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. Author manuscript; available in PMC: 2015 Dec 11.
Published in final edited form as: Cell Rep. 2014 Dec 4;9(5):1718–1728. doi: 10.1016/j.celrep.2014.11.011

A distal locus element mediates IFN-γ priming of LPS-stimulated TNF gene expression

Nancy A Chow *, Luke D Jasenosky *, Anne E Goldfeld *,^
PMCID: PMC4268019  NIHMSID: NIHMS642189  PMID: 25482561

SUMMARY

IFN-γ priming sensitizes monocytes/macrophages to lipopolysaccharide (LPS) stimulation, resulting in augmented expression of a set of genes including TNF. Here, we demonstrate that IFN-γ priming of LPS-stimulated TNF transcription requires a distal TNF/LT locus element 8 kb upstream of the TNF transcription start site (hHS-8). IFN-γ stimulation leads to increased DNase I accessibility of hHS-8 and its recruitment of IRF1, and subsequent LPS stimulation enhances H3K27 acetylation and induces enhancer RNA synthesis at hHS-8. Ablation of IRF1 or targeting the hHS-8 IRF1 binding site in vivo with Cas9 linked to the KRAB repressive domain abolishes IFN-γ priming while LPS induction of the gene is unaffected. Thus, IFN-γ poises a distal enhancer in the TNF/LT locus by chromatin remodeling and IRF1 recruitment, which then drives enhanced TNF gene expression in response to a secondary TLR stimulus.

INTRODUCTION

Produced by natural killer cells and activated Th1 lymphocytes, IFN-γ sensitizes circulating monocytes and tissue-resident macrophages leading to augmentation of macrophage activation after microbial recognition and toll-like receptor (TLR) signaling (Murray, 1988; Schwartz and Svistelnik, 2012). This phenomenon, known as IFN-γ priming, results in enhanced gene expression of inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-12, and IL-6 (Lorsbach et al., 1993; Ma et al., 1996; Pace et al., 1983; Sanceau et al., 1991). In the case of TNF, de novo transcription of TNF is enhanced in human monocytes primed by IFN-γ and then stimulated by LPS (Hayes and Zoon, 1993). However, the molecular mechanisms that control IFN-γ priming, and whether these mechanisms are gene-specific, are poorly understood.

The TNF gene and the genes encoding lymphotoxin-α and -β (LTA and LTB) comprise the ~20 kb TNF/LT locus region, which lies within the histocompatibility locus on human chromosome 6 and mouse chromosome 17. TNF is highly and rapidly expressed in both lymphocytes and monocytes (Goldfeld and Maniatis, 1989; Goldfeld et al., 1990; Goldfeld et al., 1993), and its transcriptional regulation occurs in a cell type- and inducer-specific manner. Distinct sets of transcription factors and co-activators including chromatin modifying enzymes, are recruited to DNA elements in the TNF promoter depending on the type of cell and the type of stimulus received (Falvo et al., 2000a; Falvo et al., 2000b; Tsai et al., 2000, Tsytsykova and Goldfeld, 2000). Furthermore, the formation of higher-ordered structures, or enhanceosomes, is required for TNF gene expression in specific cell types (Tsytsykova and Goldfeld, 2002; Barthel et al., 2003). Moreover, distal hypersensitive (DH) elements upstream and downstream of the TNF transcription start site (TSS) have been identified in the TNF/LT locus. A subset of these DH sites also varies by cell type (Barthel et al. 2003, Tsytsykova et al., 2007, Taylor et al., 2008; Biglione et al., 2011). For example, DH sites ~9 kb upstream and ~3 kb downstream of the murine gene act as NFATp-dependent enhancers in T cells and participate in activation-induced intrachromosomal interactions with the promoter (Tsytsykova et al., 2007), while a myeloid-specific DH site ~7 kb upstream of the TSS functions as a matrix attachment region (Biglione et al., 2011).

In this study, we show that a DH site ~8 kb upstream of the human TNF TSS (hHS-8 for human hypersensitive site -8 kb), is required for, and mediates IFN-γ-stimulated augmentation of LPS-induced TNF gene expression in human monocytes/macrophages. The highly conserved hHS-8 noncoding element exhibits increased nuclease accessibility in response to IFN-γ stimulation and IRF1 is recruited. Upon subsequent LPS stimulation of IFN-γ primed cells, there is increased acetylation of H3K27 and synthesis of enhancer RNA (eRNA) at hHS-8. IFN-γ priming of TNF is abrogated with the ablation of IRF1, disrupting the IRF1 site in reporter assays, or by targeting the IRF1 binding element in hHS-8 with the catalytically inactive form of Cas9 linked to the Krüppel-associated box (KRAB) domain of Kox1 (Margolin et al., 1994; Gilbert et al., 2013) in human monocytic cells. Thus, IRF1 expression and an intact hHS-8 IRF1 binding element is required for IFN-γ priming of TNF in vivo.

These experiments expand the functional role of distal regulatory elements in the innate immune response to IFN-γ priming and highlight the potential of CRISPR/Cas9 technology as a tool for interrogation of the function of distal regulatory elements in human hematopoietic cells.

RESULTS

IFN-γ promotes chromatin accessibility at hHS-8 in the TNF/LT locus

As a single stimulus, LPS significantly induces TNF mRNA levels whereas IFN-γ alone is not sufficient to induce TNF gene expression in human monocytic THP-1 cells (Fig. 1A). However, priming of cells by pre-treatment with IFN-γ for 2 hours before LPS stimulation significantly enhances TNF mRNA levels compared to stimulation by LPS alone (Fig. 1A). This observation supported our hypothesis that IFN-γ poises the TNF gene for enhanced transcription in response to LPS by stimulating chromatin remodeling at the TNF/LT locus.

Figure 1.

