Transcriptional Regulation of Clr-b (Clec2d) in Health and Disease

Innate immunity provides the first line of defense against infectious disease and cancer. Natural killer (NK) cells are innate lymphocytes that recognize and eliminate pathological cells. Target cell recognition is facilitated by germline-encoded NK cell receptors, including the NKR-P1B inhibitory receptor. The NKR-P1B-ligand, Clr-b, is broadly expressed by normal healthy cells, yet rapidly downregulated during pathological alterations, resulting in NK cell disinhibition via NKR-P1B. This thesis investigates transcriptional regulation of Clr-b during health and disease. A whole-genome lentiviral shRNA screen was used to identify novel transcription factors involved in the normal expression of the Clr-b gene (Clec2d). MeCP2 was identified and validated as a transcription factor that represses Clr-b expression. TBP and CBP were also identified as ubiquitous transcription factors required for normal basal Clr-b surface levels. Accordingly, a core Clec2d promoter with an inverted TATA motif was identified ~20bp upstream of the transcriptional start site (TSS) as sufficient to drive basal Clec2d expression. During NIH3T3 cell infection using MCMV-GFP, infected cells (GFP+) lost Clr-b expression, while uninfected ‘bystander’ cells (GFP–) upregulated Clr-b levels. This was due to reciprocal regulation of the Clec2d promoter and nascent transcripts in virus-infected versus bystander cells exposed to paracrine type-I interferon (IFN). Infected cells downregulated Clec2d nascent transcripts, mainly due to absence of RNAPII recruitment to the Clec2d promoter. Moreover, a cell-autonomous role for the MCMV ie3 gene product was also discovered, whereby ectopic expression of IE3 directly repressed Clec2d activity. In uninfected cells, paracrine or exogenous type-I IFN upregulated Clec2d promoter activity via canonical IFNAR-mediated signaling. This was dependent upon recruitment of STAT2 to a consensus cluster of IRF motifs (IRF3/7/9, termed the IRFC) located ~200bp upstream of the Clec2d TSS. Clec2d induction by type-I IFN was IRF9- and STAT1-dependent, abrogated in IRF9–/– or STAT1–/– MEF or upon mutation of the IRFC. Thus, in addition to Clr-b being an MHC-I-independent ‘missing-self’ marker on virus-infected cells, Clec2d is an interferon-stimulated gene on uninfected bystander cells, collectively facilitating greater dynamic range of NKR-P1B-dependent self-nonself discrimination during virus infection.

Department dc:contributor.department

Immunology

Year dc:date.issued

2017

Author dc:creator

• Kirkham, Christina

Advisor dc:contributor.advisor

• Carlyle, James R

• Clr-b (Clec2d)
• Innate Immunity
• MCMV
• Natural Killer Cells
• Transcriptional Regulation
• Type-I Interferon

Handle dc:identifier.uri

http://hdl.handle.net/1807/79022

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/79022