A Barrier-Type Insulator Forms a Boundary between Active and Inactive Chromatin at the Murine TCRβ Locus

This information is current as Juan Carabana, Akiko Watanabe, Bingtao Hao and Michael of September 27, 2021. S. Krangel J Immunol 2011; 186:3556-3562; Prepublished online 11 February 2011; doi: 10.4049/jimmunol.1003164

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Barrier-Type Insulator Forms a Boundary between Active and Inactive Chromatin at the Murine TCRb Locus

Juan Carabana,1,2 Akiko Watanabe,2 Bingtao Hao,2 and Michael S. Krangel

In CD42CD82 double-negative thymocytes, the murine Tcrb locus is composed of alternating blocks of active and inactive chromatin containing Tcrb segments and trypsinogen , respectively. Although chromatin structure is appreciated to be critical for regulated recombination and expression of Tcrb gene segments, the molecular mechanisms that maintain the integrity of these differentially regulated Tcrb locus chromatin domains are not understood. We localized a boundary between active and inactive chromatin by mapping chromatin modifications across the interval extending from Prss2 (the most 39 trypsinogen gene) to Db1. This boundary, located 6 kb upstream of Db1, is characterized by a transition from repressive (histone H3 9 dimethylation [H3K9me2]) to active (histone H3 acetylation [H3ac]) chromatin and is marked by a peak of histone H3

lysine 4 dimethylation (H3K4me2) that colocalizes with a retroviral long terminal repeat (LTR). Histone H3 lysine 4 dimethylation Downloaded from is retained and histone H3 lysine 9 dimethylation fails to spread past the LTR even on alleles lacking the Tcrb enhancer (Eb) suggesting that these features may be determined by the local DNA sequence. Notably, we found that LTR-containing DNA functions as a barrier-type insulator that can protect a transgene from negative chromosomal position effects. We propose that, in vivo, the LTR blocks the spread of heterochromatin, and thereby helps to maintain the integrity of the Eb-regulated chromatin domain. We also identified low-abundance, Eb-dependent transcripts that initiate at the border of the LTR and an adjacent long

interspersed element. We speculate that this transcription, which extends across Db,Jb and Cb gene segments, may play an http://www.jimmunol.org/ additional role promoting initial opening of the Eb-regulated chromatin domain. The Journal of Immunology, 2011, 186: 3556– 3562.

n their cell surface, ab T lymphocytes express a unique (2–4). An accessible chromatin configuration, in turn, is directed TCR composed of TCRa- and TCRb-chains that is ac- by TCR locus promoter and enhancer elements. O quired during T cell development in the thymus. The The murine Tcrb locus is roughly 700 kb in length and contains genetic loci encoding these chains (Tcra and Tcrb) consist of 21 functional Vb gene segments, all but one of which is located variable (V), diversity (D), and joining (J) gene segments that are upstream of two tandem Db-Jb-Cb gene segment clusters (Fig. by guest on September 27, 2021 assembled through V(D)J recombination. This recombination re- 1A). The sole exception is the Vb14 gene segment, which lies action is initiated by the lymphoid-specific recombinase downstream of the Cb2 gene segment at the very 39 end of the RAG1 and RAG2, which recognize and generate DNA double- locus. A special feature of the Tcrb locus is the arrangement of 19 strand breaks at recombination signal sequences that flank V, D, functional Vb gene segments in a central cluster that is separated and J gene segments (1). V(D)J recombination occurs in a de- from the 59 distal Vb2 gene segments by ∼150 kb containing velopmental stage-specific manner, with developmental control seven trypsinogen genes and gene fragments, and from the Db- exerted in large measure through regulated access of the recom- Jb-Cb clusters by a 250-kb region comprising 13 trypsinogen binase to selected recombination signal sequences in chromatin genes and gene fragments. Notably, whereas Vb,Db, and Jb gene segments are all transcribed and undergo recombination in CD42 CD82 double-negative (DN) thymocytes, trypsinogen genes are Department of Immunology, Duke University Medical Center, Durham, NC 27710 not expressed and adopt a relatively closed chromatin conforma- 1Current address: Department of Ophthalmology, University of North Carolina at tion in the same cells (5). Hence the locus displays a modular Chapel Hill, Chapel Hill, NC. structure with alternating regions of active and inactive chromatin 2 J.C., A.W., and B.H. contributed equally to this work. in DN thymocytes. Received for publication September 23, 2010. Accepted for publication January 12, A Tcrb enhancer (Eb) located between Cb2 and Vb14 works in 2011. collaboration with promoters associated with Db1 and Db2to This work was supported by the National Institutes of Health (Grant AI35748 to activate the chromatin associated with Db and Jb gene segments M.S.K.). (6–10). Deletion of this enhancer severely reduces Db-Jb-Cb Address correspondence and reprint requests to Dr. Michael S. Krangel, Department of Immunology, Campus Box 3010, Duke University Medical Center, Durham, NC germline transcription and chromatin modifications, and nearly 27710. E-mail address: [email protected] abolishes Tcrb locus recombination, including the initial Db-to-Jb The online version of this article contains supplemental material. step and the subsequent Vb-to-DbJb step (6, 7, 9, 10). However, Abbreviations used in this article: ChIP, chromatin immunoprecipitation; CTCF, deletion of Eb does not influence transcription and chromatin CCCTC-binding factor; D, diversity; DN, double-negative; Eb, Tcrb enhancer; Ed, structure of unrearranged Vb gene segments, which are activated Tcrd enhancer; ERVK, endogenous retrovirus K; H3ac, histone H3 acetylation; H3K4me2, histone H3 lysine 4 dimethylation; H3K4me3, histone H3 lysine 4 trime- independently (10). Similarly, Eb does not appear to influence thylation; H3K9me2, histone H3 lysine 9 dimethylation; HS, hypersensitive site; J, transcription and chromatin structure of the trypsinogen genes, joining; LINE, long interspersed element; LTR, long terminal repeat; PDb1, promoter because these genes are inactive in DN thymocytes (5). Rather, the Db1; PVd1, promoter Vd1; V, variable. influence of Eb is limited to no more than 25 kb encompassing the Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 two Db-Jb-Cb clusters near the 39 end of the Tcrb locus (10). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003164 The Journal of Immunology 3557

