicating that cloning of an MHC class II transactivator mutated in hereditary MHC class II these mice had a functional immune system (data not shown). deficiency (or bare lymphocyte syndrome). Cell 75: 135–146. 4. Silacci, P., A. Mottet, V. Steimle, W. Reith, and B. Mach. 1994. Developmental ZBTB32ko mice developed wild-type levels of anti-influenza Abs

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Supplemental Figures

Supplemental Figure 1. ZBTB32ko mice form wild-type immune responses to influenza,

Listeria monocytogenes, and LPS. (A) 1x106 splenocytes from wild-type (n=4), ZBTB32ko

(n=4), and Blimp-1cko (n=4) were stained to CD19, I-Ab, B220, CD5 and IgM and analyzed by flow cytometry to assess B cell subsets in the spleen. No significant differences in

B cell populations were observed between the three mouse lines. (B) ZBTB32ko (n=7) and wild-type littermates (n=8) were infected with a 0.1 x LD50 dose of influenza (A/PR/8/34 strain) intranasally. Influenza-specific antibody levels were monitored by hemagglutination inhibition assay (HAI) at the indicated days post-infection. The HAI titer represents the reciprocal of the highest dilution of serum that conferred inhibition of hemagglutination. The values are presented as the geometric mean, +/- standard error of the mean. (C) ZBTB32ko (n=5) and wild-type littermates (n=5) were challenged with 9.8x106 cfu of Listeria monocytogenes i.p in 200 μl PBS.

Infected animals were monitored for weight loss over 14 days post-infection. Data were plotted with respect to the percent change from the initial weight of the animal. (C) ZBTB32ko (n=3),

Blimp-1cko (n=2) and wild type (n=3) mice were injected retro-orbitally with 50 μg LPS or PBS control. Three days post-injection total splenocytes were isolated and stained with anti-B220 and anti-CD138 antibodies and analyzed by flow cytometry to monitor plasmablast formation.

Supplemental Table 1. Primers for RT-real time PCR and Chromatin IP-coupled real time

PCR

Supplemental Table 2. Genes changed more than two fold by cDNA microarray analysis

1 Supplemental Figure 1

A Wild type MZ/FO B cells 100 MZ/FO 88% 86% 80 B1 87% 91% 60

B220 CD19 MHCII 40 CD5 % IgM+ cells 20 ZBTB32ko MZ/FO 87% 81% 0 Wt ZBTB32ko Blimp-1cko B1 75% 82% 10.0 B1 B cells B220 CD19 MHCII CD5 7.5 Blimp-1cko 5.0 MZ/FO 89% 81% % IgM+ cells B1 2.5

85% 92% 0.0 B220 CD19 MHCII Wt ZBTB32ko Blimp-1cko CD5 B C 120 256 9.8x106 cfu Wild type 128 ZBTB32ko Wild type ZBTB32ko 64 100 32 GMT + 95% CI 16 % Weight Loss

8 80 Day 2 Day 5 Day 28 0 2 3 4 5 6 7 8 9 10 11 13 14 Days post-infection

D PBS 50 g LPS Wild type Wild type ZBTB32ko Blimp-1cko

0.23% 4.29% 4.22% 0.86% B220 CD138 Supplement Table 1 RT-real time PCR (Mus musculus ) Genes Sequence 18S rRNA FF 5-GTAACCCGTTGAACCCCATT RR 5-CCATCCAATCGGTAGTAGCG BCL6 FF 5-CACACTCGAATTCACTCTG RR 5-TATTGCACCTTGGTGTTGG Blimp-1 FF 5-TTCAAGTGCCAGACCTGCAACAAG RR 5-TCCAGAATGCAATCGAAGGTGGGT CIITA FF 5-GCAGCTGGACTACAGACGTTAC RR 5-GGAGTTCCAGGTAGCTGCCCTCTG c-Myc FF 5-GGGCCAGCCCTGAGCCCCTAGTGC RR 5-ATGGAGATGAGCCCGACTCCGACC EBF1 FF 5-TCCCAGCCCTTGCTAACAC RR 5-CTTGCAGGCTGTTCCCGTT EZH2 FF 5-CAGTTCGTGCCCTTGTGTGATAGC RR 5-CACTCTCGGACAGCCAGGTAGC IAb FF 5-GACGCAGCGCATACGGCTC RR 5-CCGCCGCAGGGAGGTGCT MTA3 FF 5-CCGAGAGCAAATTGAAACAAG RR 5-AGGAATACCACTGATGAGACTG Pax5 FF 5-CTCCCAGATGTAGTCCGCCAAAG RR 5-GCTTGATGCTTCCTGTCTCATAATACC SpiB FF 5-AGGACTTCACCAGCCAGACC RR 5-TCGGAGCCAGCCAAGAGG SpiC FF 5-AACACTCGCTGAACTCTGG RR 5-GCCTCGCTGAACTGGTAG Syndecan-1 FF 5-GCCACAGGTGCTTCTCAGAG RR 5-TGCCTTCGTCCTTCTTCTTCATG TfDP1 FF 5-AGAGGAGGCTGGAGAGG RR 5-GGTGTTGACAATGATGAAGG XBP1 FF 5-GGGCATTCTGGACAAGTTGGAC RR 5-AGAGAAAGGGAGGCTGGTAAGG ZBTB32 FF 5-CGTCTGGGCAAGGGTTCAC RR 5-CGAGAGCGAGCAGGAGAGG Smyd2 FF 5-CGACCTCCTCTTCTCC RR 5-TTTGACAATCCTTCTTTCC CD19 FF 5-AGGACTGGAAGAAGAAGG RR 5-TCATAAGACTCAGCATTGG

RT-real time PCR (Homo sapiens ) Genes Sequence 18S rRNA FF 5-GTAACCCGTTGAACCCCATT RR 5-CCATCCAATCGGTAGTAGCG CIITA FF 5-GGGGAGCTGAGGGCACG RR 5-GAAGCTCCAGGTAGCCACCTTCTA HLA-DRA FF 5-GAGTTTGATGCTCCAAGCCCTCTCCCA RR 5-CAGAGGCCCCCTGCGTTCTGCTGCAAT MTA3 FF 5-TGTAAGATGCTTTTAAATTCTTAACC RR 5-GTACACAAGAACTTATGTTTATTGC BCL6 FF 5-AATCTGTGGCACCCGTTTCC RR 5-GACAGGTTACACTTCTCACAATGG

