Interplay between Chromatin Remodeling and Epigenetic Changes during Lineage-Specific Commitment to Expression This information is current as of September 27, 2021. Torsten Juelich, Elissa Sutcliffe, Alice Denton, Yiqing He, Peter C. Doherty, Christopher Parish, Steven J. Turner, David Tremethick and Sudha Rao J Immunol 2009; 183:7063-7072; Prepublished online 13 November 2009; Downloaded from doi: 10.4049/jimmunol.0901522 http://www.jimmunol.org/content/183/11/7063 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2009/11/13/jimmunol.090152 Material 2.DC1 References This article cites 56 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/183/11/7063.full#ref-list-1

Why The JI? Submit online. by guest on September 27, 2021

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/184/3/1653.full.pdf

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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Interplay between Chromatin Remodeling and Epigenetic Changes during Lineage-Specific Commitment to Granzyme B Expression1

Torsten Juelich,* Elissa Sutcliffe,* Alice Denton,‡ Yiqing He,* Peter C. Doherty,‡ Christopher Parish,* Steven J. Turner,‡* David Tremethick,† and Sudha Rao2‡

The role of chromatin remodeling and histone posttranslational modifications and how they are integrated to control ex- pression during the acquisition of cell-specific functions is poorly understood. We show here that following in vitro activation of .CD4؉ and CD8؉ T lymphocytes, both cell types show rapid histone H3 loss at the granzyme B (gzmB) proximal promoter region However, despite the gzmB proximal promoter being remodeled in both T cell subsets, only CD8؉ T cells express high levels of gzmB and display a distinct pattern of key epigenetic marks, notably differential H3 acetylation and methylation. These data Downloaded from suggest that for high levels of transcription to occur a distinct set of histone modifications needs to be established in addition to histone loss at the proximal promoter of gzmB. The Journal of Immunology, 2009, 183: 7063–7072.

pon primary activation, naive T lymphocytes undergo a functions (1). Although CTL can secrete a range of cytokines, such as program of proliferation and differentiation that results IFN-␥, TNF-␣, and IL-2 (6), a predominant function of CTL is lysis 3

in the acquisition of lineage-specific T cell functions (1, of virally infected cells or tumor cells (7). Granzyme B (gzmB) is http://www.jimmunol.org/ U ϩ 2). Naive CD4 T cells are capable of differentiating into separate known to play an integral part in the induction of programmed cell Th (Th) lineages characterized by specific patterns of cytokine death in target cells, where it activates a cascade of events, ultimately production (2). For example, the Th1 lineage is characterized by leading to of these cells and clearance of intracellular patho- IFN-␥ production, the Th17 lineage by IL-17 and IL-21 secretion, gens (8). The gzmB pathway is normally associated with NK cell and and the Th2 lineage by IL-4, IL-5, and IL-13 production. There is CD8ϩ T cell responses, but not usually with CD4ϩ Th cell function increasing evidence that Th subset-specific functions are regulated (9). Intriguingly, early experiments indicated that granzymes can and maintained by epigenetic mechanisms (3). Thus, compared sometimes be expressed in both murine CD4ϩ and CD8ϩ T lympho- with naive T cells, Th1 and Th2 cells exhibit significant alterations cytes, albeit at differing levels, depending on the conditions of stim- in both chromatin structure (4) and biochemical modifications within ulation and developmental stages of these T cell subsets (10). Re- by guest on September 27, 2021 the IFN-␥ and IL-4 loci (4, 5). For example, under sustained Th1 cently, gzmB was shown to possess a second function besides its role differentiating conditions, acetylation of the H3 and H4 histones is in target cell killing, being possibly involved in regulating the ho- enriched within the IFN-␥ locus and diminished within the IL-4 locus meostasis of activated Th2 T cells (11). Specific transcription factors (5). In contrast, under continued Th2 differentiating conditions, this (TFs) required for the regulation of gzmB in NK cells and CD8ϩ T pattern is reversed, with enrichment of H3/H4 acetylation being ob- cells have been studied in some detail earlier (12–14). It has also been served within the IL-4, and not the IFN-␥ locus (5). These data sug- established that the proximal promoter region of up to 208 bp up- gest a model of naive T cell differentiation where Th lineage-specific stream of the transcriptional start site (TSS) is both necessary and function is largely determined by the epigenetic marks within specific sufficient for highly inducible gzmB expression. This region contains gene loci that control gene expression. canonical TF binding sites for AP-1, Ikaros, CBF family members, Following primary activation, naive Ag-specific CD8ϩ T cells dif- CREB, NFAT, as well as ETS proteins (reviewed in Ref. 15). ferentiate into CTL, a process which results in extensive changes in Despite there being considerable information available about the transcriptional activity, signature gene expression, and CTL-specific TFs that control gzmB expression, surprisingly little is known about epigenetic and chromatin-dependent processes involved in the tran- scriptional regulation of gzmB (16). In the nucleus of eukaryotic cells, *Division of Immunology and Genetics and †Division of Molecular Biosciences, John gene transcription takes place in the context of a multicomponent Curtin School of Medical Research, Australian National University, Canberra, Aus- DNA/protein structure known as chromatin. The fundamental unit of tralia; and ‡Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia chromatin is the nucleosome, which is composed of an octamer of Received for publication May 15, 2009. Accepted for publication August 16, 2009. histone proteins enclosed in two helical turns of DNA (17). There are three key mechanisms by which chromatin structure can be modu- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance lated. First, chromatin remodeling where a promoter-bound histone with 18 U.S.C. Section 1734 solely to indicate this fact. octamer is removed or repositioned by ATP-dependent chromatin 1 This work is supported by Australian National Health and Medical Research Coun- cil Project Grant 454455, National Health and Medical Research Council Program Grants 299907 and 455395, a National Health and Medical Research Council Dora 3 Abbreviations used in this paper: gzmB, granzyme B; gzmN, granzyme N; TF, Lush Postgraduate Fellowship awarded to A.D., and a Pfizer Senior Research Fel- transcription factor; Pol II, polymerase II; ChIP, chromatin immunoprecipitation; lowship awarded to S.J.T. CHART, chromatin accessibility and real time; Ct, threshold cycle; Mnase, micro- 2 Address correspondence and reprint requests to Dr. Sudha Rao, Division of Immu- coccal nuclease; H3K9acet, H3K9 acetylation; TSS, transcriptional start site; PTM, nology and Genetics, John Curtin School of Medical Research, Australian National posttranscriptional modification E-Pol II. University, GPO Box 334, Canberra City ACT 2601 Australia. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901522 7064 gzmB TRANSCRIPTION IN CD4ϩ AND CD8ϩ T CELLS

remodelers (17–21). Second, changes in histone composition by in- formed in 96- or 384-well plates (PerkinElmer) with an Applied Biosys- terchanging core histones with histone variants. Third, epigenetic tems PRISM 7900 Real-Time System (PerkinElmer/PE Biosystems) at the modification of the N termini of the histone tails mediated by enzymes Biomolecular Resource Facility (John Curtin School of Medical Research, Australian National University, Canberra, Australia). To correlate the recruited to gene regulatory regions. Posttranscriptional modification threshold cycle (Ct) values from the cDNA amplification plots to fold E-Pol II (PTMs) can function either by directly changing chromatin increases in starting material, the ⌬⌬Ct method was applied, with addition structure or via recruitment of other factors (22–24). of conversion into arbitrary copy numbers, using the formula: arbitrary 5 (Ct value Ϫ 17) From recent genome-wide studies in a broad range of different copies ϭ 10 /(2 ), after verification that amplification efficien- cies were optimal and equal between primers used in one experiment. species, a picture is emerging about the complex interplay of hi- For granzyme mRNA expression experiments, TaqMan gene expression stone modifications, nucleosome position, and recruitment of the assays were purchased and performed according to the manufacturer’s general transcriptional machinery in regulating gene expression. guidelines (Applied Biosystems). Importantly, these studies indicate that transcriptional activation or repression of a gene is determined by a combination of distinct CHART assay epigenetic events rather than by a single nucleosome modification The CHART assay was performed as described originally (24). For each (25–28) (25–27), as has often been proposed in previous studies. batch of micrococcal nuclease (Mnase), digestion of gDNA was optimized 4 Despite this wealth of knowledge, the complex interplay between as shown in supplemental Fig. S3. All samples were digested equally, a process that was verified by the bioanalyzer results obtained for each sam- chromatin remodeling and the modification of histone tails is ple on multiple occasions (supplemental Fig. S3). Since there were no poorly understood with regard to their relative importance in the differences in the actual digestion efficiencies, the differences observed can gene activation process. only be explained by changes in nucleosomal positioning across the spe-