In eukaryotic cells, active and inactive genes can coexist and PCR kit (Qiagen). Ratios of bound to input for modifications H3ac and b maintain differential regulation despite their proximity to multiple H3K4me2 were normalized to those for 2-microglobulin; ratios for sets of regulatory elements. Insulators prevent the misregulation H3K9me2 were normalized to those for MageA2. PCR reaction conditions were as follows: 5 min at 95˚C, followed by 45 cycles of 1 s at 95˚C, 5 s at of neighboring genes by delimiting the chromatin domains over 60–64˚C (depending of the primer pair), 7 s at 72˚C. PCR primers (Sup- which regulatory elements can function. Two types of insulator plemental Table I) matched perfectly only a single Tcrb locus site in the elements are involved in the establishment of genomic domains: C57BL/6 genome and were judged to amplify unique, single-copy se- enhancer-blocking elements that interfere with the ability of en- quences on the basis of melting curve analysis and amplification efficiency from input chromatin. hancers to activate the promoters of neighboring inactive genes, For analysis of CTCF binding, cross-linked chromatin was prepared and and barrier elements that block the spread of repressive hetero- used for ChIP as described previously (25) using 5 mg anti-CTCF Ab (no. chromatin into regions containing active genes (11, 12). In ver- 07-729; Upstate Biotechnology-Millipore). Bound and input materials tebrates, enhancer-blocking is mediated by the DNA-binding were quantified as described earlier. A known CTCF binding site in the CTCF (CCCTC-binding factor), which is thought to func- c-myc locus insulator (26) was used as a positive control. PCR primers are shown in Supplemental Table II. tion by promoting the formation of chromatin loops (13–15). Sev- eral mechanisms have been proposed to explain barrier activity, but 59 RACE a common theme is the recruitment of histone-modifying enzymes 2 2 59 RACE was performed on thymocyte RNA isolated from a Rag2 / Tcrb that deliver activating chromatin modifications that can interrupt the transgenic mouse using a GeneRacer Kit (Invitrogen) according to the propagation of silent chromatin (16–19). Although transcriptional manufacturer’s instructions. Strand-specific primers were used for PCR activity has been demonstrated for many yeast insulators with amplification of cDNA products. PCR products were purified by agarose gel barrier function, transcription is not an absolute requirement for electrophoresis and cloned using a TOPO TA Cloning Kit (Invitrogen). The Downloaded from 9 this function (20, 21). anti-sense primer tested was 5 -AGCCACAGTGTGTGGTCTGCTACAG- 39. Two sense primers (59-CAAAGACTCAGAGAAGGTTGTTCACTC-39 To ask whether insulator activity helps to maintain the integrity and 59-GATCAGTTGGAGTAACAGGAAAGAGAG-39) were also tested of adjacent regulatory domains of the Tcrb locus, we mapped but did not amplify specific products. chromatin modifications to define the border between active Db-Jb-Cb and inactive trypsinogen chromatin. Remarkably, this cDNA amplification and analysis border, situated ∼6 kb upstream of promoter Db1 (PDb1), cor- RNA was extracted using TRIzol reagent (Invitrogen) following the manu- http://www.jimmunol.org/ responded to an endogenous long terminal repeat (LTR). This facturer’s specifications. Three micrograms RNA were digested with 3 U element was shown to have bona fide barrier activity, because it RNase-free DNase (Sigma) in 33 ml digestion buffer (20 mM Tris-HCl, pH could insulate an integrated reporter construct from chromosomal 8.0, 50 mM KCl, 2 mM MgCl2) for 15 min at 23˚C. After addition of EDTA to a final concentration of 2.5 mM and incubation at 68˚C for 10 min, the position effects in transfected cells. We discuss the implications of RNA was split in two aliquots that were incubated with SuperScript III our observations in the context of Tcrb locus activation during (Invitrogen) or mock incubated, according to the manufacturer’s instruc- thymocyte development. tions. The resulting cDNAs were diluted 1:10 (or 1:100 for Actb analysis) and used for conventional PCR using primers P1 (59-GGCTATCTGCA- ACACAGATCTT-39) and P3 (59-TGATGGCTCAAACAAGGAGAC-39) or quantitative real-time PCR using primers P1 and P2 (59-AAGCCA-

