TGF-ß and retinoic acid induce the microRNA miR-10a, which targets Bcl-6 and constrains the plasticity of helper T cells.

Distinct CD4(+) T cell subsets are critical for host defense and immunoregulation. Although these subsets can act as terminally differentiated lineages, they have been increasingly noted to demonstrated plasticity. MicroRNAs are factors that control T cell stability and plasticity. Here we report that naturally occurring regulatory T cells (T(reg) cells) had high expression of the microRNA miR-10a and that miR-10a was induced by retinoic acid and transforming growth factor-ß (TGF-ß) in inducible T(reg) cells. By simultaneously targeting the transcriptional repressor Bcl-6 and the corepressor Ncor2, miR-10a attenuated the phenotypic conversion of inducible T(reg) cells into follicular helper T cells. We also found that miR-10a limited differentiation into the T(H)17 subset of helper T cells and therefore represents a factor that can fine-tune the plasticity and fate of helper T cells.

WHSC1 links transcription elongation to HIRA-mediated histone H3.3 deposition.

Actively transcribed genes are enriched with the histone variant H3.3. Although H3.3 deposition has been linked to transcription, mechanisms controlling this process remain elusive. We investigated the role of the histone methyltransferase Wolf-Hirschhorn syndrome candidate 1 (WHSC1) (NSD2/MMSET) in H3.3 deposition into interferon (IFN) response genes. IFN treatment triggered robust H3.3 incorporation into activated genes, which continued even after cessation of transcription. Likewise, UV radiation caused H3.3 deposition in UV-activated genes. However, in Whsc1(-/-) cells IFN- or UV-triggered H3.3 deposition was absent, along with a marked reduction in IFN- or UV-induced transcription. We found that WHSC1 interacted with the bromodomain protein 4 (BRD4) and the positive transcription elongation factor b (P-TEFb) and facilitated transcriptional elongation. WHSC1 also associated with HIRA, the H3.3-specific histone chaperone, independent of BRD4 and P-TEFb. WHSC1 and HIRA co-occupied IFN-stimulated genes and supported prolonged H3.3 incorporation, leaving a lasting transcriptional mark. Our results reveal a previously unrecognized role of WHSC1, which links transcriptional elongation and H3.3 deposition into activated genes through two molecularly distinct pathways.

The small ubiquitin-like modifier-deconjugating enzyme sentrin-specific peptidase 1 switches IFN regulatory factor 8 from a repressor to an activator during macrophage activation.

Macrophages, when activated by IFN-γ and TLR signaling, elicit innate immune responses. IFN regulatory factor 8 (IRF8) is a transcription factor that facilitates macrophage activation and innate immunity. We show that, in resting macrophages, some IRF8 is conjugated to small ubiquitin-like modifiers (SUMO) 2/3 through the lysine residue 310. SUMO3-conjugated IRF8 failed to induce IL12p40 and other IRF8 target genes, consistent with SUMO-mediated transcriptional repression reported for other transcription factors. SUMO3-conjugated IRF8 showed reduced mobility in live nuclei and bound poorly to the IL12p40 gene. However, macrophage activation caused a sharp reduction in the amount of SUMOylated IRF8. This reduction coincided with the induction of a deSUMOylating enzyme, sentrin-specific peptidase 1 (SENP1), in activated macrophages. In transfection analysis, SENP1 removed SUMO3 from IRF8 and enhanced expression of IL12p40 and other target genes. Conversely, SENP1 knockdown repressed IRF8 target gene expression. In parallel with IRF8 deSUMOylation, macrophage activation led to the induction of proteins active in the SUMO pathway and caused a global shift in nuclear protein SUMOylation patterns. Together, the IRF8 SUMO conjugation/deconjugation switch is part of a larger transition in SUMO modifications that takes place upon macrophage activation, serving as a mechanism to trigger innate immune responses.

Gamma interferon triggers interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase HDAC3 to the interferon-responsive element.