Recently, a study has examined the epigenetic changes at the cific regions being measured. For Mnase accessibility time-course exper- Downloaded from human gzmB regulatory regions occurring in memory CD8ϩ T iments, the data were calculated as a percentage of the accessibility ob- served in the unstimulated digested DNA sample, which was set to 100%. cells, correlating acetylation of H3K9 with an increase in gzmB For basal accessibility studies, the data were graphed as percent genomic expression in activated memory T cells compared with acti- DNA signal observed in the undigested sample. Regarding the actual val- vated naive T cells (16). In this study, we show that major ues, they are not calculated according to an end point of digestion (which histone H3 loss occurs from the gzmB proximal promoter during was set at 100%), but against the accessibility of each region being exam- ϩ ϩ ined in the unstimulated T cell samples.

the in vitro activation of both naive CD4 and CD8 T cells. http://www.jimmunol.org/ However, despite this histone loss in T cell subsets, polymerase ChIP assay II (Pol II) recruitment and high levels of chromatin accessibility ChIP analysis was performed according to the standard Upstate Biotechnology and corresponding gzmB transcription only occur in activated ␮ ϩ ChIP protocol. After sonication, samples were precleared with 60 l of salmon CD8 T cells. Furthermore, to uncover the chromatin regula- sperm DNA-protein A-agarose (Upstate Biotechnology) and subsequently in- tory signatures associated with this efficient transcriptional ac- cubated overnight with rotation at 4°C with one of the following Abs per 106ˆ tivation, we performed a detailed analysis of specific H3 mod- cells starting material: 20 ␮g of anti-histone H3 (Abcam ab1791), 5 ␮gof ␮ ifications before and after in vitro stimulation. Specifically, we anti-H3K9acet (Upstate Biotechnology 06-942), 7 l of anti-H3K4-dimethyl (Upstate Biotechnology 07-030), 5 ␮l of anti-H3K4-trimethyl (Upstate Bio- found that H3K9 acetylation (H3Kacet) is directly and strongly technology 07-473), 5 ␮g of anti-H3K9-trimethyl (Upstate Biotechnology 07-

ϩ by guest on September 27, 2021 correlated with high levels of gzmB transcription in CD8 T 442), 4 ␮g of anti-TFIID (Santa Cruz Biotechnology sc-204), and 4 ␮gof cells. Taken together, this work proposes a new step in the anti-Pol II (Abcam ab817) or no Ab as a specificity control. Briefly, Ct values transcriptional activation process, a “prepoised” step, which in- from the PCR amplification plots were converted to arbitrary copy numbers. volves substantial chromatin remodeling resulting in the loss of Sample data were then normalized to the corresponding total input before fold ϩ ϩ change above the average value of the control samples (without Ab) was histones in both CD4 and CD8 T cells. We propose that it is calculated to give the ChIP enrichment ratio as previously described (29). ChIP the efficient recruitment of RNA Pol II and the acquisition of enrichment ratios greater than 2 are considered to be significant binding above specific PTMs that drive high levels of gzmB transcription only background. All ChIP assays were performed in duplicate or triplicates as in activated CD8ϩ T cells. indicated, and all PCRs were run in duplicate. Materials and Methods Flow cytometry ϩ ϩ Primary T cell preparation and activation Naive and stimulated CD4 and CD8 T cells were harvested and washed with 0.1% BSA-containing PBS. For cell surface staining, cells were in- All mice were maintained in a pathogen-free environment in barrier facil- cubated with PBS and various Abs for 30 min on ice. The Abs used were ities. Spleens were isolated from C57BL/6 mice (4–6 wk old). The CD4 PE-CD3, FITC-CD8, and allophycocyanin/Cy7-CD4. Cells were analyzed and CD8 cells were purified using MACS CD4 (LT34) beads and MACS on a FACSCalibur (BD Biosciences). CD8 beads, respectively, according to the manufacturer’s guidelines (Miltenyi Biotec). The cells were subsequently stained and analyzed by Confocal microscopy flow cytometry with T cell populations shown to be 90–95% pure using ϩ ϩ Abs against CD4 T cells and CD8 T cells. Primary mouse CD4 and CD8 T cells were isolated and cultured in To activate freshly isolated cells, 6-well plates were coated with anti- MLC medium supplemented with 10% FCS and antibiotics. Cells were ␧ CD3␧ (553058, 10 ␮g/ml; BD PharMingen) for 24 h at 4°C and then maintained at 37°C and in 5% CO2. Cells were stimulated with anti-CD3 washed three times with cold PBS before cells were added. Anti-CD28 Ab (10 ␮g/ml; BD Pharmingen) and anti-CD28 Abs (5 ␮g/ml; BD Pharmin- (553295; BD Pharmingen) was added directly to the cells at a concentra- gen) for 3 days. After rinsing in PBS briefly, cells were fixed with 2% tion of 5 ␮g/ml before activation. formaldehyde in PBS for 10 min at room temperature. After washing cells in PBS three times, they were transferred to 0.1% poly-L-lysine (Sigma- RNA extraction and cDNA synthesis Aldrich)-coated round coverslips (ø ϭ 13 mm). Coverslips were then in- cubated with 1% Triton X-100 for 15 min and blocking solution (1% BSA Total RNA was extracted from stimulated and unstimulated T cells using and 0.1% Tween 20 in PBS) for 45 min at room temperature. Cells were RNA TRIzol reagent (Molecular Research Centre). The DNase I-treated incubated with FITC-conjugated anti-mouse gzmB-specific Ab as de- total RNA was reverse transcribed using 50 U of Superscript II reverse scribed previously (1/50; eBioscience 11-8822-80 (30)) for 60 min at 37°C transcriptase (Invitrogen) as detailed in the manufacturer’s guidelines. and washed repeatedly with blocking solution. Nuclei were counterstained SYBR Green PCR amplification and TaqMan gene expression with 0.1 ␮g/ml 4Ј,6-diamidino-2-phenylindole for 7 min and washed with assay blocking solution once and then twice with distilled water. Coverslips with SYBR Green RealTime PCR for gDNA (chromatin immunoprecipitation (ChIP)/chromatin accessibility and real-time (CHART) assays) were per- 4 The online version of this article contains supplemental material. The Journal of Immunology 7065 cells were added with 5 ␮l of mounting medium (H-1000; Vector Labo- ratories) and sealed with nail polish. Immunofluorescent images were col- lected using a laser scanning microscopy (Olympus IX71). Merged images were generated in Adobe Photoshop CS. Tetramer and protein staining Purified T cells were stained with either anti-CD8-FITC, anti-CD3-PE, or anti-CD4-allophycocyanin-Cy7 (BD Pharmingen). Unstimulated and stim- ulated cells were stained as above but also fixed and permeabilized using a BD Pharmingen Cytofix/Cytoperm kit. Intracellular gzmB was detected using anti-human gzmB-allophycocyanin (clone GB12; Caltag Laborato- ries). The flow cytometry analysis utilized a FACSCalibur (BD Immuno- cytometry Systems) and CellQuest software. For isolation of influenza A virus-specific CTL, splenic populations were stained with DbNP366-PE or DbPA224-allophycocyanin tetramers for1hinsort buffer (PBS/0.1% BSA). Cells were washed twice with sort buffer and stained with anti-CD8- FITC for 30 min on ice. The cells were then washed, resuspended in sort buffer, and transferred to polypropylene tubes for sorting. Isolation of CD8ϩDbNP366ϩ and CD8ϩDbPA224ϩ cells was conducted using a Mo- Flo high-speed cell sorter and summit software (DakoCytomation).

