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. Author manuscript; available in PMC: 2015 Oct 15.
Published in final edited form as: J Immunol. 2014 Sep 15;193(8):3959–3965. doi: 10.4049/jimmunol.1401099

Regulation of the Th1 genomic locus from Ifng through Tmevpg1 by T-bet

Sarah P Collier *, Melodie A Henderson †, John T Tossberg †, Thomas M Aune *,†
PMCID: PMC4185266  NIHMSID: NIHMS621674  PMID: 25225667

Abstract

Long noncoding RNAs (lncRNA), critical regulators of protein-coding genes, are likely to be co-expressed with neighboring protein-coding genes in the genome. How the genome integrates signals to achieve co-expression of lncRNA genes and neighboring protein-coding genes is not well understood. The lncRNA Tmevpg1 (NeST, Ifng-AS1) is critical for Th1-lineage specific expression of Ifng and is co-expressed with Ifng. Here, we show that T-bet guides epigenetic remodeling of Tmevpg1 proximal and distal enhancers leading to recruitment of stimulus-inducible transcription factors, NF-κB and Ets-1, to the locus. Activities of Tmevpg1- specific enhancers and Tmevpg1 transcription are dependent upon NF-κB. Thus, we propose that T-bet stimulates epigenetic remodeling of Tmevpg1 specific enhancers and Ifng specific enhancers to achieve Th1-lineage specific expression of Ifng.

Introduction

The cytokine, IFN-γ, plays a critical role in protecting vertebrate organisms from invading pathogens, including bacteria and viruses, as well as preventing tumor development (1–3). Naïve CD4+ T cells do not efficiently produce IFN-γ in response to antigenic stimulation but differentiate into cells termed effector Th1 cells in the presence of antigenic stimulation and cytokine stimulation, such as IL-12, capable of producing high levels of IFN-γ in response to antigenic stimulation. This process is governed by transcription factors Stat1 and Stat4 that promote expression of the Th1 transcription factor, T-bet. This combination of transcription factors promotes epigenetic remodeling at the Ifng locus (4–8). A general view is that this remodeling process allows for efficient transcription of Ifng mRNA in response to secondary antigenic stimulation (9–12).

Long non-coding RNAs (lncRNAs) are newly discovered species of regulatory RNAs (13, 14). Thousands of lncRNAs have been discovered but functions of only a few have been determined. In certain instances genes encoding lncRNAs are positioned adjacent in the genome to the protein coding genes these lncRNAs regulate (15, 16). Examples are HOTAIR and HOTTIP that regulate the locus of developmentally regulated HOX genes (17, 18). One mechanism by which these lncRNAs target the HOX protein-coding genes is by acting as scaffolds to recruit histone-modifying complexes to target gene loci to produce either repressing (HOTAIR) or activating (HOTTIP) epigenetic modifications. Thus, these lncRNAs appear to function by remodeling the epigenetic landscape rather than by directly repressing or activating transcription of protein coding genes (19). In other instances, lncRNAs genes are adjacent to protein-coding genes in the genome and are co-expressed with these adjacent protein-coding mRNAs but have regulatory functions distinct from regulating expression of the adjacent protein-coding gene. An example is lincRNA-Cox2 and Ptgs2 (gene that encodes Cox-2) (20). Other examples exist where adjacent lncRNA coding genes and protein-coding genes are co-expressed in response to different stimuli (13, 16, 21). Thus it seems that co-expression of adjacent lncRNAs coding genes and protein-coding genes is a common property, little is known about the underlying epigenetic and transcriptional mechanisms that produce this phenomenon.

The lncRNA transcript, Tmevpg1, also named NeST and Ifng-AS1, is encoded by a gene adjacent to Ifng in both human and mouse genomes, is induced in response to the Th1 differentiation program by mechanisms dependent upon Stat4 and T-bet (22–24). Tmevpg1 cooperates with T-bet to promote Ifng transcription. One mechanism by which Tmevpg1 functions is to bind to WDR5, a component of the histone H3 lysine 4 (H3K4) methyltransferase complex and enhance marking of the Ifng locus with H3K4 methylation. Importantly, studies in transgenic mice demonstrate that Tmevpg1 (NeST, Ifng-AS1) plays a critical role in establishing resistance to Salmonella enterica pathogenesis and preventing clearance of Theiler’s virus.