Figure 1

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IFN-γ priming promotes chromatin accessibility at hHS-8 in the TNF/LT locus. (A) IFN-γ priming enhances TNF mRNA levels in THP-1 cells stimulated with LPS. Cells were stimulated with IFN-γ alone for 3h, LPS alone for 1h, and both IFN-γ and LPS (IFN-γ for 2h followed by LPS for 1h). TNF mRNA levels were measured post LPS stimulation by q-PCR. (**) p ≤ 0.01, data is represented as mean ± SEM. (B, C) IFN-γ increases chromatin accessibility hHS-8. DHAs using the restriction enzyme ScaI (B) and BamHI (C) allowed for examination of hHS-8 and the TNF promoter, respectively, in resting and IFN-γ-treated THP-1 cells. (D) IFN-γ priming enhances TNF mRNA levels in primary human MDMs stimulated with LPS. MDMs were stimulated as in (A) and RNA was collected 1h after LPS stimulation. Data from 3 separate donors, (*) p ≤ 0.05, data is represented as mean ± SEM. (E, F) DHAs were performed in resting and IFN-γ-treated primary human MDMs as in (B, C). (G) Map of the human TNF/LT locus. DH sites and positions and directions of transcription of the TNF, LTA, and LTB genes are shown.

Positions of the parental ScaI, parental BamHI, and DNase I digestion products for the DHAs are indicated. (H, I) IFN-γ and LPS decreases nucleosome occupancy at the TNF promoter and hHS-8. ChIP using THP-1 cells (H) and primary human MDMs (representative donor, I) measures nucleosome occupancy (total H3 levels) at both the TNF promoter and hHS-8. Although not significant, IFN-γ alone decreases total H3 levels at the TNF promoter (p=0.054) in THP-1 cells; this was not repeated in the MDM donor. Data from 3 separate experiments, (*) p ≤ 0.05 and (***) p ≤ 0.001, data are represented as mean ± SEM.

In order to test this idea, we performed a DNase I hypersensitivity (DH) assay comparing the landscape and intensity of hypersensitive sites across the TNF/LT locus in IFN-γ-treated and untreated THP-1 cells. IFN-γ treatment of THP-1 cells promoted chromatin accessibility at a DH site located ~8 kb upstream of the TNF TSS (hHS-8), as evidenced by the IFN-γ-dependent enhancement of the DNase I-generated band corresponding to hHS-8 (Fig. 1B, compare lane 4 to lane 8). Thus, in a population of unstimulated human monocytic cells, hHS-8 is constitutively present and, upon IFN-γ treatment, the proportion of cells in which hHS-8 becomes accessible to DNase I increases. We also observed a smaller increase in DNase I cleavage at the TNF promoter as compared to hHS-8 in response to IFN-γ (Fig. 1C, compare lane 4 to lane 8).

To extend our findings to human primary cells, we examined the effects of IFN-γ priming on monocyte-derived macrophages (MDMs) and confirmed that IFN-γ pre-treatment significantly enhanced TNF mRNA levels as compared to stimulation by LPS alone (Fig. 1D). Similar to what we observed in THP-1 cells, IFN-γ treatment increased DNase I cleavage at hHS-8 in primary human MDMs (Fig. 1E, compare lane 3 to lane 6). Furthermore, in both cell types, IFN-γ priming prior to LPS stimulation led to enhanced DNase I cleavage as compared to LPS stimulation alone at hHS-8 (compare lanes 12 and 16 of Fig. 1B, and lanes 9 and 12 of Fig. 1E). We note that examination of data from the ENCODE database (Thurman et al., 2012) revealed that a constitutive DH site ~8 kb upstream of the TNF TSS was present in resting primary human monocytes in this data set (Fig. S1), providing confirmation of our detection of a DH site at this location in resting monocytic cells. In contrast to THP-1 cells, we observed no change in DNase I cleavage at the TNF promoter upon IFN-γ stimulation (Fig. 1F, compare lane 3 to lane 6). Restriction sites and probe positions for DH analysis of both hHS-8 and the TNF promoter are shown in Figure 1G.

A decrease in total H3 levels is generally reflective of enhanced chromatin accessibility (Reinke and Horz, 2004). In order to confirm chromatin remodeling of the TNF/LT locus after IFN-γ stimulation, we next measured total H3 levels at hHS-8 and the TNF promoter by chromatin immunoprecipitation (ChIP) analysis in THP-1 cells and primary human MDMs under the same conditions used for the DH analysis. Consistent with the DHA findings, we observed a significant reduction in total H3 levels at hHS-8 in response to IFN-γ in both THP-1 cells (Fig. 1H) and in primary MDMs (Fig. 1I), but not at the TNF promoter (Fig. 1H and 1I). Furthermore, LPS alone and IFN-γ + LPS stimulation caused a significant loss in total H3 levels at both hHS-8 and the TNF promoter in both the THP-1 cells and MDMs (Figs. 1H and 1I). Taken together, these findings show that sole stimulation with IFN-γ remodels the TNF/LT locus at the distal DNA element hHS-8 to increase nuclease accessibility, consistent with hHS-8 playing a role in IFN-γ mediated TNF transcriptional augmentation. In the case of LPS, nuclease accessibility is increased after stimulation at both the TNF promoter and hHS-8.

IRF1 binds to hHS-8 in an IFN-γ-inducible manner

IFN-γ is a potent inducer of the transcription factor IRF1 (Pine et al., 1990). Examination of the TNF/LT locus for sequences resembling the IRF consensus binding sequence 5′-AANNGAAANGAA-3′ (Tamura et al., 2008) revealed putative IRF sites in both the TNF promoter and in hHS-8 (Fig. 2A). To determine whether IRF1 is capable of binding to these sequences, we first performed quantitative DNase I footprinting analysis with recombinant IRF1 (rIRF1) and found that the protein binds to the TNF proximal promoter at the 5′ boundary of the predicted site, which lies within a composite binding site of the TNF enhanceosome that also binds Sp1, Egr1, NFATp and Ets in a cell type-specific manner (Tsai et al., 2000) (Fig. 2B). Moreover, rIRF1 also binds to hHS-8 at an IRF binding motif containing three 5′-GAAA-3′ motifs (Fig. 2C). Using ChIP we confirmed that IRF1 binds to hHS-8 in vivo in primary human MDMs and that its binding is significantly enhanced upon IFN-γ stimulation in vivo. By contrast, IRF1 recruitment to the promoter was minimal. After LPS treatment of IFN-γ-primed primary human MDMs, IRF1 recruitment to the TNF promoter increased while IRF1 binding at hHS-8 declined, but IRF1 binding at hHS-8 remained significantly elevated as compared to non-stimulated conditions (Fig. 2D).