Results Downloaded from Comparison of gzmB expression after in vitro stimulation of CD4ϩ and CD8ϩ T cells The cellular differentiation pathways as well as the effector functions of committed CD4ϩ and CD8ϩ subsets have been studied in great detail (31), thus making them an ideal system to study chromatin- based mechanisms associated with lineage commitment. For the full http://www.jimmunol.org/ manifestation of CTL activity by CD8ϩ T cells, expression of the effector function gene gzmB is generally regarded as crucial (7–9). We first assessed the gzmB expression patterns within purified CD4ϩ and CD8ϩ T cell populations after in vitro stimulation with Abs directed against CD3 and CD28. As expected, both nonstimulated CD4ϩ and CD8ϩ T cells exhibited similar low amounts of gzmB mRNA (sup- plemental Fig. S1b) and no protein expression (Fig. 1, b and c)as measured by quantitative PCR and immunofluorescence, respectively. Interestingly, increased levels of gzmB mRNA were first detected in by guest on September 27, 2021 both CD4ϩ and CD8ϩ T cells 12 h after initial activation (Fig. 1a). gzmB mRNA levels continued to increase up to 48 h after stimulation, with levels in the CD8ϩ T cells increasing ϳ1200-fold compared with nonstimulated cells (Fig. 1a). Surprisingly, CD4ϩ T cells also showed an ϳ400-fold increase in gzmB mRNA content at the 48-h time point compared with nonstimulated cells. This increase was significantly FIGURE 1. Kinetics of gzmB expression in activated CD4ϩ ϩ ϩ and CD8 smaller than in activated CD8 T cells (Fig. 1a) and cannot be at- T cells. a, mRNA expression of gzmB as measured by TaqMan quantita- tributed to any differences in mRNA recovery due to normalization tive PCR. gzmB mRNA kinetics were measured in resting and anti-CD3/ with the L32 control gene (supplemental Fig. S1a). This suggests that CD28 Ab-stimulated CD4ϩ and CD8ϩ T cells for the time points indicated. CD8ϩ T cells possess specific regulatory mechanisms for high-level mRNA levels for gzmB are expressed as fold change relative to nonstimu- gzmB mRNA expression, either at the single-cell or cell population lated samples. Data shown are the mean Ϯ SE of three replicate experi- ments. b, Flow cytometry analysis of gzmB protein expression by 48- and level. ϩ ϩ To examine gzmB protein expression at the cellular level, flow 72-h-activated CD4 (green) and CD8 (black) T cells, with gzmB protein expression being detected by an anti-gzmB-FITC Ab. c, gzmB protein cytometry (Fig. 1b) and immunofluorescence microscopy (Fig. 1c) expression, as measured by confocal laser-scanning microscopy using non- studies were performed. gzmB mRNA production was mainly asso- stimulated (NS) and 48-h anti-CD3/CD28 Ab-stimulated (ST) CD4ϩ and ciated with high levels of protein expression, with the majority CD8ϩ ϩ ϩ - ϩ T cells. CD4 and CD8 T cells were fixed and intracellular stain (ϳ90%) of 48-h activated CD8 T cells producing gzmB, but only ing was performed using an anti-gzmB-FITC Ab and 4Ј,6-diamidino-2- ϩ 10% of CD4 T cells expressing gzmB at levels detectable above the phenylindole (DAPI) as a nuclear DNA stain. background autofluorescence of the cells (Fig. 1b). Close examination of the anti-gzmB staining of the 48-h activated CD4ϩ T cells re- vealed, however, a small fluorescence shift in the entire T cell pop- ulation, suggesting that low-level production of gzmB occurs in all of 32–35). To investigate changes in the chromatin structure of the the activated CD4ϩ T cells. Taken together, these data suggest that gzmB gene in activated CD4ϩ and CD8ϩ T cells, a Mnase)- gzmB expression is likely to be regulated at the transcriptional level based CHART-PCR assay was performed (33). Mnase digestion is in CD8ϩ T cells, which is the focus of the present study. an ideal tool to measure chromatin accessibility, since it cuts pref- erentially in regions of DNA that are nucleosome free. Primer sets Mnase accessibility changes across the gzmB proximal ϩ ϩ were designed across the proximal gzmB promoter, spanning a promoter in CD4 and CD8 T cells region of ϳ400 bp upstream of the TSS (Fig. 2a). Initial basal Chromatin accessibility is now well established as playing a chromatin accessibility measurements revealed that there was crucial role in transcriptional regulation of inducible (18, comparable low-level (ϳ10–15%) accessibility of the gzmB 7066 gzmB TRANSCRIPTION IN CD4ϩ AND CD8ϩ T CELLS Downloaded from

FIGURE 2. Chromatin remodeling across the proximal promoter region of gzmB. a, Schematic representation of the gzmB proximal promoter region indicating the locations of the three primer sets A, B, and C spanning a 400-bp region upstream of the gzmB TSS. b, Changes in chromatin accessibility of the proximal gzmB promoter in activated splenic CD4ϩ and CD8ϩ T cells. CHART assays were performed using the accessibility agent Mnase and nuclei generated from nonstimulated and anti-CD3/CD28 Ab-stimulated CD4ϩ and CD8ϩ T cells for the time points indicated. Genomic DNA was subsequently analyzed by SYBR Green real-time PCR using primer sets A, B, and C that target the gzmB proximal promoter region. Data are graphed as percentage accessibility with respect to the nonstimulated sample. Data are representative of three independent experiments. c, Remodeling of the gzmB http://www.jimmunol.org/ promoter in CD8ϩ T cells as a function of TCR signal strength. CHART assays were performed using the accessibility agent Mnase and nuclei isolated from CD8ϩ T cells either nonstimulated or stimulated for 2 days with different concentrations (0.5–10 ␮g/ml) of an anti-CD3 Ab and a constant concentration (5 ␮g/ml) of an anti-CD28 Ab. Accessibility changes were determined by real-time PCR using primer sets A, B, and C that target the gzmB proximal promoter region. Data are graphed as percentage accessibility with respect to the nonstimulated sample.