Whereas epigenetic remodeling and transcriptional regulation of lineage-specific protein coding gene loci has been an area of significant investigation, less is known about the roles of epigenetic remodeling and transcriptional regulation of gene loci that encode lineage-specific lncRNAs. Here we show that T-bet binds to the Tmevpg1 gene promoter/proximal enhancer as well as upstream distal enhancers in developing and differentiated effector Th1 cells. T-bet also plays a critical role in epigenetic remodeling of these genomic regions. These genomic sites actively recruit inducible transcriptional regulators, such as NF-κB and Ets-1, which is largely dependent upon T-bet. Stimulus-dependent expression of Tmevpg1 is also dependent upon activity of NF-κB. These findings expand our current understanding of the multiple mechanisms by which T-bet regulates Th1 lineage commitment and underlying mechanisms that contribute to co-expression of adjacent lncRNAs coding genes and protein-coding genes.

Materials and Methods

Mice

BALB/cJ and DO11.10 mice were obtained from Jackson Laboratory (Bar Harbor, ME). All mice were bred in the Vanderbilt University animal facilities. Research using mice complied with all relevant institutional and federal guidelines and policies. Human IFNG BAC transgenic mice contain a 190 kb transgene that contain the IFNG and IL26 genes but not the TMEVPG1 gene have been previously described and were backcrossed more than 12 generations to the C57BL/6 genetic background (25, 26).

Cultures

Primary BALB/cJ or DO11.10 splenocytes (1 × 106 cells/ml) were stimulated with immobilized anti-CD3 (2C11; American Type Culture Collection) or OVA323–339 peptide antigen (10 µg/ml; InvivoGen) respectively, under neutral Th0 (10 µg/ml anti-IFN-γ and 10 µg/ml anti–IL-4), Th1 (10 µg/ml anti-IL-4, 11B11; American Type Culture Collection) or Th2 (10 ng/ml IL-4 and 10 µg/ml anti-IFN-γ, R4-642; American Type Culture Collection) polarizing conditions for 3 days generating primary cultures. Effector cultures were generated by restimulation for an additional 48 hours with immobilized anti-CD3. Jurkat T lymphocytes (American Type Culture Collection) were maintained in complete RPMI at a density of 5×105 cells/ml. CD4+ T cells and NK cells were purified by negative magnetic selection (Miltenyi Biotec).

RNA isolation and RT-PCR

Total RNA was isolated with TRI Reagent (Ambion). cDNA was synthesized with the SSRIII kit (Invitrogen). Tmevpg1 (Life Technologies TaqMan assay ID: Mm01161206_m1) and Ifng (TaqMan, Life Technologies) transcript levels were measured by RT-PCR and calculated relative to Gapdh (TaqMan, Life Technologies) by the delta-delta Ct method.

Chromatin immunoprecipitation (ChIP)

ChIP procedures were followed as described (11, 26) using the following antibodies: anti-H4Ac (06-866, Millipore), T-bet (4B10, sc-21749, Santa Cruz), Ets-1 (C-20, sc-350, Santa Cruz), NF-κB p65 (ab7970, Abcam), and anti-rabbit IgG (Sigma). Primers used to amplify DNA across the Tmevpg1 locus and conserved DNA sequences at DNase hypersensitivity sites (27) are listed in Supplementary Table 1. Real-time PCR with SYBR green was used to measure DNA after ChIP and DNA purification. Melt curves after amplification showed that primer pairs generated single products. Quantitative ChIP assays were performed in triplicate and results from two or three independent experiments were averaged. Samples with a difference less than 2.0 in mean Ct value for specific and nonspecific (isotype Ig control) immunoprecipitation were considered nonspecific. Standard curves from the input DNAs were also generated using the different primer pairs. Slopes of these curves were essentially identical. Therefore, we elected not to include input DNA results in our calculations since this would only serve to add an additional source of error. Relative enrichment was determined using the following formula: 2(Ct (Ig control)−Ct (antibody-specific IP)), where Ct (Ig control) is the Ct obtained from the IgG isotype control ChIP and Ct (antibody-specific IP) is the Ct obtained from the antibody-specific ChIP. For example, a mean Ct value of 31 for the isotype control ChIPs and a mean Ct value of 26 for the antibody-specific ChIPs yielded a relative enrichment of 25 or 32.

