Anjum Zafar

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.

Unexpected new roles for heparanase in Type 1 diabetes and immune gene regulation

Heparanase (Hpse) is an endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS) in basement membranes (BMs) to facilitate leukocyte migration into tissues. Heparanase activity also releases HS-bound growth factors from the extracellular matrix (ECM), a function that aids wound healing and angiogenesis. In disease states, the degradation of HS in BMs by heparanase is well recognized as an invasive property of metastatic cancer cells. Recent studies by our group, however, have identified unexpected new roles for heparanase and HS. First, we discovered that in Type 1 diabetes (T1D) (i) HS in the pancreatic islet BM acts as a barrier to invading cells and (ii) high levels of HS within the insulin-producing islet beta cells themselves are critical for beta cell survival, protecting the cells from free radical-mediated damage. Furthermore, catalytically active heparanase produced by autoreactive T cells and other insulitis mononuclear cells was shown to degrade intra-islet HS, increasing the susceptibility of islet beta cells to free radical damage and death. This totally novel molecular explanation for the onset of T1D diabetes opens up new therapeutic approaches for preventing disease progression. Indeed, administration of the heparanase inhibitor, PI-88, dramatically reduced T1D incidence in diabetes-prone NOD mice, preserved islet beta cell HS and reduced islet inflammation. Second, in parallel studies it has been shown that heparanase and HS can be transported to the nucleus of cells where they impact directly or indirectly on gene transcription. Based on ChIP-on-chip studies heparanase was found to interact with the promoters and transcribed regions of several hundred genes and micro-RNAs in activated Jurkat T cells and up-regulate transcription, with many of the target genes/micro-RNAs being involved in T cell differentiation. At the molecular level, nuclear heparanase appears to regulate histone 3 lysine 4 (H3K4) methylation by influencing the recruitment of demethylases to transcriptionally active genes. These studies have unveiled new functions for heparanase produced by T lymphocytes, with the enzyme mediating unexpected intracellular effects on T cell differentiation and insulin-producing beta cell survival in T cell-dependent autoimmune T1D.

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.

Chromatinized Protein Kinase C-θ Directly Regulates Inducible Genes in Epithelial to Mesenchymal Transition and Breast Cancer Stem Cells

ABSTRACT Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor β (TGF-β) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer.

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 role of protein kinase-C theta in control of epithelial to mesenchymal transition and cancer stem cell formation.

The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) induces transition of the epithelial MCF-7 cell line to a mesenchymal phenotype. A subset of the resulting mesenchymal cells has surface markers characteristics of a cancer stem cell (CSC) population. We profiled the transcriptome changes associated with the epithelial to mesenchymal transition and those that occurred in the CSC subset. Using a siRNA knockdown strategy, we examined the extent to which these changes were dependent on the PKC family member, PKC-θ. The importance of the cytoplasmic signaling role of this kinase is well established and in this study, we have shown by PKC-θ ChIP-sequencing analysis that this kinase has a dual role with the ability to also associate with chromatin on a subset of PKC-θ dependent genes. In the associated manuscript (Zafar et al., 2014 [5]) we presented evidence for the first time showing that this nuclear role of PKC-θ is also important for gene induction and mesenchymal/CSC phenotype. Here we describe the analysis associated with the transcriptome and ChIP-seq data presented in Zafar et al. (2014) [5] and uploaded to NCBI Gene Expression Omnibus (GSE53335).

Identification of chromatin accessibility domains in human breast cancer stem cells

ABSTRACT Epithelial-to-mesenchymal transition (EMT) is physiological in embryogenesis and wound healing but also associated with the formation of cancer stem cells (CSCs). Many EMT signaling pathways are implicated in CSC formation, but the precise underlying mechanisms of CSC formation remain elusive. We have previously demonstrated that PKC is critical for EMT induction and CSC formation in inducible breast EMT/CSC models. Here, we used formaldehyde-assisted isolation of regulatory elements-sequencing (FAIRE-seq) to investigate DNA accessibility changes after PKC activation and determine how they influence EMT and CSC formation. During EMT, DNA accessibility principally increased in regions distant from transcription start sites, low in CpG content, and enriched with chromatin enhancer marks. ChIP-sequencing revealed that a subset of these regions changed from poised to active enhancers upon stimulation, with some even more acteylated in CSCs. While regions with increased accessibility were enriched for FOX, AP-1, TEAD, and TFAP2 motifs, those containing FOX and AP-1 motif were associated with increased expression of CSC-associated genes, while those with TFAP2 were associated with genes with increased expression in non-CSCs. Silencing of 2 members of the FOX family, FOXN2 and FOXQ1, repressed CSCs and the mesenchymal phenotype and inhibited the CSC gene signature. These novel, PKC-induced DNA accessibility regions help explain how the epigenomic plasticity of cells undergoing EMT leads to CSC gene activation.

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

ABSTRACT 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. Summary: Memory T cells have a rapid transcriptional program upon re-stimulation. Chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit this transcriptional memory in T cells.

PKCθ and HIV-1 Transcriptional Regulator Tat Co-exist at the LTR Promoter in CD4+ T Cells

PKCθ is essential for the activation of CD4+ T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4+ T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T538 residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4+ T cells and could be used as a therapeutic target.

Multi-Layered Epigenetic Regulatory Mechanisms Mediate Epithelial toMesenchymal Transition in Cancer

Epithelial to Mesenchymal Transition (EMT) is a central feature of embryonic development and is also a critical early event in cancer progression and metastasis. Our understanding of the complexity of the chromatin platform and the epigenetic mechanisms that contribute to transcriptional control has expanded dramatically in recent years. These mechanisms include the presence/absence of histone modifications, which form epigenetic signatures that mark active or inactive genes. These signatures are dynamically added or removed by a wide variety of histonemodifying epigenetic enzymes, which more recently have been found to include chromatin-associated signalling kinases. Here, we discuss the multi-layered regulation of gene transcription during EMT in cancer. Given that epigenetics-based therapeutics are showing promise for the treatment of cancer, unravelling the detail of these epigenetic signatures during EMT is crucial to the development of novel therapeutic strategies that exploit these mechanisms.