proximal promoter in both resting CD4ϩ and CD8ϩ T cells the greatest overall increase in accessibility was observed in the (supplemental Fig. S1c). Ϫ34 bp to Ϫ300 bp region of the proximal promoter, with no CD4ϩ and CD8ϩ T cells were then stimulated with anti-CD3 significant change in accessibility being observed in the distal re- by guest on September 27, 2021 and anti-CD28 Abs and harvested at different time points to es- gion of the proximal promoter (Ϫ300 bp to Ϫ420 bp). These re- tablish the kinetics of chromatin accessibility. Following stimula- sults suggest that the degree of chromatin accessibility of the gzmB tion increased DNA accessibility was observed in both the CD4ϩ promoter depends upon TCR signaling strength. and CD8ϩ T cells across the entire 400-bp region of the gzmB Loss of histone H3 from the gzmB promoter following proximal promoter (Fig. 2b). Closer examination revealed that re- ϩ ϩ gions B and C, corresponding to the regions within 300 bp of the activation of CD4 and CD8 T cells TSS, exhibited the greatest relative increase in accessibility in both It has been shown for both yeast (36) and mammalian genes (37) CD4ϩ and CD8ϩ T cells (Fig. 2b). Changes in chromatin structure that loss of the core histone H3 at the proximal promoter region of were observed as early as 12 h after activation (Fig. 2b), correlat- genes is often associated with chromatin remodeling and transcrip- ing with the early onset of transcriptional activity (Fig. 1a). How- tional activation. To examine whether histones are lost from the ever, there were differences in both the extent and rapidity of chro- gzmB promoter during activation of naive CD4ϩ and CD8ϩ T matin accessibility between the CD4ϩ and CD8ϩ T cells (Fig. 2b). cells, a ChIP assay was performed using a specific Ab raised Maximum chromatin accessibility was observed 24–48 h after ac- against the C-terminal region of histone H3. As shown in Fig. 3a, tivation of the CD8ϩ T cells, whereas changes in chromatin struc- nonstimulated CD4ϩ and CD8ϩ T cells showed comparable levels ture within the gzmB promoter of activated CD4ϩ T cells was of H3 enrichment using three primer sets which span the ϳ400-bp highest at 72 h after stimulation. Although, the same regions of the region of the gzmB promoter. Following activation, however, the proximal gzmB promoter within both CD4ϩ and CD8ϩ T cells entire promoter region showed a rapid reduction in H3 enrichment were altered following activation, the overall extent of chromatin in both CD4ϩ and CD8ϩ T cells, with the decrease being observed accessibility was 3- to 4-fold higher in activated CD8ϩ T cells as early as 4 h after stimulation. Interestingly, CD4ϩ T cells compared with CD4ϩ T cells. This difference in chromatin acces- showed a steady decline in H3 enrichment up to 24 h after stim- sibility closely parallels the differential mRNA levels observed in ulation, whereas with CD8ϩ T cells, maximum H3 loss occurred CD4ϩ and CD8ϩ T cells. 12 h after stimulation. Therefore, chromatin remodeling as mea- To determine whether the extent of chromatin accessibility sured by histone H3 loss across the gzmB promoter (in particular across the proximal gzmB promoter was dependent on TCR-me- around Ϫ200 from TSS) occurs at a comparable level in both diated signal strength, naive CD8ϩ T cells were stimulated, as CD4ϩ and CD8ϩ T cells, despite a major difference in the level of described earlier, with increasing concentrations of anti-CD3 Ab transcription. (0.5–10 ␮g/ml) in the presence of a constant concentration of anti- CD28 Ab (5 ␮g/ml). Increasing levels of anti-CD3-mediated TCR H3K9acet and RNA Pol II recruitment stimulation resulted in increasing levels of chromatin accessibility Epigenetic modifications across the regulatory regions of inducible within the proximal gzmB promoter (Fig. 2c). As observed earlier, genes, including acetylation of lysine 9 in histone H3 (H3K9acet), The Journal of Immunology 7067 Downloaded from http://www.jimmunol.org/

FIGURE 3. Distinct patterns of histone H3 deposition, H3K9 acetylation (H3K9acet), and Pol II recruitment occur following CD4ϩ and CD8ϩ T cell by guest on September 27, 2021 activation. Remodeling of the gzmB promoter region following anti-CD3/28 Ab stimulation of CD4ϩ and CD8ϩ T cells as measured by (a) histone H3 deposition, (b) H3K9acet enrichment, and (c) Pol II recruitment. ChIP assays were performed on CD4ϩ and CD8ϩ T cells either nonstimulated or stimulated with anti-CD3/CD28 Abs for the time points indicated. Enrichment of specific DNA sequences in H3-specific, H3K9acet-specific, and Pol II-specific chromatin immunoprecipitates was determined by real-time PCR using primer sets A, B, and C that target the gzmB proximal promoter region. Data are calculated from the ratio of immunoprecipitated DNA minus the no Ab control and normalized against the total input. The results represent the mean Ϯ SE of two independent experiments. are well-established hallmarks of transcriptional activation (20). To ascertain whether the increase in H3K9acet is associated Acetylation of this residue is associated with lineage-specific gene with transcriptional activity, a ChIP assay was performed using an expression within Th1 and Th2 cells (5). Given that there was no Ab against the active form of Pol II. Similar to the large differences apparent difference in the extent of histone H3 loss at the proximal observed in H3K9acet between CD4ϩ and CD8ϩ T cells, Pol II promoter region, it was of interest to determine whether the dif- was essentially only recruited to the gzmB promoter in activated ference in gzmB transcription between activated CD4ϩ and CD8ϩ CD8ϩ T cells, but not in activated CD4ϩ T cells (Fig. 3c). Spe- T cells was associated with changes to the level of H3K9acet. ChIP cifically, activated CD8ϩ T cells displayed rapid recruitment of analyses utilizing an Ab specific for H3K9acet revealed a striking Pol II to the proximal gzmB promoter, with the greatest enrichment difference in the level of H3K9acet within the proximal gzmB pro- being at 4–12 h after activation (Fig. 3c). Furthermore, after 24 h moter between the two T cell subsets (Fig. 3b). Activated CD8ϩ T of activation, Pol II recruitment decreased to levels equivalent to cells displayed substantial levels of H3K9acet across the proximal those before induction (Fig. 3c); a result that is consistent with the gzmB promoter, especially within the region 300 bp upstream of decrease in Mnase accessibility at this time point in CD8ϩ T cells the TSS (primer B, Fig. 3b). It is important to note that this same (Fig. 2b). promoter region exhibited a substantial increase in chromatin ac- Collectively, these data demonstrate that H3 histone loss occurs cessibility following T cell activation (Fig. 2). Furthermore, in a comparable manner for both CD4ϩ and CD8ϩ T cells, H3K9acet was sustained in activated CD8ϩ T cells as it was max- whereas substantial H3K9acet and Pol II recruitment appear to be imal 24 h after activation. In contrast, activated CD4ϩ T cells hallmarks of the gzmB promoter in activated CD8ϩ T cells. exhibited minimal levels of H3K9acet across the gzmB promoter (Fig. 3b). Therefore, efficient gzmB transcription in CD8ϩ T cells Histone H3 methylation patterns and transcriptional activation is associated with a high level of H3K9acet across the gzmB prox- Next, we investigated the relationship between specific histone H3 imal promoter region. These findings are in line with a previous methylation marks with the significantly different gzmB mRNA study conducted in yeast that showed histone loss occurs before or expression patterns observed in CD4ϩ and CD8ϩ T cells. To this independently of histone acetylation (38). end, we performed ChIP assays utilizing specific Abs against the 7068 gzmB TRANSCRIPTION IN CD4ϩ AND CD8ϩ T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Kinetics and patterns of histone H3 methylation across the promoters and transcribed regions of the gzmB and gzmN genes in activated CD4ϩ and CD8ϩ T cells. ChIP assays were performed on CD4ϩ and CD8ϩ T cells either nonstimulated (0 days) or stimulated with anti-CD3/CD28 Abs for 1, 2, or 3 days. a, Schematic representation of the gzmB proximal promoter and transcribed region indicating the locations of the two promoter primer sets B and C, the TSS, and the three transcribed region primer sets 5Ј,M,and3Ј. Enrichment of specific DNA sequences in (b) H3K4me3-specific, (c) H3K4me2-specific, and (d) H3K9me3-specific chromatin immunoprecipitates was determined by real-time PCR using primer sets B and C that target the gzmB proximal promoter region and primer sets 5Ј, middle (M), and 3Ј that target the transcribed region of gzmB and gzmN, with bar graphs being shown for the 0 (black)-, 1 (red)-, 2 (blue)-, and 3 (green)-day time points for each primer set. Data are calculated from the ratio of immunoprecipitated DNA minus the no Ab control and normalized against the total input. The results represent the mean Ϯ SE of two independent experiments. methylation marks H3K4me3, H3K4me2, and H3K9me3, with region kept increasing up to 3 days after stimulation, reaching subsequent quantitative PCR analysis using primers for both pro- maximum levels of ϳ500-fold enrichment. (Fig. 4b). A similar moter and transcribed regions (Fig. 4a). Previously, it was shown H3K4me3 pattern was observed for CD4ϩ T cells, but the av- that H3K4me2 marks active as well as inactive euchromatin genes, erage levels of H3K4me3 enrichment were 10-fold lower (Fig. while H3K4me3 directly correlates with the transcriptional process 4b). To compare H3K4me3 levels with that of an inactive gene, (39). In contrast, H3K9me3 has traditionally been associated with the promoter and transcribed region of the granzyme N (gzmN) repression of transcription, involving the recruitment of the het- gene were included in the analysis. On average, the H3K4me3 erochromatin protein 1 (40). levels were Ͻ10 in both CD4ϩ and CD8ϩ T cells (Fig. 4b), Consistent with the histone H3K9acet results (Fig. 3b), consistent with the view that this histone modification does in- higher levels of H3K4me3 in the transcribed region of the gzmB deed play an activation-specific role. gene were observed in activated CD8ϩ T cells (Fig. 4b), in Next, we investigated the distribution of H3K4me2 across the particular downstream of the TSS (primer 5Ј, Fig. 4a), which is promoter and transcribed region of the gzmB gene (Fig. 4c). For in agreement with recent genome-wide studies (25, 26). In both CD4ϩ and CD8ϩ T cells, H3K4me2 enrichment at the CD8ϩ T cells, maximum levels of H3K4me3 recruitment within gzmB promoter and transcribed regions was already observed in the promoter region were reached within the first 24 h following nonstimulated cells, with comparable levels of methylation in stimulation, whereas H3K4me3 levels in the gzmB-transcribed both regions for both cell types. Following activation of CD8ϩ The Journal of Immunology 7069