Materials and Methods by guest on September 27, 2021 9 Mice CAGTGTGTGGTCTG-3 ). Experimental PCR values were normalized to those for Actb (primers 59-TCGTAGATGGGCACAGTGTG-39 and 59- 2/2 2/2 2/2 Rag2 mice (22), Rag1 Eb mice (10) (kind gift from P. Ferrier, GGTCAGAAGGACTCCTATGTG-39). To quantify transcripts detected by Centre d’Immunologie Marseille-Luminy, Marseille, France), and Rag22/2 3 the P1-P2 primer set relative to total germline Tcrb transcripts, we conducted Tcrb transgenic mice (23) were used in accordance with protocols approved amplifications with P1-P2 and amplifications with a Cb primer set (59- by the Duke University Animal Care and Use Committee. AAAAGGCTACCCTCGTGTGC-39 and 59-CTGTGGACCTCCTTGCCATT- 39) from both cDNA and genomic DNA. Percent germline transcripts using Chromatin immunoprecipitation the 470-bp exon was estimated as follows: (P1-P2cDNA/P1-P2genomic DNA)/ (Cb /Cb ) 3 100. Histone modifications were analyzed by chromatin immunoprecipitation cDNA genomic DNA (ChIP) using antiacetylated histone H3 (anti-H3ac; no. 06599; Upstate Barrier assay plasmids Biotechnology-Millipore), antidimethylated H3 lysine 4 (anti-H3K4me2; no. 07030; Upstate Biotechnology-Millipore), antitrimethylated H3 ly- To create a reporter construct that would be sensitive to chromosomal sine 4 (anti-H3K4me3; no. 04745; Upstate Biotechnology-Millipore), and position effects, we generated a pGL3 (Promega)-based vector that ex- antidimethylated histone H3 lysine 9 (anti-H3K9me2; no. Ab1220; pressed GFP under the control of a weak promoter (human Vd1 promoter Abcam). ChIP with rabbit IgG (no. AB-105-C; R&D Systems) served as [PVd1]) and enhancer (human Tcrd enhancer [Ed]). The 1.8-kb PVd1 a control. Chromatin was prepared by one of two methods. For some fragment was excised from plasmid EVNS’ (27) with SalI and HindIII, and experiments, chromatin was prepared in large scale by micrococcal nu- cloned into the pGL3-promoter vector digested with XhoI and HindIII. clease digestion and purification of mononucleosomes or a mixture of The luciferase gene was then excised by XbaI digestion, followed by dinucleosomes and trinucleosomes by sucrose gradient fractionation as Klenow treatment to obtain a blunt end, and subsequent digestion with described previously (24). For other experiments, chromatin was prepared NcoI. The GFP gene, isolated from plasmid MSCV-IRES-GFP by HindIII in small scale by micrococcal nuclease digestion as follows: 1 3 107 digestion, Klenow treatment, and digestion with NcoI, was then introduced thymocytes were washed and lysed as described previously (24). Nuclei into the pGL3 vector in place of luciferase. A 380-bp Ed fragment, were then resuspended in 200 ml digestion buffer, and 5 U micrococcal obtained from plasmid EVNS’ by digestion with XbaI and EcoRI, fol- nuclease (Worthington) was added for 5 min at 37˚C to generate a mixture lowed by Klenow treatment, was then cloned downstream of GFP by consisting primarily of mononucleosomes with a small fraction of dinu- ligation into a BamHI site rendered blunt by Klenow treatment. The cleosomes. The reaction was stopped by addition of 300 ml of 10 mM Tris- resulting plasmid, pGL3-PVd1-GFP-Ed, was used to assay fragments for HCl, pH 8.0, 5 mM EDTA, 10 mM sodium butyrate, after which the barrier activity, with two copies of each test fragment introduced both sample was sonicated on ice for eight 15-s intervals using a Sonicator 3000 upstream of the promoter and downstream of the enhancer. Test fragments (Misonix) with the output set to 3.0. After centrifugation, the supernatant were amplified by PCR to introduce terminal MluI and NheI sites or NheI was harvested and Triton X-100 was added to a concentration of 1% (v/v). and XhoI sites, and the two PCR fragments were then subcloned stepwise Chromatin was precleared with protein A-Sepharose/salmon sperm DNA into the multiple cloning site of pGL3-promoter. For cloning into the slurry (Upstate Biotechnology-Millipore) and was incubated with the ap- upstream site, the fragments were excised as a dimer by XhoI digestion, propriate specific Ab overnight at 4˚C followed by addition of protein A- Klenow treatment, and MluI digestion. The excised dimer was then cloned Sepharose/salmon sperm DNA slurry for 1 h. Immunoprecipitates were into MluI and SmaI digested pGL3-PVd1-GFP-Ed. For cloning into the washed vigorously and DNA was purified. Bound and input fractions were downstream site, the dimer was excised by digestion with XhoI and MluI, quantified by quantitative real-time PCR using a LightCycler and FastStart followed by Klenow treatment, and then introduced into the AfeI site of DNA Master SYBR Green I kit (Roche) or a QuantiFast SYBR Green the plasmid. Test fragments were: HS4, a 1.2-kb fragment containing the 3558 A TCRb LOCUS BOUNDARY ELEMENT chicken b-globin 59 hypersensitive site (HS) 4 insulator (positive control, region spanning from the silent Prss2 gene (the most 39 - 9 9 2/2 amplified using 5 -TGAGAACGCGTCCGCGGATCTCACGGGGACAG-3 ogen gene) to the active PDb1 in DN thymocytes of Rag2 mice 9 9 and 5 -AGAGAGCTAGCTCACTGACTCCGTCC-3 ); fragment A, a 1.5-kb (Fig. 1A,1B). We detected a signature of highly active chromatin fragment spanning from 7.6 to 6.1 kb upstream of Db1 (MMAE000665 145657–147165, amplified using primers 59-ATGCACGCGTACGCATCA- at PDb1 (H3ac and H3K4me2) and of inactive chromatin at Prss2 GAAGAGGGCATC-39 and 59-ATGCGCTAGCGATCTGCAGTTAGCTC- (H3K9me2). Notably, there was a sharp transition from H3K9me2 ACTGG-39); and fragment B, a 1.5-kb fragment spanning from 6.9 to 5.4 kb to H3ac over a 1-kb region situated between 5.5 and 6.5 kb up- upstream of Db1 (MMAE000665 146357–147868, amplified using primers stream of Db1 and between 2 and 3 kb downstream of the Prss2 59-ATGCACGCGTACTCAGAGAAGGTTGTTCACTC-39 and 59-ATGCG- CTAGCAAGCCACAGTGTGTGGTCTG-39). polyadenylation signal. Not surprisingly, we detected elevated H3K4me2 that mapped to PDb1 (31). However, we also noted Barrier assay a peak of H3K4me2, centered 6.2 kb upstream of Db1, that Jurkat cells were maintained in RPMI 1640 medium supplemented with mapped to the transition between H3K9me2 and H3ac. This 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. The cells suggested that chromatin-modifying enzymes may be recruited to 3 6 (1 10 ) were transfected with 1 pmol linearized test plasmid plus 0.33 this site by an active cis-regulatory element. pmol of a linearized puromycin resistance plasmid by electroporation using an Amaxa Nucleofector system (Lonza), as instructed by the man- Repressive chromatin, as reflected by the H3K9me2 mark, may ufacturer. Transfected Jurkat cells were then cultured and expanded in spread until opposed by chromatin-modifying activities that deliver nonselective medium. After 6 d of culture, cells expressing intermediate activating histone modifications such as H3ac and H3K4me2 (32, levels of GFP were sorted on a Beckman-Coulter MoFlo and plated at 33). If this is the case at the Prss2-Db1 boundary, the activity of a density of 13 105/ml in selective medium containing 0.2 mg/ml puro- mycin. After 2 wk of selection, GFP+ cells were resorted using a BD Eb itself could be critical to prevent the encroachment of het- Biosciences FACSVantage (defined as day 0) and cultured in the presence erochromatin into the Db-Jb-Cb region. Previous studies showed Downloaded from or absence of puromycin for up to 42 d to monitor the loss of GFP ex- that, in the absence of Eb, the Db-Jb-Cb region is characterized by pression using a BD Biosciences FACSCanto II. a loss of germline transcription that is associated with reduced histone acetylation, increased DNA methylation, and resistance to Results nuclease digestion (10). Increased H3K9me2 was also detected at A59 boundary of the Eb-regulated chromatin domain both Db1 and Cb1 (31).

