Shonagh Munro

Selective inhibition of BET bromodomains

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic ‘writers’ and ‘erasers’. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein–protein interactions of epigenetic ‘readers’, and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Arginine methylation controls growth regulation by E2F‐1

E2F transcription factors are implicated in diverse cellular functions. The founding member, E2F‐1, is endowed with contradictory activities, being able to promote cell‐cycle progression and induce apoptosis. However, the mechanisms that underlie the opposing outcomes of E2F‐1 activation remain largely unknown. We show here that E2F‐1 is directly methylated by PRMT5 (protein arginine methyltransferase 5), and that arginine methylation is responsible for regulating its biochemical and functional properties, which impacts on E2F‐1‐dependent growth control. Thus, depleting PRMT5 causes increased E2F‐1 protein levels, which coincides with decreased growth rate and associated apoptosis. Arginine methylation influences E2F‐1 protein stability, and the enhanced transcription of a variety of downstream target genes reflects increased E2F‐1 DNA‐binding activity. Importantly, E2F‐1 is methylated in tumour cells, and a reduced level of methylation is evident under DNA damage conditions that allow E2F‐1 stabilization and give rise to apoptosis. Significantly, in a subgroup of colorectal cancer, high levels of PRMT5 frequently coincide with low levels of E2F‐1 and reflect a poor clinical outcome. Our results establish that arginine methylation regulates the biological activity of E2F‐1 activity, and raise the possibility that arginine methylation contributes to tumourigenesis by influencing the E2F pathway.

Lysine methylation regulates the pRb tumour suppressor protein

The pRb tumour suppressor protein has a central role in coordinating early cell cycle progression. An important level of control imposed on pRb occurs through post-translational modification, for example, phosphorylation. We describe here a new level of regulation on pRb, mediated through the targeted methylation of lysine residues, by the methyltransferase Set7/9. Set7/9 methylates the C-terminal region of pRb, both in vitro and in cells, and methylated pRb interacts with heterochromatin protein HP1. pRb methylation is required for pRb-dependent cell cycle arrest and transcriptional repression, as well as pRb-dependent differentiation. Our results indicate that methylation can influence the properties of pRb, and raise the interesting possibility that methylation modulates pRb tumour suppressor activity.

Arginine Methylation-Dependent Reader-Writer Interplay Governs Growth Control by E2F-1

The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.

Interplay between lysine methylation and Cdk phosphorylation in growth control by the retinoblastoma protein

As a critical target for cyclin‐dependent kinases (Cdks), the retinoblastoma tumour suppressor protein (pRb) controls early cell cycle progression. We report here a new type of regulation that influences Cdk recognition and phosphorylation of substrate proteins, mediated through the targeted methylation of a critical lysine residue in the Cdk substrate recognition site. In pRb, lysine (K) 810 represents the essential and conserved basic residue (SPXK) required for cyclin/Cdk recognition and phosphorylation. Methylation of K810 by the methyltransferase Set7/9 impedes binding of Cdk and thereby prevents subsequent phosphorylation of the associated serine (S) residue, retaining pRb in the hypophosphorylated growth‐suppressing state. Methylation of K810 is under DNA damage control, and methylated K810 impacts on phosphorylation at sites throughout the pRb protein. Set7/9 is required for efficient cell cycle arrest, and significantly, a mutant derivative of pRb that cannot be methylated at K810 exhibits compromised cell cycle arrest. Thus, the regulation of phosphorylation by Cdks reflects the combined interplay with methylation events, and more generally the targeted methylation of a lysine residue within a Cdk‐consensus site in pRb represents an important point of control in cell cycle progression.

Diversity within the pRb pathway: is there a code of conduct?

The failure of cell proliferation to be properly regulated is a hallmark of tumourigenesis. The retinoblastoma protein (pRb) pathway represents a key component in the regulation of the cell cycle and tumour suppression. Recent findings have revealed new levels of complexity reflecting a repertoire of post-translational modifications that occur on pRb together with its key effector E2F-1. Here we provide an overview of the modifications and consider the possibility of a ‘code’ that endows pRb with the ability to function in diverse physiological settings.

Structural analysis of human KDM5B guides histone demethylase inhibitor development.

Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe(2+)-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25-100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design.

NEDDylation regulates E2F-1-dependent transcription

The ubiquitin‐like molecule NEDD8 modifies cullin‐RING ubiquitin E3 ligases. NEDD8 has been shown to have a few additional substrates, but the extent to which this modification targets non‐cullins and the functional significance of such modifications remain unclear. Here, we demonstrate that the cell‐cycle‐regulating transcription factor E2F‐1 is a substrate for NEDD8 post‐translational modification. NEDDylation results in decreased E2F‐1 stability, lower transcriptional activity and slower cell growth. The lysine residues in E2F‐1 targeted for NEDDylation can also be methylated, pointing to a possible interplay between these modifications. These results identify a new mode of E2F‐1 regulation and highlight the emerging role of NEDD8 in regulating transcription factor stability and function.

Lysine methylation-dependent binding of 53BP1 to the pRb tumor suppressor

Significance The retinoblastoma protein (pRb) is a key regulator of cell cycle progression and the DNA damage response. Its importance in these processes is highlighted by the fact that it is mutated or functionally inactivated in almost all human tumors. Its activity is finely regulated by a number of post-translational modifications, including phosphorylation and methylation, which act to recruit “reader” proteins that mediate signaling events. Here, to our knowledge for the first time, we describe the methyl-dependent interaction between pRb and the tudor domain containing tumor protein p53 binding protein 1 (53BP1) and describe how this interaction integrates pRb cell cycle control with the DNA damage response. Our results therefore widen the repertoire of cellular targets for 53BP1 and suggest a new role in regulating pRb tumor suppressor activity. The retinoblastoma tumor suppressor protein pRb is a key regulator of cell cycle progression and mediator of the DNA damage response. Lysine methylation at K810, which occurs within a critical Cdk phosphorylation motif, holds pRb in the hypophosphorylated growth-suppressing state. We show here that methyl K810 is read by the tandem tudor domain containing tumor protein p53 binding protein 1 (53BP1). Structural elucidation of 53BP1 in complex with a methylated K810 pRb peptide emphasized the role of the 53BP1 tandem tudor domain in recognition of the methylated lysine and surrounding residues. Significantly, binding of 53BP1 to methyl K810 occurs on E2 promoter binding factor target genes and allows pRb activity to be effectively integrated with the DNA damage response. Our results widen the repertoire of cellular targets for 53BP1 and suggest a previously unidentified role for 53BP1 in regulating pRb tumor suppressor activity.

Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response

PAD4-mediated citrullination of E2F-1 transcription factor and its interplay with acetylation affects inflammatory gene expression. Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.