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Revisiting the specificity of the MHC class?II transactivator CIITA in classical murine dendritic cells in vivo.

ABSTRACT: Ciita was discovered for its role in regulating transcription of major histocompatibility complex class II (MHCII) genes. Subsequently, CIITA was predicted to control many other genes based on reporter and ChIP-seq analysis but few such predictions have been verified in vivo using Ciita-/- mice. Testing these predictions for classical dendritic cells (cDCs) has been particularly difficult, since Ciita-/- mice lack MHCII expression required to identify cDCs. However, recent identification of the cDC-specific transcription factor Zbtb46 allows the identification of cDCs independently of MHCII expression. We crossed Zbtb46gfp mice onto the Ciita-/- background and found that all cDC lineages developed in vivo in the absence of Ciita. We then compared the complete transcriptional profile of wild-type and Ciita-/- cDCs to define the physiological footprint of CIITA for both immature and activated cDCs. We find that CIITA exerts a highly restricted control over only the MHCII, H2-DO and H2-DM genes, in DC1 and DC2 cDC subsets, but not over other proposed targets, including Ii. These findings emphasize the caveats needed in interpreting transcription factor binding sites identified by in-vitro reporter analysis, or by ChIP-seq, which may not necessarily indicate their functional activity in vivo.

SUBMITTER: Anderson DA

PROVIDER: S-EPMC5757839 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Revisiting the specificity of the MHC class II transactivator CIITA in classical murine dendritic cells in vivo.

Anderson David A DA Grajales-Reyes Gary E GE Satpathy Ansuman T AT Vasquez Hueichucura Carlos E CE Murphy Theresa L TL Murphy Kenneth M KM

European journal of immunology 20170714 8

Ciita was discovered for its role in regulating transcription of major histocompatibility complex class II (MHCII) genes. Subsequently, CIITA was predicted to control many other genes based on reporter and ChIP-seq analysis but few such predictions have been verified in vivo using Ciita<sup>-/-</sup> mice. Testing these predictions for classical dendritic cells (cDCs) has been particularly difficult, since Ciita<sup>-/-</sup> mice lack MHCII expression required to identify cDCs. However, recent ...[more]

PMID: 28608405

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Project description:BackgroundWe previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells.MethodsU937 Plus cells stably expressing CIITA were generated and HLA-II positive clones were selected by cell sorting and cloning. HLA and CIITA proteins were analyzed by cytofluorometry and western blotting, respectively. HLA-II DR and CIITA mRNAs were quantified by qRT-PCR. Tat-dependent transactivation was assessed by performing the HIV-1 LTR luciferase gene reporter assay. Cells were infected with HIV-1 and viral replication was evaluated by measuring the RT activity in culture supernatants.ResultsCIITA was expressed only in HLA-II-positive U937 Minus cells, and this was strictly correlated with inhibition of Tat-dependent HIV-1 LTR transactivation in Minus but not in Plus cells. Overexpression of CIITA in Plus cells restored the suppression of Tat transactivation, confirming the inhibitory role of CIITA. Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells. This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.ConclusionsU937 Plus and Minus cells represent an interesting model to study the role of CIITA in HIV-1 restriction in the monocytic/macrophage cell lineage. The differential expression of CIITA in CIITA-negative Plus and CIITA-positive Minus cells correlated with their capacity to support or not HIV-1 replication, respectively. In Minus cells CIITA targeted the viral transactivator Tat to inhibit HIV-1 replication. The generation of Plus-CIITA cells was instrumental to demonstrate the specific contribution of CIITA in terms of inhibition of Tat activity and HIV-1 restriction, independently from other cellular factors, including TRIM22. Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

Dataset Information

Revisiting the specificity of the MHC class?II transactivator CIITA in classical murine dendritic cells in vivo.

Publications

Revisiting the specificity of the MHC class II transactivator CIITA in classical murine dendritic cells in vivo.

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