Figure 2.

Figure 2

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IRF1 binds to hHS-8 in an IFN-γ-inducible manner. (A) TNF/LT locus with partial sequences and binding site positions of transcription factors for both the TNF promoter and hHS-8. (B) rIRF1 binds to the TNF promoter. Quantitative DNase I footprinting analysis of the TNF promoter (−200 to +1) and increasing concentrations of rIRF1. Sense and anti-sense strand with G/A ladder and BSA control. Bars mark areas of rIRF1 binding at −172 to -136. (C) rIRF1 binds to hHS-8. Quantitative DNase I footprinting analysis of hHS-8 (−7031 to −6782) was performed as in (B). Bars mark areas of rIRF1 binding at −6833 to −6782. (D) IRF1 is recruited to hHS-8 in an IFN-γ-inducible manner. ChIP using primary human MDMs and analyzing IRF1 recruitment to the TNF promoter and hHS-8. Data from 3 separate donors, (*) p ≤ 0.05 and (**) p ≤ 0.01, data is represented as mean ± SEM. (E) IRF1 binding sites in hHS-8 are highly conserved in all primate species examined. Critical 5′-GAAA-3′ motifs for IRF1 binding are highlighted. (F, G) Enhanced TNF expression induced by IFN-γ priming is abrogated in IRF1-deficient murine BMDMs. Wild-type (F) and Irf1−/− (G) BMDMs were stimulated, and TNF protein levels in supernatants were measured by ELISA post LPS stimulation. Data from 3 separate experiments each with N=3, (*) p ≤ 0.05 and (***) p ≤ 0.001, data is represented as mean ± SEM. (H) IRF1 mRNA levels induced by IFN-γ are silenced by IRF1 shRNA. THP-1 cells that constitutively express lentivirally delivered shRNA targeting IRF1 or control shRNA encoding a scrambled sequence were stimulated with IFN-γ alone for 3h. Data from 3 separate experiments, (*) p ≤ 0.05, data is represented as mean ± SEM. (I) Enhanced TNF gene expression induced by IFN-γ priming is abrogated in human monocytes where IRF1 expression is silenced. THP-1 cells expressing IRF1 and control shRNA were stimulated, and TNF mRNA levels were measured (shown relative to LPS values). Data from 3 separate experiments, (*) p ≤ 0.05, data is represented as mean ± SEM.

We note that previously, through comparative analyses of TNF noncoding sequences 5′ of the TSS in the primate lineage we delineated phylogenetic footprints that matched and were predictive of important TNF regulatory elements (Leung et al., 2000; Baena et al., 2007). When we specifically focused on the hHS-8 ~50 bp IRF1 binding element and compared it to corresponding sequences in the primate lineage and to the murine sequence, we found that the core 5′-GAAA-3′ motifs were completely conserved in the primate lineage representatives down to Callithrix jacchus, the common marmoset (Fig. 2E). Furthermore, even the differences observed between the mouse and human sequences did not impact IRF1 binding to the site (Fig. S2A). Thus, the exquisite level of sequence conservation of the IRF1 phylogenetic footprint in hHS-8 strongly suggested that there is an important function related to the conservation of these specific sequences. Notably, when we examined the sequence conservation of the entire 1.3 kb hHS-8 element, we found it to be ~70% conserved between human and mouse (Fig. S2B), further supporting it having an important role in TNF gene regulation.

IRF1 is required for enhanced TNF expression in IFN-γ-primed monocytes and macrophages

IRF1 is a member of the nine member IRF family of transcription factors, which all share a cognate binding motif (Tamura et al., 2008). Although the transcription of IRF1, IRF8, and IRF9 is induced by IFN-γ treatment, IRF1 is thought to be the dominant IFN-γ-inducible IRF family member (Tamura et al., 2008). To test for a specific and non-redundant functional role of IRF1 in IFN-γ-induced enhancement of TNF expression, we examined bone marrow-derived macrophages (BMDMs) from wild-type and IRF1-deficient (Irf1−/−) mice (Fig. 2F and 2G). BMDMs from wild-type control mice responded to IFN-γ priming and secreted significantly higher levels of TNF protein after LPS stimulation as compared to cells stimulated with LPS alone (Fig. 2F and 2G). By contrast, while LPS-induced TNF protein production in Irf1−/− BMDMs was similar to protein levels in wild-type BMDMs, priming by IFN-γ pre-treatment was eliminated in the IRF1-deficient cells.

To extend this finding to human monocytic cells, we next constructed lentiviral expression vectors encoding an shRNA targeting IRF1 transcripts or a non-specific control shRNA and demonstrated that IFN-γ-induced IRF1 mRNA levels were significantly inhibited in the cells carrying the shRNA targeting IRF1 (Fig. 2H). We then tested the ability of IFN-γ priming to effect LPS-induced TNF mRNA expression in the IRF1-deficient cells and found that IFN-γ priming was abrogated, whereas LPS induction of TNF transcription was not affected (Fig. 2I). These experiments thus demonstrated that: (i) IRF1 recruitment is significant to the highly conserved hHS-8 element upon sole IFN-γ stimulation in vivo, but not to the human TNF promoter, and that (ii) IRF1 is necessary for IFN-γ priming of LPS-stimulated TNF gene expression in both murine macrophages and human monocytic cells and thus other IRF family members cannot compensate for its loss in IFN-γ priming of TNF.

hHS-8 functions as an IFN-γ-inducible, IRF1-dependent enhancer of TNF gene expression

