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. Author manuscript; available in PMC: 2018 Aug 1.
Published in final edited form as: J Immunol. 2017 Jun 21;199(3):1131–1141. doi: 10.4049/jimmunol.1700146

Activation of Mouse Tcrb: Uncoupling RUNX1 Function from its Cooperative Binding with ETS11

Jiang-yang Zhao *, Oleg Osipovich *, Olivia I Koues *, Kinjal Majumder *, Eugene M Oltz *,#
PMCID: PMC5526733  NIHMSID: NIHMS880211  PMID: 28637900

Abstract

T lineage commitment requires the coordination of key transcription factors (TFs) in multi-potent progenitors (MPP) that transition them away from other lineages and cement T cell identity. Two important TFs for the MPP to T lineage transition are RUNX1 and ETS1, which bind cooperatively to composite sites throughout the genome, especially in regulatory elements for genes involved in T lymphopoiesis. Activation of the T cell receptor β (Tcrb) locus in committed thymocytes is a critical process for continued development of these cells, and is mediated by an enhancer, Eβ, which harbors two RUNX-ETS composite sites. An outstanding issue in understanding T cell gene expression programs is whether RUNX1 and ETS1 have independent functions in enhancer activation that can be dissected from cooperative binding. We now show that RUNX1 is sufficient to activate the endogenous mouse Eβ element and its neighboring 25 kb region by independently tethering this TF without coincidental ETS1 binding. Moreover, RUNX1 is sufficient for long-range promoter-Eβ looping, nucleosome clearance, and robust transcription throughout the Tcrb recombination center (RC), spanning both DβJβ clusters. We also find that a RUNX1 domain, termed the negative regulatory domain for DNA binding (NRDB), can compensate for the loss of ETS1 binding at adjacent sites. Thus, we have defined independent roles for RUNX1 in the activation of a T cell developmental enhancer, as well as its ability to mediate specific changes in chromatin landscapes that accompany long-range induction of RC promoters.

Introduction

Cell fate commitment is coordinated largely by the temporal regulation of lineage-specifying transcription factors (TFs). During hematopoiesis in mammals, precursor cells in the thymus maintain developmental plasticity until they extinguish “master” TFs for the myeloid and B cell lineages, including SPI1 (1). Conversely, these precursor cells must induce or maintain expression of TFs that control key aspects of T-lineage expression programs, including BCL11b, TCF1, RUNX1, and ETS1 (1). Among the genomic targets for lineage-specifying factors are regulatory regions, dubbed super-enhancers (SEs), which are densely-spaced collections of enhancer elements associated with genes important for cell identity and function (2).

For T cell precursors, one such SE is associated with the locus encoding T cell receptor β (Tcrb) (3), which has a powerful enhancer (Eβ) situated near its 3′ terminus. Eβ is essential for generating the SE, which spans both clusters of DβJβ gene segments and neighboring promoters, activating their expression as sterile transcripts in CD4−CD8−, double-negative (DN) thymocytes (4–6). Potent transcription and deposition of activating histone modifications over the DβJβ-Eβ super-enhancer (7, 8), render it a prime target for RAG-1 and RAG-2, the V(D)J recombinase. Indeed, the Tcrb-SE has been designated as a “recombination center (RC)” due to its high load of RAG-1/2 in DN thymocytes that, in turn, triggers rapid recombination of DβJβ gene segments, an essential first step in the assembly of complete Tcrb genes (9, 10).

The critical regulatory element for formation of the Tcrb-RC is Eβ, which, like many enhancers that contribute to T-lineage SEs, has adjacent binding sites for the RUNX and ETS family of TFs (7, 11, 12). The biological significance of recurrent ETS/RUNX motifs is further underscored by their enrichment in T cell SEs harboring SNPs associated with auto-inflammatory conditions (13). The RUNX and ETS families are defined by their respective DNA binding regions, termed Runt and ETS domains, respectively (14, 15). Although these TFs can bind to isolated sites in the genome, compound ETS-RUNX motifs are commonly employed to attain tissue-specific cooperativity between family members in a number of cell lineages. For example, RUNX1 and the ETS member, SPI1, cooperatively regulate a gene encoding the M-CSF receptor in myeloid cells (16), while ETS1 and RUNX2 bind cooperatively to an element controlling the osteopontin gene in colorectal cancer cells (17). In T-lineage cells, RUNX1 and ETS1 target Eβ to generate the Tcrb super-enhancer and initiate its assembly (7).

At the biochemical level, both RUNX1 and ETS1 harbor domains that partially inhibit their binding to single, cognate sites, a feature that is overcome when the two bind cooperatively to composite sites (18–21). Specifically, regions flanking the ETS1 DNA binding domain undergo a significant conformational change when its exon-VII domain interacts with RUNX1, resulting in a higher affinity of ETS1 binding to composite sites (19, 20). RUNX1 is stabilized by its interaction with a second protein, CBFβ, which protects RUNX1 from proteolysis and induces conformational changes in its Runt domain that enhance DNA binding (21, 22). However, the Runt domain of RUNX1 binds poorly to its cognate sites in chromatin until a region termed the “Negative Regulatory DNA-Binding” (NRDB) interacts with ETS1 (20, 21). Once bound to genomic sites, the transactivation domains (TADs) of ETS1 and RUNX1 recruit distinct sets of downstream factors that potentiate enhancer activities. Importantly, the TADs of both ETS1 and RUNX1 interact with the histone acetyltransferases p300/CBP, key enhancer-associated factors that alter epigenetic landscapes for gene activation (23, 24).

