Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes.

Studies in yeast demonstrate that signaling kinases have a surprisingly active role in the nucleus, where they tether to chromatin and modulate gene expression programs. Despite these seminal studies, the nuclear mechanism of how signaling kinases control transcription of mammalian genes is in its infancy. Here, we provide evidence for a hitherto unknown function of protein kinase C-theta (PKC-θ), which physically associates with the regulatory regions of inducible immune response genes in human T cells. Chromatin-anchored PKC-θ forms an active nuclear complex by interacting with RNA polymerase II, the histone kinase MSK-1, and the adaptor molecule 14-3-3ζ. ChIP-on-chip reveals that PKC-θ binds to promoters and transcribed regions of genes, as well as to microRNA promoters that are crucial for cytokine regulation. Our results provide a molecular explanation for the role of PKC-θ not only in normal T cell function, but also in circumstances of its ectopic expression in cancer.

Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4(+) T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells.

The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.

Follicular helper T (Tfh) cells provide selection signals to germinal center B cells, which is essential for long-lived antibody responses. High CXCR5 and low CCR7 expression facilitates their homing to B cell follicles and distinguishes them from T helper 1 (Th1), Th2, and Th17 cells. Here, we showed that Bcl-6 directs Tfh cell differentiation: Bcl-6-deficient T cells failed to develop into Tfh cells and could not sustain germinal center responses, whereas forced expression of Bcl-6 in CD4(+) T cells promoted expression of the hallmark Tfh cell molecules CXCR5, CXCR4, and PD-1. Bcl-6 bound to the promoters of the Th1 and Th17 cell transcriptional regulators T-bet and RORgammat and repressed IFN-gamma and IL-17 production. Bcl-6 also repressed expression of many microRNAs (miRNAs) predicted to control the Tfh cell signature, including miR-17-92, which repressed CXCR5 expression. Thus, Bcl-6 positively directs Tfh cell differentiation, through combined repression of miRNAs and transcription factors.

The microRNA-9/B-lymphocyte-induced maturation protein-1/IL-2 axis is differentially regulated in progressive HIV infection.

The fine control of T-cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B-lymphocyte-induced maturation protein-1 (Blimp-1/Prdm1) is highly expressed in CD4(+) T cells from chronically HIV-infected (CHI) patients compared to cells from long-term nonprogressors or healthy controls. Stimulation through the T-cell receptor in the presence of IL-2 induces Blimp-1 protein expression. We show here that Blimp-1 levels are translationally regulated by microRNA-9 (miR-9). Overexpression of miR-9 induces Blimp-1 repression, restoring IL-2 secretion in CD4(+) T cells via reduction in the binding of Blimp-1 to the il-2 promoter. In CHI patients where IL-2 expression is reduced and there is generalized T-cell dysfunction, we show differential expression of both miR-9 and Blimp-1 in CD4(+) cells compared with levels in long-term nonprogressors. These data identify a novel miR-9/Blimp-1/IL-2 axis that is dysregulated in progressive HIV infection.

Interplay between chromatin remodeling and epigenetic changes during lineage-specific commitment to granzyme B expression.

The role of chromatin remodeling and histone posttranslational modifications and how they are integrated to control gene expression during the acquisition of cell-specific functions is poorly understood. We show here that following in vitro activation of CD4(+) and CD8(+) T lymphocytes, both cell types show rapid histone H3 loss at the granzyme B (gzmB) proximal promoter region. However, despite the gzmB proximal promoter being remodeled in both T cell subsets, only CD8(+) T cells express high levels of gzmB and display a distinct pattern of key epigenetic marks, notably differential H3 acetylation and methylation. These data suggest that for high levels of transcription to occur a distinct set of histone modifications needs to be established in addition to histone loss at the proximal promoter of gzmB.

Chromatinized Protein Kinase C-θ: Can It Escape the Clutches of NF-κB?

We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer.

The endoglycosidase heparanase enters the nucleus of T lymphocytes and modulates H3 methylation at actively transcribed genes via the interplay with key chromatin modifying enzymes.