Chromatin Immunoprecipitation Region Sequence CIITA pIII FF 5-CATGGGCAAATTAGAGGGTATCC RR 5-GCAAAGAAAGCGAGCAAGGG CIITA Control Site FF 5-CTGGCACCGTGAAGTAACC RR 5-GGACACTTGTCACTTTGATTTAGG ProbeID Symbol Fold Change (Treatment/Control) 844045 Rog 102.225 342663 1700018O18Rik 92.291 879746 Ptgir 68.058 900319 null 64.295 308384 null 42.110 579729 Cdc6 29.967 631939 Exo1 29.722 921504 Mcm10 29.518 503985 Il2ra 28.968 924312 Il6 27.226 553698 null 24.628 448632 Bub1 24.425 793711 2810417H13Rik 24.169 741436 Melk 23.743 643994 Ccna2 23.461 339138 null 23.028 785382 Slc7a3 22.997 640536 Rad51 22.584 638386 Plk1 21.659 920709 2610019I03Rik 20.886 358792 null 20.727 891902 2610034E18Rik 20.352 623101 Pbk 19.341 499488 Rrm2 19.128 794903 Tpx2 18.893 894894 null 18.544 762434 Chek1 18.472 869355 Shcbp1 18.452 840505 null 18.258 918258 Gcat 18.013 500059 Pask 17.253 578430 Clspn 17.110 535303 null 17.036 906073 Cdc45l 17.023 886555 2810417H13Rik 16.775 626312 null 16.737 510606 Birc5 16.448 332733 null 16.045 863113 Rad54l 16.015 311758 Batf 15.940 546148 Chaf1a 15.716 619055 2410030K01Rik 15.524 512087 Fignl1 15.241 687672 Kif11 15.089 734358 BC048355 15.040 616112 F630043A04Rik 14.992 906272 Nek2 14.912 599490 Kif2c 14.654 620108 2310031L18Rik 14.296 560198 Pycr1 14.251 734718 null 14.236 686672 null 14.093 328175 null 13.832 506357 Cdc25c 13.731 312029 2600005O03Rik 13.644 526689 Bcat1 13.629 898210 G1p2 13.517 619764 Top2a 13.492 829558 Ckap2 13.462 901949 null 13.413 790941 null 13.349 678570 Grm6 13.303 802374 Cdca3 13.243 928630 Umpk 13.135 357387 Tmepai 13.077 870834 Car13 12.853 868516 Dhfr 12.690 907537 C330027C09Rik 12.678 682431 Calmbp1 12.624 582194 Mybl2 12.546 733915 null 12.154 666838 Sytl3 11.930 416412 null 11.858 634388 null 11.768 826570 Idi1 11.534 475404 1700022C02Rik 11.503 886579 null 11.202 532035 null 11.119 335286 Pole2 11.115 861380 Sgol2 11.032 906635 Cenph 11.031 673998 null 11.012 548048 Aurka 10.973 565056 Idi1 10.954 513334 2610510J17Rik 10.883 932873 Nusap1 10.694 918096 Prc1 10.677 540278 Bcl2l1 10.591 735288 null 10.515 913394 Cdc20 10.456 806203 null 10.448 822973 Esco2 10.439 562974 Brrn1 10.433 741669 1700013H19Rik 10.420 765925 Cdca1 10.356 727669 null 10.327 812986 Gmnn 10.312 573318 null 10.292 591231 null 10.259 651391 null 10.210 427906 9630033F20Rik 10.103 326239 Aurkb 10.088 920120 Psmc3ip 10.083 741266 null 10.052 765900 Xrcc3 9.962 650716 Dlg7 9.857 761513 5830426I05Rik 9.844 903874 Cks1b 9.761 907969 Hells 9.751 563249 Pole 9.523 759886 null 9.519 389989 null 9.469 742256 Nlgn2 9.442 596934 Stard4 9.387 894728 null 9.366 926789 Uchl1 9.354 841989 2810047L02Rik 9.313 534344 Smyd2 9.284 597024 null 9.238 630135 Anln 9.216 426714 Cdr2 9.189 324997 Dctd 9.091 547181 Cdca8 9.091 383828 null 9.071 749727 null 9.013 898223 Stmn1 8.993 454474 Mns1 8.951 896093 Rangnrf 8.927 766109 Gfi1b 8.908 638897 2610318C08Rik 8.876 590437 C030025P15Rik 8.873 364783 Hk2 8.766 694869 null 8.759 453309 Ccnd2 8.739 902956 null 8.714 828947 null 8.685 898849 null 8.668 516708 null 8.664 674865 9530058B02Rik 8.661 512122 Pyp 8.588 797667 null 8.554 529335 Sqle 8.552 395412 Cenpe 8.547 774391 null 8.449 533701 null 8.345 555651 null 8.335 586296 Gbp1 8.326 607214 null 8.287 371577 Tcf19 8.247 913580 E130303B06Rik 8.220 895554 null 8.005 854585 null 7.946 478962 Ccnb2 7.914 336961 Shmt1 7.870 361838 Nmt2 7.860 912101 Mthfd2 7.859 450069 Hic1 7.829 467529 Hmgn3 7.787 898407 1810014L12Rik 7.781 452235 2610016C23Rik 7.714 685710 Cetn4 7.698 735112 Tnmd 7.649 932567 Nup43 7.628 311449 MGC65590 7.600 355315 null 7.570 892804 null 7.559 902241 Hspd1 7.527 685000 null 7.526 916448 Dut 7.526 640447 Dhcr24 7.520 791114 Tfdp1 7.473 686820 null 7.469 376244 Mcm5 7.442 557978 Adk 7.407 592921 Pfkp 7.370 763321 Recql4 7.222 327034 Slc39a4 7.191 919600 Tipin 7.176 901980 Srm 7.152 784383 Kifc1,Kifc5a 7.134 381222 null 7.133 920113 Slc7a5 7.113 782101 C86302 7.105 647686 2510040D07Rik 7.096 466841 Ccne1 6.996 628939 Cpox 6.983 375582 Neil3 6.983 893211 null 6.912 533605 Prdm16 6.908 443685 Racgap1 6.905 804474 Cbx5 6.887 628682 Bbc3 6.871 914355 Dna2l 6.813 895391 null 6.781 512605 null 6.776 620528 null 6.737 551844 null 6.732 772486 Iqcg 6.728 307095 Ccbl2 6.690 765151 Esrrbl1 6.634 379245 Slc43a3 6.629 797171 null 6.612 780225 Traf1 6.589 900801 Kpna2 6.559 427106 2410015N17Rik 6.555 485202 Acp1 6.548 933097 Serf1 6.528 667857 null 6.501 658612 Ezh2 6.467 924562 Oasl1 6.452 831955 Tk1 6.435 659722 null 6.435 674325 Ahrr 6.402 597826 Bach 6.399 511025 Zmynd19 6.328 532793 Kif23 6.275 906716 Dorz1 6.266 363112 6820428D13 6.265 581603 null 6.228 611050 Rfc4 6.221 557646 Camkk1 6.210 920912 Prim1 6.209 611175 Mcm6 6.187 917597 Tacc3 6.170 911031 Mcm4 6.130 368164 Atp10a 6.128 360661 null 6.125 594573 Hesx1 6.120 916661 Psat1 6.097 887390 null 6.088 626096 null 6.072 332903 Cbx2 6.007 603698 null 5.997 917926 Acat2 5.984 923925 Orc6l 5.979 731398 null 5.949 303484 Adh5 5.948 916326 4921516M08Rik 5.942 901543 null 5.934 743155 Ranbp1 5.931 895153 Hist1h2an 5.879 893894 Psat1 5.870 736750 Bub1b 5.828 745122 Hdhd3 5.787 922169 Tubb5 5.781 909303 Coq4 5.759 551930 Stard4 5.755 743006 Ctps 5.725 648897 1110019C08Rik 5.716 374467 null 5.698 587689 null 5.681 730737 Acat2,Acat3 5.670 923068 Chc1 5.664 909892 Hmgcs1 5.656 621280 null 5.638 841819 Mthfd1 5.629 900209 Timm8a 5.614 469328 Tnfrsf22 5.612 918397 Mxd3 5.580 473479 Gsg2 5.561 353849 9530077C05Rik 5.443 579825 null 5.440 648457 Ms4a6c 5.407 921580 2610207P08Rik 5.381 812909 2810028N01Rik 5.371 862993 Ase1 5.367 892092 Hspd1 5.367 882247 null 5.348 713706 Bysl 5.348 310752 null 5.319 316250 Traip 5.315 549862 3110082I17Rik 5.307 513645 Kif18a 5.283 477588 Gmds 5.280 919540 Nudcd2 5.247 351910 null 5.225 662947 BC006933 5.209 437927 Siva 5.203 466290 D2Ertd750e 5.184 544530 2210013M04Rik 5.183 930118 Cdc25a 5.172 634387 Cyp51 5.159 916660 Arl6 5.157 906600 Nudt1 5.115 924003 Pecr 5.112 309759 1110001A07Rik 5.079 918108 Timeless 5.070 343919 Chchd4 5.070 715000 null 5.065 401199 Gypc 5.051 631704 null 5.038 409046 Fads1 5.033 524960 null 5.029 781027 null 4.993 926089 Pxmp2 4.993 434321 null 4.986 739760 4932411G06Rik 4.982 900707 Timm8a 4.968 453860 null 4.946 644189 null 4.945 470889 null 4.943 932283 2010204K13Rik 4.920 815016 Plscr1 4.916 895998 null 4.912 618403 null 4.901 446807 Usp18 4.900 894173 Mad2l1 4.864 903015 null 4.855 921193 2810027O19Rik 4.851 757700 Bdh 4.847 916992 Sdf2l1 4.842 906981 Incenp 4.841 755735 null 4.838 429529 Lsm2 4.834 439826 Ranbp5 4.807 807657 Cdkn2b 4.800 804312 Stxbp1 4.783 846150 null 4.768 770406 Fah 4.767 454605 2010305C02Rik 4.766 480631 Pdxk 4.761 698794 2010005O13Rik 4.753 