During T-lineage commitment, multi-potent progenitors in the thymus constitutively express RUNX1 but not ETS1 (25). Upon signaling through Notch and other receptor pathways, these progenitors induce expression of BCL11b and TCF1, which, in turn, activate the Ets1 gene (1, 25, 26). Thus, ETS1 expression is a key development switch, pairing with RUNX1 to target cohorts of regulatory elements that control genes important for the T cell program, including the Tcrb-RC (27). Indeed, inactivation of either TF in mice leads to profound defects in T cell development and activation (28, 29). Because of its central role in T cell development, Eβ and its pair of ETS-RUNX composite sites have been studied extensively, especially with regard to activation of the Tcrb super-enhancer (7). Mutation of both ETS-RUNX composite sites within Eβ phenocopies a deletion of the entire enhancer (7, 20, 30, 31), including the loss of: (i) looping to promoters near the Dβ1 and Dβ2 gene segments (7, 32), (ii) transcription derived from these promoters (7, 31, 32), (iii) active histone modification over both DβJβ clusters (7, 32–34), and (iv) DβJβ recombination (7, 31, 32). Thus, Eβ provides an excellent model to test mechanisms by which RUNX1 and ETS1 coordinate activation of a developmentally important super-enhancer, the Tcrb-RC, either through their individual or cooperative functions.

In this regard, we previously proposed a stepwise model for Tcrb activation in which promoter-enhancer holocomplex formation precedes nucleosome clearance, transcription, and activation (32). However, the unique roles for ETS1 and RUNX1 in each of these steps at Tcrb or any locus remains unclear due primarily to the requirement for cooperative binding of the TFs at their composite enhancer sites. Moreover, most cellular systems in which these mechanistic questions can be addressed either rely on extrachomosomal reporters or asynchronous activation of an endogenous gene (20, 35). Here, we report unique and cooperative functions of RUNX1 during activation of the endogenous Tcrb-RC using precursor T cells in which a Gal4 fusion of this TF is inducibly targeted to Eβ. We find that, when uncoupled from its need to bind cooperatively with ETS1, RUNX1 is sufficient to induce promoter-enhancer looping and transcription throughout the Tcrb-RC. Long-range transactivation by RUNX1 is uncoupled from extensive revisions to the chromatin landscape, features normally associated with promoter-driven transcription. Thus, although expression of RUNX1 is sufficient for transcriptional activation of the Tcrb-RC, ETS1 co-recruitment during T-lineage commitment may be required to boost Tcrb’s epigenetic status to that of a super-enhancer, ensuring its efficient assembly and persistent expression.

Materials and Methods

shRNA depletion of ETS1 and RUNX1

Previously reported shRNA target sequences for Ets1 and Runx1 (36, 37) were cloned into the pSIREN shuttle vector (Clontech), excised as BglII-MluI fragments, and inserted into pcDNA3.1 containing a truncated human CD4 cDNA (38). P5424 cells were cultured as described (39) and were transfected by electroporation (Biorad, 250 V/960 μF ) with control (scrambled GFP 5′-AAGCTGGAGTACAACTACA-3′), ETS1-specific (target sequence 5′-GCAGACAGACTACTTTGCCAT-3′), or RUNX1-specific shRNA vectors (target sequence 5′-GCCCTCCTACCATCTATACTA-3′). Transfected cells were purified 48 hours post-transfection with an EasySep Human CD4+ T Cell Enrichment Kit (Stem Cell Technologies #19052).

Construction of cell models

P5424 cells were engineered to harbor only a single allele of the Tcrb-RC, termed O3-73 clone, by concurrent expression of two Zinc Finger Nucleases (ZFNs; Sigma). Each pair of ZFNs targets a DNA break to a site in Tcrb, one located upstream of Dβ1 (ZFN1 pair, target 5′-GGACTTGTCCTTAACTCCCTTTACCTAGCAAGATAGG-3′) and a second, located downstream of Eβ (ZFN2 pair, target 5′-GTCTGTCTGTCCTGCATTCATTGGCTGGCTTTCGCT-3′). 5×106 cells were transfected with 5 μg of each ZFN expression plasmid using electroporation (250 V/960 μF). After two days of recovery, cells were cloned by serial dilution and screened by PCR assays for the large Tcrb-RC deletion (Table S1). Clone O3-73 was selected as a parental cell line for future experiments (allele 1: intact Tcrb-RC, allele 2: deletion of Dβ1-Dβ2-Eβ).

Targeting vectors for mutation of Eβ were generated as follows. Gal4 or TF site mutations were first introduced into a 1.85 kb fragment spanning Eβ (HpaI-HindIII), using PCR-assisted mutagenesis with a high-fidelity polymerase according to the manufacturer’s protocol (Phusion, NEB M0503). Oligonucleotide sequences used for site-directed mutagenesis and cloning are provided in Table S1. All Eβ mutations were verified by sequencing. Mutagenesis was performed sequentially to alter both ETS-RUNX sites in Eβ. Targeting vectors were generated in pBS by sequentially cloning a 3.2 kb 5′ homology arm (BamHI-HpaI), a 0.7 kb mutant Eβ (HpaI-MluI), a 1.9 kb loxP:PGK-promoter:Puro:loxP cassette (MluI-NheI), and a 2.1 kb 3′ homology arm (NheI-NotI). For the ΔEβ targeting vector, a hygromycin-resistant cassette flanked by FRT sites was used for drug selection.

For targeting, 5×106 O3-73 cells were co-transfected with 20 μg of a targeting vector and 5 μg each of expression plasmids for the ZFN2 pair, which introduce a DNA break ~200 bp downstream of Eβ. Two days post-electroporation, cells were selected with puromycin (1 μg/mL) or hygromycin (100 μg/mL) and cultured for 5–6 d before sub-cloning by limiting dilution. Initially, single cell clones were screened by a PCR assay in which insertion of the targeting vector disrupts amplification (Table S1). Candidate recombinant clones were then screened by a long-range PCR assay for proper integration (Table S1), which was verified by sequencing. Successfully targeted clones were transfected with 1 μg of human CD4 expression plasmid and 10 μg of CRE or FLP expression plasmids and purified for CD4 expression. After one week, cultures were subcloned, screened for deletion of the drug marker cassette, and mutant alleles were verified by sequencing.