The methylation of histones is a fundamental epigenetic process regulating gene expression programs in mammalian cells. Dysregulated patterns of histone methylation are directly implicated in malignant transformation. Here, we report the unexpected finding that the invasive extracellular matrix degrading endoglycosidase heparanase enters the nucleus of activated human T lymphocytes and regulates the transcription of a cohort of inducible immune response genes by controlling histone H3 methylation patterns. It was found that nuclear heparanase preferentially associates with euchromatin. Genome-wide ChIP-on-chip analyses showed that heparanase is recruited to both the promoter and transcribed regions of a distinct cohort of transcriptionally active genes. Knockdown and overexpression of the heparanase gene also showed that chromatin-bound heparanase is a prerequisite for the transcription of a subset of inducible immune response genes in activated T cells. Furthermore, the actions of heparanase seem to influence gene transcription by associating with the demethylase LSD1, preventing recruitment of the methylase MLL and thereby modifying histone H3 methylation patterns. These data indicate that heparanase belongs to an emerging class of proteins that play an important role in regulating transcription in addition to their well-recognized extra-nuclear functions.

Duplicity of protein kinase C-θ: Novel insights into human T-cell biology.

We recently reported on a new wrinkle of complexity in how eukaryotic genes are regulated by providing evidence for a hitherto unknown nuclear function of the signaling kinase, Protein Kinase C-theta (PKC-θ). This chromatin-anchored complex positively regulates inducible immune genes and negatively regulates target miRNA genes. These data challenge the traditional view of mammalian signaling kinases and provides new avenues for therapeutic drug design.

Dynamic histone variant exchange accompanies gene induction in T cells.

Changes in chromatin composition are often a prerequisite for gene induction. Nonallelic histone variants have recently emerged as key players in transcriptional control and chromatin modulation. While the changes in chromatin accessibility and histone posttranslational modification (PTM) distribution that accompany gene induction are well documented, the dynamics of histone variant exchange that parallel these events are still poorly defined. In this study, we have examined the changes in histone variant distribution that accompany activation of the inducible CD69 and heparanase genes in T cells. We demonstrate that the chromatin accessibility increases that accompany the induction of both of these genes are not associated with nucleosome loss but instead are paralleled by changes in histone variant distribution. Specifically, induction of these genes was paralleled by depletion of the H2A.Z histone variant and concomitant deposition of H3.3. Furthermore, H3.3 deposition was accompanied by changes in PTM patterns consistent with H3.3 enriching or depleting different PTMs upon incorporation into chromatin. Nevertheless, we present evidence that these H3.3-borne PTMs can be negated by recruited enzymatic activities. From these observations, we propose that H3.3 deposition may both facilitate chromatin accessibility increases by destabilizing nucleosomes and compete with recruited histone modifiers to alter PTM patterns upon gene induction.

Regulation of mouse Heparanase gene expression in T lymphocytes and tumor cells.

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate (HS) and plays an important role in tumor metastasis, angiogenesis and inflammation. The regulation of HPSE expression and function is tightly controlled and the increasing use of the mouse as an animal model to define the role of HPSE in many physiological and pathological settings, makes understanding the regulatory mechanisms of HPSE in this species of fundamental importance. However, the expression distribution of the mouse Hpse gene and the mechanisms that regulate its transcription are poorly defined. In this study, the mouse Hpse gene was determined to encode for two mRNA transcripts of 1.9 and 3.2 kb in length with identical open reading frames that showed similar tissue expression distribution to the human HPSE. The mouse Hpse promoter was cloned and a 478-bp minimal promoter was identified that contained regulatory elements responsible for both basal promoter activity in mouse tumor cells as well as inducible activity in T cells. Mutagenesis and transactivation studies identified a functional site in the minimal promoter region for the transcription factor Early growth response gene 1 (Egr1). Interestingly, Egr1 acted differentially in mouse tumor cells, functioning in an activating or repressive manner in breast carcinoma or melanoma cells, respectively. Furthermore, the proximal region of the promoter, identified as important in the regulation of Hpse transcription, was shown to become accessible in T cells upon cell activation. Significantly, the maximal accessibility of the promoter occurred at 16 h post-stimulation, which correlated with the induction kinetics of Hpse mRNA expression. In summary, this study demonstrates that mouse Hpse is expressed and regulated in a similar manner to human HPSE and also provides some novel insights into mechanisms of Hpse gene regulation that are likely to be relevant to control of the human gene.