T cells, significant enrichment of H3K4me2 occurred in the promoter, in the 5Ј, and in particular in the 3Ј-transcribed re- gions of the gzmB gene. Such major changes were not observed in activated CD4ϩ T cells. In contrast, the silent gene gzmN exhibited less pronounced differences. Moreover, increases in H3K4me2 only occurred on the gzmB promoter in CD8ϩ T cells upon transcriptional activation. Finally, we investigated whether H3K9me3 played a role in the transcriptional regulation of gzmB in CD4ϩ and CD8ϩ T cells. The H3K9me3 levels were generally higher in CD4ϩ compared with CD8ϩ T cells (Fig. 4d), in particular across the gzmB promoter region. Following activation, H3K9me3 enrich- ment levels appeared to increase across the gzmB promoter in CD4ϩ T cells, whereas this mark increased in the transcribed region of gzmB in CD8ϩ T cells (Fig. 4d). These findings are reminiscent of a recent report indicating that H3K9me3 may influence the termination of inducible transcription (41). In con- trast, low levels of H3K9me3 were detected across the gzmN promoter and transcribed regions in both T cell subsets (ϳ2- to Downloaded from 3-fold), despite gzmN being a transcriptionally inactive gene. This raises the possibility that H3K9me3 plays only a minor role in the long-term silencing of this gene. FIGURE 5. Chromatin remodeling and epigenetic modifications of the gzmB gene in in vivo-activated CTL. Mice previously primed (at least 6 Taken together, lineage-specific epigenetic changes mark the ϩ wk) with the influenza virus strain A/PR8 were challenged intranasally gzmB gene in activated CD8 T cells, which include increases with the influenza strain A/HKx31. Eight days after infection, enriched ϩ in H3K9acet, H3K4me3, and H3K4me2. In contrast, there is an CD8 T cells from spleens were stained with DbNP366- and DbPA224- http://www.jimmunol.org/ ϩ increase in H3K9me3 in CD4 T cells, which may help keep specific tetramers, with tetramer-positive and -negative cells being sepa- the gzmB gene in a more repressed state. rated by cell sorting. a, Chromatin remodeling at the gzmB proximal pro- moter, as determined by CHART, in tetramer-negative (TetϪ) and Chromatin remodeling and epigenetic modification of the gzmB DbNP366 tetramer-positive (NP366ϩ) CD8ϩ T cell populations using in vivo primer sets A (Ⅺ),B(f), and C (u) across the gzmB proximal promoter Our in vitro data suggest that the acquisition of gzmB transcrip- as shown in Fig. 2a. Shown is the average accessibility of pooled samples tion by CD8ϩ T cells is associated with increased Mnase ac- from five mice. b, Deposition of H3K9acet across the most proximal pro- moter region of the gzmB gene in tetramer-negative and DbNP366ϩ or cessibility followed by a dramatic increase in H3K9acet within ϩ ϩ DbPA224(PA244 ) tetramer-positive CD8 T cell populations, as re- by guest on September 27, 2021 the proximal promoter and transcribed region of gzmB.Toex- vealed by ChIP and real-time PCR (primer set C). Shown are individual amine whether the observed in vitro chromatin changes also experiments of pooled groups of five mice per experiment. occur in an in vivo context, we used a well-characterized model of influenza A virus infection to induce a de novo virus-specific CTL response. Influenza A virus infection induces CTL re- ϩ sponses via a number of different peptide Ags, with the pre- served for in vitro-activated CD8 T cells, both Ag-specific dominant responses being directed against H-2Db-binding pep- CTL populations showed enrichment of H3K9acet (Fig. 5b) tides derived from the viral nucleoprotein (aa NP366–374; within the proximal gzmB promoter (primer C, region Ϫ34 to Ϫ termed NP366) (42) and the acid polymerase A subunit (aa 191). This supports the notion that H3K9acet is intricately

PA224–233; termed PA224) (43). To obtain the required num- linked with activation of inducible genes such as gzmB and can ϩ ber of Ag-specific CD8ϩ T cells for CHART and ChIP analysis, persist in CD8 T cells for long periods after activation. b b we used H-2D tetramer staining to isolate D NP366- and b D PA224-specific CTL from mice that were initially primed Discussion with the A/PR8 virus strain and then challenged 8 days later gzmB, unlike many other inducible genes in T cells, is not re- with the A/HKx31 influenza strain. We have previously dem- quired to be expressed immediately following T cell activation onstrated that CTL isolated at this time point exhibit potent because it is an effector molecule involved in target cell lysis. cytotoxic capacity (44) and significant gzmB expression (45). It Furthermore, gzmB exhibits specific expression, being predom- should be noted that the virus-specific CTL are derived from inantly activated in CD8ϩ compared with CD4ϩ T cells. Thus, memory CD8ϩ T cells that have been re-exposed to specific Ag we proposed that chromatin regulatory mechanisms may play a 8 days earlier. Thus, only chromatin changes that are required key role in this differential expression of gzmB. Indeed, this is for long-term gzmB transcription will be evident. As was ob- the case, with a number of important transcriptional features ϩ b served with in vitro-activated CD8 T cells, D NP366-specific being identified, which are depicted in Fig. 6. We show that CTL exhibited an extensive increase in Mnase accessibility histone H3 loss at the proximal promoter region of gzmB ap- within the proximal gzmB promoter region (Fig. 5a) when com- pears to be an essential event that accompanies gzmB transcrip- pared with naive (tetramer negative) CD8ϩ T cells. Impor- tion in both CD4ϩ and CD8ϩ T cells (prepoised chromatin tantly, the same regions of the gzmB proximal promoter that state). Significant chromatin accessibility and the corresponding showed increased accessibility after in vitro activation were high levels of gzmB transcription observed specifically in also more accessible in DbNP366-specific CTL (i.e., Ϫ34 to CD8ϩ T cells appear to require 1) the recruitment of substantial Ϫ281 bp upstream of the TSS; Fig. 5a). Pol II on the promoter of this gene (poised chromatin state) and The extent of H3K9acet was then characterized for both 2) the existence of a specific epigenetic signature, namely, b b D NP366- and D PA224-specific CTL (Fig. 5b). As was ob- H3K9acet, H3K4me3, and H3K4me2 (active chromatin state). 7070 gzmB TRANSCRIPTION IN CD4ϩ AND CD8ϩ T CELLS

