Chromatin Targeting of HIPK2 Leads to Acetylation-Dependent Chromatin Decondensation

Abstract

The protein kinase homeodomain-interacting protein kinase 2 (HIPK2) plays an important role in development and in the response to external cues. The kinase associates with an exceptionally large number of different transcription factors and chromatin regulatory proteins to direct distinct gene expression programs. In order to investigate the function of HIPK2 for chromatin compaction, HIPK2 was fused to the DNA-binding domains of Gal4 or LacI, thus allowing its specific targeting to binding sites for these transcription factors that were integrated in specific chromosome loci. Tethering of HIPK2 resulted in strong decompaction of euchromatic and heterochromatic areas. HIPK2-mediated heterochromatin decondensation started already 4 h after its chromatin association and required the functionality of its SUMO-interacting motif. This process was paralleled by disappearance of the repressive H3K27me3 chromatin mark, recruitment of the acetyltransferases CBP and p300 and increased histone acetylation at H3K18 and H4K5. HIPK2-mediated chromatin decompaction was strongly inhibited in the presence of a CBP/p300 inhibitor and completely blocked by the BET inhibitor JQ1, consistent with a causative role of acetylations for this process. Chromatin tethering of HIPK2 had only a minor effect on basal transcription, while it strongly boosted estrogen-triggered gene expression by acting as a transcriptional cofactor.

Expression of human HIPKs in Drosophila demonstrates their shared and unique functions in a developmental model

“…There is support for this model, since HIPK2 can associate with the Polycomb protein Pc2/CBX4, which is part of the Polycomb repressive complex 1 (PRC1) ( Roscic et al 2006 ). Another recent study used HIPK2 tethered to chromatin to directly address its ability to modulate chromosome compaction, where chromatin bound HIPK2 led to decreased Histone 3 Lysine 27 trimethylation (H3K27me3), an epigenetic mark normally associated with transcriptional repression and formation of heterochromatin ( Haas et al 2020 ). Thus, our findings of phenotypes associated with dysfunction of TrxG/PcG and previous work suggest that Hipks may play roles in regulating chromatin condensation.…”

Functional characterization of human Homeodomain-interacting protein kinases (HIPKs) inDrosophila melanogasterreveal both conserved functions and differential induction of HOX gene expression

“…Previous research has shown that hHIPK2 can interact with and phosphorylate CBX4/Pc2, a PRC1 component homologous to D. melanogaster Polycomb (Pc), in the early response to DNA damage, however this interaction was found to promote transcriptional silencing in this context [57]. More recently, the same group has found that under otherwise normal conditions, targeting hHIPK2 to specific DNA regions causes de- condensation and de-repression of chromatin at those genomic loci [58]. The latter result supports our finding of normally repressed homeotic genes becoming de-repressed in response to hHIPK expression, however interactions between Hipks and PRC1 or PRC2 components that are important in the context of normal development have not yet been described.…”

SIAH ubiquitin E3 ligases as modulators of inflammatory gene expression

“…After reaching 60–70% confluence Lipofectamine transfection with siScramble (10 nM) (D-001810-10-05, Dharmacon) and siRNAs targeting SIAH1 (10 nM) (#4427037, Thermo Fisher) and/or SIAH2 (10 nM) (#L-006561-00-0010, Dharmacon) was carried out for 48 h. One day after transfection in DMEM containing 10% FCS without penicillin/streptomycin, medium was exchanged to full media. The plasmids encoding HA-tagged p300, SIAH1 and SIAH2 [ 27 ] and GFP-lacI [ 31 ] were described and GFP-lacI-p300 (Addgene ID: 179270) was created. These plasmids were transiently transfected using linear PEI as published [ 32 ].…”

“…Cells were transfected as displayed in the figure legends. After 24 h cells were washed with PBS and fixed with 3.7% (v/v) formaldehyde for 10 min and further processed as described [ 31 ]. Nuclei were stained with Hoechst 33324 (Invitrogen).…”