To determine if hHS-8 could function as an IFN-γ-inducible enhancer element, we inserted the 1.3 kb hHS-8 element upstream of the TNF promoter in a luciferase reporter construct and compared its transcriptional activity to the activity of a reporter construct containing only the human TNF promoter (with sequences up to 982 bp upstream of the TNF TSS). As shown previously (Tsai et al., 2000), the human TNF promoter alone is LPS inducible (Fig. 3A). Consistent with our findings of the endogenous TNF gene (see Fig. 1), sole treatment with IFN-γ did not activate expression of the TNF promoter-reporter construct (Fig. 3A). Furthermore, IFN-γ priming did not enhance LPS-induced transcriptional activity (Fig. 3A, p=0.653). Thus, the TNF promoter alone was not sufficient to mediate IFN-γ priming of transcription. By contrast, when the 1.3 kb hHS-8 sequence was inserted upstream of the TNF promoter, we found a significant enhancement of IFN-γ-primed, LPS-stimulated reporter expression (Fig. 3A, p=0.001). Strikingly, the introduction of mutations that disrupt IRF1 binding within the context of the otherwise isogenic 1.3 kb hHS-8 element (Fig. 3B) completely abolished IFN-γ priming of LPS-driven TNF promoter activity (p=0.001), but did not impair LPS induction of the gene (Fig. 3A, p=0.222). We also note that consistent with the regulation of the endogenous gene in monocytic cells (Fig. 1A and 1D) sole treatment with IFN-γ did not activate expression of the wild-type TNF promoter + hHS-8-reporter construct (Fig. 3A). These data thus demonstrate that the TNF promoter alone is unable to drive enhanced transcription in response to IFN-γ priming, but gains this capacity when linked to the IRF1-dependent inducible hHS-8 regulatory element. This is in contrast to the IL12A promoter, which contains an IRF1 binding site and is sufficient for IFN-γ priming of LPS-driven transcriptional activation (Liu et al., 2003).

Figure 3.

Figure 3

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hHS-8 functions as an IFN-γ-inducible, IRF1-dependent enhancer of TNF gene expression. (A) Disruption of IRF1 binding to hHS-8 abolishes inducible enhancer function and thus enhanced TNF gene expression induced by IFN-γ priming. Constructs using the pGL3-Basic luciferase vector were transfected into J774 cells and stimulated with IFN-γ alone for 8h, LPS alone for 6h, and both IFN-γ and LPS (IFN-γ 2h followed by LPS for 6h). “TNF” is the TNF promoter, “hHS-8” is the entire sequence of hHS-8 (1250bp), and “muthHS-8” is hHS-8 with mutations that disrupt IRF1 binding. Data from 3 separate experiments; (***) indicates p ≤ 0.001; data is represented as mean ± SEM. (B) Nucleotide changes in the critical 5′-GAAA-3′ motifs disrupt rIRF1 binding. EMSA was performed with rIRF1 and wild-type and mutant radiolabeled P32 oligonucleotides (sequences for positions −6838 to −6785). (C) Activation of hHS-8 enhancer function corresponds with increased H3K27ac prevalence at hHS-8. ChIP analyzing H3K27ac prevalence was performed using THP-1 cells and analyzing H3K27ac prevalence at the TNF promoter and hHS-8. Data from 3 separate experiments, (*) p ≤ 0.05, data is represented as mean ± SEM. (D, E) IFN-γ + LPS induces hHS-8 eRNA transcription. hHS-8 eRNA (RNA sequence containing IRF1 binding sites) were measured post LPS stimulation in THP-1 cells (D) and primary human MDMs (representative donor, E) Data from 3 separate experiments; (*) indicates p ≤ 0.05; data are represented as mean ± SEM.

IFN-γ priming enhances H3K27 acetylation at hHS-8 upon LPS stimulation

In addition to increased chromatin accessibility, activated enhancers are associated with enrichment in H3K27ac levels (Calo and Wysocka, 2013). We thus next investigated whether H3K27ac levels were altered at hHS-8 or at the TNF promoter during IFN-γ priming. At the TNF promoter, while we saw no change in H3K27ac levels in response to IFN-γ alone, H3K27ac enrichment increased dramatically after the single LPS stimulus (Fig. 3C), and there was no further enhancement in H3K27ac levels in response to IFN-γ priming followed by LPS stimulation (Figure 3C). By contrast, solitary LPS or IFN-γ treatment of THP-1 cells did not cause an increase in H3K27ac levels at hHS-8, however H3K27ac enrichment increased significantly at hHS-8 in cells that had first been primed with IFN-γ and then stimulated with LPS (Fig. 3C). We note that the acetyltransferases CBP/p300 are inducibly recruited to the TNF promoter after LPS stimulation (Tsai et al., 2000), and that IFN-γ stimulation alone is not sufficient to induce p300 recruitment to hHS-8, which requires LPS stimulation (Fig. S3), consistent with the pattern of enhanced H3K27ac levels after dual IFN-γ and LPS treatment,

Enhancer RNA is synthesized at hHS-8 during IFN-γ priming

Since enhancer RNA (eRNA) production is associated with functional enhancer elements (Jiao and Slack, 2013), we next investigated whether hHS-8 eRNA was transcribed during IFN-γ priming and LPS stimulation of THP-1 cells and primary human MDMs. In both cell types, similar to our findings with TNF gene transcription initiated at the promoter, IFN-γ as a single stimulus did not induce transcription of hHS-8 eRNA (Fig. 3D and 3E). However, IFN-γ priming of THP-1 cells prior to LPS stimulation significantly enhanced hHS-8 eRNA synthesis as compared to stimulation by LPS alone (Fig. 3D), and a similar effect was seen in MDMs from a representative donor (Fig. 3E).

hHS-8 is required for IFN-γ augmentation of TNF gene expression in vivo

Finally, to demonstrate the functional role of hHS-8 in IFN-γ priming within the endogenous chromatin environment of the TNF/LT locus, we employed CRISPR/Cas9 technology. We used the catalytically “dead” version of codon-optimized Cas9 (dead Cas9 or dCas9) linked to the KRAB repressive domain (Gilbert et al., 2013) to specifically target the IRF1 binding element within hHS-8. We modified the lentiCRISPR lentiviral vector developed by Zhang and colleagues (Shalem et al., 2014) to encode the far-red reporter E2-Crimson, and made nucleotide changes to the Cas9 sequence to introduce the D10A and H840A mutations to generate dCas9. To enhance the targeting strategy, we incorporated two human pol III promoters into this lentivirus to drive expression of two unique guide RNAs in order to cover the entire 50 bp IRF1 binding element (CRISPR-hHS-8). As a positive control, we designed a lentivirus encoding two guide RNAs directed against the TATAA box and a core Sp1 site within the TNF core promoter (CRISPR-TNFp). As a negative control, we generated a lentivirus encoding two guide RNAs that contain at least two mismatches with any human genomic sequence and for which the closest genomic targets lack the required NGG protospacer adjacent motif (PAM) at their 3′ ends (CRISPR-Ctrl) (sequences and strategy are shown in Fig. S4 and in Table S1). After transduction of THP-1 cells with each lentivirus, we enriched for E2-Crimson+ cells by two rounds of sorting, which resulted in >95% E2-Crimson+ cells at the time of experimental analysis (Fig. 4A).