IL-12p40 (35, 48). Furthermore, an earlier study detected a hy- persensitivity site at approximately Ϫ150 bp from the TSS of the gzmB gene (12), which overlaps with the region covered by primers sets B and C in this study. Our finding that chromatin accessibility changes coincide well with gene transcription in both CD4ϩ and CD8ϩ T cells is not surprising, given that ac- cessibility changes can be attributed to an array of factors. These include histone variant exchange (46), nucleosome slid- ing, the binding of transcriptional activators/loss of repressors, and chromatin conformational alterations as a consequence of histone modifications (35, 48). Many early inducible genes have been shown to have Pol II already bound to their regulatory regions before gene transcription, a state termed “poised” to indicate the ability for rapid transcrip- tional activation (49–51). Interestingly, we show here that Pol II is recruited rapidly and robustly in a transient manner to the gzmB promoter in activated CD8ϩ T cells, but not CD4ϩ T cells. This event appears to be closely coupled to histone loss across this promoter following activation, suggesting that this chromatin con- Downloaded from formation represents a prepoised state, which may have to be es- tablished before the recruitment of Pol II and establishment of a “poised chromatin state.” This additional mechanistic step could account for the delay in the activation of the gzmB gene. This step from a prepoised to a poised state may occur in a lineage-depen- dent manner and is likely to be inefficient in cell types not appro- http://www.jimmunol.org/ priately primed for high-level gzmB transcriptional activation, such as CD4ϩ T cells (Fig. 6). One possible explanation for this lineage-specific behavior could be the lack of cell-specific gzmB transcriptional activators, such as Eomesodermin (52), or the pres- FIGURE 6. Preliminary model of the associations and interplay be- ence of gzmB transcriptional repressors, including Bcl-6 (53). tween chromatin remodeling and histone modifications. State 1, Within a Taken together, these results lend support to a model whereby the population of naive CD8ϩ and CD4ϩ T cells, TF binding sites across the loss of histones in the proximal promoter establishes a distinct

gzmB proximal promoter are likely to be occupied by a loosely packed prepoised chromatin state before the recruitment of Pol II. Con- by guest on September 27, 2021 nucleosomal array (inherited during T cell lineage development, and version of this prepoised state to an “active state” appears to re- judged by the basal Mnase sensitivity of the proximal gzmB promoter), thus quire changes in the modification status of histone H3 in CD8ϩ T preventing the assembly of the general TF machinery, and keeping the cells. Specifically, our findings illustrate that lineage-specific epi- gzmB gene in a transcriptionally inactive state (state 2). Upon cell activa- genetic changes mark the gzmB gene in activated CD8ϩ T cells. tion, the gzmB promoter region undergoes rapid and substantial nucleoso- ϩ mal remodeling within the majority of both CD8ϩ and CD4ϩ T cells, with High levels of gzmB transcription in activated CD8 T cells a simultaneous loss of the core histone H3 without, however, changes in correlated with dramatic increases in H3K9acet upstream of the histone tail modifications, resulting in a transcriptionally prepoised state gzmB TSS, a result that is in agreement with a recently pub- (state 3). The gzmB gene is now ready to move from this prepoised into a lished study of human CD8ϩ T cells (16). The H3K9acet find- poised state (as indicated by the recruitment of Pol II to the proximal ings also appear to be relevant for long-term in vivo-activated promoter), a process that appears to be occurring with high efficiency in ϩ ϩ CD8 T cells, since H3K9acet is also enriched within the gzmB activated CD8 T cells (likely due to the presence of positive regulatory promoter of virus-specific CTL generated during a secondary factors), but not, however, in CD4ϩ T cells (possibly due to the presence ϩ immune response to a viral infection, a result which is in agree- of repressors) (state 4). In CD8 T cells, the gzmB gene can now move into a state of active transcription, as indicated by the accumulation of a specific ment with a recently published study (16). In addition, distinct combination of histone H3 acetylation and methylation patterns (font size and specific H3 methylation changes occur at different regions of histone modifications correspond to ChIP enrichment levels), resulting of the gene, which correlate well with the extent of gzmB tran- in the Pol II efficiently escaping from the promoter, leading to high-level scription. Methylation of histone H3K4 is currently thought to gzmB transcription. The higher levels of inhibitory histone methylation be exclusively associated with active transcription of inducible ϩ across the gzmB promoter in CD4 T cells may be an indication of neg- genes (39). Indeed, we observed significant enrichment of ative factors outweighing positive transcriptional factors, thus favoring re- H3K4me3 in the promoter and transcribed region of the gzmB pression of high-level transcription. gene after in vitro activation of CD8ϩ T cells. A similar pattern was observed for H3K4me2, albeit with the greatest enrichment Our findings concur with previous studies which demon- detected in the 3Ј-transcribed region. These patterns are in strated that histone loss accompanies inducible gene transcrip- agreement with previous studies on the genome-wide distribu- ϩ tion (35–37, 46, 47). Although both activated CD4ϩ and CD8ϩ tion of modifications of activated genes (25–27). When CD4 ϩ T cells undergo extensive histone loss at the gzmB proximal and CD8 T cells exist in a basal state, the level of H3K4me2 promoter, only CD8ϩ T cells are able to establish and maintain at the promoter region of gzmB in both T cell populations was high levels of gzmB transcription. Indeed, chromatin accessi- similar. Interestingly, the H3K4me3 and H3K4me2 marks bility correlated well with the gzmB transcriptional profiles in across the gzmB gene were generally higher than those in the both CD4ϩ and CD8ϩ T cells. This finding is consistent with inactive gzmN gene, indicating that these PTMs may have a role previous observations with inducible genes, such as IL-2 and in facilitating lineage-specific gzmB transcription. The Journal of Immunology 7071