Cell Transfection and Luciferase Assays

Human HS elements were amplified with the primers listed in Supplemental Table 1 from Jurkat T cell genomic DNA and cloned into the minimal promoter-luciferase or promoter-less luciferase construct (pGL4.24 and pGL4.10, respectively; Promega). The NFAT-IL4-Pr-luciferase construct has been previously described (28). Briefly the construct contains three copies of the distal NFAT binding site within the Il4 promoter, termed P1, linked to a minimal promoter and the firefly luciferase gene. Jurkat T cells were transfected with luciferase constructs by the DEAE transfection method (29) at 1 µg of plasmid DNA per 106 cells. After overnight recovery, cells were stimulated with 50 ng/ml PMA and 1 µM ionomycin per for 6 hours before luciferase activity was measured with the luciferase activity system (Promega). Cells were treated with BAY 11-7085 NF-κB inhibitor (5µM, Sigma) for one hour (30). Promoter truncations were generated with the primers listed in Supplementary Table 1 and cloned into the pGL4.10 construct.

Statistical analysis

Statistical significance was determined by a Student’s t test using Prism software. A P value of less than 0.05 was considered statistically significant.

Results

T-bet physically associates with and regulates epigenetic modifications at the Tmevpg1 locus

Th1 polarization promotes the expression of IFN-γ while suppressing Th2-specific genes, a process orchestrated in part by transcription factor recruitment, such as with T-bet, to noncoding regulatory elements (10). T-bet is known to associate with the Ifng promoter and numerous sites along a 140 kb intergenic distance between Ifng and Tmevpg1. Further, T-bet is required for the formation, spreading, and maintenance of certain histone modifications across this region(10). As Tmevpg1 expression is also dependent upon T-bet, we investigated T-bet association with the Tmevpg1 locus. Splenic cultures were polarized under Th1 conditions for three days to generate primary Th1 cultures and for five days with an additional 48 hours of stimulation with immobilized anti-CD3 to generate effector Th1 cultures. T-bet binding was assessed from +3.0 kb upstream to −160 bp downstream of the Tmevpg1 transcriptional start site (TSS) by ChIP. T-bet associated with chromatin mapping to the Tmevpg1 locus in primary Th1 cells as well as in effector Th1 cells (Fig. 1). T-bet was similarly found to associate with the Ifng promoter in both populations; however, was markedly enriched in effector Th1 cultures. For comparison, a conserved DNA element located 40kb upstream of Ifng previously determined not to recruit T-bet (10), also did not recruit T-bet in our experiments, thus serving as a negative control. Moreover, the majority of T-bet association in the primary Th1 cultures was within ± 100 base pairs of the Tmevpg1 TSS whereas T-bet association was found to spread across the entire 3 kb region in effector Th1 cells. Further, T-bet did not associate with the Tmevpg1 locus under Th2 culture conditions. Thus, T-bet bound to the Tmevpg1 promoter in primary Th1 cells and spread across up to +3 kilobases of the Tmevpg1 region as cells further differentiated into effector Th1 cells.

FIGURE 1.