Posttranslational modifications of histone tails can reflect the To determine whether these changes reflected the spreading http://www.jimmunol.org/ transcriptional status and overall configuration of chromatin: open of heterochromatin from the 59 boundary, we compared histone and transcriptionally active euchromatin is typically marked by modifications across this region in DN thymocytes of Rag22/2 acetylation of histone H3 on 9 and 14 and dimethylation of mice in the presence or absence of Eb.Eb deletion abolished H3ac histone H3 on lysine 4, whereas condensed and transcriptionally in the immediate vicinity of Db1 and partially reduced H3ac 5.5 silent heterochromatin is typically enriched in histone H3 dime- and 3.4 kb upstream of Db1 (Fig. 2A). Eb deletion also abolished thylated at lysine 9 (17, 28–30). To define the edge of the Eb- regulated chromatin domain, we used ChIP to analyze an 11-kb by guest on September 27, 2021

FIGURE 1. A boundary between active and inactive chromatin upstream of Db1. A, Schematic diagram of the Tcrb locus, including functional Tcrb gene segments (black rectangles) and intervening regions containing try- psinogen genes and gene fragments (gray bars). The Tcrb enhancer (Eb) (filled oval) and germline PDb1 (bent arrow) are also identified. B, A map of H3ac, H3K4me2, and H3K9me2 histone modifications across the Prss2- FIGURE 2. The boundary is maintained in the absence of Eb. Histone 2/2 Db1 interval in DN thymocytes. H3ac and H3K4me2 were analyzed by modifications were analyzed on wild-type (WT) alleles in Rag2 DN 2/2 2/2 ChIP from pure mononucleosomes, and H3K9me2 was analyzed by ChIP thymocytes and on Eb-deleted alleles (DEb)inRag1 Eb DN thy- from pure dinucleosomes and trinucleosomes. Real-time PCR was used mocytes. A, H3ac was analyzed by ChIP from small-scale chromatin prep- to evaluate fold enrichment relative to input, with normalization to b2- arations. Data are the mean 6 SE of three independent experiments. B, microglobulin (B2m) for H3ac and H3K4me2 (right y-axis) and to MageA2 H3K4me2 was analyzed by ChIP from mononucleosomes. Data are the for H3K9me2 (left y-axis). Data are the mean 6 SE of two independent mean 6 SE of two independent experiments. Data for WT were derived experiments. Relative positioning of Db1, Prss2 (Trypsinogen 20), and from Fig. 1. C, H3K9me2 was analyzed by ChIP from small-scale chromatin previously described DNase HSs (45) are indicated below the graph. preparations. Data are the mean 6 SE of three independent experiments. The Journal of Immunology 3559

H3K4me2 at Db1, but this modification was unperturbed at the 59 products indicative of sense transcription initiating within a 50-bp boundary (Fig. 2B). Of note, Eb-deficient alleles displayed a small region ∼5.7 kb upstream of Db1 (Fig. 3A). increase in H3K9me2 at PDb1, but no increase in H3K9me2 in the Analysis of the DNA sequence around the start sites revealed the region between the 59 boundary and PDb1 (Fig. 2C). This indi- presence of an endogenous retroviral LTR (LTR BglII family cates that Eb plays no role in halting the spread of heterochro- endogenous retrovirus K [ERVK]) (Fig. 3A). This retroviral LTR is matin. Rather, if such an activity is present, it is likely mediated by 397 bp in length and contains several putative CCAAT and TATA an autonomous element that maintains an active chromatin con- boxes. It is flanked by a 750-bp long interspersed element (LINE) figuration at the boundary. Mur3 L1 fragment that is affixed at its 39 end to another 365-bp LTR11 family ERVK. The detected transcripts initiate near the 9 An endogenous retroviral LTR at the 5 boundary junction between the 59 LTR and the LINE element, whereas the Although a barrier effect may be achieved by disruption of a nu- peak of H3K4me2 is centered further upstream at the 59 edge of cleosome array (34) or attachment to nuclear structures (35), many the 59 LTR. of the best described barriers including the silent mating-type loci To more fully characterize the structure of these transcripts, we HMR and HML in yeast (16, 36) and chicken b-globin 59 HS4 carried out RT-PCR using a forward primer (P1) that anneals (18) share the property that they are highly enriched in histone across the LTR-LINE junction (to ensure specificity) and a reverse modifications associated with transcriptional activation (H3ac, primer (P3) that anneals to Cb1 (Fig. 3B). We identified two major H4ac, and H3K4me2). However, transcription itself has been products that were shown by cloning and sequencing to corre- shown not to be necessary for barrier activity (21). spond to differentially spliced versions of primary transcripts

