Abstract 2597: The Cyclin D1 carboxyl terminus encodes an epigenetic reader domain

Abstract

Abstract Purpose of the study: The cyclin D1 gene has been shown to promote chromosomal instability CIN) in cultured cells and in vivo using transgenic mice, through a kinase-independent mechanism. Correlative studies suggest the transcriptional function of cyclin D1 governs the induction of CIN. The cyclin D1 gene encodes a regulatory subunit of a holoenzyme complex that phosphorylates and inactivates the retinoblastoma protein (Rb) and the nuclear respiratory factor 1 (NRF1) proteins to thereby regulate DNA synthesis and mitochondrial metabolism. In addition, cyclin D1 regulates gene expression. Cyclin D1 promotes oncogene-dependent growth, and genetic ablation of the cyclin D1 gene in mouse models produces mice resistant to Ras or ErbB2-induced mammary tumorigenesis and APC-induced gastrointestinal tumorigenesis. The abundance of cyclin D1 is increased early in the malignant process and cyclin D1 abundance is regulated at different molecular levels, (transcription, translation and post-translational). In our previous studies, we identified, via a genome wide ChIP-Seq analysis, cyclin D1 binding regions in genes governing CIN including Top2A, AurkB, Cenpp, Mlf1ip, Zw10, Ckap2. Experimental procedures: Cyclin D1 promotes CIN via accessing gene regulatory regions at the local chromatin level. We deployed a histone peptide array and a peptide pull-down assays. Subsequent, we analyzed the biochemistry of the specific interactions between cyclin D1 and histone modifications via Surface Plasmon Resonance (SPR). Results: Cyclin D1 selectively recognized histone residues of H2B and H3 that were acetylated, methylated, phosphorylated or citrullinated (H2B-S14pho, H2B-K15ac), (H3R26Cit H3K273me). Mutational and deletion analysis showed that the cyclin D1 C terminus was sufficient to augment transcription of CIN genes and cell proliferation and that the E-box region (aa272-aa280) was necessary for cyclin D1 binding to modified histones. Cyclin D1 ΔE mutant (lacking the E-box motif) abolished the interaction with modified histones and abolished the transcription response and the augmentation of cell proliferation. In the peptide pull-down assay, we confirmed the specific physical interaction between cyclin D1 and H2B-S14pho. We further investigated this strength of the interaction using SPR. This approach showed a strong binding between cyclin D1 and H2B-S14pho and a decreased affinity for the unmodified histone H2B. The interaction was proven to be abolished between H2B-S14pho and cyclin D1 when the cyclin D1 ΔE mutant was introduced. Conclusion: Collectively these findings are consistent with a model in which cyclin D1 is recruited into chromatin through binding directly to an epigenetic histone code. Such binding may tether cyclin D1 to participate in transcriptional activity at specific target genes. Citation Format: Gabriele Di sante, Agnes Tantos, Mathew Craig Casimiro, Peter Tompa, Richard G. Pestell. The Cyclin D1 carboxyl terminus encodes an epigenetic reader domain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2597.