FIGURE 1

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T-bet associates with the Tmevpg1 locus in primary and effector Th1 cell cultures. T-bet relative enrichment across the Tmevpg1 proximal promoter/enhancer, at the Ifng promoter and at a site 40 kb upstream of Ifng was determined by ChIP assay in primary and effector Th1 cultures. Results are expressed as the mean ± SEM of triplicate PCR determinations from three independent experiments. * P<0.05, comparing antibody-specific immunoprecipitations to isotype control immunoprecipitations at each genomic position

Given the above results, we next examined T-bet-dependent H4Ac patterns around the Tmevpg1 TSS. Spleen cell cultures from DO11.10 wildtype or DO11.10.Tbx21−/− mice were polarized under Th1 conditions for three days to generate primary Th1 cultures and five days with 48 hours additional stimulation with immobilized anti-CD3 to generate effector Th1 cultures. H4Ac histone modifications were assessed by ChIP spanning +2.3 kb upstream to −165 bp downstream of the Tmevpg1 TSS. In primary Th1 cultures, H4Ac modifications were enriched at genomic regions surrounding the Tmevpg1 TSS and these modifications required the presence of T-bet (Fig. 2A). H4Ac modifications were also enriched in effector Th1 cells and these were also dependent upon T-bet (Fig. 2B). Further, the level of H4Ac modifications was higher in effector Th1 cultures than primary Th1 cultures and H4Ac modifications appeared to spread across this ~3 kb genomic region. Histone H4Ac modifications at these sites in primary and effector Th2 cells were essentially undetectable and were equivalent to isotype controls. These findings indicate that T-bet associated with sites along the Tmevpg1 TSS and was necessary for seeding and spreading of H4Ac marks across this genomic region. Alternatively, T-bet may play a critical but indirect role to stimulate formation of these histone modifications by promoting Th1 differentiation through mechanisms still incompletely defined.

FIGURE 2.

FIGURE 2

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Seeding and spreading of histone H4Ac marks across the Tmevpg1 proximal promoter/enhancer is dependent upon T-bet in primary and effector Th1 cells. H4Ac enrichment was determined by ChIP in (A) primary and (B) effector Th1 and Th2 cultures. Results are expressed as the mean ± SEM of triplicate PCR determinations from three independent experiments. * P<0.05, comparing antibody-specific immunoprecipitations to isotype control immunoprecipitations at each genomic position

Tmevpg1 is surrounded by functional enhancer elements dispensable for Ifng expression

Identification of DNase 1 hypersensitivity sites (HS) is one method to identify potential functional genomic enhancer elements. Genome-wide analysis of DNase 1 HS in human Th1 and Th2 cells has been performed and is available on the UCSC Genome Browser (31, 32). Inspection of these tracks within the genomic region between Ifng and Tmevpg1 (Fig. 3A, adapted from the UCSC Genome Browser) shows the presence of five Th1-specific DNase 1 hypersensitivity sites indicating that these areas represent accessible, open chromatin. Using an unbiased deletion strategy to identify noncoding regions conferring Th1-specific Ifng expression, we examined the requirement of two large deletions of the 190 kilobase BAC transgene mapping to the region between IFNG and TMEVPG1. This BAC transgene contains the complete IFNG gene but does not contain the TMEVPG1 gene. CD4+ T cells were purified and cultured under neutral (Th0), Th1 and Th2 conditions for five days to generate primary Th1 cells or were restimulated for 48 hours with immobilized anti-CD3 to generate effector Th1 cells. Compared to the full BAC transgene, deletion 1 and deletion 2 cultures exhibited equivalent abundance of IFNG message relative to Gapdh in Th0, Th1 and Th2 primary and effector cultures (Fig. 3B&C) coinciding with levels of IFN-γ in culture supernatants (25). These results support the notion that this intergenic region is dispensable for IFNG expression. Therefore, if these HS sites possess transcriptional enhancer activity, it may be that their function is to enhance TMEVPG1 transcription rather than IFNG transcription.

FIGURE 3.

