Nuclear protein Kinase C-theta and permissive Chromatin states co-ordinate transcriptional memory responses in human T cells

Abstract

Memory T cells have the selective ability to up-regulate gene transcription much more rapidly and at higher levels than naive T cells. Histone modifications are known to provide permissive Chromatin structure that facilitates this process of transcriptional memory. Protein Kinase C (PKC) family members have recently emerged as a new class of enzymes that function as both cytoplasmic signalling kinases and chromatin-modifiers that regulate gene transcription. PKC-theta is predominately expressed in T cells but not in B cells and it plays an integral part in the T cell immunological synapse, which leads to the activation of Activator Protein-1 (AP-1), Nuclear Factor of Activated T cells (NFAT) and Nuclear Factor-kappaB (NF-KB) for the expression of immune responsive genes. Its importance has also been demonstrated previously in antigen recalling responses in memory T cells. At the level of chromatin regulation, PKC-theta has been shown to be essential in mediating the formation of an active transcription complex for IL2 gene expression, a gene known to have transcriptional memory response. To determine the role of PKC-theta in the establishment of transcriptional memory, an in vitro Jurkat T cell model was developed. Whole-transcriptome microarray expression analysis of this model identified groups of genes with shared characteristic transcriptional responses to repeated stimulation, namely transcriptional memory responsive, primary-specific responsive, secondary-specific responsive and stimulation compliant genes. These gene cohorts could be distinguished on the basis of the number of consensus motifs shared across their gene promoters, with transcriptional memory responsive and secondary-specific responsive genes being the richest in transcription factor binding sites compared to the other groups. Genome-wide ChIP-sequencing of the cytoplasmic-nuclear signalling kinase, PKC-theta in ex vivo derived T cells showed that it was significantly enriched in the chromatin of effector and activated memory CD4+ T cells compared to naive T cells. The knockdown of PKC-theta impaired the expression of genes with a transcriptional memory response but not the Activation Compliant genes in ex vivo derived activated memory CD4+ T cells. Gene-specific ChIP-PCR demonstrated the recruitment of PKC-theta to promoters of actively transcribed genes in Jurkat T cells during both primary and secondary activation. In the ex vivo derived naive and memory T cells, a consistent level of PKC-theta enrichment was detected at the promoter of CD69, a transcriptional memory non-responsive gene but rapid IL2 transcriptional response was associated with a gene-specific increase of nuclear PKC-theta at its proximal promoter in activated memory CD4+ T cells. Furthermore, nuclear PKC-theta was recruited to a regions with a permissive chromatin state, defined by high levels of the activating marks, H3K4me3 and H3K9ac and low levels of the repressive mark, H3K27me3. ChIP-PCR analysis of the IL2 promoter also revealed enrichment of the histone demethylase LSD1 in these cells. Collectively, these results indicate that chromatin-tethered PKC-theta forms a part of the permissive chromatin state that co-ordinates rapid transcriptional programs in memory T cells. Show more