438817 Blm 4.750 634992 Nme6 4.733 495463 null 4.723 484926 null 4.699 893502 Fdft1 4.693 528616 Gpr56 4.675 543499 Pmf1 4.673 478404 Top1mt 4.667 909447 Nola2 4.663 616642 Hsp105 4.660 900610 Ahcy 4.647 900361 null 4.619 421573 Pitrm1 4.603 561240 Tfrc 4.599 878246 Atic 4.596 430706 null 4.590 538384 null 4.561 896653 Mad2l1 4.540 696218 Hat1 4.532 346803 Pold2 4.512 877508 null 4.500 687391 Lactb2 4.500 633417 Txnl1 4.489 309470 null 4.479 542220 Sc4mol 4.456 510362 Atad2 4.445 895165 Tuba6,Tuba2 4.435 553944 Mre11a 4.408 918052 Col4a6 4.395 629558 Rnmtl1 4.376 392163 Tbc1d19 4.370 899390 Mif 4.369 905986 Mrps28 4.366 884118 Gzmb 4.365 929926 Dtymk 4.357 737877 null 4.352 803370 Tada2l 4.346 557165 Mrps33 4.344 729309 Hcr 4.313 729603 Brp17 4.308 919849 1110025F24Rik 4.297 827068 null 4.292 902972 null 4.271 493908 Rpp40 4.270 473288 null 4.266 421496 1810003N24Rik 4.252 417446 Pgk1 4.244 362187 null 4.244 604511 2410127E18Rik 4.243 747549 E2f6 4.230 433268 C030036P15Rik 4.210 735439 Wdr12 4.188 532882 null 4.183 459310 MGC47262 4.164 656030 null 4.164 911822 Echdc1 4.164 435645 Hsd17b7 4.153 372084 null 4.146 366298 null 4.145 546383 Orc2l 4.126 450459 1810054D07Rik 4.126 300312 null 4.119 927036 Dscr2 4.111 656907 Wdr4 4.111 773450 4933434G05Rik 4.110 925148 1700022L09Rik 4.109 919526 6330503K22Rik 4.109 529323 null 4.105 557237 null 4.103 365892 null 4.099 892563 null 4.076 559603 Mvd 4.073 490831 null 4.073 911598 C1qbp 4.071 647423 1810044O22Rik 4.061 931116 Kifap3 4.054 761831 Nolc1 4.045 549097 null 4.036 872360 Cdk5r1 4.035 436575 Gclm 4.033 556873 5730438N18Rik 4.019 744941 Wdr18 4.008 557227 Dnahc11 4.005 838809 null 3.998 903368 null 3.995 895753 null 3.982 862616 null 3.978 335619 null 3.969 709270 null 3.969 656624 D13Wsu177e 3.959 334822 Sfxn1 3.948 793338 Rad18 3.943 378511 Shmt2 3.939 609753 6720467C03Rik 3.922 529145 null 3.921 903671 null 3.921 879898 null 3.909 900427 null 3.909 489566 null 3.909 905997 Prim2 3.900 328460 null 3.894 496310 BC050092 3.889 913252 Fen1 3.885 890996 Impa2 3.883 392984 Bard1 3.880 902264 null 3.872 763444 Tmem20 3.860 836724 Ruvbl2 3.854 844157 Rasl2-9 3.842 714716 Gpatc4 3.835 896822 null 3.831 629624 null 3.829 334079 A130010J15Rik 3.823 547262 null 3.813 795197 Tbl3 3.810 632672 2610029G23Rik 3.807 726921 1110004E09Rik 3.799 649700 Ipo11 3.797 489051 2610529H08Rik 3.789 704736 Cdca2 3.781 461851 Tex292 3.776 674858 Rnu3ip2 3.770 855683 Mrps22 3.768 628393 AU067695 3.764 523427 Gart 3.764 856164 Polr3k 3.763 517154 Tnpo1 3.758 362780 null 3.757 537229 Hist1h2aa 3.748 662028 null 3.747 911061 Myohd1 3.746 812709 D10Ertd214e 3.746 902940 null 3.733 781531 null 3.726 406199 Rpusd2 3.723 308790 null 3.713 904753 Ccdc2 3.713 922379 Slc19a1 3.708 712337 Olfr663 3.703 455381 Nudt2 3.702 718537 Rps27l 3.700 897385 Atp5g1 3.696 893727 Csnk2a1 3.685 714790 Bzw2 3.680 362464 Ubl4 3.662 911668 425O18-1 3.659 510118 null 3.648 894005 Cycs 3.640 587499 Timm13a 3.632 530529 Gas2 3.622 680985 Zwint 3.620 710672 2810430M08Rik 3.619 459404 Nme1 3.615 789576 null 3.613 805956 null 3.607 548757 Tubg1 3.598 895161 Phgdh 3.590 631038 Lap3 3.589 856332 V1rc16 3.575 588131 Mtap 3.572 467179 Cct6a 3.569 825717 Nol5 3.567 839537 null 3.565 893264 Pcna 3.560 913940 Csda 3.558 341773 null 3.552 614386 null 3.548 830078 null 3.547 756734 D4Ertd22e 3.541 902685 Tuba6 3.540 504772 Zdhhc21 3.533 542487 null 3.530 894795 null 3.527 880784 null 3.522 810417 9430097H08Rik 3.518 624336 Elovl6 3.513 648105 Mrpl1 3.511 820967 Bing4 3.510 560146 null 3.497 306708 AF233884 3.496 892649 Prdx1 3.492 743549 Gnl3 3.491 763298 Tbc1d7 3.487 817839 null 3.486 900401 Cycs 3.479 897674 Ldh1 3.479 479599 null 3.478 885791 Ifrd2 3.477 640297 null 3.477 303435 2310010B21Rik 3.476 804789 null 3.468 896174 null 3.447 610478 Gstt3 3.444 493273 null 3.440 893075 Hist2h2ac,Hist1h 3.439 454844 0610016J10Rik 3.437 664698 ICRFP703B1614 3.435 898460 D10Ertd322e 3.426 541892 null 3.423 459783 Cltb 3.419 718549 Ube2n 3.414 922648 Nudc 3.411 594543 Pgea1 3.411 760792 AK122525 3.410 900983 Nudc 3.410 847876 Gbp2 3.407 861845 Mrps5 3.402 900592 Amd1 3.398 440444 null 3.397 913776 Pard6g 3.392 691042 9630054F20Rik 3.392 507308 Ppid 3.392 704921 null 3.385 331697 2610510H01Rik 3.384 658449 Hrmt1l2 3.377 906458 Abhd5 3.376 857532 null 3.370 432477 1700011F14Rik 3.366 898825 null 3.365 905667 Stip1 3.357 908629 Aven 3.346 688390 null 3.338 631876 null 3.334 919030 Grwd1 3.331 331036 null 3.330 541128 Prkr 3.326 922492 Idh3a 3.319 816659 BC022692 3.316 864858 Spg20 3.313 896798 2810025M15Rik 3.313 760299 1110051M20Rik 3.312 931571 null 3.309 449526 null 3.303 903569 Rrs1 3.300 887708 Slc39a14 3.299 429119 null 3.299 900064 null 3.292 896515 Tubb3 3.290 881483 2810453L12Rik 3.289 573975 Gemin5 3.287 613644 2700097O09Rik 3.283 456571 Trip3 3.280 493129 AI326906 3.280 850381 Tnfsf5ip1 3.278 876623 AB112350 3.278 739970 Mrps25 3.275 767453 null 3.268 368438 Pole3 3.259 864371 null 3.257 902474 Ruvbl1 3.256 392895 Ero1l 3.256 331954 Paf53 3.252 453393 Polr3f 3.252 741552 Bst2 3.248 855066 Slco3a1 3.248 320576 Nup133 3.245 833148 Smn1 3.237 898626 Mrpl47 3.236 903921 null 3.227 548465 Rps24 3.221 552143 Cct3 3.216 894280 Mphosph6 3.213 712928 null 3.205 812255 null 3.204 695039 1500011K16Rik 3.200 704039 Agpat5 3.192 355299 null 3.191 855911 3830402I07Rik 3.168 346055 null 3.168 920081 Aprt 3.168 613798 2310042G06Rik 3.165 569199 null 3.164 907805 Snrpa1 3.162 897209 Ppih 3.161 726213 null 3.160 745835 Herc5 3.148 783758 Ran 3.142 741949 Lzic 3.142 687981 2310061C15Rik 3.139 790415 null 3.137 433814 null 3.132 407388 null 3.126 916109 1700021F05Rik 3.124 813812 null 3.121 871089 Thoc4 3.111 693365 null 3.110 503883 null 3.110 887462 Rbmx2 3.106 907017 Ncl 3.097 892386 Ube2n 3.094 900558 null 3.092 865413 null 3.092 453158 null 3.085 864277 null 3.085 906999 Mtx2 3.083 782419 null 3.080 494056 null 3.080 850083 Hist1h2bk 3.075 645357 null 3.074 918832 null 3.069 316311 null 3.066 669114 Snrpd3 3.065 760949 null 3.064 901343 Cbx3 3.063 669762 null 3.062 900091 Trim13 3.060 905459 Hbb-bh1 3.054 351003 Mad2l2 3.053 873436 null 3.051 798923 Sap30 3.049 304158 Rbl1 3.043 881667 Rfwd3 3.031 732795 Sfrs3 3.022 601714 Acy1 3.021 706194 1810060D16Rik 3.019 456269 Casp3 3.017 528718 null 3.013 564267 Lnp 3.009 442170 null 3.009 686285 Mrpl18 3.006 337412 2610528H13Rik 3.004 925883 Impdh2 3.004 916039 AI429152 3.004 723269 null 3.000 903736 Phgdh 2.996 578498 null 2.992 722633 Pcnt1 2.991 371033 Slfn1 2.985 563142 Pphln1 2.983 547313 null 2.972 849739 Ppil1 2.972 621903 Ebna1bp2 2.971 508037 null 2.970 633117 null 2.968 900896 null 2.964 904245 null 2.954 930477 Fdx1 2.949 921013 null 2.943 