The H3K9me3 PTM has typically been associated with tran- 16. Araki, Y., M. Fann, R. Wersto, and N. P. Weng. 2008. Histone acetylation fa- scriptional repression (40). Our in vitro analysis demonstrated cilitates rapid and robust memory CD8 T cell response through differential ex- pression of effector molecules (eomesodermin and its targets: perforin and gran- that H3K9me3 was enriched across the gzmB promoter and zyme B). J. Immunol. 180: 8102–8108. transcribed region of both activated CD4ϩ and CD8ϩ T cells, 17. Luger, K., and T. J. Richmond. 1998. The histone tails of the nucleosome. Curr. compared with naive T cells. In general, CD4ϩ T cells dis- Opin. Genet. Dev. 8: 140–146. 18. Becker, P. B., and W. Horz. 2002. ATP-dependent nucleosome remodeling. played greater enrichment of H3K9me3 following induction, Annu. Rev. Biochem. 71: 247–273. raising the possibility that these repressive marks may be spe- 19. Bird, A. P., and A. P. Wolffe. 1999. Methylation-induced repression–belts, cifically targeted to ensure that gzmB is not expressed. braces, and chromatin. Cell 99: 451–454. 20. Cosgrove, M. S. 2007. Histone proteomics and the epigenetic regulation of nu- Our study may provide a molecular explanation for previous cleosome mobility. Expert Rev. Proteomics 4: 465–478. ϩ observations showing that under certain circumstances, CD4 T 21. Hake, S. B., and C. D. Allis. 2006. Histone H3 variants and their potential role cell have the potential to express gzmB (11, 54). Upon in vitro in indexing mammalian genomes: the “H3 barcode hypothesis.” Proc. Natl. Acad. ϩ ϩ Sci. USA 103: 6428–6435. activation, the proximal gzmB promoter of both CD4 and CD8 22. Berger, S. L. 2007. The complex language of chromatin regulation during tran- T cells undergoes significant H3 loss and an increase in chromatin scription. Nature 447: 407–412. accessibility. Since these events are occurring in a lineage-inde- 23. Kouzarides, T. 2007. Chromatin modifications and their function. Cell 128: 693–705. pendent fashion, this proposed prepoised chromatin state supports ϩ 24. Strahl, B. D., and C. D. Allis. 2000. The language of covalent histone modifi- the notion that CD4 T cells have the potential to express gzmB cations. Nature 403: 41–45. depending upon additional intrinsic signals (11, 55, 56). 25. Pokholok, D. K., C. T. Harbison, S. Levine, M. Cole, N. M. Hannett, T. I. Lee, G. W. Bell, K. Walker, P. A. Rolfe, E. Herbolsheimer, et al. 2005. Genome-wide In conclusion, our study provides new insights into the mecha- map of nucleosome acetylation and methylation in yeast. Cell 122: 517–527. nisms underpinning lineage-specific transcriptional activation. We 26. Schones, D. E., and K. Zhao. 2008. Genome-wide approaches to studying chro- Downloaded from propose that high levels of gzmB transcription in CD8ϩ T cells are matin modifications. Nat. Rev. Genet. 9: 179–191. 27. Wang, Z., C. Zang, J. A. Rosenfeld, D. E. Schones, A. Barski, S. Cuddapah, driven by the coordinated interplay of histone loss, substantial pro- K. Cui, T. Y. Roh, W. Peng, M. Q. Zhang, and K. Zhao. 2008. Combinatorial moter-bound Pol II, together with specific PTM signatures distrib- patterns of histone acetylations and methylations in the . Nat. uted across the gzmB gene. Genet. 40: 897–903. 28. Wei, G., L. Wei, J. Zhu, C. Zang, J. Hu-Li, Z. Yao, K. Cui, Y. Kanno, T. Y. Roh, W. T. Watford, et al. 2009. Global mapping of H3K4me3 and H3K27me3 reveals ϩ Acknowledgments specificity and plasticity in lineage fate determination of differentiating CD4 T http://www.jimmunol.org/ We thank Dr. P. Milburn and Dr. B. Quah for excellent technical assistance cells. Immunity 30: 155–167. 29. Pokholok, D. K., J. Zeitlinger, N. M. Hannett, D. B. Reynolds, and R. A. Young. and Prof. Anne Kelso for critical reading of this manuscript. We thank Drs. 2006. Activated signal transduction kinases frequently occupy target genes. Sci- Damien Purcell, Nicole La Gruta, and Katherine Kedzierska for helpful ence 313: 533–536. discussion. 30. Kilinc, M. O., K. S. Aulakh, R. E. Nair, S. A. Jones, P. Alard, M. M. Kosiewicz, and N. K. Egilmez. 2006. Reversing tumor immune suppression with intratu- moral IL-12: activation of tumor-associated T effector/memory cells, induction of Disclosures T suppressor apoptosis, and infiltration of CD8ϩ T effectors. J. Immunol. 177: The authors have no financial conflict of interest. 6962–6973. 31. Lee, G. R., S. T. Kim, C. G. Spilianakis, P. E. Fields, and R. A. Flavell. 2006. T helper cell differentiation: regulation by cis elements and epigenetics. Immunity References 24: 369–379. by guest on September 27, 2021 1. Kaech, S. M., S. Hemby, E. Kersh, and R. Ahmed. 2002. Molecular and func- 32. Agarwal, S., J. P. Viola, and A. Rao. 1999. Chromatin-based regulatory mech- tional profiling of memory CD8 T cell differentiation. Cell 111: 837–851. anisms governing cytokine gene transcription. J. Allergy Clin. Immunol. 103: 2. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman. 990–999. 1986. Two types of murine helper T cell clone: I. Definition according to profiles 33. Rao, S., E. Procko, and M. F. Shannon. 2001. Chromatin remodeling, measured of lymphokine activities and secreted proteins. J. Immunol. 136: 2348–2357. by a novel real-time polymerase chain reaction assay, across the proximal pro- 3. Ansel, K. M., D. U. Lee, and A. Rao. 2003. An epigenetic view of helper T cell moter region of the IL-2 gene. J. Immunol. 167: 4494–4503. differentiation. Nat. Immunol. 4: 616–623. 34. Ward, S. B., G. Hernandez-Hoyos, F. Chen, M. Waterman, R. Reeves, and 4. Agarwal, S., and A. Rao. 1998. Modulation of chromatin structure regulates E. V. Rothenberg. 1998. Chromatin remodeling of the interleukin-2 gene: distinct cytokine gene expression during T cell differentiation. Immunity 9: 765–775. alterations in the proximal versus distal enhancer regions. Nucleic Acids Res. 26: 5. Avni, O., D. Lee, F. Macian, S. J. Szabo, L. H. Glimcher, and A. Rao. 2002. Th 2923–2934. cell differentiation is accompanied by dynamic changes in histone acetylation of 35. Weinmann, A. S., D. M. Mitchell, S. Sanjabi, M. N. Bradley, A. Hoffmann, cytokine genes. Nat. Immunol. 3: 643–651. H. C. Liou, and S. T. Smale. 2001. Nucleosome remodeling at the IL-12 p40 6. Slifka, M. K., F. Rodriguez, and J. L. Whitton. 1999. Rapid on/off cycling of Nat. Immunol. ϩ promoter is a TLR-dependent, Rel-independent event. 2: 51–57. cytokine production by virus-specific CD8 T cells. Nature 401: 76–79. 36. Workman, J. L. 2006. Nucleosome displacement in transcription. Genes Dev. 20: 7. Trapani, J. A., and M. J. Smyth. 2002. Functional significance of the perforin/ 2009–2017. granzyme cell death pathway. Nat. Rev. Immunol. 2: 735–747. 37. Chen, X., J. Wang, D. Woltring, S. Gerondakis, and M. F. Shannon. 2005. Hi- 8. Trapani, J. A. 2001. Granzymes: a family of lymphocyte granule serine proteases. stone dynamics on the interleukin-2 gene in response to T-cell activation. Mol. Genome Biol. 2: REVIEWS3014. Cell. Biol. 25: 3209–3219. 9. Shresta, S., C. T. Pham, D. A. Thomas, T. A. Graubert, and T. J. Ley. 1998. How 38. Guyon, J. R., G. J. Narlikar, S. Sif, and R. E. Kingston. 1999. Stable remodeling do cytotoxic lymphocytes kill their targets? Curr. Opin. Immunol. 10: 581–587. of tailless nucleosomes by the human SWI-SNF complex. Mol. Cell. Biol. 19: 10. Fruth, U., F. Sinigaglia, M. Schlesier, J. Kilgus, M. D. Kramer, and M. M. Simon. 2088–2097. 1987. A novel serine proteinase (HuTSP) isolated from a cloned human CD8ϩ ϩ ϩ 39. Santos-Rosa, H., R. Schneider, A. J. Bannister, J. Sherriff, B. E. Bernstein, cytolytic T cell line is expressed and secreted by activated CD4 and CD8 N. C. Emre, S. L. Schreiber, J. Mellor, and T. Kouzarides. 2002. Active genes are lymphocytes. Eur. J. Immunol. 17: 1625–1633. tri-methylated at K4 of histone H3. Nature 419: 407–411. 11. Devadas, S., J. Das, C. Liu, L. Zhang, A. I. Roberts, Z. Pan, P. A. Moore, G. Das, and Y. Shi. 2006. Granzyme B is critical for T cell receptor-induced cell death of 40. Bannister, A. J., P. Zegerman, J. F. Partridge, E. A. Miska, J. O. Thomas, type 2 helper T cells. Immunity 25: 237–247. R. C. Allshire, and T. Kouzarides. 2001. Selective recognition of methylated 12. Babichuk, C. K., B. L. Duggan, and R. C. Bleackley. 1996. In vivo regulation of lysine 9 on histone H3 by the HP1 chromo domain. Nature 410: 120–124. murine granzyme B gene transcription in activated primary T cells. J. Biol. Chem. 41. Vakoc, C. R., S. A. Mandat, B. A. Olenchock, and G. A. Blobel. 2005. Histone ␥ 271: 16485–16493. H3 lysine 9 methylation and HP1 are associated with transcription elongation 13. Prendergast, J. A., C. D. Helgason, and R. C. Bleackley. 1992. A comparison of through mammalian chromatin. Mol. Cell 19: 381–391. the flanking regions of the mouse cytotoxic cell proteinase genes. Biochim. Bio- 42. Townsend, A. R., J. Rothbard, F. M. Gotch, G. Bahadur, D. Wraith, and phys. Acta 1131: 192–198. A. J. McMichael. 1986. The epitopes of influenza nucleoprotein recognized by 14. Wargnier, A., S. Legros-Maida, R. Bosselut, J. F. Bourge, C. Lafaurie, cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 44: C. J. Ghysdael, M. Sasportes, and P. Paul. 1995. Identification of human gran- 959–968. zyme B promoter regulatory elements interacting with activated T-cell-specific 43. Belz, G. T., N. S. Wilson, C. M. Smith, A. M. Mount, F. R. Carbone, and ϩ proteins: implication of Ikaros and CBF binding sites in promoter activation. W. R. Heath. 2006. Bone marrow-derived cells expand memory CD8 T cells in Proc. Natl. Acad. Sci. USA 92: 6930–6934. response to viral infections of the lung and skin. Eur. J. Immunol. 36: 327–335. 15. Glimcher, L. H., M. J. Townsend, B. M. Sullivan, and G. M. Lord. 2004. Recent 44. Stambas, J., P. C. Doherty, and S. J. Turner. 2007. An in vivo cytotoxicity thresh- developments in the transcriptional regulation of cytolytic effector cells. Nat. Rev. old for influenza A virus-specific effector and memory CD8ϩ T cells. J. Immunol. Immunol. 4: 900–911. 178: 1285–1292. 7072 gzmB TRANSCRIPTION IN CD4ϩ AND CD8ϩ T CELLS

