•The activity of writers and erasers of chromatin marks is coupled to the function of reader modules.•Reader domains regulate recruitment, substrate specificity and catalysis of chromatin modifying ...enzymes.•Histone and DNA methylation patterns are tightly coordinated.
DNA and histone lysine methylation are dynamic chemical modifications that play a crucial role in the establishment of gene expression patterns during development. Both types of genomic methylation patterns are enzymatically regulated by the opposing activities of enzymes that introduce and remove these marks, known as methylation ‘writers’ and ‘erasers’, respectively. The appropriate localization and activity of these enzymes on chromatin is, in part, regulated by chromatin ‘readers’, protein modules that recognize histone and DNA modifications. Such reading modules are either encoded within the same polypeptide as the catalytic domains of writers and erasers, or present in protein partners that associate with them. Here, we review recent structural, biochemical and biological studies that demonstrate that there are multiple mechanisms by which reader domains can regulate the writers and erasers of histone and DNA methylation.
Understanding the transcriptional changes that are engaged in stress resilience may reveal novel antidepressant targets. Here we use gene co-expression analysis of RNA-sequencing data from brains of ...resilient mice to identify a gene network that is unique to resilience. Zfp189, which encodes a previously unstudied zinc finger protein, is the highest-ranked key driver gene in the network, and overexpression of Zfp189 in prefrontal cortical neurons preferentially activates this network and promotes behavioral resilience. The transcription factor CREB is a predicted upstream regulator of this network and binds to the Zfp189 promoter. To probe CREB-Zfp189 interactions, we employ CRISPR-mediated locus-specific transcriptional reprogramming to direct CREB or G9a (a repressive histone methyltransferase) to the Zfp189 promoter in prefrontal cortex neurons. Induction of Zfp189 with site-specific CREB is pro-resilient, whereas suppressing Zfp189 expression with G9a increases susceptibility. These findings reveal an essential role for Zfp189 and CREB-Zfp189 interactions in mediating a central transcriptional network of resilience.
Histone demethylase KDM5A removes methyl marks from lysine 4 of histone H3 and is often overexpressed in cancer. The in vitro demethylase activity of KDM5A is allosterically enhanced by binding of ...its product, unmodified H3 peptides, to its PHD1 reader domain. However, the molecular basis of this allosteric enhancement is unclear. Here we show that saturation of the PHD1 domain by the H3 N-terminal tail peptides stabilizes binding of the substrate to the catalytic domain and improves the catalytic efficiency of demethylation. When present in saturating concentrations, differently modified H3 N-terminal tail peptides have a similar effect on demethylation. However, they vary greatly in their affinity towards the PHD1 domain, suggesting that H3 modifications can tune KDM5A activity. Furthermore, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiments reveal conformational changes in the allosterically enhanced state. Our findings may enable future development of anti-cancer therapies targeting regions involved in allosteric regulation.
Development of tool molecules that inhibit Jumonji demethylases allows for the investigation of cancer-associated transcription. While scaffolds such as 2,4-pyridinedicarboxylic acid (2,4-PDCA) are ...potent inhibitors, they exhibit limited selectivity. To discover new inhibitors for the KDM4 demethylases, enzymes overexpressed in several cancers, we docked a library of 600 000 fragments into the high-resolution structure of KDM4A. Among the most interesting chemotypes were the 5-aminosalicylates, which docked in two distinct but overlapping orientations. Docking poses informed the design of covalently linked fragment compounds, which were further derivatized. This combined approach improved affinity by ∼3 log-orders to yield compound 35 (K i = 43 nM). Several hybrid inhibitors were selective for KDM4C over the related enzymes FIH, KDM2A, and KDM6B while lacking selectivity against the KDM3 and KDM5 subfamilies. Cocrystal structures corroborated the docking predictions. This study extends the use of structure-based docking from fragment discovery to fragment linking optimization, yielding novel KDM4 inhibitors.
The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to ...its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism--enabled by the functional coupling between a reader and a catalytic domain in KDM5A--suggests a model for the spread of demethylation on chromatin.
Jumonji histone demethylases catalyze removal of methyl marks from lysine residues in histone proteins within nucleosomes. Here, we show that the catalytic domain of demethylase JMJD2A (cJMJD2A) ...utilizes a distributive mechanism to remove the histone H3 lysine 9 trimethyl mark. By developing a method to assess demethylation of homogeneous, site-specifically methylated nucleosomes, we determined that the kinetic parameters for demethylation of nucleosomes by cJMJD2A are comparable to those of peptide substrates. These findings imply that other domains of the demethylase or its protein partners may contribute to nucleosome recognition in vivo and, in this way, may further regulate demethylation activity and processivity. The quantitative assays of nucleosome demethylation developed in our work provide a platform for future work with complex chromatin substrates and full-length demethylases.
Display omitted
► Catalytic domain of JMJD2A (cJMJD2A) removes methyl marks in a distributive manner ► Homogeneously methylated nucleosomes were used as substrates ► Quantitative assay for nucleosome demethylation has been developed
Jumonji histone demethylases are critical transcriptional regulators, but major mechanistic details are missing. Shiau et al. reveal the intrinsic distributive nature of the catalytic domain of JMJD2A and suggest that JMJD2A’s activity and processivity is regulated by the auxiliary domains and protein partners.
Histone lysine methylation plays an essential role in a variety of cellular processes including transcriptional regulation and cell differentiation. The retinoblastoma binding protein KDM5A is a ...histone H3 lysine 4 (H3K4) demethylase, capable of removing the trimethyl mark on K4 all the way to zero methylation. As trimethylation of H3K4 (H3K4me3) is often associated with promoters of actively transcribed genes, removal of H3K4me3 by KDM5A is believed to promote transcriptional repression of its target genes. Mis-regulation of KDM5A has been shown to contribute to pathogenesis of lung and gastric cancer, as well as to the resistance to receptor tyrosine kinase inhibitors in non-small cell lung cancer. Yet, the mechanisms by which KDM5A is recruited to its target loci and how it functions on chromatin are largely unknown. In addition to its catalytic jumonji C (JmjC) eraser domain, KDM5A contains three PHD reader domains that are hypothesized to regulate the biological function of KDM5A. It is unknown, however, if and how these reader domains play a functional role in the demethylation reaction of KDM5A. In this thesis, using an array of biochemical and biophysical techniques, we show that the PHD1 domain specifically recognizes an unmethylated histone H3 tail, which interestingly is the final product of KDM5A. We also show that occupancy of PHD1 by its ligand allosterically stimulates the demethylase activity of the demethylase on histone tail peptides and on in vitro reconstituted nucleosome substrates. Together, these observations reveal an unprecedented allosteric regulation of a histone demethylase by one of its auxiliary non-catalytic domain and, challenges the prevailing model that PHD domains solely act to recruit chromatin-modifying enzymes to their target sites. Moreover, our findings suggest a model by which demethylation could spread on chromatin through a positive feedback-based mechanism. Furthermore, this mode of regulation provides new alternative mechanisms for the development of cancer therapies to specifically target RBP2 activity when mis-regulated in lung and gastric cancer. Here, we will also present data toward our initial efforts for the development of a small molecule allosteric modulator of KDM5A as well as our initial attempts to determine structural features of the enzyme by electron microscopy EM.
PDF