FIGURE 3

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Enhancers in the intergenic region between IFNG and TMEVPG1 are not required for IFNG expression. (A) Human genomic locus on chromosome 12 containing TMEVPG1, IFNG, and IL26 genes is from the UCSC genome browser (GRCg37/hg19 build). Peaks represent DNase I hypersensitivity sites in polarized human Th1 and Th2 cultures obtained form the UCSC genome browser, references 31 and 32. Above these tracks are the genomic locations of the BAC used to make transgenic mice and the locations of 40-kb BAC deletions (del1 and del 2) also used to generate human IFNG transgenic mice (note that the human BAC transgene does not contain TMEVPG1). Distances of each HS site (HS1-HS5) from IFNG are shown below the DNase 1 HS tracks. (B&C) Human IFNG expression by (B) primary and (C) effector un-polarized (Th0), Th1 and Th2 cultures from BACdel1, BACdel2 or the full-length human 190 kb BAC transgene. Results are expressed as mean ± the SEM of human IFNG expression relative to murine Gapdh expression.

To explore this hypothesis further, we assessed enhancer activity of each HS. Each HS, numbered one through five, was cloned into the pGL4.24 vector construct containing a minimal promoter followed by the luciferase gene. The constructs were transfected into Jurkat T lymphocytes at 1 µg per 106 cells. After overnight recovery, cells were stimulated with 50 ng/ml PMA and 1 µM ionomycin for six hours, lysed and assayed for luciferase activity. Transfection with HS1-, HS2-, HS3- or HS4-pGL4.24 constructs resulted in a significant increase in luciferase activity relative to vector alone indicating that these DNA elements possessed enhancer activity (Fig. 4A). Similar levels of enhancer activity were observed in HS1 and HS3 while HS4 exhibited markedly greater enhancer activity relative to the other HS elements. In contrast, HS5 exhibited a 10-fold decrease in luciferase activity relative to vector alone (Fig. 4B). These findings indicated that HS1, HS2, HS3 and HS4 are functional enhancer elements while HS5 appeared to have suppressive function.

FIGURE 4.

FIGURE 4

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Enhancer and promoter activity of genomic regions marked by Th1-specific Dnase hypersensitivity (27). (A&B) Human HS elements were cloned into the pGL4.24-luciferase construct under the control of a general minimal promoter. Constructs were transfected into Jurkat T cells via DEAE transfection. Results are shown as the mean ± SEM of triplicate determinations from two independent transfections. (C) HS1 promoter activity was evaluated by cloning into the promoter-less pGL4.10 construct in Jurkat T cells. Results are expressed as the mean of triplicate determinations from three independent experiments ± SEM. (D) Truncations of HS1 to identify regions contributing to promoter activity was evaluated by cloning fragments into the pGL4.10 construct in Jurkat T cells. Results are expressed as the average of triplicate determinations from three independent experiments ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 comparing stimulated versus unstimulated cultures

In addition to having enhancer activity, the HS1 element aligns with the TMEVPG1 TSS, thus we evaluated HS1 for promoter activity. To do so, HS1 was cloned into the pGL4.10 promoterless-luciferase construct and transfected into Jurkat T lymphocytes at 1 µg per million cells. After overnight recovery, cells were stimulated with 50 ng/ml PMA and 1 µM ionomycin for 6 hours and assayed for luciferase activity. A three-fold increase in luciferase activity was observed relative to the vector control supporting the fact that in addition to enhancer activity, HS1 also contains the Tmevpg1 promoter (Fig. 4C).

The HS1 element contains two distinct peaks of Th1-specific DNase I hypersensitivity, either of which could contribute to enhancer as well as promoter activity. Therefore, we employed a traditional promoter analysis truncation approach to identify regions of HS1 that contributed to the observed promoter activity. The HS1 region was divided into four segments, cloned into the pGL4.10 vector construct and assayed for promoter activity relative to the full HS1 construct alone (Fig. 4D). Greatest promoter activity was found at the more distal regions of HS1; −281 bp to −11 bp and +274 bp to +518 bp while constructs with intervening sequences possessed lower promoter activity (compare activity of the +274 bp to +518bp construct to activity of the +7 bp to +518 bp construct) indicating the presence of both enhancer activity and repressor activity in HS1.