45. Jenkins, M. R., K. Kedzierska, P. C. Doherty, and S. J. Turner. 2007. Heteroge- control genes in the Drosophila melanogaster embryo. Nat. Genet. 39: neity of effector phenotype for acute phase and memory influenza A virus-specific 1512–1516. CTL. J. Immunol. 179: 64–70. 52. Pearce, E. L., A. C. Mullen, G. A. Martins, C. M. Krawczyk, A. S. Hutchins, 46. Sutcliffe, E. L., I. A. Parish, Y. Q. He, T. Juelich, M. L. Tierney, D. Rangasamy, V. P. Zediak, M. Banica, C. B. DiCioccio, D. A. Gross, C. A. Mao, et al. 2003. P. J. Milburn, C. R. Parish, D. J. Tremethick, and S. Rao. 2009. Dynamic histone Control of effector CD8ϩ T cell function by the transcription factor Eomesoder- variant exchange accompanies gene induction in T cells. Mol. Cell. Biol. 29: min. Science 302: 1041–1043. 1972–1986. 47. Weinmann, A. S., S. E. Plevy, and S. T. Smale. 1999. Rapid and selective re- 53. Yoshida, K., A. Sakamoto, K. Yamashita, E. Arguni, S. Horigome, M. Arima, modeling of a positioned nucleosome during the induction of IL-12 p40 tran- M. Hatano, N. Seki, T. Ichikawa, and T. Tokuhisa. 2006. Bcl6 controls granzyme ϩ scription. Immunity 11: 665–675. B expression in effector CD8 T cells. Eur. J. Immunol. 36: 3146–3156. 48. Rao, S., S. Gerondakis, D. Woltring, and M. F. Shannon. 2003. c-Rel is required 54. Casazza, J. P., M. R. Betts, D. A. Price, M. L. Precopio, L. E. Ruff, for chromatin remodeling across the IL-2 gene promoter. J. Immunol. 170: J. M. Brenchley, B. J. Hill, M. Roederer, D. C. Douek, and R. A. Koup. 2006. 3724–3731. Acquisition of direct antiviral effector functions by CMV-specific CD4ϩ T lym- 49. Guenther, M. G., S. S. Levine, L. A. Boyer, R. Jaenisch, and R. A. Young. 2007. phocytes with cellular maturation. J. Exp. Med. 203: 2865–2877. A chromatin landmark and transcription initiation at most promoters in human 55. Sharma, V., M. Delgado, and D. Ganea. 2006. Granzyme B, a new player in cells. Cell 130: 77–88. 50. Liang, M. D., Y. Zhang, D. McDevit, S. Marecki, and B. S. Nikolajczyk. 2006. activation-induced cell death, is down-regulated by vasoactive intestinal peptide The interleukin-1␤ gene is transcribed from a poised promoter architecture in in Th2 but not Th1 effectors. J. Immunol. 176: 97–110. monocytes. J. Biol. Chem. 281: 9227–9237. 56. Zhang, Y., H. Shin, J. S. Song, Y. Lei, and X. S. Liu. 2008. Identifying positioned 51. Zeitlinger, J., A. Stark, M. Kellis, J. W. Hong, S. Nechaev, K. Adelman, nucleosomes with epigenetic marks in human from ChIP-Seq. BMC Genomics 9: M. Levine, and R. A. Young. 2007. RNA polymerase stalling at developmental 537. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 The Journal of Immunology

Corrections

Juelich, T., E. Sutcliffe, A. Denton, Y. He, P. C. Doherty, C. Parish, S. J. Turner, D. Tremethick, and S. Rao. 2009. Interplay between chromatin remodeling and epigenetic changes during lineage-specific commitment to granzyme B expression. J. Immunol. 183: 7063–7072.

The middle initials were omitted for the second, sixth, and eighth authors. In addition, the first name of the seventh author was published incorrectly. The corrected author line is shown below.

Torsten Juelich, Elissa L. Sutcliffe, Alice Denton, Yiqing He, Peter C. Doherty, Christopher R. Parish, Stephen J. Turner, David J. Tremethick, and Sudha Rao

In addition, one of the corresponding authors was omitted from the second footnote. The corrected footnote is below.

2 Address correspondence and reprint requests to Dr. Sudha Rao, Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601 Australia; E-mail address: [email protected] or Assoc. Prof. Stephen J. Turner, Department of Microbiology and Immunology, University of Melbourne, Melbourne, VIC 3010, Australia. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.0990115

Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00