531320 null 2.942 866664 null 2.942 929950 5730596K20Rik 2.942 345274 null 2.940 558132 Sf3b4 2.937 621410 Exosc8 2.934 612791 Fbl 2.933 901753 Ppid,Lamp3 2.931 920159 Uchl5 2.930 394027 Prps1 2.926 369930 Ercc8 2.924 693435 2310043N13Rik 2.921 725563 Cops3 2.918 795835 null 2.915 602438 Exosc2 2.914 691280 null 2.913 780272 6230416J20Rik 2.913 793761 Rpl14 2.907 382804 Thumpd1 2.905 305157 null 2.901 584877 null 2.898 783567 2410075D05Rik 2.898 879787 null 2.897 310484 null 2.896 588923 null 2.895 446434 null 2.895 326200 Psmd12 2.894 918657 4122402O22Rik 2.894 839914 2900010M23Rik 2.893 916253 Sepw1 2.891 764339 Rdbp 2.884 831167 Rpp30 2.879 659648 null 2.877 809776 null 2.874 321940 Rbbp7 2.873 903349 null 2.867 335030 Hdac2 2.867 928939 Spata5 2.865 842988 Gtf2h4 2.865 676587 null 2.865 656522 null 2.862 904194 null 2.859 911702 Armet 2.857 309186 2410080P20Rik 2.854 468332 Hnrpab 2.851 330594 Cops5 2.849 537276 2410002O22Rik 2.849 653356 null 2.846 903894 Hnrpa3 2.844 451990 Polr2h 2.842 921350 Gadd45gip1 2.841 694718 Rcl1 2.839 887925 Prkar2a 2.838 775191 null 2.837 689028 null 2.836 916971 Ccdc5 2.833 576306 null 2.829 526667 Mars 2.827 608198 null 2.824 370884 Olfr1106 2.822 914514 Mtbp 2.822 912671 2510038A11Rik 2.819 899204 Ube2v2 2.818 906740 2310057D15Rik 2.818 903599 null 2.812 787545 4930415J21Rik 2.807 813895 null 2.806 486818 Lipc 2.803 799772 D15Ertd682e 2.802 906254 2510005D08Rik 2.801 339470 Olfr290,Olfr291 2.799 365566 null 2.789 352380 D2Wsu81e 2.787 903346 Hspe1 2.785 577273 Kpnb1 2.780 639591 null 2.776 682171 0610042I15Rik 2.775 300605 Exosc9 2.772 745959 null 2.770 433323 null 2.767 913978 1700019E19Rik 2.766 899637 Ppp1r14b 2.765 893253 null 2.765 800513 null 2.762 902773 null 2.754 905543 0610009I22Rik 2.753 392854 null 2.751 366637 null 2.749 714679 null 2.749 831702 Mrpl35 2.747 896326 Npm3 2.736 375614 Apex2 2.732 902190 null 2.730 420488 null 2.727 384420 null 2.725 678695 null 2.723 562661 null 2.722 784384 2810437L13Rik 2.717 932428 Anp32e 2.715 579707 Mbnl3 2.714 927388 Mki67ip 2.710 392509 Ppp1r7 2.708 456417 null 2.708 892972 Gapd,LOC14433 2.702 931802 null 2.694 410126 1700009P17Rik 2.693 339449 U2af1 2.690 929933 1810037G04Rik 2.690 660657 null 2.687 316944 3010026O09Rik 2.685 897650 Snrpd2 2.684 667865 Il12rb1 2.681 915586 Banf1 2.679 658631 Gphn 2.678 379870 Mrps7 2.674 903753 null 2.673 347051 Msh6 2.670 460900 AA959742 2.667 398302 D7Wsu128e 2.667 574392 Myef2 2.666 925369 Arl6ip6 2.665 587998 null 2.660 343858 Galk1 2.659 597734 Hist1h1a 2.658 705526 null 2.652 324166 null 2.648 930927 Zcchc10 2.648 902994 Cd2bp2 2.646 719517 Ap1s1 2.645 921070 Rars 2.644 315886 0610009F02Rik 2.639 440209 Dars 2.638 438164 Zcwpw1 2.638 897487 Nme2 2.634 783167 Olfr54 2.633 580322 Smfn 2.633 451704 Arbp 2.633 897021 null 2.632 620920 null 2.632 896957 Iars 2.631 389366 2210404G23Rik 2.630 927483 Lmnb2 2.627 723934 Pola2 2.626 828518 AW742319 2.623 894735 Hnrpa1 2.619 817890 BC066028 2.617 378176 Itpa 2.617 561277 Alg8 2.616 762530 Supt16h 2.614 891684 Psmb5 2.613 459636 Thy28 2.612 517788 D19Bwg1357e 2.610 329038 2610204M08Rik 2.608 912244 Ndufa5 2.606 798728 Sephs1 2.603 918644 Gtf3c5 2.603 903301 null 2.602 465532 BC034664 2.602 725956 null 2.599 382361 Nans 2.598 596044 Fasn 2.597 778267 null 2.595 899243 null 2.593 847838 Arl2 2.593 537344 Cct8 2.590 829442 Fkbp1a 2.590 654850 Mftc 2.590 444821 null 2.589 900999 Snrpa 2.589 378405 2700083B06Rik 2.588 925720 Mrpl2 2.587 928678 Itgb1 2.587 922864 Tnnt1 2.586 643007 null 2.585 898562 null 2.584 667619 Kars 2.583 697021 Med9 2.583 898843 Npm1 2.582 361288 Ftsj3 2.581 392862 Phb 2.579 382291 Ppif 2.572 657300 Ddx39 2.571 690330 null 2.566 552853 Dusp12 2.566 652224 1600013P15Rik 2.564 716182 null 2.562 900433 null 2.554 453584 Acsl3 2.554 502017 Aebp2 2.553 898848 Ssfa1 2.552 910155 null 2.547 546500 Dhdds 2.545 827126 null 2.541 903234 null 2.541 714520 Kdelc1 2.540 932509 A930011F22Rik 2.539 901777 null 2.539 817387 Pms1 2.538 719085 Zfp106 2.538 457575 Fancl 2.537 830517 Psme3 2.534 926171 Eif3s9 2.533 580691 Lsm3 2.531 323923 Ftsj1 2.529 901613 null 2.524 416021 Mrpl16 2.524 422898 null 2.519 366364 Lsm7 2.514 435099 Eif2b1 2.512 915410 Rbm19 2.512 375231 null 2.511 930137 null 2.511 925205 9130416N05Rik 2.510 557563 Ncbp2 2.505 919754 Pwp2h 2.502 930983 Dtnbp1 2.502 320617 Psma7 2.501 487523 Nars 2.501 549233 Bhlhb9 2.499 321715 null 2.497 374230 null 2.496 463797 null 2.492 755101 Hus1 2.489 902389 null 2.487 903688 Eif4a1 2.485 923632 Ogg1 2.484 780705 null 2.483 854747 Dek 2.482 652873 null 2.478 449900 null 2.477 724513 Mrpl55 2.477 731074 Nsun2 2.473 656797 null 2.470 353640 null 2.467 702134 Pinx1 2.463 765507 null 2.463 914732 2810441K11Rik 2.461 429201 Ankrd32 2.458 804551 null 2.456 454017 Rpa2 2.455 926146 Mrps31 2.455 896148 null 2.454 813115 null 2.450 332242 Cugbp1 2.450 322776 null 2.448 513530 Pgm1 2.447 388026 null 2.444 744878 Smyd3 2.442 705143 Mrpl28 2.441 785462 Samd8 2.439 361626 Rcn1 2.437 918866 Mrpl50 2.432 686212 3110010F15Rik 2.432 400247 G22p1 2.430 442221 Mrpl36 2.429 588026 null 2.425 899685 Snrpd2 2.421 725464 Sema7a 2.414 585468 D3Ertd194e 2.413 897538 Cdc34 2.412 776862 Ap2b1 2.411 919369 1700030A21Rik 2.409 460924 Gas8 2.405 658889 null 2.399 639587 Gng12 2.399 868644 null 2.398 522574 2900091E11Rik 2.396 916041 Aaas 2.396 638629 Cpsf5 2.395 464798 Nsbp1 2.392 924321 Rangap1 2.382 855531 Nek4 2.380 761899 null 2.375 371326 null 2.374 544680 Fcrh3 2.372 901896 null 2.371 895264 Socs2 2.370 848564 2610027O18Rik 2.369 685948 Rtn4ip1 2.368 872644 null 2.364 801860 null 2.360 741835 Agpat4 2.360 752232 null 2.358 646239 Mrpl14 2.357 412751 Tkt 2.357 893296 Hnrpa1 2.357 360210 Bop1 2.355 366084 4933425I22Rik 2.355 383055 null 2.355 921888 2310047O13Rik 2.354 789567 Smc4l1 2.351 534311 null 2.351 740280 Psip1 2.350 635042 Trnt1 2.349 627586 null 2.349 767843 Pdhx 2.348 665747 Tbrg4 2.347 750973 Rpe 2.345 437843 Gca 2.342 386178 null 2.341 496013 3110003A22Rik 2.335 378072 2010208K18Rik 2.333 642833 null 2.332 422109 Tcp1 2.328 419682 Mrps18b 2.328 932045 2410195B05Rik 2.326 835347 Klhdc4 2.325 807992 Ddx27 2.325 650993 Arl10c 2.324 414755 Sip1 2.322 595386 9130401M01Rik 2.319 913261 2400006A19Rik 2.319 930840 Uble1a 2.318 743028 null 2.317 919794 Psmc6 2.317 781737 null 2.316 909335 B130055D15Rik 2.314 763533 Abce1 2.313 561492 null 2.312 928852 null 2.311 765669 Arhgap11a 2.310 892644 null 2.305 760080 null 2.302 904196 Psma5 2.301 574092 Hrpt2 2.301 566447 Nomo1 2.298 899317 Anp32b 2.296 896953 null 2.290 922033 null 2.287 643809 Noc4 2.285 576909 Paxip1 2.283 331133 null 2.283 896014 Eif2s3x 2.282 362457 1190005P17Rik 2.280 924567 Hist1h4m,Hist1h4 