Figure 4.

Figure 4

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hHS-8 IRF1 binding sites are required for IFN-γ priming of TNF gene expression in vivo. (A) Flow cytometry data demonstrating that >95% of THP-1 cells were successfully transduced with the CRISPR-Ctrl, CRISPR-TNFp, and CRISPR-hHS-8 lentiviruses at the time of experimental analysis. (B) Targeting of the TNF promoter with dCas9-KRAB. CRISPR-Ctrl and CRISPR-TNFp THP-1 cells were mock-stimulated or stimulated with LPS for 1h and TNF and IL-6 mRNA were quantitated after normalization to the housekeeper cyclophilin B. Data from at least 3 independent experiments are shown; (***) indicates p ≤ 0.001; data are represented as mean ± SD. (C) Targeting hHS-8 with dCas9-KRAB blocks priming of TNF. CRISPR-Ctrl and CRISPR-hHS-8 THP-1 cells were mock-stimulated, stimulated with LPS for 1h, or stimulated with IFN-γ for 2h and LPS for 1h. For analysis of TNF expression, data are presented as fold inductions over unstimulated TNF mRNA levels to control for baseline constitutive TNF transcription in THP-1 cells, while for analysis of IL6 expression data are presented as fold induction of primed versus non-primed conditions due to the absence of detectable IL-6 transcripts in the absence of stimulation. Data from 3 independent experiments are presented; (**) indicates p<0.01; data are represented as mean ± SD

Consistent with the requirement of the TATAA box and the core TNF promoter Sp1 site for LPS-driven TNF transcription (Goldfeld et al., 1990; Falvo et al., 2000), LPS-stimulated TNF gene expression was ablated (>98%) in the CRISPR-TNFp cells as compared to the CRISPR-Ctrl cells (Fig. 4B, left panel). Furthermore, we showed that TNF transcriptional repression was specific to the TNF gene, since induction of IL-6 mRNA synthesis by LPS was highly inducible in both the CRISPR-Ctrl cells and CRISPR-TNFp cells (Fig. 4B, right panel).

We then examined the impact of targeting the hHS-8 IRF1 binding element with dCas9-KRAB upon IFN-γ priming of LPS-stimulated TNF gene transcription. As shown in the left panel of Figure 4C, there was no difference between the CRISPR-Ctrl and the CRISPR-hHS-8 cells in their transcriptional response to LPS, and the gene was highly inducible in both, indicating that the dCas9-KRAB fusion did not have a general repressive effect upon TNF activation in a stimulus-independent manner. However, when the CRISPR-hHS-8 cells were primed with IFN-γ prior to LPS stimulation, augmentation of TNF gene expression was abolished, while it proceeded normally in the CRISPR-Ctrl cells (Fig. 4C, left panel). As a control for specificity, we also examined IFN-γ priming of the endogenous IL6 gene in the CRISPR-hHS-8 cells and found that IFN-γ priming of IL6 gene transcription was not affected in the CRISPR-hHS-8 cells (Fig. 4, right panel), indicating that loss of IFN-γ priming at the TNF/LT locus was specific, and not due to off-target effects of the hHS-8 guide RNAs. These findings provide the fundamental functional demonstration that hHS-8 is required for IFN-γ priming of LPS-induced TNF gene expression in vivo.

DISCUSSION

We have demonstrated that IFN-γ priming of TNF requires an exquisitely conserved distal regulatory element, hHS-8, which lies ~8 kb upstream of the TNF transcription start site. Upon exposure to IFN-γ, hHS-8 becomes more accessible to DNase I and IRF1 is recruited. Once the LPS signal occurs, levels of H3K27ac are enriched, and eRNA is transcribed, which corresponds to augmented TNF gene expression. Ablation of IRF1 in murine macrophages or human monocytic cells, or the targeting of the endogenous IRF1 binding element in hHS-8 with a dCas9-KRAB fusion protein in human monocytic cells, all abolish IFN-γ priming of LPS-stimulated TNF transcription. Thus, a combination of IFN-γ-induced chromatin accessibility and IRF1 binding at the distal hHS-8 enhancer poise the TNF/LT locus for augmented TNF gene expression in response to the TLR signal. These experiments thus provide the fundamental functional demonstration that a distal regulatory element enhances expression of a specific gene during classical macrophage activation.

A major question regarding the epigenetic and transcriptional mechanisms underlying IFN-γ priming at the TNF/LT locus is whether similar mechanisms are involved in the priming of other inflammatory genes, or whether they are unique to TNF. By contrast to our findings of the necessity for a distal enhancer element in priming of TNF, it has previously been shown that the IL12A promoter is sufficient for IFN-γ priming of LPS-induced IL12A gene expression (Liu et al., 2003), and the IL6 promoter is also sufficient for IFN-γ priming of IL6 in response to stimulation by TNF (Sanceau et al., 1995).

A recent study reported that IFN-γ + M-CSF treatment of primary human monocytes for 24 hours led to increased histone acetylation and STAT1 recruitment at the promoters and distal sites upstream of the IL6, IL12B, and TNF genes as compared to M-CSF treatment alone, and also reported that subsequent LPS activation after IFN-γ + M-CSF pre-treatment led to enhanced H3K27ac enrichment at the promoters and distal sites upstream of the IL6, IL12B, and TNF genes. Synthesis of eRNA at the IL6 and IL12 upstream sequences was also reported; the regions upstream of the TNF gene were not examined for eRNA production, IRF1 binding, or enhancer function (Qiao et al., 2013). The authors mined the ENCODE database (http://genome.usc.edu) and found that DNase I hypersensitive sites identified in CD14+ human monocytes corresponded to the general upstream regions where increased histone acetylation and STAT1 recruitment was detected by ChIP-Seq, leading them to conclude that these distal noncoding elements are involved in IFN-γ augmentation of LPS-induced gene expression, although no functional analyses were shown that link epigenetic modifications and changes in transcription factor recruitment to gene regulation (Qiao et al., 2013). Indeed, only marginal (and non-significant) enhancement of SV40 promoter-driven expression by upstream sequences from the IL6 and IL12B loci in reporter assays in response to IFN-γ + LPS activation was observed.

