DYRK1A phoshorylates histone H3 to differentially regulate the binding of HP1 isoforms and antagonize HP1-mediated transcriptional repression

Abstract

Heterochromatin protein 1 (HP1) proteins are chromatin-bound transcriptional regulators. While their chromodomain binds histone H3 methylated on lysine 9, their chromoshadow domain associates with the H3 histone fold in a region involved in chromatin remodeling. Here, we show that phosphorylation at histone H3 threonine 45 and serine 57 within this latter region differentially affects binding of the three mammalian HP1 isoforms HP1α, HP1β and HP1γ. Both phosphorylation events are dependent on the activity of the DYRK1A kinase that antagonizes HP1-mediated transcriptional repression and participates in abnormal activation of cytokine genes in Down's syndrome-associated megakaryoblastic leukemia.

Keywords: chromatin silencing; cytokine; epigenetic; histone code; inflammation.

Figure 1. Phospho-mimic mutations in the histone H3 ShaDock modifies binding of HP1 isoforms

A Diagram of the B10-tagged recombinant histone H3 (rH3) and the rH3(25-66) peptide. B Indicated GST fusions bound to agarose beads were incubated with rH3 then washed. Western blotting with anti-B10 antibody revealed retained proteins. The bottom panel shows the GST fusion proteins stained with Ponceau S. C GST fusions bound to agarose beads were incubated with the indicated rH3(25-66) peptides and washed. As in (B), retained peptides were revealed with anti-B10 antibody. Source data are available online for this figure.

Figure 2. DYRK1A phosphorylates histone H3

A NIH3T3 cells were transfected with expression vectors for either EGFP-histone H1, EGFP-DYRK1A, or mutant EGFP-DYRK1A(K188R) fusion proteins. After 48h, cells were fixed and indirectly labeled with anti-H3T45p antibody. DNA was stained with DAPI. B Total protein extracts from NIH3T3 cells transfected with the indicated expression vectors were immunoblotted with indicated antibodies. C, D NIH3T3 cells were transfected with expression vectors for either EGFP-DYRK1A or mutant EGFP-DYRK1A(K188R) fusion proteins as in (A). After 48 h, cells were fixed and indirectly labeled with anti-Brg1 (C) or anti-HP1α (D) antibody. DNA was stained with DAPI. In each panel, inset shows an EGFP-positive nucleus magnified 2-fold. E WT or indicated mutant rH3(25–66) peptides were incubated with either purified GST or GST-DYRK1A fusion proteins and detected with the indicated antibodies. Source data are available online for this figure.

Figure 3. Antagonistic effects of DYRK1A and HP1 proteins on inducible genes

A HeLa cells were transfected with the indicatedsiRNAs. After 48 h, levels of mRNA for the indicated genes were measured by RT-qPCR. B Total protein extracts from a representative experimentwere immunoblotted with indicated antibodies. C Expression of WT DYRK1A or mutant DYRK1A(K188R) was induced by ponasterone in engineered HEK293 cells. After 48 h, levels of mRNA for the indicated genes were measured by RT-qPCR. D Total protein extracts from a representative experimentwere immunoblotted with indicated antibodies. E HeLa cells were transfected with the indicatedsiRNAs. After 48 h, levels of mRNA for the indicated genes were measured by RT-qPCR. F Total protein extracts from a representative experimentwere immunoblotted with indicated antibodies. Data in histograms are presented relative to levels of Ribosomal Protein, Large, P0 (RPLP0) mRNA considered as invariant in the experimental conditions. Data information: Data are means ± s.e.m. from three independent experiments. Significance of the differences was estimated using Student’s t-test. ***P < 0.001, 0.001 < **P < 0.01, 0.01 < *P < 0.05.

Figure 4. Depletion of DYRK1A antagonizes open chromatin marks at inducible genes

A–I HeLa cells were transfected with indicated siRNAs. Accumulation of the indicated proteins or histone modifications at the promoter of the listed genes were evaluated by ChIP analysis followed by qPCR. Data are normalized to the values obtained with either non-immune IgGs or anti-histone H3 antibodies as indicated. Data shown are means ± s.e.m. from three independent experiments. In each panel, significance of the differences was estimated using Student’s t-test. ***P < 0.001, 0.001 < **P < 0.01, 0.01 < *P < 0.05.

Figure 5. DYRK1A in leukemia cells

A Total RNA from 22 human tissues and the indicated cell lines were assayed by RT-qPCR for levels of DYRK1A mRNA. Values were normalized to levels 18S RNA in each sample. Data shown are means ± s.e.m. from two independent experiments. The level of mRNA detected in prostate was similar to the mean of all levels and was chosen as a reference (set to 1). B CMK cells were cultured in the presence of 0, 1, or 10 μM DYRK1A inhibitor L41. After 72 h, levels of mRNA for the indicated genes were measured by RT-qPCR. Data shown are means ± s.e.m. from three independent experiments. Significance of the differences was estimated using Student’s t-test (***P < 0.001, 0.001 < **P < 0.01, 0.01 < *P < 0.05). C HL-60 cells were transfected with indicated siRNAs. After 48 h, levels of mRNA for the indicated genes were measured by RT-qPCR. Data shown are means ± s.e.m. from two independent experiments. Significance was estimated as in (B).