NF-κB and Ets-1 recruitment to the Tmevpg1-Ifng locus and function

We inspected the HS1 DNA sequence for potential transcription factor binding sites and identified both Ets-1 and NF-κB binding motifs. Ets-1 is a transcription factor predicted to function with T-bet in the Th1 polarization pathway (33, 34). These results indicate that TMEVPG1 is surrounded by functional enhancer sequences while HS1 also possesses promoter activity. Whereas IFN-γ production by primary and effector Th1 cells is dependent upon T-bet, it is known that T-bet is dispensable for Ifng expression by memory cells (35). Inducible transcription factors such as NF-κB, in particular, contribute to rapid IFN-γ responses by effector-memory Th1 cells(36). As such, we examined NF-κB and Ets-1 recruitment to the Tmevpg1 locus. Spleen cell cultures from DO11.10 wildtype mice were polarized under Th1 or Th2 conditions for three days for primary cultures and five days with 48 hours additional stimulation with immobilized anti-CD3 to generate effector cultures. NF-κB and Ets-1 recruitment to Tmevpg1 was assessed by ChIP assay spanning +2.3 kb upstream to −165 bp downstream of the Tmevpg1 TSS. NF-κB recruitment to Tmvepg1 was relatively low across the locus particularly in primary Th1 cultures (Fig. 5A). However, a distinct peak of NF-κB binding was seen at −70 bp downstream of the Tmevpg1 TSS in effector Th1 cultures. Conversely, in these same samples, Ets-1 was recruited at multiple HS1 sites in both primary and effector Th1 cultures (Fig. 5B). These same transcription factors were not recruited to HS1 in primary or effector Th2 cultures. ChIP determinations at the B2m promoter (37) and the Cd3e promoter (38) for NF-kB and Ets-1, respectively, served as positive signals not dependent upon Th1/Th2 differentiation. Thus, both NF-κB and Ets-1, along with T-bet, are recruited to specific sites Tmevpg1 HS1 in primary and effector Th1 cells but not effector Th2 cells.

FIGURE 5.

FIGURE 5

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NF-κB and Ets-1 associate with the Tmevpg1 proximal promoter/enhancer in primary and effector Th1 cells. (A) NF-κB and (B) Ets-1 associations to chromatin from primary and effector Th1 cultures or effector Th2 cultures were determined by ChIP. Genomic positions are shown relative to Tmevpg1 TSS. Recruitments of NF-kB to the known B2m promoter NF-kB binding site (reference 37) and Ets-1 to the known Cd3e promoter Ets-1 binding site were also determined as positive controls. Results are expressed as the mean fold enrichment relative to isotype control chromatin immunoprecipitations and are the average of triplicate determinations from three independent experiments ± SEM. * P<0.05, comparing antibody-specific immunoprecipitations to isotype control immunoprecipitations at each genomic position

As both NF-κB and Ets-1 associated with Tmevpg1 genomic promoter/enhancers, we examined recruitment of these transcription factors to murine HS enhancer elements that were identified by sequence conservation to the above human HS elements (the HS2 element is not conserved between humans and mice). To address this question, spleen cell cultures from DO11.10 Tbx21+/+ or DO11.10.Tbx21−/− mice were polarized under Th1 or Th2 conditions for three days for primary cultures and five days with 48 hours additional stimulation with immobilized anti-CD3 to generate effector Th1 or Th2 cultures. We found that NF-κB was enriched at these conserved HS regions in primary and effector Th1 cultures but not Th2 cultures and recruitment was largely T-bet dependent (Fig. 6A&B). The magnitude of recruitment to mHS3 and mHS4 was greater than recruitment to the Ifng promoter. Ets-1 was also recruited to each of the mHS enhancer sequences in primary and effector Th1 cultures but not Th2 cultures and was found to be largely, but not absolutely, dependent upon the presence of T-bet (Fig. 6C&D), similar to the pattern observed for NF-κB enrichment. Lastly, Ets-1 associated with the Ifng promoter in a T-bet dependent manner in effector cultures. These results support that T-bet is required, in part, for recruitment of the inducible transcription factors, Ets-1 and NF-κB, to these intergenic enhancers.

FIGURE 6.