By contrast, we saw enhanced H3K27 acetylation at hHS-8 only after IFN-γ + LPS stimulation, not after IFN-γ treatment alone. Furthermore, we observed that hHS-8 dramatically and significantly enhanced TNF promoter-driven reporter expression in response to IFN-γ + LPS versus LPS alone, and conferred priming capacity on the otherwise ‘non-primeable’ TNF promoter (Fig 3A). Moreover, disruption of the IRF1 binding element in hHS-8 abolished the ability of this 1.3 kb enhancer to augment TNF promoter-driven reporter expression. Finally, precise targeting of dCas9-KRAB to the hHS-8 IRF1 binding element within the endogenous chromatin environment inhibited IFN-γ priming of LPS-induced TNF gene expression, clearly demonstrating the importance of this upstream region for priming of this critical inflammatory gene.

Thus, whereas the TNF hHS-8 functions as an essential priming enhancer element, the sites upstream of IL6 and IL12B have no demonstrated functional role in the regulation of these genes to date. It will be of interest to determine in future studies whether the regions upstream of the IL6 and IL12B genes identified by Qiao et al play a role in IFN-γ priming of LPS-induced gene expression at these loci, or whether the epigenetic and other changes seen at these sites in response to IFN-γ stimulation are bystander marks of a localized, “primed” chromatin environment.

Our data suggests the possibility that IRF1 may function at hHS-8 as a “pioneer factor” (Zaret et al., 2011) for enhanced TNF gene expression in primed monocytes and macrophages. In this scenario, IRF1 binding to hHS-8 would promote increased DNase I accessibility at hHS-8 and the recruitment of chromatin-remodeling complexes and additional factors that poise this element for rapid activation in response to the LPS signal. Indeed, several studies have identified a class of enhancers, termed ‘poised enhancers’, that are linked to inactive genes and are distinguished by the absence of H3K27ac (Zentner et al., 2011; Creyghton et al., 2010; Rada-Iglesias et al., 2011; Cotney et al., 2012). When activated, these poised enhancers become enriched in H3K27ac (Rada-Iglesias et al., 2011). This is reminiscent of what we observed at hHS-8 upon LPS stimulation of IFN-γ primed cells. We note that IRF1 has previously been shown to recruit CBP/p300 (Marsili et al., 2004) and PCAF (Masumi et al., 1999). We imagine that, upon IFN-γ stimulation of monocytes/macrophages, there is an increase in the percentage of cells in the sample population in which hHS-8 is ‘open’ and associated with IRF1, resembling a poised enhancer ready for activation. Indeed, IRF1 could function as a beacon for enhanced recruitment of CBP/p300 to hHS-8 following IFN-γ + LPS stimulation, leading to H3K27 acetylation and the commissioning of hHS-8 as an active enhancer to augment transcription of TNF.

Finally, by demonstrating that specific targeting of dCas9-KRAB to the IRF1 binding element in hHS-8 within its endogenous chromatin environment abrogates IFN-γ augmentation of LPS-induced TNF transcription in human monocytic cells, we have confirmed that hHS-8 is required for priming of this critical early response gene during classical macrophage activation. Our data also suggest that applying dCas9-KRAB technology to the functional interrogation of global data sets like ENCODE would be of particular value. Furthermore, probing the function of distal elements linked to specific genes can achieve a fundamental understanding of the role of long-range interactions in control of cell type- and/or stimulus-specific gene expression.

EXPERIMENTAL PROCEDURES

Cell culture and stimulations

THP-1 cells were maintained in RPMI-1640 supplemented with 10% FBS. J774 cells were maintained in DMEM supplemented with 10% FBS. For primary human MDMs, enriched populations of human monocytes were isolated from healthy human donor buffy coats using a CD14+ positive selection kit (Stemcell). MDMs were obtained after 6d of culture in RPMI-1640 medium supplemented with 5% human serum AB (Gemcell) and GM-CSF (50ng/ml; Peprotech). Cells were treated with IFN-γ (100ng/ml; R&D) and LPS (100ng/ml; Sigma E. coli O111:B4).

RNA extraction and quantitative real-time RT-PCR

Total RNA was extracted from cells with a QuickRNA Mini kit (Zymo) and treated with Turbo DNA-free kit (Invitrogen). cDNA was synthesized from total RNA with M-MLV reverse transcriptase (Invitrogen) and 20-residue oligo (dT) (Invitrogen). eRNA was synthesized from total RNA with hHS-8 eRNA reverse primer. TNF mRNA levels were measured by the change-in-threshold (ΔΔCt) method based on quantitative real-time PCR in an iCycler iQ (Bio-rad) with SyberGreen Master Mix (Invitrogen) and primers recognizing exon 4 and exon 3 of the human TNF gene, the human GAPDH gene, and the human IRF1 gene. Primers used for ChIP and cDNA measurements by quantitative real-time PCR are shown in Table S1.

DNase I Hypersensitivity Assay (DHA)

DHAs were performed using both THP-1 cells and primary human MDMs. MDMs were detached from culture surface using TrypLE™ (Life Technologies). Cells were harvested, washed with PBS, and resuspended in RSB buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, and 3mM MgCl2). Cells were lysed with lysis buffer (0.5% NP-40 in 1x RSB buffer) on ice for 5min. Resuspended nuclei in RSB buffer were treated with DNase I (40ng/ul) at 37C for 5min. DNase I activity was quenched upon addition of stop solution (0.6M NaCl, 20mM Tris-HCl pH 8.0, 10mM EDTA, and 1% SDS). Samples were treated with Proteinase K at 56C O/N. DNA was digested with ScaI and BamHI restriction enzymes and analyzed by Southern blotting using a radiolabeled P32 probe corresponding to the coding region of LTA. 10ug of DNA was used for each lane.