Gal4 fusion proteins

Expression vectors for all Gal4-RUNX1 fusion proteins were prepared from an acceptor plasmid containing a cDNA for the Gal4 DNA-binding domain (DBD, aa 1–147), into which was cloned, in-frame, cDNAs for portions of murine RUNX1. Each of these RUNX1 cDNAs was generated by PCR using primers provided in Table S1 (RUNX1GNT aa 184–451, RUNX1GN aa 182–292, and RUNX1GT aa 291–451). The final fused Gal4-RUNX1 cDNAs were then excised and cloned into pcDNA3.1. Each version was validated by sequencing and protein expression in 293T cells. For inducible expression, each variant was shuttled into the pFLRU:Thy1.1 tet-responsive lentiviral vector. Viruses containing each Gal4-RUNX1 expression cassette were packaged as described previously (40) and used to spin-infect 5×106 O3-73 derivative cells. Infected cells were cultured for 10 days, sorted for high Thy1.1 expression by flow cytometry (Anti-Mouse/Rat CD90.1 APC, eBioscience 17-0900-82), expanded, and induced with 1 μg/mL doxycycline (Dox) for 24 hours prior to molecular analysis.

RT-qPCR

Total RNA was extracted by TRIzol (Roche #1697478) from O3-73 and its derivatives. In each reaction, 1 μg total RNA was digested by RQ1 RNase-free DNase (Promega M199A) in 20 μL total volume, as recommended by the manufacturer. The treated RNA was split equally for cDNA preparation with M-MuLV Reverse Transcriptase (NEB M0253) or used as a no RT control, with random hexamers. Real-time qPCR was performed with SYBR Green JumpStart Taq ReadyMix (Sigma S4438) using primers and conditions provided in Table S1.

Chromatin Immunoprecipitation

ChIP assays were performed as described previously (41). Sheared chromatin from O3-73 and its derivatives were prepared after fixation with 1% formaldehyde and sonication with Diagenode Bioruptor Pico for 20 cycles (30 s each, vortexed every 5 cycles). ChIP antibodies of rabbit IgG isotypes were coupled to Protein A Dynabeads and were obtained from the following sources: RUNX1 C terminus was provided by Dr. Takeshi Egawa (42), ETS-1 C20 (Santa Cruz sc-350×), normal rabbit IgG (Santa Cruz sc-2027×), Gal4DBD (Santa Cruz sc-577×), total Histone H3 (Abcam ab1791), H3K4Me3 (Abcam ab8580) and H3K27Ac (Abcam ab4729). ChIP antibodies of mouse IgG isotypes were coupled to Protein G Dynabeads and were obtained from the following sources: p300 (Santa Cruz sc-584×) and normal mouse IgG (Santa Cruze sc-2025). Eluted ChIP and input DNAs were purified with Qiagen QIAquick PCR purification kit and analyzed by qPCR using primers and conditions provided in Table S1.

Chromosome Conformation Capture assays

3C assays were performed on 107 O3-73 cells and its derivatives fixed with 2% formaldehyde. Chromatin was isolated and digested with HindIII, as described (43, 44). Bacterial artificial chromosomes (BACs) spanning the entire Tcrb locus and a control ERCC3 locus were digested with HindIII, ligated, and used as controls for Taqman qPCR efficiency in all 3C analyses (44). Primers and probes for these Taqman assays are listed in Table S1.

IP- and conventional western blotting

O3-73 cell lysates were prepared in RIPA buffer (2×107 cells in 300 μL) and sonicated for 40 cycles (30s, vortexed each 10 cycles, Diagenode Bioruptor Pico). For IP-westerns, each lysate was incubated with rabbit anti-Gal4DBD antibody (1 μg, Santa Cruz sc-577×) attached to Dynabeads M-280 sheep anti-rabbit IgG (50 μL) as described by the bead manufacturer. For conventional western blotting, sonicated lysates were used directly (10 μL). Whole cell or IP lysates were fractionated on 12% PAGE gels, transferred to PVDF membranes, incubated with detection antibodies and visualized by ECL substrate (Thermo Fisher Scientific #34087). Primary antibodies were used at a concentration of 1 μg/mL for protein detection and obtained from the following sources: mouse anti-Gal4DBD antibody (clone RK5C1; Santa Cruz sc-510), rabbit anti-ETS1 (clone N276; Santa Cruz sc-111), mouse anti-GAPDH (clone G-9; Santa Cruz sc-365062), rabbit anti-RUNX1 C terminus (Dr. Takeshi Egawa) (42), mouse anti-RUNX1 NRDB domain (clone A-2; Santa Cruze sc-365644). Secondary antibodies, diluted 1:104, were goat anti-rabbit IgG-HRP (Santa Cruz sc-2004) and goat anti-mouse IgG-HRP (Promega W4021).

Results

Eβ function requires both ETS1 and RUNX1 in precursor T cells

The critical element for conferring SE status to Tcrb is its conventional enhancer, Eβ (8), which contains a pair of composite ETS-RUNX binding motifs as its core (Fig. 1A). To dissect the individual functions of these TFs in driving Eβ activation, we required a genetically tractable system for introducing mutations at the endogenous element. Such an approach in primary thymocytes would be unwieldy, since these cells fail to grow in long-term cultures. Thus, genetic and biochemical dissection of ETS-RUNX function at Eβ would require in vivo studies of multiple mutant mouse lines. To circumvent this obstacle, we selected a cell line that serves as an excellent model for precursor T cells based on transcriptome-wide data, as well as the transcriptional and epigenetic landscape spanning the Tcrb recombination center (3, 45). The cell line, P5424, is a thymoma derived from RAG1/P53-deficient mice, which retains a germline but transcriptionally active Tcrb locus (45). To serve as an appropriate model for dissecting ETS-RUNX cooperativity, Eβ function in P5424 must depend on these individual transcription factors. Indeed, depletion of either ETS1 or RUNX1 using shRNAs significant decreased germline Dβ1 or Dβ2 transcripts in P5424 (Figs. 1B and C).