2.278 805560 1200014J11Rik 2.275 920381 Hs1bp1 2.275 630180 null 2.273 686105 null 2.271 929523 Txnrd3 2.270 666365 Ppan 2.269 374955 Dhx33 2.268 729339 null 2.268 681625 null 2.267 481346 Mulk 2.267 446187 Psma6 2.266 834065 Psmc5 2.265 303226 null 2.261 552458 null 2.253 425912 null 2.252 754013 Arl3 2.250 929416 Cct2 2.248 640249 Mrpl32 2.243 339417 null 2.243 411258 AL033314 2.238 748512 Tars 2.236 369506 null 2.233 791646 Vegfb 2.233 576001 null 2.233 904594 Psmd2 2.233 766610 null 2.232 920354 Magoh 2.231 652602 D330028D13Rik 2.230 299116 Rpp14 2.229 745505 null 2.228 907607 Rnmt 2.228 503224 Sc5d 2.227 452127 Dgcr6 2.227 369139 Tomm70a 2.226 462877 1810043M15Rik 2.225 346999 null 2.225 439528 Srp19 2.222 381336 null 2.220 892057 0 2.219 492890 2410091C18Rik 2.218 338441 Mut 2.218 552055 BC021395 2.217 892920 Eif3s10 2.216 894189 null 2.216 896494 null 2.215 739996 Gfer 2.211 925929 Hnrpd 2.209 893322 null 2.206 452189 Gtf2e2 2.205 595676 Fubp1 2.205 329248 null 2.203 924534 null 2.202 811742 null 2.202 334723 Nrbf1 2.199 680078 Ddx21 2.195 413650 Psmc2 2.193 829303 Sec24a 2.192 342005 2700038L12Rik 2.189 388007 Ces2 2.188 390775 2610027L16Rik 2.186 305378 Ahsa1 2.183 925081 Chchd1 2.182 577661 Slc2a6 2.182 901333 Tdg 2.179 421788 null 2.175 661344 null 2.171 840801 null 2.171 493945 null 2.170 908506 Slc39a11 2.167 825683 null 2.166 896749 1110014J01Rik 2.166 896685 Pabpc4 2.164 345537 Atpaf1 2.162 903851 Atp5o 2.162 461890 null 2.160 894817 null 2.158 748291 Tnip1 2.157 377118 null 2.154 396711 null 2.153 897017 Ifitm2 2.150 907246 Hprt1 2.150 797342 Psmb7 2.148 623205 Gmppb 2.146 892545 1810056O20Rik 2.145 406166 D1Wsu40e 2.145 694977 BC003885 2.145 673962 Enc1 2.143 403894 null 2.142 466910 Slc31a1 2.141 918134 1110025L05Rik 2.137 447645 Blmh 2.134 576286 Cct5 2.131 818156 Gtf2f1 2.131 899527 null 2.129 562048 null 2.128 932500 null 2.128 582956 Phf6 2.126 591700 null 2.125 854843 Ash2l 2.121 768759 null 2.119 867868 Phf10 2.117 536661 null 2.110 823005 9430083G14Rik 2.109 665063 Emb 2.108 902024 Sod1 2.105 360511 Ndufb3 2.105 580194 null 2.104 485549 2810422B04Rik 2.104 921676 Pus1 2.102 783349 Sh2d2a 2.100 569681 null 2.099 929202 1200009K13Rik 2.098 559349 Eif1ay 2.098 901544 Snrp1c 2.097 894157 2600013N14Rik 2.096 547782 Rwdd1 2.096 880049 2010003J03Rik 2.094 831979 Afg3l1 2.093 415222 Ndufa9 2.093 643313 null 2.091 893635 Hmgn2 2.091 456651 null 2.088 326972 Mov10 2.087 641765 2210415M20Rik 2.087 924631 Hspcb 2.084 883725 1110020P15Rik 2.082 678418 Ptpn12 2.079 537632 Park7 2.079 633303 Tmem38b 2.072 365301 Anapc10 2.072 892769 Pdcd5 2.071 897700 Uqcr 2.069 893834 null 2.062 554463 Serpine2 2.061 705975 Mrpl39 2.060 409582 Pfdn1 2.059 397805 null 2.058 908272 Pkp4 2.056 784094 1810044O22Rik 2.055 665176 Mnat1 2.054 742632 Cct4 2.053 532062 2610528A15Rik 2.050 904214 null 2.049 898453 Ndufc2 2.049 897170 2010100O12Rik 2.047 301240 Slc7a6 2.045 343526 Mrpl54 2.045 554699 null 2.044 743668 Rbmxrt 2.042 742742 null 2.041 901167 Phf5a 2.038 572105 null 2.037 435967 null 2.037 931085 Arts1 2.035 874504 null 2.029 349989 null 2.027 675389 null 2.026 879563 Oas1f 2.023 480641 null 2.022 875318 null 2.021 710677 Hrb2 2.019 901208 null 2.018 654873 Itpk1 2.018 851942 null 2.015 301035 Mrps12 2.012 755394 Actl6a 2.012 631370 Utp14a 2.011 603516 null 2.011 924639 Rpa1 2.007 466277 Ampd2 2.007 497004 Kctd5 2.005 693022 F830014G06Rik 2.005 901549 null 2.005 572511 Tceb3 2.004 896960 null 2.003 798805 null 0.500 888902 Olfr312 0.500 429239 null 0.499 438101 null 0.499 443584 Krt2-7 0.498 913373 null 0.498 463865 null 0.498 497010 null 0.497 826419 null 0.497 918064 0610007C21Rik 0.497 914947 Vps54 0.496 703853 2610024E20Rik 0.496 692121 Dnchc1 0.496 631331 null 0.496 723617 Dap 0.496 671932 null 0.496 405189 null 0.495 568880 Olfr1049 0.495 577279 null 0.495 447853 LOC56628 0.493 919480 null 0.493 393647 Snx5 0.492 590082 null 0.492 745234 null 0.492 389735 Zfp276 0.492 897948 null 0.491 363983 null 0.491 726641 Mark2 0.491 533945 Csad 0.490 328805 Aldh3b1 0.490 330820 Papolg 0.490 697673 null 0.489 914762 Gpr108 0.489 835249 null 0.489 388471 Ccnt2 0.489 707068 Mbc2 0.488 896523 null 0.488 628307 null 0.487 901690 Map1lc3b 0.487 788029 Dgat1 0.487 930156 1810059G22Rik 0.486 334840 null 0.486 797234 null 0.486 666425 Dexi 0.485 673600 null 0.485 588156 null 0.484 902232 null 0.484 740768 null 0.484 920968 H2-Eb1 0.483 865317 null 0.482 479671 null 0.482 380936 null 0.482 719851 null 0.482 379196 null 0.482 761785 null 0.482 602516 AI450540 0.481 818523 Pax5 0.479 931074 Elf2 0.479 557371 null 0.477 894009 H3f3a 0.477 919244 Crlf3 0.477 388152 null 0.477 427729 null 0.476 707522 1810038H16Rik 0.476 924020 Plac8 0.476 892170 Ccni 0.476 806556 null 0.475 900977 null 0.475 882835 null 0.475 907602 Csnk1g2 0.475 771602 null 0.475 736226 null 0.474 663313 B3gat3 0.474 738581 1110014K08Rik 0.474 914005 5730453I16Rik 0.473 635703 4933421G18Rik 0.473 523804 null 0.472 857032 null 0.472 387887 Ms4a1 0.472 442062 null 0.471 726622 Capg 0.471 932761 Cklfsf6 0.471 479437 null 0.471 857639 null 0.471 580842 Scel 0.470 918558 Drd1ip 0.470 581847 null 0.469 298770 Hist2h2bb 0.468 400498 null 0.468 909602 Pdlim3 0.468 371253 Lamp1 0.468 635079 null 0.467 565091 Snx8 0.467 393745 Lrp10 0.466 603345 Gripap1 0.466 302328 Kctd2 0.466 529703 null 0.466 615665 null 0.466 785909 BC025458 0.466 340636 Il10ra 0.465 661801 null 0.465 372826 null 0.465 570468 Arrdc1 0.464 442475 null 0.463 741113 null 0.463 899036 null 0.463 305669 null 0.463 927740 Clcn7 0.462 406844 Snx14 0.462 402065 null 0.462 808065 null 0.461 650375 null 0.461 914100 null 0.461 358271 Pou2af1 0.460 463221 E030024M05Rik 0.460 381105 null 0.460 437192 null 0.460 830805 null 0.460 338867 null 0.459 783097 null 0.458 421110 null 0.458 579218 null 0.458 569489 null 0.458 298527 Nek9 0.458 886330 null 0.458 530006 Galnact2 0.457 531414 Hemp1 0.457 778564 null 0.456 789466 null 0.455 455840 Mefv 0.455 680222 null 0.455 432244 Ddb2 0.455 331677 Arid4b 0.454 872778 null 0.454 928460 Amfr 0.454 872375 Spry2 0.454 446655 Zfp513 0.454 865647 Ap4b1 0.454 806549 6330583M11Rik 0.454 630663 null 0.454 658871 null 0.453 885398 Tram1 0.453 904213 null 0.453 575175 1300018J18Rik 0.453 838078 Rnf111 0.453 532159 null 0.452 483305 null 0.451 894506 BC002236 0.451 889212 Rab3il1,C76746 0.451 928427 Lmo6 0.451 455696 null 0.451 898975 null 0.450 318496 null 0.450 821216 LOC434320 0.449 542925 Tex27 0.449 625863 Gp5 0.448 822241 Arsa 0.447 493546 null 0.447 348117 null 0.447 742436 Inpp5a 0.447 337107 null 0.446 826423 Fez2 0.446 639983 null 0.446 658873 null 0.445 433192 Ttyh3 0.445 358886 Als2cr2 0.445 898855 null 0.444 383522 