Novel Roles for the Transcriptional Repressor PRDM1 in Human Natural Killer Cells and Identification of an Inhibitor of its Interacting Methyltransferase G9a.

摘要

The studies presented within this dissertation provide the first description of PRDM1 (also known as Blimp-1 or PRDI-BF1) function in natural killer cells. NK cells are major effectors of the innate immune response via antigen-independent cytotoxicity and link to the adaptive immune response through cytokine release. Molecular mechanisms mediating NK activation are relatively well-studied; however, much less is known about the mechanisms that restrain activation.;In the first study, the transcriptional repressor PRDM1 is shown to be a critical negative regulator of NK function. Microarray analysis was used to characterize transcriptional changes associated with cytokine-mediated activation. PRDM1 is expressed at low levels in resting NK cells and three distinct PRDM1 isoforms are selectively induced in the CD56dim NK population in response to activation. PRDM1 coordinately suppresses the production of IFNgamma, TNF&agr; and TNFbeta through direct binding to multiple conserved regulatory regions. Ablation of PRDM1 expression leads to enhanced production of IFNgamma and TNF&agr; but does not alter cytotoxicity, whereas over-expression blocks cytokine production. PRDM1 response elements are defined at the IFNG and TNF loci.;To further delineate the targets of PRDM1-mediated regulation in NK cells, global approaches were utilized. Experiments utilizing chromatin immunoprecipitation coupled to promoter tiling arrays identified 292 novel direct targets of PRDM1 binding. These studies revealed widespread binding of PRDM1 to the genome, which was not limited to proximal promoter regions. Furthermore, microarray analysis of stimulated NK cells combined with PRDM1 knockdown has enabled the identification of genes responsive to PRDM1 knockdown using primary cells. Collectively, these experiments identify both direct and indirect targets of PRDM1 regulation and help define a PRDM1-centered gene regulatory network in NK cells.;Data presented in the final chapter pertains to an independent project aimed at identifying small molecule inhibitors of the methyltransferase G9a, which is recruited by PRDM1 and is required for silencing of target genes. A mass spectrometry-based assay was developed and used to screen a small molecule library. Several hits were identified and combinatorial chemistry yielded several compounds with ≤ 20microM IC50 values. In cell-based assays, however, treatment with the small molecules had limited efficacy, indicating additional chemical modifications are necessary to yield bioactive compounds.;The data presented here demonstrate a key role for PRDM1 in the negative regulation of NK activation and position PRDM1 as a common regulator of the adaptive and innate immune response.