Figure 1. Eβ activity is dependent on ETS1 and RUNX1 in P5424 cells.

Figure 1

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(A) Schematic representation of mouse Tcrb-RC showing promoters (pDβ1 and pDβ2), D elements (Dβ1 and Dβ2), J elements (Jβ1 and Jβ2 clusters), constant regions (Cβ1 and Cβ2) and the enhancer (Eβ). TF binding sites in Eβ identified by in vivo footprinting are highlighted below (34), including a pair of composite ETS-RUNX sites (black bars). (B) P5424 cells were transfected with shRNAs targeting ETS1, RUNX1, or control transcripts (GFP). Shown are ETS1 and RUNX1 mRNA levels measured by RT-qPCR and normalized to Actb transcripts. (C) Levels of spliced Jβ1-Cβ1 and Jβ2-Cβ2 germline transcripts in the indicated knock-downs as measured by RT-qPCR. In B and C, data are presented as the average values of three PCR replicates (±S.D.) and are derived from one of two biological replicates (see also Figs. S1 A and B), ** denotes statistical significance (p<0.01), as determined by Student’s t-test.

These data; however, do not rule out the possibility that ETS1 or RUNX1 regulate Tcrb transcription independent of their effects on Eβ, including a potential role at the Dβ1 or Dβ2 promoters (5, 6). To definitively assess whether these factors are essential for Eβ function, we targeted the endogenous enhancer in P5424, introducing mutations at the ETS or RUNX motifs. For this purpose, we used a version of P5424, called O3-73, in which we had deleted the Tcrb-RC (both DβJβ clusters and Eβ) from one allele (Fig. 2A, see Materials and Methods). Initially, we made three mutant versions of Eβ in O3-73 using targeting vectors in combination with a zinc finger nuclease that introduces double-strand breaks at a site ~200 bp downstream of the core enhancer. The three mutant Tcrb alleles harbored (i) a complete Eβ deletion (ΔEβ), (ii) a replacement of both ETS1 sites in Eβ with Gal4 binding motifs (EG), or (iii) a replacement of both RUNX1 sites in the enhancer with Gal4 binding motifs (RG, Fig. 2A). All three of these targeted mutations were verified by sequencing. Replacement of either the ETS or RUNX motifs with Gal4 sites abolished binding of ETS1 and RUNX1 to the enhancer, respectively (Fig. 2B), as measured by chromatin immunoprecipitation (ChIP). Consistent with cooperative binding (11, 20, 21), the ETS mutations also abolished RUNX1 binding to the enhancer, while destruction of the RUNX motifs dramatically attenuated ETS1 binding to Eβ. Importantly, mutation of either the ETS or RUNX binding sites in Eβ crippled germline transcription throughout the recombination center in P5424 (Fig. 2C), phenocopying the ΔEβ allele in this cell line or RUNX site mutations at endogenous Eβ in primary thymocytes (7). We conclude that Eβ function in this precursor T cell model depends completely on its two ETS-RUNX composite motifs.

Figure 2. Mutation of ETS or RUNX motifs abolishes Eβ function.

Figure 2

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(A) Schematic showing intact and mutant Tcrb alleles in O3-73 (top). As indicated, the entire RC, including Eβ, was deleted from the mutant allele. The intact allele was further targeted to generate a variety of mutant Eβ versions (bottom): EG (both ETS-RUNX sites converted to ETS1/Gal4), RG (ETS1/RUNX1 to Gal4/RUNX1 sites), EmRG (ETS1/RUNX1 to mutant ETS1/Gal4 sites). (B) ETS1 and RUNX1 binding at different versions of Eβ(WT, EG, and RG) measured by ChIP-qPCR. The antibodies used for all ChIP experiments recognize either the ETS1-DBD or the RUNX1-TAD. Control ChIPs with a non-specific IgG antibody are shown for WT cells. (C) Unspliced germline transcripts corresponding to Jβ1.2 and Jβ2.1 gene segments in cells with different versions of Eβ (WT, Δ, EG, and RG), as measured by RT-qPCR. For panels B and C, data are presented as the average values of three PCR replicates (±S.D.) and are derived one of two biological replicates (see also Figs. S1 C and D), ** denotes statistical significance (p<0.01), as determined by Student’s t-test.

Tethering of RUNX1 rescues mutant Eβ function

Replacement of the two RUNX motifs in Eβ with Gal4 sites allowed us to recruit mutant versions of RUNX1 to the enhancer for functional dissection. As a proof of concept, we introduced into O3-73 mutants a lentiviral vector that is inducible by doxycycline (Dox) for expression of a fusion protein in which the Runt domain of RUNX1 was replaced with the Gal4 DNA binding domain (RUNX1GNT, Fig. 3A). The RUNX1GNT fusion retains both the putative ETS1 binding domain (NRDB) and its transactivation domain. Induced levels of RUNX1GNT protein were similar in all O3-73 variants, and did not impact the expression of endogenous ETS1 (Fig. 3B). Gal4-RUNX1 fusions efficiently targeted the mutant Eβ enhancer (RG), partially restoring ETS1 binding as measured by ChIP (Fig. 3C). Importantly, tethering of RUNX1GNT was sufficient to rescue transcription of germline Tcrb alleles harboring the RG mutant enhancer, reaching levels comparable to those observed in the parental O3-73 line (Fig. 3D). As a control, the Gal4DBD failed to rescue ETS1 or RUNX1 binding at Eβ, nor did it rescue transcription of the Tcrb-RC. Moreover, germline Dβ1/Dβ2 transcription remained silent when RUNX1GNT was expressed in O3-73 lines lacking the enhancer element (ΔEβ), excluding potential indirect effects of the fusion protein. These data demonstrate that recruitment of RUNX1 proteins containing only its NRDB and TAD portions is sufficient to restore Eβ function when binding of endogenous RUNX1 is precluded.