null 0.444 829690 Cd84 0.443 431322 Gba2 0.443 371448 null 0.443 457775 Grcc10 0.443 893142 null 0.443 920749 null 0.442 587616 Fhod1 0.441 824077 null 0.441 754894 Rhbdf1 0.441 404821 null 0.441 678826 null 0.441 560400 null 0.441 553636 Lnk 0.440 409843 8430411H09Rik 0.440 747095 null 0.440 869794 null 0.439 659293 Polm 0.439 375473 null 0.439 638154 Mical1 0.438 489722 Ogt 0.438 788603 null 0.437 411187 null 0.437 574167 Herpud1 0.437 331406 null 0.436 391537 Doc2g 0.435 388536 null 0.435 921760 Tpd52 0.435 742698 null 0.434 375969 null 0.434 522483 null 0.434 716720 H2-Q7 0.434 823261 null 0.433 632659 Cybasc3 0.433 587251 null 0.432 892758 null 0.432 652926 Gba 0.432 573670 null 0.432 758320 Clk2 0.432 652598 null 0.430 734954 null 0.430 437166 Flcn 0.430 797351 null 0.430 502664 null 0.429 691011 Fchsd2 0.429 925688 Camta2 0.429 323988 null 0.429 459528 1300002F13Rik 0.428 924582 Capn1 0.428 856087 null 0.428 697495 null 0.428 607662 null 0.428 360591 null 0.427 596021 null 0.427 348873 Limd1 0.427 561061 null 0.427 897277 Arpc5 0.427 885628 Ripk4 0.427 789841 1810031K17Rik 0.426 525826 Tulp4 0.426 394475 null 0.425 904028 null 0.425 661185 Tcirg1 0.425 558144 Gimap6 0.425 633131 BC012278 0.425 906047 Ccndbp1 0.424 544993 null 0.424 607891 null 0.424 891891 null 0.424 700649 4833420G17Rik 0.424 872475 Abcd1 0.424 628711 null 0.423 327517 Tgfbr2 0.423 394336 null 0.423 520843 Ceacam1 0.423 651771 null 0.423 778341 null 0.423 899560 BC002236 0.422 355965 Glul 0.422 301005 null 0.422 924237 Pold4 0.422 803192 null 0.422 527243 null 0.422 862842 null 0.422 469428 null 0.421 363984 null 0.421 424942 null 0.421 894874 null 0.420 923624 Ccnl2 0.420 894095 Glcci1 0.420 411078 null 0.419 332421 null 0.419 856654 Klf7 0.419 589177 Galnt1 0.419 930444 5530601I19Rik 0.418 852412 ORF18 0.417 831790 Ralgps2 0.417 351250 BC006909 0.417 846189 null 0.417 823783 null 0.416 906617 2610318G18Rik 0.416 486493 Gtlf3b 0.416 848841 Birc3 0.416 404184 9830124H08Rik 0.416 788195 null 0.416 699693 Mybpc2 0.415 317748 null 0.415 671051 4732481H14Rik 0.415 815742 null 0.415 640616 null 0.415 914040 Waspip 0.415 932416 null 0.414 440329 null 0.414 446731 Mapk12 0.414 646949 Ids 0.414 332283 null 0.413 625407 Map3k3 0.412 738393 Oxt 0.412 924203 Usp52 0.412 913609 Vamp8 0.411 313669 Dock7 0.410 443708 null 0.410 632372 null 0.409 497635 null 0.408 460155 null 0.408 913567 null 0.408 894160 null 0.408 435912 null 0.407 335818 null 0.407 705537 null 0.407 739022 D930015E06Rik 0.407 318088 Itgb1bp2 0.407 921519 Zfp307 0.407 904212 null 0.406 574582 null 0.406 311257 null 0.406 302259 null 0.406 651503 4432416J03Rik 0.406 432693 4833425H18Rik 0.405 856428 Anxa6 0.405 903737 null 0.405 310400 Wbscr5 0.405 467222 Blvrb 0.404 837391 Mmp13 0.404 648688 Rbm5 0.404 681342 null 0.403 531622 null 0.403 913882 A230020G22Rik 0.402 829669 5730593F17Rik 0.402 392440 Vgll4 0.402 798479 null 0.402 752953 Rasgrp2 0.401 826151 BC004728 0.401 914828 0610041B22Rik 0.401 426371 Unc84a 0.401 915102 Plp2 0.400 770867 0 0.399 926891 Cnn2 0.399 739642 null 0.399 932017 Jarid2 0.399 445181 null 0.398 337007 null 0.398 852296 null 0.398 924808 Trpv2 0.397 407732 null 0.397 353855 null 0.396 459869 Tnfsf13 0.396 798238 null 0.396 475429 null 0.394 892566 null 0.393 674333 Rab24 0.393 520249 null 0.393 378126 1810031K02Rik 0.392 429279 Prkab2 0.392 928577 null 0.391 569766 null 0.391 755470 2900024C23Rik 0.391 568715 B230104P22Rik 0.390 585837 Brdt 0.390 926460 Zyx 0.390 603625 Tax1bp1 0.389 732943 null 0.389 726777 null 0.389 316223 null 0.388 415592 null 0.388 500438 null 0.388 657549 null 0.388 928973 Lgals4 0.387 912960 2810407K09Rik 0.387 919247 Cklfsf3 0.387 918718 Numa1 0.386 926411 Ing4 0.386 878918 Sdc3 0.386 523258 Gcipip 0.385 402720 Mbl1 0.385 560783 null 0.385 391768 null 0.385 707054 null 0.385 544494 Arid1a 0.385 637138 4631402N15Rik 0.385 629859 null 0.384 400504 null 0.384 548044 null 0.384 778137 null 0.384 653387 Vezf1 0.384 913904 null 0.384 786647 null 0.383 405680 Hip1r 0.383 773976 Bbs2 0.383 676273 Myo1c 0.382 583766 null 0.382 733267 Fmnl1 0.381 881509 Cd81 0.381 895717 null 0.381 601381 Ryr1 0.380 479319 1200014P03Rik 0.380 671722 null 0.379 359219 null 0.379 444945 Il11ra1 0.379 562320 Tyk2 0.379 713976 Gpr124 0.379 533479 null 0.379 590220 Ciz1 0.378 395749 null 0.378 413216 Zfp99 0.378 773069 null 0.378 851333 1700102P08Rik 0.377 809227 Ches1 0.377 846355 null 0.376 463956 Mast1 0.376 672765 Tde1 0.376 455071 Cdc25b 0.376 905489 5330414D10Rik 0.376 390019 Hps3 0.376 339905 null 0.375 918928 null 0.375 760762 Uckl1 0.375 472388 Arhgap15 0.375 421825 null 0.374 656780 Ebf1 0.373 872227 null 0.373 614673 Usp53 0.373 408020 null 0.373 466919 null 0.373 741072 null 0.371 904995 Ddx5 0.371 361141 Stxbp3 0.371 845799 1700026P10Rik 0.371 337899 Hipk1 0.370 548160 Nrbp 0.370 649333 null 0.369 532500 BC029214 0.369 576556 null 0.368 332928 Sh3bp2 0.368 333827 Malt1 0.368 905715 Bat5 0.368 612699 Maf1 0.368 464035 Flt3l 0.367 540563 null 0.367 389652 null 0.367 640625 2510006D16Rik 0.367 321945 Olfr671,Olfr675 0.366 721129 null 0.365 546424 Il11ra2,Il11ra1 0.365 423483 null 0.365 670301 BC017643 0.364 927958 Ly6e 0.364 334859 null 0.363 929387 null 0.363 324461 Nbr1 0.363 691599 null 0.363 351212 null 0.363 892824 null 0.362 356009 null 0.362 397862 null 0.362 689888 Anxa5 0.362 926335 null 0.361 830017 null 0.361 721938 Rdh1 0.361 763602 Mkrn1 0.360 347705 null 0.360 595619 null 0.360 762217 null 0.360 743157 4930553F24Rik 0.359 395134 null 0.359 863053 Gpr49 0.358 433639 Mnt 0.358 427568 null 0.358 905141 Mgat1 0.358 726181 AI428795 0.357 513208 Dpp7 0.357 430244 null 0.356 897711 null 0.356 425013 1110018J12Rik 0.356 699838 Zmynd11 0.356 308525 Rassf5 0.356 597545 Evi2a 0.356 763326 Dnase1l3 0.356 378529 null 0.355 656351 null 0.355 579693 null 0.355 390343 Emid1 0.355 382729 Tas2r136 0.355 662015 null 0.355 313644 null 0.354 753672 Lpp 0.354 519556 Itga9 0.354 480144 Art4 0.353 511364 null 0.353 427311 4732429D16Rik 0.353 604826 Rbl2 0.353 612709 null 0.353 733081 null 0.352 489010 null 0.351 488156 null 0.350 650597 P2rx4 0.350 800828 null 0.350 656759 Zhx2 0.350 585421 null 0.349 875823 null 0.349 645596 Ucp3 0.349 498083 Cd79b 0.349 901337 null 0.348 904233 null 0.348 732153 null 0.348 865313 Rnf123 0.348 882394 Phemx 0.348 848264 null 0.348 534547 null 0.347 706195 BC021921 0.347 665530 Csf2ra 0.347 573628 null 0.347 830062 null 0.346 486792 null 0.346 924896 Plekhg3 0.346 366498 null 0.344 433425 null 0.344 504932 Ndst1 0.344 713476 null 0.343 841253 Gprk6 0.343 915148 Unc93b1 0.343 743442 Ubp1 0.342 911768 Cln2 0.342 690974 Tob1 0.342 383575 null 0.342 681688 MGC30332 0.341 922280 