The adaptor protein ARA55 and the nuclear kinase HIPK1 assist c-Myb in recruiting p300 to chromatin

Abstract

LIM-domain proteins, containing multiple cysteine-rich zinc finger-like motifs, have been shown to play diverse roles in several cellular processes. A common theme is that they mediate important protein-protein interactions that are key to their function. Androgen receptor-associated protein 55 (ARA55) belongs to this family of bridging proteins containing four C-terminal LIM domains. It has a dual role with functions both at focal adhesions and in the nucleus, apparently shuttling between the two compartments. In the present work, we have expanded our understanding of its nuclear functions by showing that it interacts with three nuclear regulators not previously linked to ARA55. We first identified ARA55 as a novel interaction partner of the nuclear kinase HIPK1 and found that ARA55, like HIPK1, also interacts with the transcription factor c-Myb. In search of a function for these associations, we observed that the coactivator p300 not only binds to c-Myb, but to ARA55 as well. When combined, c-Myb, p300, HIPK1 and ARA55 caused strong synergistic activation of a chromatinized reporter gene. In parallel, all partners, including p300, were efficiently recruited to chromatin at the c-Myb-bound promoter. Consistent with this cooperation, we found that c-Myb and ARA55 share a common set of target genes in an osteosarcoma cellular context. We propose that ARA55 and HIPK1 assist c-Myb in recruiting the coactivator and acetyltransferase p300 to chromatin.

The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

“…The HD-11 cell line was transfected with plasmids encoding TY-tagged c-Myb variants (empty vector, 3xTY1-c-Myb, 3xTY-c-Myb D152V) described in [ 13 ]. The ChIP analysis was performed essentially as previously described [ 53 , 54 ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac. The anti-Ty1 monoclonal mouse antibody was produced in our lab from a hybridoma cell line [ 55 ].…”

Chromatin Targeting of HIPK2 Leads to Acetylation-Dependent Chromatin Decondensation

“…While these data show that HIPK2 by itself has only a very low gene-activating capacity it was interesting to investigate whether it can support gene expression triggered by other transcription factors. As a coactivator function of HIPK2 for androgen receptors has already been described ( Imberg-Kazdan et al, 2013 ; Bengtsen et al, 2017 ), we tested a possible contribution of HIPK2 to gene induction by a different hormone receptor. Toward this goal we created a plasmid where a lacO binding site allowing the tethering of GFP-LacI-HIPK2 was inserted upstream from three copies of an estrogen receptor response element (ERE) and a luciferase reporter gene.…”

“…Also other members of the HIPK family can associate with p300. It was shown that c-Myb associates with the coactivator androgen receptor-associated protein 55 (ARA55) to form a complex with HIPK1/p300, thus strongly enhancing c-Myb transcriptional activity ( Bengtsen et al, 2017 ). The association of HIPK2 with HATs and HDAC7 was independent from the chromatin state, as we observed co-localization between these proteins also in CHO RREB1 cells harboring lacO repeats in an euchromatic region ( Supplementary Figure S8 ).…”

Structural Insights in Multifunctional Papillomavirus Oncoproteins

“…Remarkably, the best characterized LxxLL-containing E6 targets, i.e., E6AP [ 121 ], p53 [ 122 ], MAML1 [ 123 ], IRF3 [ 124 ], paxillin [ 125 ] and its close paralog ARA55 (Androgen Receptor Associated protein 55) [ 126 ], hADA3 (human Transcriptional Adapter 3) [ 127 ] and CCR4-Not complex [ 128 ], are all involved in transcriptional activation or co-activation. Moreover, most of them have been shown to interact with the CBP-p300 protein [ 121 , 122 , 123 , 124 , 126 , 127 , 128 ], which is itself a direct target of some E6 proteins, such as those of HPV16 and β1 HPVs [ 36 , 129 ]. CBP-p300 is a central transcriptional co-activator with acetyltransferase activity involved in numerous functions, including the host innate antiviral response [ 124 , 130 ].…”