Figure 3. GAL4-RUNX tethering fully restores function to Eβ mutants lacking RUNX motifs.

Figure 3

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(A) Cartoon depictions of domains in full-length ETS1 and RUNX1 proteins, as well as in Gal4-RUNX1 fusions. ETS1 consists of an N terminus transactivation domain (TAD), exon VII domain (VII), and ETS DNA binding domain (ETS). RUNX1 consists of an N terminus Runt DNA binding domain (Runt), a negative regulatory region of DNA binding domain (NRDB), and a transactivation domain (TAD). (B) IP- and conventional western blotting analysis for expression of fusion proteins. Transduced cells were treated with 1 μg/mL Dox for 24 hours, and expression levels of Gal4 fusion proteins were detected by IP-western blotting. ETS1 and GAPDH expression were measured by conventional western blotting using the same cell lysates. A representative of three independent experiments is shown. (C) ChIP-qPCR assays for ETS1 and RUNX1 binding to the RG mutant enhancer upon expression of RUNX1GNT. Control ChIPs with a non-specific IgG antibody are shown for WT cells. TF denotes Gal4DBD and Gal4-RUNX1 fusion proteins. (D) Unspliced germline transcripts corresponding to the Jβ1.2 and Jβ2.1 gene segments induced by RUNX1GNT in cells with the RG mutant enhancer. For panels C and D, data are shown as averages (± S.E.M.) from three biological replicates, ** denotes statistical significance (p<0.01), as determined by Student’s t-test.

ETS1 binding is dispensable for restoration of enhancer function by RUNX1

One hypothetical model for ETS-RUNX cooperativity is that either transcription factor alone may be sufficient for enhancer activation, but stable binding of either to its target sites requires NRDB/exon VII interactions and co-occupancy of composite sites. Although tethering of RUNX1GNT to the RG mutant enhancer fully rescued Tcrb germline transcription, the fusion also recruited significant amounts of ETS1 to Eβ. To test whether RUNX1 binding to Eβ is sufficient for enhancer activation in the absence of ETS1 occupancy, we generated a fourth version of O3-73, in which the RUNX motifs of Eβ were replaced with Gal4 sites and the two adjacent ETS motifs were destroyed (EmRG, see Fig. 2A). We then introduced RUNX1GNT and Gal4-DBD expression vectors into the mutant O3-73 cells, which exhibited comparable steady-state levels of each protein (Fig. 4A). Additional mutation of the ETS motifs did not impair binding of RUNX1GNT at Eβ (Fig. 4B, top panel, compare RG and EmRG). We point out that no clear correlations were observed in independent experiments between differences in levels of Gal-RUNX1 fusions and functional readouts (e.g., transcription), likely because even low levels of the fusion proteins saturate their binding to Eβ. In contrast, destruction of the ETS motifs completely compromised ETS1 binding to the enhancer, despite elevated levels of RUNX1GNT (Fig. 4B, bottom panel). These data indicate that the NRDB domain of RUNX1 cannot recruit ETS1 to Eβ independently of the ETS sites in its composite motifs. Although the EmRG enhancer lacked detectable ETS1, recruitment of RUNX1GNT completely restored the expression of germline DβJβ transcripts (Fig. 4C). Moreover, the tethered RUNX1GNT fusion recruited near wild type levels of the p300 acetyltransferase to Eβ in the presence or absence of ETS1 (Fig. S2A). We conclude that ETS1 is dispensable for Eβ function if RUNX1 recruitment is uncoupled from its cooperative binding to composite ETS-RUNX motifs.

Figure 4. ETS1 is dispensable for Eβ activation by RUNX1.

Figure 4

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(A) Western blotting analysis for expression of Gal4-RUNX1 fusion proteins. Transduced cells were treated with 1 μg/mL Dox for 24 hours, and expression levels of Gal4 fusions or GAPDH were detected by IP- or conventional western blotting, respectively. A representative of three independent experiments is shown. (B) ChIP-qPCR assays for ETS1 and RUNX1 binding to the RG and EmRG mutant enhancers upon expression of RUNX1GNT, as described in Fig. 3C. (C) Unspliced germline transcripts for Jβ1.2 and Jβ2.1 segments induced by RUNX1GNT in cells with RG and EmRG mutant enhancers, as described in Fig. 3D. All cells for ChIP- and RT-qPCR assays were cultured in parallel with those shown in Fig. 3, the data from which are included again for direct comparisons. For panels B and C, data are shown as averages (± S.E.M.) from three biological replicates, ** denotes statistical significance (p<0.01), as determined by Student’s t-test.

Dissection of RUNX1 functional domains for Eβ activation

The RUNX1 fusion that was sufficient to fully activate Eβ contained two previously defined functional domains (20). The NRDB has been shown in vitro to interact with exon VII domain of ETS1, presumably stabilizing the binding of both factors to composite sites (20). However, our data suggest that ETS1 recruitment is dispensable for activation by the tethered RUNX1GNT fusion (Fig. 4). The transactivation domain (TAD) of RUNX1, therefore, may be sufficient to support Eβ function if recruited to the enhancer independently of ETS1.