Chromatin immunoprecipitation (ChIP)

ChIP assays were performed with anti-IRF1 (H-205, Santa Cruz), Rb IgG (Diagenode), anti-H3K27me3 (C36B11, Cell Signaling), anti-H3K27ac (Active Motif), and anti-H3 (Abcam). THP-1 cells and primary human MDMs were treated (IFN-γ 100ng/ml, LPS 100ng/ml), fixed with 10% formaldehyde for 15min, treated with 2.5M glycine for 5min, harvested, washed with PBS, lysed with 0.25% Triton X-100 and 0.5% NP-40 for 5min, centrifuged at 1200rpm for 10min, resuspended in 1% SDS lysis buffer, and sonicated 5min for 4 cycles in a Biorupter. Sonicated DNA was set up for immunoprecipitation O/N and DNA-protein complexes were recovered by adding Protein A/G Plus Agarose Beads (Thermo Scientific) for 3h. Samples were washed 6 times with 1ml of wash buffer and treated with proteinase K at 65C O/N. Samples were treated with phenol/chloroform before O/N ethanol precipitation. DNA fragments for IRF1 recruitment were analyzed by quantitative real-time PCR with Sybergreen master mix (Invitrogen) and primer sets for regions −244 to −82 (promoter) relative to the TNF TSS and −6842 to −6737 (hHS-8) relative to the TNF TSS (Table S1). Rb IgG percent input values were subtracted from IRF1 percent input values. DNA fragments for H3K27me3 and H3K27ac analysis were analyzed by quantitative real-time PCR using Jumpstart™ Taq ReadyMix™ For Quantitative PCR (Sigma) and primer/probe sets for the TNF promoter and hHS-8 (Table 1). H3K27me3 and H3K27ac percent input values were normalized to H3 percent input values.

DNase I Footprinting Assay

Radiolabeled P32 fragments of the TNF promoter (−200 to +1) and hHS-8 (−7031 to −6782) regions were incubated with recombinant IRF1 protein (Abcam) and treated with diluted DNase I at RT for 5s before quenching enzyme activity with stop solution (0.13mM EDTA, 0.5% SDS, and tRNA). Samples were treated with phenol chloroform and DNA was precipitated O/N at −20C. G/A ladder was treated with 4% formic acid, radiolabeled with P32, treated with 1M piperdine, and precipitated with n-butanol. DNA fragments were separated by electrophoresis on an 8% sequencing gel.

Mice

6–8 week old C57BL/6J mice and B6.129S2-Irf1tm1Mak/J mice were purchased from Jackson Laboratories. Experimental procedures were done in accordance with the Institutional Animal Care and Use Committee (IACUC) and the Harvard Medical Area Standing Committee on Animals (HMA IACUC).

Isolation, culture, and stimulation of murine BMDMs

For the generation of murine bone marrow-derived macrophages (BMDMs), bone marrow cells of wild-type or Irf1−/− mice (6–8wks) were cultured in DMEM medium supplemented with 10% FBS, 10% L929 cell conditioned medium (LCCM), 100U/ml penicillin, 100ug/ml streptomycin, and 2mM L-glutamine. Cells were fed on day 5 and media was changed on day 7, 3h before mIFN-γ (100ng/ml; R&D) and LPS (100ng/ml; Sigma E. coli O111:B4) treatment. Supernatant was collected 2, 4, and 6h after treatment.

Short hairpin RNA

The lentiviral plasmid pLKO.1 expressing shRNA targeting human IRF1 was purchased from the RNAi Consortium (TRC) Lentiviral shRNA library (Thermo Scientific). Clone TRCN0000014668 with a target sequence of 5′-CGTGTGGATCTTGCCACATTT-3′ was validated in our laboratory. Control shRNA encodes a scrambled sequence. Lentiviruses encoding shRNA sequences were generated by transfecting the packaging cell line HEK-293T with the shRNA-encoding pLKO.1 plasmids in combination with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G using Effectene transfection reagent (Qiagen). Supernatants were collected 48h post-transfection, clarified by centrifugation, and stored at −80C. THP-1 cells were transduced with the lentiviral particles by culturing the cells with supernatants from the virus-producing cells in the presence of 8ug/ml polybrene (Millipore) and spinoculation for 2h at 2000 RPM. Successfully transduced cells were selected and expanded by treatment with 0.8ug/ml puromycin.

Electrophoretic Mobility Shift Assay (EMSA)

Radiolabeled 32P oligonucleotides were added to THP-1 nuclear extracts or recombinant IRF1 protein (Abcam) in a binding buffer solution (10mM Tris-HCl pH 7.5, 53mM NaCl, 1mM DTT, 0.01% Nonidet-P40, 5% glycerol, and 0.05ug/ul of double-stranded poly(dI-dC)) at RT for 30min. In super-shift experiments, samples were incubated with 2ug of anti-IRF1 (H-205, Santa Cruz Biotechnology). Protein-DNA complexes were separated by electrophoresis on a 5% PAAG gel.

Sequencing

Procurement of cell lines and samples of blood or DNA from representative individuals of the primate species and subspecies was previously described (Leung et al., 2000; Baena et al., 2007). Genomic DNA was isolated using QIAamp DNA Blood kit (Qiagen). Sequence alignments were performed using ClustalW2 multiple sequence alignment provided by EMBL-EBI.

Plasmids

Construction of the TNF promoter-driven luciferase reporter was previously described (Tsai et al., 2000). The TNF promoter with hHS-8 plasmid was constructed by inserting nucleotides −7833 to −6583 relative to the TNF TSS into the TNF promoter-driven luciferase reporter construct using MluI and NheI restriction enzyme sites. The TNF promoter with mutated hHS-8 plasmid was constructed by circular site-directed mutagenesis.

Luciferase Reporter Assay

J774 cells were transfected with luciferase reporter constructs using an Effectene Transfection Reagent kit (Qiagen). Cells were treated with mIFN-γ (100ng/ml; R&D) and LPS (100ng/ml, Sigma E. coli O111:B4). Luciferase assays were performed 8h after treatment under the Dual Luciferase Reporter Assay System (Promega) using a Dynex luminometer and Renilla luciferase (pRL-TK) as a control.

