IKZF1 modulates PP2Ac expression through an intronic binding site

Kamalpreet Nagpal¹, Katsue Sunahori², George C. Tsokos¹

¹Department of Medicine, Division of Rheumatology, Beth Israel Deaconess Medical

Center, Harvard Medical School, Boston, Massachusetts 02115.

²Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan

Background/Purpose:

Protein phosphatase 2A (PP2A) is a highly conserved and ubiquitous serine/threonine phosphatase.  We have previously shown that mRNA and protein, as well as the activity of the catalytic subunit (PP2Ac) of PP2A are increased in T-cells of SLE patients as compared to healthy controls.  This increased expression and activity of PP2A plays a central role in the molecular pathogenesis of SLE, including reduced production of the cytokine IL-2.  Although the control of PP2Ac expression has been the focus of many studies, many aspects of its regulation still remain poorly understood.  In this study, we identify the transcription factor Ikaros (IKZF1) as a novel regulator of PP2Ac expression.

Methods:

CD3 (+)ve T-cells from healthy individuals were transfected with Ikaros expression plasmid using AMAXA nucleoporation.  RNA and protein were isolated and Real-Time PCR and western blotting were used to analyze gene expression. Biotinylated oligos spanning the putative Ikaros binding site or a non-specific control oligo were used in pull-down assays followed by western blotting with Ikaros specific antibody to examine the binding of Ikaors. EMSA with Jurkat cells nuclear protein extract and radio-labeled oligos was used to confirm this binding.  Binding of endogenous Ikaros to the PP2Ac intron region was validated by ChIP using a commercially available kit.

Results:

An online in-silico transcription factor search identified the transcription factor Ikaros as having a binding site in the first intron of PPP2AC gene.  To examine this binding, biotinylated oligos spanning this region in the intron were used in a pull-down assay with nuclear proteins extracted from Jurkat cells.  As compared to the control oligo, the specific oligo exhibited significant binding to Ikaros.  EMSA with radio-labeled oligos as well as ChIP assays in primary T-cells further validated the binding of Ikaros to this region.  Exogenous expression of Ikaros in T-cells led to diminished PP2Ac expression. Conversely, siRNA enabled knockdown of Ikaros enhanced the expression of PP2Ac, suggesting that Ikaros acts as a suppressor of PP2Ac gene expression.  We also attempt to delineate the mechanism of Ikaros mediated PP2Ac gene suppression. We report that Ikaros recruits the histone deacetylase HDAC1 as well as DNA methyltransferase DNMT1 to this intronic site, thus mediating epigenetic alterations leading to reduced gene expression.

Conclusion:

We show for the first time that the transcription factor Ikaros can regulate the gene expression of PP2Ac by binding to a site in the first intron.  Ikaros seems to mediate this suppression by modulating chromatin modifications at this site.  Our study thus identifies a novel means of control of PP2A, a critical molecule involved in lupus pathogenesis.