To further define functions of the two RUNX1 domains, we first expressed a Gal4 fusion of the NRDB (RUNX1GN, Fig. 3A) in O3-73 cells with mutant versions of Eβ (Fig. 5A). The RUNX1GN fusion was expressed at a much lower level than endogenous RUNX1 (Fig. S2B) but was recruited to Eβ as efficiently as RUNX1GNT, which has both the NRDB and TAD (Fig. 5B). However, only the TAD-containing fusion permitted recruitment of ETS1 to its neighboring sites in the RG mutant version of Eβ. These data indicate that the NRDB of RUNX1 is insufficient for ETS1 recruitment, perhaps due to a lack of chromatin accessibility at the mutant enhancer (see below). Consistent with these results, RUNX1GN failed to activate germline transcription from the distal Dβ1-Jβ1 cluster, while it induced only modest levels of Dβ2-Jβ2 transcripts (Fig. 5C). Thus, the NRDB of RUNX1 is insufficient for ETS1 recruitment to Eβ and activation of this enhancer element.

Figure 5. The TAD of RUNX1 is sufficient for short- but not long-range activation of the Tcrb-RC.

Figure 5

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(A) Western blotting analysis for expression of indicated Gal4-RUNX1 fusion proteins, as described in Fig. 4A. A representative of three independent experiments is shown. (B) ChIP-qPCR assays for ETS1 and Gal4DBD binding to the RG and EmRG mutant enhancers upon expression of RUNX1GN, as described in Fig. 3C. (C) Unspliced germline transcripts for Jβ1.2 and Jβ2.1 segments induced by RUNX1GN in cells with RG and EmRG mutant enhancers (see Fig. 3D). (D) ChIP-qPCR assays for ETS1 and RUNX1 binding to the RG and EmRG mutant enhancers upon expression of RUNX1GT. (E) Unspliced germline transcripts corresponding to Jβ1.2 and Jβ2.1 segments induced by RUNX1GT in cells with RG and EmRG mutant enhancers. For panels B–E, data are shown averages (± S.E.M.) of three biological replicates, n.s. (non-significant), ** (p<0.01), as determined by Student’s t-test. All cells for ChIP- and RT-qPCR assays were cultured in parallel with those shown in Figs. 3&4, the data from which are included again for direct comparisons.

To test whether the TAD of RUNX1 is sufficient for Eβ activation, we expressed a Gal4-TAD fusion (RUNX1GT, Fig. 3A) that was tethered to mutant enhancers with or without intact ETS motifs (Fig. 5A). Despite its low level of steady-state expression compared with endogenous RUNX1 (Fig. S2C), the RUNX1GT fusion tethered efficiently to both the RG and EmRG enhancer mutants (Fig. 5D), likely due to the high affinity of dimerized Gal4 for its binding sites. As expected, ETS1 was absent from the EmRG enhancer even after RUNX1GT recruitment. In contrast, RUNX1GT permitted binding of ETS1 to the RG enhancer, despite its lack of an NRDB domain, which is thought to be essential for cooperative binding (Fig. 5D). This finding may reflect the ability of RUNX1GT to endow Eβ with sufficient chromatin accessibility, allowing for a substantial, albeit sub-wild type, levels of ETS1 binding to its exposed site (see below).

Importantly, the TAD of RUNX1 was sufficient to fully activate germline transcription of the Tcrb-RC when tethered to the RG enhancer, to which ETS1 also binds (Fig. 5E). In sharp contrast, when ETS1 binding was precluded at the EmRG enhancer, RUNX1GT failed to activate transcription from the distal Dβ1 promoter. The Gal4-TAD fusion; however, supported modest expression levels from the more proximal Dβ2 promoter, when tethered to the EmRG enhancer (Fig. 5E). Together, these data provide several new insights into the function of RUNX1 domains at RUNX-ETS composite sites in Eβ, including (i) the TAD is sufficient for enhancer activation when modest levels of ETS1 are present, but is insufficient in the absence of ETS1 at the enhancer, (ii) the NRDB is dispensable for some ETS1 recruitment to these composite sites, (iii) the NRDB, in some manner, must compensate for an absence of ETS1 given that RUNX1GNT fully activates the EmRG enhancer, and (iv) when activated solely by the TAD, enhancer function is limited spatially to the most proximal Dβ2 promoter. Thus, either ETS1 or efficient binding of RUNX1 by itself is required for maximal, long-range activation by Eβ.

Epigenetic mechanisms for short-versus long-range activation of the Tcrb-RC

Initiation and augmentation of transcription by distal enhancers is a multi-step process involving revisions to the regional epigenetic landscape and conformational changes that drive enhancer-promoter contacts (46, 47). Alterations in the epigenetic landscape germinate at the enhancer and may spread into neighboring regions, permitting access to nuclear factors (48). Enhancer-promoter looping is thought to deliver nucleosome remodelers and components of the basal transcription machinery to the promoter after initial recruitment to an activated/accessible enhancer (32, 49).

Given that the RUNX1 TAD can partially activate the proximal Dβ2, but not the more distal Dβ1 region, we tested whether specific aspects of multi-step promoter and SE activation are supported by this domain when compared with the complete RUNX1 fusion. As shown in Fig. 6A, the H3K27ac activation mark, a hallmark of SEs, is at least partially restored at the proximal Dβ2Jβ2 cluster when any TAD-containing version of RUNX1 fusions are recruited to Eβ, paralleling transcriptional activation of this region. Indeed, only modest levels of both H3K27ac and Dβ2Jβ2 transcription are observed when the RUNX1GT is recruited to the EmRG enhancer lacking ETS1. By comparison, mutant versions of Eβ had little impact on levels of a promoter activation mark, H3K4me3 (Fig. 6A). Complete deletion of Eβ further reduced each of the activation marks, but at Dβ2Jβ2, H3K27ac and H3K4me3 remained above background levels observed in 3T3 fibroblasts (Figs. S3 B and C). These data indicate that mutant versions of Eβ (RG and EmRG) retain some ability to modify chromatin and that the Dβ2 region has an inherent, Eβ-independent function to partially activate chromatin in T-lineage cells.