CRISPR/dCas9 analysis

The plasmids pCas9_GFP (Addgene plasmid 44719; deposited by Kiran Musunuru) and LentiCRISPR (Addgene plasmid 49535; deposited by Feng Zhang) were obtained from Addgene. D10A and H840A substitutions were introduced into the Cas9 coding region of pCas9_GFP by overlapping PCR in order to generate the catalytically inactive dCas9 as described previously (Qi et al., 2013). The KRAB coding sequence was ordered as a gBlock fragment from Integrated DNA Technologies and cloned in-frame at the 3′ end of the dCas9 coding sequence. After the E2-Crimson coding sequence (Clontech), preceded by the P2A self-cleaving peptide DNA sequence, was substituted for the 2A-puromycin resistance gene in LentiCRISPR, the dCas9-KRAB sequence was amplified and substituted for Cas9 in LentiCRISPR upstream and in-frame with the 2A-E2-Crimson sequence. For guide RNA generation, we first cloned a cassette containing the tracr RNA sequence from LentiCRISPR followed by a TTTTTTT termination signal and the 98 bp H1 promoter sequence into plasmid pSP73 (Promega). This plasmid was used as template with the primers shown in Table S1 to create individual PCR products consisting of BsmBI site-20bp target#1-tracr-term-H1pro-20bp target#2-BsmBI site that could then be cloned into the BsmBI sites of the modified lentiCRISPR vector, placing this cassette after the U6 promoter and before the tracr-term sequence already present in the vector. The sequences in the TNF promoter and hHS-8 that were targeted with this dual guide RNA vector system are shown in Figure S6. For the control lentivirus, the 20 bp targets 5′gttcgtgtcgtcgtgtctta-3′ and 5′gaatctagcggtctgacatt-3′ were used, as these sequences have at least two mismatches with any 20 bp sequence in the human genome, and the closest matches in the human genome do not possess the 5′-NGG-3′ PAM sequence required for full Cas9 binding.

The CRISPR/dCas9-KRAB lentiviruses were prepared and THP-1 cells were transduced as described above for the shRNA lentiviruses, except that virus-containing medium was centrifuged over 20% sucrose at 11,500 RPM for 4h to increase the lentiviral concentration prior to spinoculation. After expansion of transduced cells, E2-Crimson+ cells were enriched by FACS at two timepoints over the course of 3 weeks. For experimental analysis, cells were seeded at 5 × 105 cells/ml and stimulated with IFN-γ and LPS, and RNA isolation, cDNA synthesis and qPCR were performed as described above using primer sets for cyclophilin B, TNF, and IL-6 as shown in Table S1.

Supplementary Material

1

Figure S1, related to Figure 1B, E. Presence of hHS-8 in primary human monocytes. DNase I hypersensitive sites in primary human cells, arrow marks hHS-8. ENCODE database http://genome.usc.edu.

Figure S2, related to Figure 2E. (A) rIRF1 binds to hHS-8 in both the human and mouse Tnf/Lt locus. hHS-8 in the mouse is called murine hypersensitive site -9 (mHS-9) because it is located in the murine genome ~9 kb upstream of the TNF TSS. EMSA was performed with rIRF1 and human and mouse radiolabeled 32P oligonucleotides of sequences for positions − 6838 to −6785. (B) hHS-8 is highly conserved between human and mouse. Alignment of human and mouse sequences of hHS-8 (~1250 kb).

Figure S3, related to Figure 3C. p300 is recruited to hHS-8 upon LPS stimulation. ChIP was performed using primary human MDMs and analyzing p300 recruitment to hHS-8. Data from 3 separate donors, (*) p ≤ 0.05 and (**) p ≤ 0.01, data is represented as mean ± SEM.

Figure S4, related to Figure 4. Sites targeted by guide RNAs encoded by the CRISPR-TNFp and CRISPR-hHS-8 lentiviruses. 20 bp targets are indicated by arrows and the protospacer adjacent motif (PAM) immediately following each target is also shown. The TATAA box in the TNF promoter and IRF1 binding element in hHS-8 are highlighted in blue and the TSS of TNF is shown in red.

2. Table S1, related to Experimental Procedures.

Primers and probes used for cloning, mRNA and eRNA measurements, ChIP, and CRISPR/Cas9 studies.

Acknowledgments

This work was supported by grants from GlaxoSmithKline and the NIH/NIGMS (GM076685) to A.E.G. We are indebted to Laurens Kruidenier and David Tough for stimulating discussions and experimental suggestions. We thank Alla Tsytsykova and Shahin Ranjbar for critical experimental advice and discussions. We also thank James Falvo for helpful discussions.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Figure S1, related to Figure 1B, E. Presence of hHS-8 in primary human monocytes. DNase I hypersensitive sites in primary human cells, arrow marks hHS-8. ENCODE database http://genome.usc.edu.

Figure S2, related to Figure 2E. (A) rIRF1 binds to hHS-8 in both the human and mouse Tnf/Lt locus. hHS-8 in the mouse is called murine hypersensitive site -9 (mHS-9) because it is located in the murine genome ~9 kb upstream of the TNF TSS. EMSA was performed with rIRF1 and human and mouse radiolabeled 32P oligonucleotides of sequences for positions − 6838 to −6785. (B) hHS-8 is highly conserved between human and mouse. Alignment of human and mouse sequences of hHS-8 (~1250 kb).

Figure S3, related to Figure 3C. p300 is recruited to hHS-8 upon LPS stimulation. ChIP was performed using primary human MDMs and analyzing p300 recruitment to hHS-8. Data from 3 separate donors, (*) p ≤ 0.05 and (**) p ≤ 0.01, data is represented as mean ± SEM.

Figure S4, related to Figure 4. Sites targeted by guide RNAs encoded by the CRISPR-TNFp and CRISPR-hHS-8 lentiviruses. 20 bp targets are indicated by arrows and the protospacer adjacent motif (PAM) immediately following each target is also shown. The TATAA box in the TNF promoter and IRF1 binding element in hHS-8 are highlighted in blue and the TSS of TNF is shown in red.

NIHMS642189-supplement-1.docx (56.9KB, docx)
2. Table S1, related to Experimental Procedures.

Primers and probes used for cloning, mRNA and eRNA measurements, ChIP, and CRISPR/Cas9 studies.

NIHMS642189-supplement-2.pdf (4.8MB, pdf)

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