Herc4 0.341 884098 null 0.340 555292 null 0.340 783232 Lrrfip1 0.340 873242 null 0.339 437815 null 0.339 844889 Ogt 0.338 548765 null 0.338 566948 null 0.338 921636 Rnf130 0.338 458858 null 0.337 351482 null 0.337 443129 Trim34 0.337 573930 null 0.337 653407 null 0.336 624539 2810052M02Rik 0.336 790169 null 0.336 463081 null 0.336 561631 AI662250 0.335 923241 D11Bwg0434e 0.335 518497 null 0.334 827587 Dgkz 0.334 780663 Sidt2 0.334 585818 Ctss 0.334 823845 null 0.334 528378 null 0.334 741384 null 0.334 458888 Nedd9 0.333 492467 null 0.333 637784 Atp13a2 0.333 763848 null 0.333 895715 null 0.333 507782 Tep1 0.333 408238 null 0.333 605387 null 0.332 367657 null 0.332 883739 null 0.332 550386 Rchy1 0.332 916637 null 0.331 692814 Samhd1 0.331 903416 null 0.331 474964 null 0.331 466569 null 0.331 923079 null 0.331 928968 Grina 0.330 901291 Ubp1 0.330 481911 Il10rb 0.330 926124 2310004L02Rik 0.329 496317 null 0.329 318130 null 0.328 880211 null 0.328 406140 null 0.327 569396 Slc24a6 0.326 744253 null 0.326 676091 null 0.326 475048 null 0.326 495918 null 0.325 536618 Lzf 0.325 372869 Tnfsf5 0.325 931795 Mgst2 0.325 922538 Tm4sf13 0.324 731452 Nmb 0.324 711766 null 0.323 352907 null 0.323 886034 Stx5a 0.323 398961 null 0.322 579573 4921509E05Rik 0.322 558496 H2-T23 0.322 383734 null 0.321 622202 Slc37a2 0.321 898766 null 0.321 742299 null 0.321 496328 null 0.321 335834 Unc119 0.320 701171 null 0.320 621080 null 0.320 603120 null 0.319 323705 null 0.318 436198 Ctdsp2 0.318 424468 null 0.318 598704 Ccm2 0.317 508992 Rnf103 0.317 535626 null 0.317 467017 2810038K19Rik 0.317 454874 Ptpn22 0.317 463135 Sgsh 0.315 906238 S100a4 0.315 726386 Pacsin1 0.315 681419 null 0.315 899632 null 0.314 727964 1810015C04Rik 0.314 407287 null 0.313 738396 Klf13 0.313 725815 Plekhg2 0.313 913980 Mcoln1 0.313 502963 null 0.313 318209 null 0.312 515794 Zfp36l1 0.312 894286 null 0.312 895722 null 0.311 916709 Hexa 0.311 872407 null 0.310 567225 null 0.310 661927 null 0.310 663774 null 0.310 592477 null 0.310 614952 null 0.310 665135 null 0.309 681760 null 0.309 819976 null 0.309 327225 null 0.309 889678 1810061M12Rik 0.308 547288 null 0.307 924461 Glcci1 0.307 601354 Stard8 0.307 469655 null 0.307 309120 2310003P10Rik 0.307 569507 Naga 0.307 813335 null 0.307 905687 null 0.306 793884 D330050I23Rik 0.306 377079 null 0.306 327248 null 0.306 386903 C85492 0.305 402090 Rtn1 0.305 564483 null 0.305 377836 null 0.305 504256 null 0.304 729846 Prkar1b 0.304 602299 null 0.303 930010 Centd2 0.303 872680 Dnase2a 0.303 921152 Mppe1 0.302 317797 null 0.302 456347 Wdrx1 0.302 309042 Ctsh 0.301 848004 Pttg1ip 0.300 520081 Cd19 0.300 895334 null 0.300 652623 Arhgef1 0.299 391089 null 0.299 618183 Kcnk13 0.298 849749 9030607L17Rik 0.298 901551 null 0.298 884267 Cd53 0.298 718788 null 0.297 780008 null 0.296 688500 Fgd2 0.296 718044 null 0.296 381010 0910001K20Rik 0.296 797860 null 0.296 303148 Tm6sf2 0.295 347681 null 0.295 862901 Olfr433 0.295 481078 null 0.295 528926 null 0.295 785602 null 0.294 391039 D7Ertd413e 0.293 636673 Gm996 0.292 336308 null 0.292 397042 Gns 0.292 505384 null 0.292 379981 Zfp353 0.292 916013 Lsp1 0.291 324024 Lrrc8 0.291 531684 Cyp39a1 0.291 523646 null 0.290 388383 Map1lc3b 0.290 605439 Cyhr1 0.289 399900 null 0.289 907765 2310026E23Rik 0.289 367887 AI429612 0.289 905318 Asahl 0.289 835161 Cd37 0.289 887284 Neurod4 0.288 917445 Pdk2 0.288 790282 Foxo1 0.287 486658 null 0.287 583869 Gpsm3 0.287 353729 9130023D20Rik 0.286 550398 V1rd4 0.286 298316 null 0.286 388826 Fuca1 0.285 484054 Iqgap1 0.282 920873 Trim11 0.282 849494 null 0.282 537423 null 0.281 698514 null 0.280 913994 BC003277 0.279 566320 null 0.278 888406 null 0.277 557556 null 0.277 846204 Ubl3 0.276 403540 null 0.276 379453 AI132321 0.276 483273 null 0.276 342708 Stk10 0.276 675227 null 0.275 656511 Ppp1r13b 0.275 797059 null 0.275 898744 null 0.275 345637 Sipa1 0.275 921477 C230093N12Rik 0.273 836251 8430432M10Rik 0.273 576927 9830002I17Rik 0.273 798188 null 0.273 380040 Mapk1ip1 0.273 633640 null 0.272 507566 null 0.272 717706 Trps1 0.272 394879 null 0.272 304456 null 0.272 644586 Olfr127 0.272 449860 null 0.271 890266 Tieg3 0.271 341470 null 0.271 578599 B230342M21Rik 0.270 487567 null 0.269 608486 Wbscr22 0.268 654956 null 0.268 360289 null 0.268 725004 Perq1 0.268 481482 Ttyh2 0.267 910217 H2-Ke6 0.267 845367 null 0.266 537265 null 0.266 819640 null 0.266 575859 Kmo 0.265 321734 null 0.264 323918 Prlpa 0.263 890056 Pcanap6 0.262 637794 Scamp5 0.262 920469 Ubd 0.262 498790 Gng2 0.262 551010 Pafah1b3 0.262 676851 Rnase4 0.262 788955 Acrbp 0.261 760346 Creg1 0.261 766550 9930012K11Rik 0.261 400553 Dnase1l1 0.261 593343 Arhgef3 0.260 461806 Cd79a 0.260 416337 Hsd11b1 0.260 319728 2610507L03Rik 0.259 660792 Trim46 0.257 548380 null 0.257 641678 Pde2a 0.257 574265 null 0.257 642547 Aph1c,Aph1b 0.256 922964 Rga 0.255 661508 null 0.255 802636 Tcfec 0.254 564703 null 0.254 697725 null 0.254 443721 Hhex 0.254 341853 null 0.254 429370 null 0.254 342988 null 0.253 374041 null 0.253 836319 null 0.253 925041 Ctsd 0.252 351069 4933437K13Rik 0.252 559240 6430510M02Rik 0.251 913657 Hs3st1 0.251 684891 BC043934 0.251 619968 null 0.250 557343 null 0.250 804355 Casp6 0.249 498840 Rasgrp1 0.249 720881 null 0.249 773945 null 0.249 315802 null 0.249 741914 null 0.248 730858 U2af1-rs1 0.248 326326 A330042H22 0.248 667289 null 0.248 822764 Cmah 0.248 677473 Itm2b 0.247 736218 Ppt1 0.247 638351 null 0.247 511508 Pecam1 0.246 797894 Lgmn 0.243 555708 Nfatc1 0.243 540181 null 0.243 771898 Rgs14 0.242 734522 null 0.242 390922 1700027A23Rik 0.242 699416 null 0.241 907925 Iigp1 0.241 750011 Cyp4f13 0.241 910950 Dtx1 0.240 742000 null 0.240 875708 null 0.240 914457 Rab32 0.239 491445 null 0.239 599856 Phemx 0.239 439359 4631426J05Rik 0.239 581699 BB219290 0.238 339856 null 0.237 761963 null 0.237 397738 null 0.237 827282 null 0.236 625547 Hck 0.235 915376 Snx24 0.235 764223 Snn 0.235 801472 Ccr6 0.235 726449 Tubg2 0.235 687873 Tnfrsf25 0.234 545251 Fprl1 0.234 913030 Rabac1 0.233 326298 null 0.232 365825 null 0.231 441133 Slc28a2 0.231 669867 null 0.231 726089 null 0.231 528994 null 0.230 427499 4632423N09Rik 0.230 463292 Abhd1 0.230 352000 null 0.230 443316 Usp11 0.229 829123 C79127 0.229 339768 Ifngr1 0.228 342098 Rassf3 0.228 372944 D7Bwg0611e 0.227 577610 null 0.227 372851 2210421G13Rik, 0.227 767850 Dip3b 0.226 922147 Pbxip1 0.226 491438 D15Ertd735e 0.226 581146 null 0.226 355289 Iap,na,Fv1,31100 0.226 553855 null 0.224 923354 null 0.224 406055 Atp6ap1 0.224 315190 null 0.224 894320 null 0.223 770847 Pcp4 0.223 802713 null 0.223 305366 null 0.223 879425 null 0.222 393948 Il17r 0.222 676021 null 0.222 517377 Btla 0.222 620032 null 0.222 