Figure 6. RUNX1 is sufficient to restore aspects of the Tcrb-RC epigenetic landscape.

Figure 6

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(A) ChIP-qPCR assays for H3K27ac and H3K4me3 levels at Jβ1 or Jβ2 following induction of the indicated Gal4-RUNX1 fusions in clones containing either the RG or EmRG mutant versions of Eβ. (B) Total histone H3 ChIPs showing changes in nucleosome occupancy induced by Gal4-RUNX1 fusions. H3 levels were monitored at pDβ1, pDβ2, and immediately upstream of Eβ (5′Eβ) in RG and EmRG mutant cells expressing the indicated RUNX1 fusion proteins. Clones containing wild-type (WT) or ΔEβ versions of Tcrb, as well as 3T3 fibroblasts are included as controls. Values for IgG control ChIPs in both A and B were significantly lower than all samples shown. (C) Promoter-enhancer interactions induced by Gal4-RUNX1 fusions at RG and EmRG versions of Eβ, as measured by 3C-qPCR (43). Data are presented as the average values of three PCR replicates (±S.D.) and are derived one of two biological replicates (see also Figs. S3 A, D, and E). For panel C, n.s. (non-significant), ** (p<0.01), as determined by Student’s t-test.

In contrast to the proximal Dβ2Jβ2 cluster, recruitment of RUNX1 fusion proteins to mutant versions of Eβ induced only modest increases in H3K27ac levels and partial restoration of H3K4me3 at the distal Dβ1Jβ1 region (Fig. 6A). Despite the minor impact on active chromatin marks, many of the RUNX1 fusions fully restored Dβ1Jβ1 germline transcription. We conclude that the long-range transactivation by RUNX1 is uncoupled from general revisions to the epigenetic landscape of Dβ1Jβ1 normally associated with SE formation and promoter-driven transcription.

A downstream effect of these epigenetic revisions may be changes in nucleosome density at promoters (33, 50), driving germline Dβ1 or Dβ2 transcription. As such, we performed ChIP assays for the H3 component of nucleosomes at the proximal Dβ2 and the distal Dβ1 promoters, as shown in Fig. 6B. Inactivating mutations of Eβ significantly increased nucleosome densities on both Dβ1 and Dβ2 promoters. Recruitment of most RUNX1 fusions partially or fully reversed the enhanced nucleosome occupancy at the promoters, compared with alleles harboring a fully active Eβ. One exception was the RUNX1GN fusion, which lacks the TAD and failed to activate any transcription from the Tcrb-RC. Nucleosome density at Dβ1 and Dβ2 promoters was also elevated when the RUNX1GT fusion was recruited to the mutant version of Eβ lacking its ETS1 binding sites (EmRG). These data suggest that full promoter activation by RUNX1 involves nucleosome clearance, a process requiring either the NRDB domain or cooperation with ETS1.

Our prior studies have shown that nucleosome clearance at Dβ1 involves Eβ looping to its promoter, which facilitates recruitment of the SWI/SNF chromatin remodeling complex (32, 33). Indeed, transcriptional activation of Dβ1 can be achieved in the absence of its promoter and Eβ looping if SWI/SNF is artificially tethered to this region (33). To examine the impact of RUNX1 fusions on promoter-enhancer loops in the Tcrb-RC, we performed chromosome conformation capture (3C) assays using Eβ as a viewpoint. As expected, inactivation of Eβ disrupts its communication with both Dβ promoter regions (Fig. 6C). Recruitment of RUNX1 to mutant enhancers with intact or disrupted ETS1 binding sites restored Eβ looping to both Dβ elements. However, tethering of only the RUNX1 TAD to the enhancer with ETS sites mutations failed to restore Eβ association with either Dβ1 or Dβ2. Collectively, chromatin and conformational data indicate that, like transcription, enhancer-promoter looping in the Tcrb-RC requires either RUNX1 with an intact NRDB, or cooperative binding of at least some ETS1. Moreover, these stringent requirements for Dβ1 activation are partly dispensable for Dβ2, which may rely on Eβ-independent chromatin modifications to offset any disruptions in looping or promoter accessibility. For long-range activation of Dβ1; however, both enhancer looping and subsequent changes in chromatin accessibility are required for promoter activation, even if histone modifications associated with SE status are not fully restored.

Discussion

Cell fate decisions during development are initiated by TF combinations that coordinate lineage-specifying gene expression programs through their binding to conventional- and super-enhancers (2). Two such TFs, ETS1 and RUNX1, are particularly important for T lineage commitment, during which, they bind in a cooperative manner to large cohorts of enhancers controlling T cell expression programs. In this study, we probed the specific functions of RUNX1 in activating a super-enhancer region for T lineage commitment, Tcrb, independent of its requirement for cooperative binding with ETS1 to its target sites in Eβ. We found that RUNX1 was sufficient to initiate both promoter-Eβ looping and germline transcription within both DβJβ clusters when tethered directly to the enhancer. These data suggest that ETS1 and its transactivation domain (TAD) are dispensable for Eβ-mediated activation of the Tcrb super-enhancer in precursor T cells. However, we cannot rule out the possibility that dimerization of Gal4-RUNX1 fusions bound to Eβ, an unavoidable feature of our experimental system, can functionally substitute for the two TADs of RUNX1 and ETS1, which may be required for super-enhancer activation in vivo.