ICSBP (IRF-8) is a transcription factor of the IRF family expressed only in the immune system. It is induced in macrophages by gamma interferon (IFN-gamma) and contributes to macrophage functions. By interacting with Ets family protein PU.1, ICSBP binds to the IRF/Ets composite element and stimulates transcription. ICSBP binds to another DNA element, the IFN-stimulated response element (ISRE), a common target of the IRF family. Limited knowledge as to how ICSBP and other IRF proteins regulate ISRE-dependent transcription in IFN-gamma-activated macrophages is available. By mass-spectrometric analysis of ISRE-bound proteins in macrophages, we identified TEL, another Ets member, as a factor recruited to the element in an IFN-gamma-dependent manner. In vitro analysis with recombinant proteins indicated that this recruitment is due to a direct interaction between ICSBP and TEL, which is enhanced by the presence of ISRE. Significantly, the interaction with TEL in turn resulted in the recruitment of the histone deacetytase HDAC3 to the ISRE, causing increased repression of IFN-gamma-mediated reporter activity through the ISRE. This repression may provide a negative-feedback mechanism operating after the initial transcriptional activation by IFN-gamma. By associating with two different Ets family proteins, ICSBP exerts a dual function in IFN-gamma-dependent gene regulation in an immune system-specific manner.

Interaction of histone acetylases and deacetylases in vivo.

Having opposing enzymatic activities, histone acetylases (HATs) and deacetylases affect chromatin and regulate transcription. The activities of the two enzymes are thought to be balanced in the cell by an unknown mechanism that may involve their direct interaction. Using fluorescence resonance energy transfer analysis, we demonstrated that the acetylase PCAF and histone deacetylase 1 (HDAC1) are in close spatial proximity in living cells, compatible with their physical interaction. In agreement, coimmunoprecipitation assays demonstrated that endogenous HDACs are associated with PCAF and another acetylase, GCN5, in HeLa cells. We found by glycerol gradient sedimentation analysis that HATs are integrated into a large multiprotein HDAC complex that is distinct from the previously described HDAC complexes containing mSin3A, Mi-2/NRD, or CoREST. This HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed in vitro. The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes in vivo.

BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones.

Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb and has been proposed to release RNA polymerase II (Pol II) from promoter-proximal pausing. We show that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function. BRD4 interacts with elongating Pol II complexes and assists Pol II in progression through hyperacetylated nucleosomes by interacting with acetylated histones via bromodomains. On active enhancers, the BET inhibitor JQ1 antagonizes BRD4-associated eRNA synthesis. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by facilitating transcript elongation both at enhancers and on gene bodies independently of P-TEFb.

Inducible deposition of the histone variant H3.3 in interferon-stimulated genes.

The H3.3 histone variant is synthesized throughout cell cycle and deposited onto chromatin in a replication-independent manner. It is enriched in transcriptionally active regions of chromatin and is implicated in epigenetic memory. The dynamics of H3.3 deposition during transcriptional activation, however, have not been fully studied so far. Here we examined H3.3 incorporation into interferon (IFN)-stimulated genes in confluent mouse NIH3T3 cells expressing H3.3 fused to the yellow fluorescent protein (YFP). Following IFN stimulation, H3.3-YFP was rapidly incorporated into all four IFN-activated genes tested, with the highest enrichment seen in the distal end of the coding region. Surprisingly, H3.3 enrichment in the coding region continued for an extended period of time, long after transcription ceased. The promoter region, although constitutively enriched with H3.3-YFP, did not show an increase in its deposition in response to IFN stimulation. Further, although H3.3-YFP deposition stably remained in non-dividing cells for days after IFN stimulation, it was rapidly diminished in dividing cells. Lastly, we examined the role of H3.3 in IFN-stimulated transcription by a short hairpin RNA approach and found that IFN-stimulated transcription was significantly impaired in H3.3 knockdown cells. Results indicate that H3.3 plays a role in IFN-mediated transcription, and its deposition leaves a prolonged post-transcriptional mark in these genes.

Selective recognition of acetylated histones by bromodomain proteins visualized in living cells.

Acetylation and other modifications on histones comprise histone codes that govern transcriptional regulatory processes in chromatin. Yet little is known how different histone codes are translated and put into action. Using fluorescence resonance energy transfer, we show that bromodomain-containing proteins recognize different patterns of acetylated histones in intact nuclei of living cells. The bromodomain protein Brd2 selectively interacted with acetylated lysine 12 on histone H4, whereas TAF(II)250 and PCAF recognized H3 and other acetylated histones, indicating fine specificity of histone recognition by different bromodomains. This hierarchy of interactions was also seen in direct peptide binding assays. Interaction with acetylated histone was essential for Brd2 to amplify transcription. Moreover association of Brd2, but not other bromodomain proteins, with acetylated chromatin persisted on chromosomes during mitosis. Thus the recognition of histone acetylation code by bromodomains is selective, is involved in transcription, and potentially conveys transcriptional memory across cell divisions.