332032 Ier2 0.220 883316 null 0.220 894317 null 0.220 396384 null 0.220 339554 Kctd12 0.220 805293 null 0.219 767228 Pde1b 0.219 585460 Csad 0.219 444840 Bcl6 0.218 603303 Apoc1 0.218 310349 Rwdd2 0.218 624929 Cxcr3 0.217 475813 null 0.217 910205 Slc26a11 0.217 905899 Tnnt3 0.216 754399 Rdh5 0.216 396981 null 0.216 634106 Cirbp 0.216 876790 null 0.214 782228 null 0.214 865102 Edg3 0.214 434211 null 0.214 529887 null 0.213 880933 Stk32c 0.213 746177 Pink1 0.213 550613 Sorl1 0.211 337053 Lincr 0.211 485872 null 0.211 880878 Hivep2 0.210 387417 Hexb 0.210 922750 Prf1 0.210 636513 null 0.210 750302 null 0.209 354837 Rdh12 0.209 446793 Ctso 0.209 469209 Cd302 0.209 339743 Gyltl1b 0.208 881491 null 0.208 704269 A630033H20Rik 0.208 588178 null 0.207 510391 null 0.206 352923 1100001H23Rik 0.206 386163 4632428N05Rik 0.205 925526 Amn 0.204 478320 null 0.204 658569 Crip1 0.204 302796 LOC280487 0.203 694329 Sprr2g 0.203 790962 Cyp4v3 0.203 610109 null 0.202 772405 Ppgb 0.201 357884 null 0.200 773044 Ppp1r14a 0.200 757884 Myadm 0.200 865358 null 0.199 637912 H2-Ab1,Rmcs1 0.198 674483 null 0.198 920753 Slc12a6 0.197 389202 null 0.196 737436 Pxk 0.195 601076 null 0.195 922519 0610039P13Rik 0.194 377424 Akap8l 0.194 418903 1810033M07Rik 0.193 919209 Klra15,Klra1 0.193 536691 Lzts2 0.192 324743 Adcy7 0.191 307853 Csf1r 0.191 298936 null 0.191 754719 null 0.191 598843 Hist3h2ba 0.190 686289 null 0.190 697664 Evl 0.190 522285 null 0.189 300547 Pde4b 0.189 640348 null 0.188 390446 null 0.187 486007 Lrrk2 0.185 432031 null 0.185 694187 null 0.185 532452 null 0.184 459859 B830021E24Rik 0.184 529379 Ccbp2 0.184 677412 Abcb1a 0.183 717929 Il5ra 0.183 340252 1110028E10Rik 0.183 347704 Cxxc5 0.183 341817 Hfe 0.183 924253 Sh2d3c 0.183 602553 null 0.183 715932 Satb1 0.182 927195 Mcoln2 0.181 430006 null 0.181 849375 null 0.179 906306 Trp53inp1 0.179 485329 null 0.178 486656 Olfr770 0.178 920779 4432409M07Rik 0.178 559176 Abtb1 0.178 470398 Lrrk2 0.177 743740 A730011L01Rik 0.177 396120 null 0.177 931538 null 0.177 904963 H2-DMb1,H2-DM 0.176 553101 Serpinb6a 0.176 434932 null 0.175 408407 null 0.174 923656 Lrpb7 0.174 629968 A930001N09Rik 0.173 767779 Stk23 0.173 465249 Armc7 0.173 333545 null 0.171 452858 null 0.171 358391 null 0.171 410905 Ezh1 0.170 735885 Add3 0.170 580847 null 0.169 854280 null 0.169 899534 null 0.169 733846 null 0.169 911875 null 0.168 618980 Apc 0.166 892269 null 0.166 445504 Hbp1 0.166 904584 null 0.164 339921 Sectm1 0.163 579797 null 0.163 925260 Fyb 0.163 745275 H2-DMa 0.162 773467 Emr1 0.162 630969 A630023P12Rik 0.162 905012 null 0.161 415662 Rgs13 0.159 474924 null 0.159 741992 null 0.159 597942 null 0.159 812669 Cd1d1 0.157 896130 null 0.157 675263 Fndc5 0.157 638449 null 0.157 613793 BC027057 0.156 878317 null 0.156 828745 Sell 0.156 307688 Retnlg 0.155 373255 null 0.155 452059 null 0.155 789191 Lhb 0.154 889776 Acas2l 0.154 914289 Metrnl 0.154 731261 null 0.154 509062 Zfp318 0.153 566227 Crip3 0.153 867236 Siglec10 0.153 522703 Rnase6 0.151 393915 null 0.150 344672 null 0.150 783471 null 0.149 845060 null 0.148 371847 null 0.148 679846 null 0.147 919536 Cd2 0.147 735924 null 0.145 903663 null 0.145 421348 null 0.144 746329 null 0.144 889509 E430019B13Rik 0.143 781669 Galnt14 0.142 819036 Rin3 0.141 908477 Slco2b1 0.141 926079 null 0.141 674422 Fgd3 0.140 522474 C2ta 0.140 351056 Tk2 0.139 612360 null 0.137 808161 BC034097 0.136 795075 Dgka 0.136 586949 H2-Oa 0.136 705442 Rasgrp2 0.136 452328 Smpdl3a 0.136 362865 Tlr3 0.135 868969 null 0.135 567873 B230315M08Rik 0.134 357994 Cerk 0.134 437857 Gsn 0.133 678233 null 0.133 861602 Crisp3 0.133 321866 null 0.133 408639 Ell3 0.132 545586 null 0.132 316945 A730041O15Rik 0.132 637069 null 0.131 304484 Rapgef4 0.131 727829 Serpinb6c 0.129 397841 null 0.128 813084 null 0.127 916589 Pira3,Pira6,Pirb,P 0.127 631715 Rgl1 0.127 340591 null 0.126 894629 Ckb 0.125 407752 Arl12 0.124 927124 Ank 0.123 603787 null 0.123 921083 Clec2i 0.123 913679 Pirb 0.123 627882 null 0.122 729052 Lair1 0.122 694880 Pira3,Pira6,Pirb,P 0.122 922016 Ypel3 0.121 621113 Frat2 0.120 346544 null 0.119 748495 Vpreb3 0.119 520471 null 0.119 765608 null 0.119 533296 null 0.118 529916 Vamp1 0.117 873788 null 0.116 904295 Ckb 0.116 883636 null 0.115 555119 4833422F24Rik 0.115 412663 Sfxn3 0.115 596063 Igsf4a 0.112 597529 null 0.111 391788 null 0.110 569169 null 0.109 800022 null 0.109 749478 Igj 0.107 888679 null 0.106 787094 null 0.105 908536 Ctsl 0.104 397261 Clec1b 0.102 923913 null 0.102 325841 null 0.099 733186 Kcnj1 0.099 354257 null 0.099 592219 Cd14 0.098 908795 Timp2 0.096 908949 Tm6sf1 0.096 535585 Cyp4f18 0.096 906209 6330406L22Rik 0.095 367165 Stab2 0.094 919635 Map1lc3a 0.094 519245 null 0.093 693359 1500004A08Rik 0.093 422307 Serpinb1a 0.093 450136 Ltb 0.093 601840 Mylip 0.092 619584 null 0.091 352965 E430026E19Rik 0.090 532780 Abcg1 0.089 384555 Cd97 0.089 323995 null 0.089 792633 null 0.088 390970 Antxr2 0.087 920596 B130017P16Rik 0.086 559867 Clec9a 0.085 774411 Alox5ap 0.084 690967 Arhgef18 0.084 781868 null 0.082 406680 Hes1 0.081 712844 null 0.079 704890 Clec12a 0.079 324258 null 0.078 832992 Txnip 0.078 803271 Cbr3 0.077 850129 C6 0.076 477181 Card4 0.074 922031 Sepp1 0.074 752295 Mrap 0.073 580861 Ear6 0.073 930150 Apob48r 0.073 300048 Pla2g7 0.072 907823 Gpr34 0.072 571611 Btg1 0.072 641341 Zfp36 0.071 827302 null 0.068 730961 Dok3 0.067 560648 H2-Ob 0.067 351046 Clec4a1 0.066 907012 Ephx1 0.064 643349 Gpr109b 0.064 834138 null 0.062 921755 Slc11a1 0.062 339263 null 0.060 920320 Clec4n 0.059 928327 Ccl6 0.058 847962 null 0.057 775646 Daf2 0.056 493734 Daf1 0.056 773688 null 0.056 907909 Edg1 0.053 712701 Rasgef1b 0.053 684165 null 0.052 452867 Igsf6 0.052 424621 Tgfb1i4 0.052 339169 Sort1 0.051 684806 Ccnd1 0.049 455908 P2ry13 0.046 880942 Trf 0.045 441728 Igsf7 0.045 317177 March1 0.044 747134 Mertk 0.041 701850 Fpr1 0.038 511891 Vcam1 0.038 648498 Cd163 0.037 472042 null 0.037 930315 Adamdec1 0.036 649745 Fcgr3 0.035 459464 Havcr2 0.034 770146 Tnfrsf1a 0.034 655400 null 0.034 719563 null 0.034 377864 null 0.032 403608 Abca1 0.031 906490 Apoe 0.030 556716 Nr1h3 0.030 928375 Cd5l 0.030 659012 Pilra 0.029 733569 Cyp27a1 0.029 731474 null 0.029 475091 Pilrb 0.029 922417 Dcir2,Clec4a2 0.027 505913 null 0.026 465504 null 0.024 809507 null 0.022 700170 Dsip1 0.022 434601 Hba-a1 0.020 347436 null 0.019 821644 Hpgd 0.019 839040 Hmox1 0.017 909409 C1qb 0.016 682726 Fcgrt 0.015 568620 Ear3 0.015 921351 Slpi 0.013 347956 Tgfbi 0.012 490408 Spic 0.012 335109 Trpm2 0.012 506800 Treml4 0.011 331550 Mrc1 0.011 578285 Slc40a1 0.010 925991 Ear10,Ear2 0.010 929684 Sgk 0.009 850619 Axl 0.009 700010 Fcna 0.009 675339 C1qg 0.004 524163 C1qa 0.002