FIGURE 6

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NF-κB and Ets-1 are enriched at Tmevpg1 enhancer elements in a T-bet dependent manner. (A&B) NF-κB and (C&D) Ets-1 binding to homologous mouse Tmevpg1 enhancer sequences and the Ifng promoter in DO11.10 Tbx21+/+, DO11.10 Tbx21−/− and primary (day3) and effector Th1 and Th2 cells (after restimulation). Cells were analyzed by ChIP for transcription factor binding. Enhancer regions are designated on the x-axis. Results are expressed as the mean fold enrichment relative to isotype control chromatin immunoprecipitations and are the average of triplicate determinations from three independent experiments ± SEM. * P<0.05, comparing antibody-specific immunoprecipitations to isotype control immunoprecipitations at each genomic position

Additionally, we assessed pharmacological inhibition of NF-κB activity by treatment with the extensively utilized I-kappa-α inhibitor BAY 11-7085. HS1-, HS2-, HS3-, HS4- and HS5-pGL4.24 constructs were transfected into Jurkat T lymphocytes at 1 µg/106 cells. After overnight recovery, cells were incubated in the presence of the inhibitor for one hour followed by stimulation with 50 ng/ml PMA and 1 µM ionomycin for six hours before determining luciferase activity. Relative to control cultures, cultures treated with the NF-κB inhibitor exhibited a marked decrease in activity of each enhancer: HS1-HS4, to varying degrees (Fig. 7A). In contrast, BAY 11-7085 did not inhibit transcriptional activity directed by a known NFAT response element from the Il4 promoter (NFAT-Il4-Pr). Further, treatment of effector Th1 cultures with BAY 11-7085 before restimulation impaired Tmevpg1 expression as well as impaired Ifng expression (Fig. 7B&C). In contrast, BAY 11-7085 did not inhibit expression of the stimulus-induced Fos gene (Fig. 7D). These results support an important role for NF-κB activation and recruitment to the Tmevpg1 proximal promoter/enhancer region as well as HS1-HS5 elements to achieve stimulus-dependent transcriptional enhancer activity and induction of Tmevpg1 expression.

FIGURE 7.

FIGURE 7

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Inhibition of NF-κB activity reduces activities of Tmevpg1 enhancers and Tmevpg1 and Ifng expression. (A) HS1-HS4-pGL4.24 or NFAT-Il4-Pr (see methods) constructs were transfected into Jurkat T cells via DEAE transfection. Cells were incubated one hour in the presence of the inhibitor before stimulation with PMA and ionomycin for six hours. Relative luciferase activity was determined. (B) Ifng, (C) Tmevpg1 and (D) Fos expression in effector Th1 cultures: effector Th1 cells were incubated one hour in the presence of inhibitor before restimulation with immobilized anti-CD3 for 48 hours. All results are mean ± SEM of triplicate measurements from two independent experiments. *P<0.05 comparing BAY 11-7085 treated and untreated cultures.

Discussion

Here, we provide evidence to extend the breadth of T-bet positive regulation of the Th1 locus from Ifng over 170 kilobases upstream to the gene encoding the Tmevpg1 lncRNA. In previous studies, we found that Tmevpg1 expression is dependent upon the master Th1 transcription factor T-bet, but this effect was poorly understood (23). To summarize our results, we find that T-bet associates with the Tmevpg1 locus promoting H4Ac marks in primary and effector Th1 cells, but not Th2 cells. T-bet is required for the formation and maintenance of H4Ac marks downstream of Ifng, however, this region is not required for IFNG expression (10, 25). The genomic region between Tmevpg1 and Ifng contains a number of Th1-specific DNase 1 HS sites to which the inducible transcription factors, NF-κB and Ets-1 are recruited in a T-bet dependent fashion. Four of these sites possess strong transcriptional enhancer activity while the fifth possesses transcriptional repressor activity. Further, pharmacological inhibition of NF-κB reduces enhancer activity of the HS1-HS4 elements supporting that enhancement from these sites is mediated, in part, by NF-κB. Taken together, our data support a mechanism by which Tmevpg1 is transcriptionally regulated by T-bet via epigenetic mechanisms enabling recruitment of inducible transcription factors to both the Tmevpg1 promoter and gene body as well as distal transcriptional enhancers and repressors (Fig. 8). Our findings not only provide a detailed description of lncRNA gene regulation as part of a developmental program but also contribute to the accepted mechanism of regulation of Ifng during Th1 lineage commitment.