Because the heterochromatin-to-euchromatin transition at the 59 that run from the LTR at least 11 kb through Cb1 (Fig. 3B,3C). Downloaded from boundary of the Eb regulatory domain coincided with a discrete The transcripts include three novel exons of 470, 780, and 72 H3K4me2 peak, we asked whether a transcription unit mapped to bp, which are defined by splice sites that conform well to the A A this region. Accordingly, we searched for transcripts that could 59 ( /CAG-GT /GAGT) and 39 (CAG-G) splice-site consensus initiate in the region by analyzing thymocyte RNA in a 59-RACE sequences (Fig. 3B,3C). The 39 end of the 780-bp exon aligns assay using a variety of specific primers. We were unable to with Jb1.2; to the best of our knowledge, the remaining spice sites

amplify PCR fragments using primers designed to detect anti- have not been described previously. http://www.jimmunol.org/ sense (relative to the orientation of Db1-Jb1-Cb1) transcripts To evaluate the regulation of transcript expression, we analyzed (data not shown). However, we did detect and clone several PCR cDNAs prepared from several RNA sources by quantitative real- by guest on September 27, 2021

FIGURE 3. Repetitive elements and transcription associated with the boundary region. A,59-RACE analysis. The diagram identifies LTR and LINE ele- ments in the vicinity of the boundary. Numbering represents distance (kb) upstream of Db1. The 59 ends of individual RACE clones are identified by bent arrows in the sequence near the junction of LTR Bgl II ERVK (white lettering on gray background) and LINE Mur3 L1 (black lettering on white background). B, Structure of boundary-associated transcripts in DN thymocytes. Transcripts running through Cb1 were amplified by PCR using primers P1 and P3 (arrowheads, left panel). Agarose gel electrophoresis and ethidium bromide staining of the predominant PCR products is shown (right panel). The splicing patterns that give rise to the two products, as deduced from sequencing, are indicated (left panel). C, DNA sequence of boundary-associated transcripts. Sequences that define the 470-, 780-, and 72-bp exons are shown. Exon sequences are capitalized and boxed; splicing signals at the intronic borders are in small letters. D, Quantification of LTR-derived transcripts. Primers P1 and P2 were used in quantitative real-time RT-PCR. RNA samples were prepared from Rag22/2 2/2 2/2 2/2 2/2 thymocytes (DN), Rag1 Eb thymocytes (DEb DN), Rag2 3 Tcrb transgenic thymocytes (double positive), and Rag2 3 Tcrb transgenic splenocytes (non-T). Values for the P1-P2 amplicon were normalized to those for Actb and are expressed relative to the value for DN thymocytes (= 1). Data represent the mean 6 SE of two to four independent experiments. E, Histone H3K4me3 was analyzed on wild-type (WT) alleles in Rag22/2 DN thy- 2/2 2/2 mocytes and on Eb-deleted alleles (DEb) in Rag1 Eb DN thymocytes by ChIP from small-scale chromatin preparations. Data are the mean 6 SE of three independent experiments. 3560 A TCRb LOCUS BOUNDARY ELEMENT time PCR using primers P1 and P2 (Fig. 3B,3D). Transcripts were detected in DN thymocytes of Rag22/2 mice and in CD4+CD8+ double-positive thymocytes of Rag22/2 mice that express a Tcrb transgene (Rag22/2Tcrb), but were detected at much reduced 2/2 2/2 levels in DN thymocytes of Rag1 Eb mice (DEb DN) and in splenocytes of Rag22/2 Tcrb mice (non-T). Thus, the tran- scripts are both T cell specific and Eb dependent. However, even in DN thymocytes of Rag22/2 mice, the abundance of these transcripts was low relative to standard germline Tcrb transcripts; by real-time PCR, transcripts detected by P1-P2 were only ∼0.1% as abundant as those detected by a pair of Cb primers (data not shown). One caveat is that because primer P1 spans the LTR- LINE junction, it can detect only a fraction of the transcripts initiating in this region (Fig. 3A). However, consistent with low- level transcription, the H3K4me3 modification, whose deposition is tightly linked to transcription (37), was readily detected at PDb1 but was barely detected above background in the LTR-LINE re- gion even on wild-type Tcrb alleles (Fig. 3E). We conclude that

H3K4me2 and H3K4me3 deposition are regulated independently Downloaded from in this region, and that neither H3K4me3 nor transcription appears to be involved in forming the identified chromatin boundary. Chromatin barrier function of the 59- LTR To determine whether the boundary region contains bona fide