Notwithstanding, the critical function of ETS1 during this lineage-commitment process may be to stabilize association of RUNX1 with the enhancer via cooperative binding. A contingent binding of RUNX1 to Eβ and other enhancers important for T lineage genes is consistent with the temporal expression of the TFs during thymocyte development (1, 26). Specifically, RUNX1 is constitutively expressed in many hematopoietic cells, including multi-potent progenitors. In these T lineage precursors, ETS1 is initially silent, precluding premature gene activation via the binding of RUNX1 to composite sites in key enhancers. When the multi-potent progenitors receive T lineage commitment cues, including Notch signaling (26), subsequent expression of ETS1 permits RUNX1 binding to composite sites, activating their target enhancers. In this way, ETS1 serves as a developmental gatekeeper for RUNX1-dependent expression programs that guide T cell development.

The RUNX1-dominant model raised additional questions about specific roles for each of its domains during Tcrb activation. Prior studies of RUNX1 and its ETS1 binding partner had mainly focused on their DNA binding and interacting domains using purified proteins and reporter assays (19, 20). These in vitro studies implicated that the NRDB of RUNX1 was essential for its association with the ETS1 exon VII domain (20). We find that the NRDB was neither necessary nor sufficient for ETS1 binding to the endogenous Eβ element. However, both of these observations may relate primarily to effects on neighboring chromatin accessibility rather than direct protein-protein contacts. Indeed, the tethered NRDB failed to clear nucleosomes from Eβ, as measured by histone H3 occupancy, likely inhibiting the recruitment of ETS1 to an adjacent site. Conversely, tethering of the RUNX1-TAD generated accessible chromatin over Eβ, which presumably led to modest binding of ETS1 at neighboring sites. The chromatin opening function of the RUNX1-TAD may be mediated by its recruitment of the histone acetyltransferase, p300 (24), leading to SWI/SNF association via its binding to acetylated histones (50).

To our knowledge, we provide the first evidence that the NRDB has any function in transcriptional activation, since it can compensate for the complete absence of ETS1 at composite RUNX-ETS sites. Specifically, we found that the TAD of RUNX1, by itself, could not activate Eβ when the neighboring ETS1 sites were mutated. Enhancer activation; however, was rescued in the absence of ETS1 when both the NRDB and TAD of RUNX1 were tethered to their native locations. Future studies using similar approaches may reveal the mechanisms by which the NRDB can compensate for enhancer-activating functions of ETS1. Taken together, our findings suggest that, in the native enhancer, the NRDB may contribute to a functional synergy with the TADs of RUNX1 and ETS1.

Like many developmentally controlled enhancers, Eβ-directed activation of the Tcrb-RC involves multiple revisions to its epigenetic and structural landscape, including histone modification, chromatin remodeling, and the formation of enhancer-promoter loops. The temporal and contingent relationships between these processes, leading ultimately to transcriptional activation, remains poorly understood at nearly all loci. Our study provided some key clues to the step-wise activation of a super-enhancer associated with the Tcrb-RC. First, we found that transcription could be largely uncoupled from acquisition of histone modifications over the super-enhancer region. This was especially true for long-range activation at the Dβ1Jβ1 cluster. Recruitment of various Gal4-RUNX1 fusions to Eβ completely restored transcription through this cluster with only modest, at best, restoration of activating histone marks, H3K27ac and H3K4me3. This finding is consistent with several reports showing that transcriptional activation of inducible genes may be uncoupled from H3K4me3 or other histone modifications at promoters (51, 52). In a broader sense, Tcrb, like many developmentally regulated genes, may be transcriptionally induced before it acquires its final epigenetic state as a super-enhancer, triggering key aspects of lineage commitment without full revisions of the epigenome (53).

In contrast to changes in histone modifications, Tcrb-RC transcription completely parallels the restoration of Eβ-promoter looping and nucleosome clearance from both types of regulatory elements, which are likely to be contingent processes. These new findings support a proposed step-wise model for activation of the Tcrb-RC (32), in which promoter-Eβ contacts generate a stable “holocomplex” to recruit additional factors, including chromatin remodelers. Indeed, recruitment of the nucleosome remodeling complex, SWI/SNF, to the Tcrb-RC, requires promoter-Eβ looping (32). In this study, we demonstrated that RUNX1 is sufficient to mediate promoter-Eβ looping, chromatin remodeling, and transcriptional activation. The RUNX1-TAD was again sufficient to mediate these processes, when modest levels of ETS1 occupied Eβ. Thus, ETS1 may support modest levels of promoter-Eβ looping in the absence of the RUNX1-NRDB, perhaps via its known interaction with ATF2 (54), a transcription factor that binds to Dβ promoters (55). In the absence of ETS1, tethering of the NRDB and RUNX1-TAD to Eβ is sufficient to drive loop formation with its distal promoters. In this case, we propose that association of RUNX1 with the nuclear factor, ALY (56), may be important to forge Eβ-promoter contacts. Since ALY is known to dimerize (56), it may form a bridge between RUNX1 molecules bound to the enhancer and Dβ promoters (39). Moreover, once the bridged promoters become even minimally active, ALY may further stabilize Eβ-promoter loops via its RNA-binding activity (57).

Together, our results demonstrate that RUNX1, when uncoupled from the requirement for cooperative ETS1 binding, is sufficient to drive long-range loop formation by Eβ, nucleosome clearance at its target promoters, and full transcriptional activation of the Tcrb-RC. Given the broad distribution of ETS-RUNX motifs in the mammalian genome, our findings with the Tcrb-RC may be applicable to the activation of super-enhancers at other key hematopoietic genes. Future studies with inducible experimental platforms, such as the one described here, will be useful to address more detailed mechanisms for long-range enhancer activation of promoters for developmentally regulated genes.

Supplementary Material

1

Acknowledgments

We thank Oltz lab members for their invaluable input, as well as Drs. B. Sleckman and T. Egawa for reagents.

Footnotes

1

This work was supported by the National Institute of Health (AI115734, AI122726, AI118852, CA188286).

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