FIGURE 8.

FIGURE 8

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Model of T-bet dependent regulation of the Ifng locus. Multiple conserved HS (orange rectangles) exist in the human genomic region from TMEVPG1 to IL26 genes, are marked by H4Ac (yellow hexagon) in response to Th1 differentiation signals. T-bet (blue diamond) is recruited to these HS sites and directs chromatin remodeling. The stimulus- activated transcription factors NF-κB (blue six-point star) and Ets-1 (green four-point star) are also recruited to these HS. These proximal and distal HS possess transcriptional enhancer activity to enhance or repress transcription of either IFNG or TMEVPG1 (indicated by the red (enhancer) or blue (repressor) arrows. The TMEVPG1 lncRNA stimulates formation of H3K4 methylation marks at the IFNG gene (green arrow) and maintains these marks in effector Th1 cells.

Our model is generally consistent with recent studies examining the requirement of Tmevpg1 for expression of Ifng in response to infection with Salmonella (24). Here, Tmevpg1 expression in CD8+ T cells promotes rapid IFN-γ expression in response to Salmonella infection through formation of covalent H3K4me3 marks at the Ifng locus correlating with active transcription and rapid expression of IFN-γ by CD8+ T cells (24). Moreover, in an in vitro assay, Tmevpg1 physically associates with the WDR5 component of the MLL/Set1 histone-modifying complex responsible for producing the H3K4me3 mark (24). These findings are generally supported by studies examining functions of other lncRNAs. Whereas repressive lncRNAs mediate epigenetic regulation of genes through association with PRC2-containing histone complexes, lncRNAs imposing positive regulation on target genes are commonly found to associate with the MLL/Set1 complex and associated their proteins.

These results combined with previous published studies support a model whereby Stat4 and T-bet play integral roles in epigenetic remodeling of the Ifng locus spanning over 300 kb. Besides activity of Ifng and Tmevpg1 proximal promoter/enhancers, distal enhancers seem to be divided into those utilized to drive Ifng expression at discrete stages of differentiation from initial Th1 polarization to responses by memory T cells and those utilized to drive Tmevpg1 expression. Transcription factors, such as NF-κB and Ets-1, which respond to TCR stimulation, are critical for Tmevpg1 expression and also Ifng expression at later stages of differentiation. Results also support the notion that Tmevpg1 recruits the MLL/SET1/WDR complex to Ifng to establish a network of H3K4 methylation marks. Finally, three principle CTCF binding sites, one at the 3’ end of the Tmevpg1 gene, one within Ifng, and one upstream of Ifng cooperate to loop this genomic region together (39, 40). It has been suggested that these CTCF binding sites serve an insulator function to protect Ifng from silencing effects of neighboring heterochromatin. We would suggest that our results are more consistent with a model whereby CTCF sites are actually utilized to bring the Tmevpg1 gene in close proximity to the Ifng gene to facilitate localization of Tmevpg1 RNA transcripts to the Ifng genomic region so Tmevpg1 RNA transcripts can carry out their function of enhancing Ifng transcription.

Supplementary Material

1

Acknowledgements

We thank Mark Boothby and Christopher Williams for providing us with Tbx21 knockout mice. We also thank Scott Collier for helpful discussions and comments.

This work was supported by National Institutes of Health Grants R01 AI044924 and T32 AR059039.

Abbreviations

lncRNA

long noncoding RNA

ChIP

chromatin immunoprecipitation

HS

DNase hypersensitivity

TSS

transcriptional start site

Footnotes

Author Contributions

SCP and TMA conceived the general experimental approach, interpreted the data and wrote the manuscript. SCP performed majority of experiments and analyzed the data. MAH and TMA assisted with experimental design and performed ChIP assays. JTT assisted with design and cloning of promoter/enhancer constructs.

Disclosures

The authors have no financial conflicts of interest

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