barrier activity, we developed a chromosomal position effect assay http://www.jimmunol.org/ similar to that used in previous studies (38, 39). This assay relies on a base plasmid in which GFP expression is driven by the human TCRd enhancer (Ed) and PVd1, with test fragments cloned as dimers both upstream and downstream of the GFP expression cassette (Fig. 4A). Test plasmids were cotransfected into Jurkat cells together with a plasmid conferring puromycin resistance. After selection and sorting for GFP expression, transfected cells were cultured for up to 6 wk in the presence or absence of pu- romycin. Negative chromosomal position effects were then scored by guest on September 27, 2021 as the loss of GFP expression in a fraction of the transfected cells. Because selection and test plasmids should cointegrate, GFP loss should not be observed with culture in the presence of puromycin. FIGURE 4. LTR-containing DNA functions as a barrier-type insulator in However, position effects should be revealed during culture in a position-effect assay. Jurkat cells were transfected with a GFP reporter the absence of puromycin, because there should be no selection plasmid in which the expression cassette was flanked by dimerized test against silencing at the integration site under these conditions. As fragments. After puromycin selection and sorting for GFP expression, cells expected, in the absence of selection, we found that GFP ex- were maintained in parallel cultures in the presence or absence of puro- pression was diminished over time in a fraction of the trans- mycin. A, The diagram (top) maps test fragments A and B relative to the fectants when no insulator was present, but was stably maintained LTR and LINE elements, with numbering representing distance (kb) up- when the expression cassette was flanked by the positive control stream of Db1. Below is a depiction of the cloning of test fragment dimers insulator chicken b-globin 59-HS4 (Fig. 4B,4C). GFP expression into the PVd1-GFP-Ed reporter construct. B, GFP fluorescence is shown was also lost over time when the expression cassette was flanked after initial sorting (d0) and at the end of the culture period (d28 or d42; by a 1.5-kb test fragment situated upstream of LTR BglII ERVK gray histogram denotes culture with selection, black line histogram de- notes culture without selection). The results of three independent experi- (fragment A). However, GFP expression was stably maintained ments are shown. C, Remaining GFP signal was calculated at d28 or d42 when the cassette was flanked by an overlapping 1.5-kb test frag- as: (GFP MFI [2selection]/GFP MFI [+selection]) 3 100, where MFI is ment that included the LTR (fragment B). Notably, this fragment mean fluorescence intensity. Data represent the mean 6 SD of three in- was as effective as chicken b-globin 59-HS4. These data support dependent experiments. **p , 0.01, ***p , 0.001, by Student t test. the argument that the LTR functions as a barrier-type insulator that can prevent repressive chromosomal position effects. der between these chromatin domains, and showed that LTR- Discussion containing DNA functions as a barrier-type insulator that can Tcrb locus organization is striking in the sense that it presents protect a transgene from negative chromosomal position effects. alternating blocks of active chromatin composed of Tcrb gene This LTR colocalizes with a peak of histone modification H3K4me2, segments and inactive chromatin composed of unrelated Prss suggesting that it may share with other barrier-type insulators the genes and gene fragments. This unique organization poses in- capacity to recruit histone-modifying enzymes that can block the trinsic regulatory problems, particularly at the borders between propagation of silent chromatin (16–19). We suggest that by func- active and inactive chromatin. Such is the case at the 39 end of the tioning in this way, this LTR helps to maintain the integrity of the locus, where a 250-kb domain of inactive trypsinogen chromatin is Eb-regulatedchromatindomaininthemurineTcrb locus. juxtaposed to a 25-kb domain of active Db-Jb-Cb chromatin that is Previous studies have revealed that some retroviral LTRs are regulated by Eb. In this article, we identified an LTR at the bor- transcriptionally active, and that they can activate the expression of The Journal of Immunology 3561 neighboring or distant genes (40–42). However, we are unaware of viral LTR situated at a distance from the human globin genes in previous instance in which a retroviral LTR was implicated in their developmental activation (42). Thus, the boundary region may chromatin barrier activity. We note that although the overall or- predispose Db-Jb-Cb chromatin to become accessible in two very ganization of the human Tcrb locus is similar to that of mouse, different ways: it may prevent invasion of repressive histone interspecies sequence conservation in this region is restricted to modifications that would act to oppose chromatin opening, and the Prss2 gene and the PDb1 only. Nevertheless, the human locus it may provide a chromatin opening function as well. That said, carries a 695-bp LTR (LTR8, family ERV1) that is located 4.6 kb a chromatin opening function has yet to be demonstrated, and it upstream of Db1. This LTR may serve a similar function in the remains to be proved whether the barrier activity exhibited in our human locus. rather artificial Jurkat position effect assay is predictive of similar Insulators can encode two separable activities: barrier activity activity in the context of endogenous cis-regulatory elements at the that prevents the spread of repressive chromatin and enhancer- murine Tcrb locus. Additional experiments will be required to blocking activity that prevents an enhancer on one side of the dissect the different functional activities of this element and to insulator from activating a promoter on the other (11, 12). The assess their biological significance in vivo. well-studied chicken 59-HS4 insulator possesses both activities, with barrier activity mediated by USF1 and enhancer-blocking activity mediated by CTCF. Although our results provide evi- Acknowledgments dence of barrier activity in the Prss2-Db1 interval, they do not We thank Hrisavgi Kondilis-Mangum and Han-Yu Shih for critical review of address directly the potential for enhancer-blocking activity. The the manuscript and Zanchun Huang for technical assistance. We also thank Nancy Martin and Lynn Martinek (Duke University Cancer Center Flow only vertebrate enhancer-blocking protein identified to date is Downloaded from Cytometry Shared Resource) for help with cell sorting. CTCF (13). We used a CTCF consensus binding-site algorithm (43, 44) to search for potential binding sites in the Prss2-Db1 interval and found two possible candidates near DNase I HSs 10 Disclosures The authors have no financial conflicts of interest. and 11 located 2.4 and 3.4 kb upstream of Db1 (45). However, as compared with positive controls including c-myc (26) and a site between Eb and Vb14, ChIP analysis revealed no signifi- References http://www.jimmunol.org/ cant binding of CTCF to these sites or to several additional 1. Schatz, D. G., and E. Spanopoulou. 2005. Biochemistry of V(D)J recombination. points tested across the chromatin boundary defined in this study Curr. Top. Microbiol. Immunol. 290: 49–85. 2. Krangel, M. S. 2003. Gene segment selection in V(D)J recombination: acces- (J. Carabana and M.S. Krangel, unpublished observations). This sibility and beyond. Nat. Immunol. 4: 624–630. suggests that the region is unlikely to contain a classical enhancer- 3. Cobb, R. M., K. J. Oestreich, O. A. Osipovich, and E. M. Oltz. 2006. Acces- blocking element. In contrast, two CTCF binding sites with sibility control of V(D)J recombination. Adv. Immunol. 91: 45–109. 4. Krangel, M. S. 2009. Mechanics of T cell receptor gene rearrangement. Curr. demonstrated enhancer-blocking activity have been defined just Opin. Immunol. 21: 133–139. upstream of DFL16.1 in the murine Igh locus (46). 5. Tripathi, R., A. Jackson, and M. S. Krangel. 2002. A change in the structure of Previous studies of barrier-type insulators HMR and HML in Vbeta chromatin associated with TCR b allelic exclusion. J. Immunol. 168:

b 9 2316–2324. by guest on September 27, 2021 yeast (20, 21) and chicken -globin 5 HS4 (18) have demonstrated 6. Bories, J. C., J. Demengeot, L. Davidson, and F. W. Alt. 1996. Gene-targeted that transcription is not required for barrier activity. Nevertheless, deletion and replacement mutations of the T-cell receptor b-chain enhancer: the we detected low-level, Eb-dependent and T lineage-specific tran- role of enhancer elements in controlling V(D)J recombination accessibility. Proc. Natl. Acad. Sci. USA 93: 7871–7876. scriptional activity associated with the barrier region, with tran- 7. Bouvier, G., F. Watrin, M. Naspetti, C. Verthuy, P. Naquet, and P. Ferrier. 1996. scripts extending for at least 11 kb across the Db1-Jb1-Cb1 cluster. Deletion of the mouse T-cell receptor b gene enhancer blocks alphabeta T-cell We do not know whether this transcription, which initiates at the development. Proc. Natl. Acad. Sci. USA 93: 7877–7881. 8. Whitehurst, C. E., S. Chattopadhyay, and J. Chen. 1999. Control of V(D)J re- LTR-LINE junction, depends on LTR or LINE sequences. Both combinational accessibility of the D b 1 gene segment at the TCR b locus by elements can have active promoters; moreover, LINE transcripts a germline promoter. Immunity 10: 313–322. are unusual because they initiate at the 59 end of the LINE and run 9. Oestreich, K. J., R. M. Cobb, S. Pierce, J. Chen, P. Ferrier, and E. M. Oltz. 2006. Regulation of TCRbeta gene assembly by a promoter/enhancer holocomplex. through a downstream LINE promoter (47, 48). Further work will Immunity 24: 381–391. be required to clarify this issue. Regardless, we suspect that this 10. Mathieu, N., W. M. Hempel, S. Spicuglia, C. Verthuy, and P. Ferrier. 2000. transcription is not important for barrier activity, because re- Chromatin remodeling by the T cell receptor (TCR)-b gene enhancer during early T cell development: implications for the control of TCR-b locus re- pressive H3K9me2 chromatin fails to spread in Eb-deficient mice, combination. J. Exp. Med. 192: 625–636. even though transcription, which is low on wild-type alleles, is 11. Gaszner, M., and G. Felsenfeld. 2006. Insulators: exploiting transcriptional and epigenetic mechanisms. Nat. Rev. Genet. 7: 703–713. even lower on Eb-deficient alleles. 12. Valenzuela, L., and R. T. Kamakaka. 2006. Chromatin insulators. Annu. Rev. We speculate that transcription initiating in the boundary region Genet. 40: 107–138. may serve a distinct and complementary function by promoting 13. Bell, A. C., A. G. West, and G. Felsenfeld. 1999. The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 98: 387–396. opening of the Eb-regulated chromatin domain. Previous work from 14. Wallace, J. A., and G. Felsenfeld. 2007. We gather together: insulators and ge- Oltz and colleagues (9) demonstrated that maximal chromatin nome organization. Curr. Opin. Genet. Dev. 17: 400–407. accessibility and histone modifications in the Db1-Jb1-Cb1 region 15. Filippova, G. N. 2008. Genetics and epigenetics of the multifunctional protein CTCF. Curr. Top. Dev. Biol. 80: 337–360. depend on functional interaction between Eb and PDb1, but that Eb 16. Donze, D., and R. T. Kamakaka. 2001. RNA polymerase III and RNA poly- possesses an intrinsic chromatin opening function that is apparent merase II promoter complexes are heterochromatin barriers in Saccharomyces in the absence of PDb1. Chromatin opening might then proceed cerevisiae. EMBO J. 20: 520–531. 17. Litt, M. D., M. Simpson, F. Recillas-Targa, M. N. Prioleau, and G. Felsenfeld. in stepwise fashion, with the initial step dependent on Eb only, 2001. Transitions in histone acetylation reveal boundaries of three separately and a subsequent step, resulting in recombinase accessibility, that regulated neighboring loci. EMBO J. 20: 2224–2235. requires activation of PDb1byEb. Because LTR-LINE transcrip- 18. West, A. G., S. Huang, M. Gaszner, M. D. Litt, and G. Felsenfeld. 2004. Re- cruitment of histone modifications by USF proteins at a vertebrate barrier ele- tion across the Db1-Jb1-Cb1regionisEb dependent, this tran- ment. Mol. Cell 16: 453–463. scription may provide PDb1-independent chromatin remodeling ac- 19. Wheeler, B. S., J. A. Blau, H. F. Willard, and K. C. Scott. 2009. The impact of tivity that might otherwise be attributed to Eb only, and that might local genome sequence on defining heterochromatin domains. PLoS Genet. 5: e1000453. support developmental activation of PDb1. In this regard, recent 20. Noma, K., H. P. Cam, R. J. Maraia, and S. I. Grewal. 2006. A role for TFIIIC studies have implicated low-level transcription initiating at a retro- transcription factor complex in genome organization. Cell 125: 859–872. 3562 A TCRb LOCUS BOUNDARY ELEMENT

21. Valenzuela, L., N. Dhillon, and R. T. Kamakaka. 2009. Transcription in- 36. Oki, M., L. Valenzuela, T. Chiba, T. Ito, and R. T. Kamakaka. 2004. Barrier dependent insulation at TFIIIC-dependent insulators. Genetics 183: 131–148. proteins remodel and modify chromatin to restrict silenced domains. Mol. Cell. 22. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, Biol. 24: 1956–1967. J. Charron, M. Datta, F. Young, A. M. Stall, et al. 1992. RAG-2-deficient mice 37. Li, B., M. Carey, and J. L. Workman. 2007. The role of chromatin during lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell transcription. Cell 128: 707–719. 68: 855–867. 38. Pikaart, M. J., F. Recillas-Targa, and G. Felsenfeld. 1998. Loss of transcriptional 23. Shinkai, Y., S. Koyasu, K. Nakayama, K. M. Murphy, D. Y. Loh, E. L. Reinherz, activity of a transgene is accompanied by DNA methylation and histone and F. W. Alt. 1993. Restoration of T cell development in RAG-2-deficient mice deacetylation and is prevented by insulators. Genes Dev. 12: 2852–2862. by functional TCR transgenes. Science 259: 822–825. 39. Recillas-Targa, F., M. J. Pikaart, B. Burgess-Beusse, A. C. Bell, M. D. Litt, 24. McMurry, M. T., and M. S. Krangel. 2000. A role for histone acetylation in the A. G. West, M. Gaszner, and G. Felsenfeld. 2002. Position-effect protection and developmental regulation of VDJ recombination. Science 287: 495–498. enhancer blocking by the chicken b-globin insulator are separable activities. 25. Carabana, J., E. Ortigoza, and M. S. Krangel. 2005. Regulation of the murine Proc. Natl. Acad. Sci. USA 99: 6883–6888. Ddelta2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb. J. 40. Buzdin, A., E. Kovalskaya-Alexandrova, E. Gogvadze, and E. Sverdlov. 2006. At Immunol. 174: 4144–4152. least 50% of human-specific HERV-K (HML-2) long terminal repeats serve 26. Gombert, W. M., S. D. Farris, E. D. Rubio, K. M. Morey-Rosler, in vivo as active promoters for host nonrepetitive DNA transcription. J. Virol. 80: W. H. Schubach, and A. Krumm. 2003. The c-myc insulator element and matrix 10752–10762. attachment regions define the c-myc chromosomal domain. Mol. Cell. Biol. 23: 41. Jern, P., and J. M. Coffin. 2008. Effects of retroviruses on host genome function. 9338–9348. Annu. Rev. Genet. 42: 709–732. 27. Zhong, X. P., and M. S. Krangel. 1997. An enhancer-blocking element between 42. Pi, W., X. Zhu, M. Wu, Y. Wang, S. Fulzele, A. Eroglu, J. Ling, and D. Tuan. alpha and delta gene segments within the human T cell receptor alpha/delta 2010. Long-range function of an intergenic retrotransposon. Proc. Natl. Acad. locus. Proc. Natl. Acad. Sci. USA 94: 5219–5224. Sci. USA 107: 12992–12997. 28. Strahl, B. D., and C. D. Allis. 2000. The language of covalent histone mod- 43. Kim, T. H., Z. K. Abdullaev, A. D. Smith, K. A. Ching, D. I. Loukinov, ifications. Nature 403: 41–45. R. D. Green, M. Q. Zhang, V. V. Lobanenkov, and B. Ren. 2007. Analysis of the 29. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science 293: 1074–1080. vertebrate insulator protein CTCF-binding sites in the . Cell 128: 30. Noma K, C. D., Allis, and S. I. Grewal. 2001. Transitions in distinct histone H3 1231–1245. Downloaded from methylation patterns at the heterochromatin domain boundaries. Science 293: 44. Jothi, R., S. Cuddapah, A. Barski, K. Cui, and K. Zhao. 2008. Genome-wide 1150–1155. identification of in vivo protein-DNA binding sites from ChIP-Seq data. Nucleic 31. Spicuglia, S., S. Kumar, J. H. Yeh, E. Vachez, L. Chasson, S. Gorbatch, Acids Res. 36: 5221–5231. J. Cautres, and P. Ferrier. 2002. Promoter activation by enhancer-dependent and 45. Chattopadhyay, S., C. E. Whitehurst, F. Schwenk, and J. Chen. 1998. Bio- b -independent loading of activator and coactivator complexes. Mol. Cell 10: chemical and functional analyses of chromatin changes at the TCR- gene locus - - + + 1479–1487. during CD4 CD8 to CD4 CD8 thymocyte differentiation. J. Immunol. 160: 32. Bannister, A. J., P. Zegerman, J. F. Partridge, E. A. Miska, J. O. Thomas, 1256–1267. 46. Featherstone, K., A. L. Wood, A. J. Bowen, and A. E. Corcoran. 2010. The

R. C. Allshire, and T. Kouzarides. 2001. Selective recognition of methylated http://www.jimmunol.org/ lysine 9 on histone H3 by the HP1 chromo domain. Nature 410: 120–124. mouse immunoglobulin heavy chain V-D intergenic sequence contains insulators 33. Nakayama, J., J. C. Rice, B. D. Strahl, C. D. Allis, and S. I. Grewal. 2001. Role that may regulate ordered V(D)J recombination. J. Biol. Chem. 285: 9327–9338. of histone H3 lysine 9 methylation in epigenetic control of heterochromatin 47. Deininger, P. L., and M. A. Batzer. 2002. Mammalian retroelements. Genome assembly. Science 292: 110–113. Res. 12: 1455–1465. 34. Bi, X., and J. R. Broach. 1999. UASrpg can function as a heterochromatin 48.Faulkner,G.J.,Y.Kimura,C.O.Daub,S.Wani,C.Plessy,K.M.Irvine, boundary element in yeast. Genes Dev. 13: 1089–1101. K. Schroder, N. Cloonan, A. L. Steptoe, T. Lassmann, et al. 2009. The regu- 35. Ishii, K., G. Arib, C. Lin, G. Van Houwe, and U. K. Laemmli. 2002. Chromatin lated retrotransposon transcriptome of mammalian cells. Nat. Genet. 41: 563– boundaries in budding yeast: the nuclear pore connection. Cell 109: 551–562. 571. by guest on September 27, 2021