Su Qin

A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1

We report the design and characterization of UNC3866, a potent antagonist of the methyl-lysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb Repressive Complex 1 to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently with a Kd of ∼100 nM for each, and is 6- to 18-fold selective versus seven other CBX and CDY chromodomains while being highly selective versus >250 other protein targets. X-ray crystallography revealed that UNC3866 closely mimics the interactions of the methylated H3 tail with these chromodomains. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform-dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, a known CBX7 phenotype, while UNC4219, a methylated negative control compound, has negligible effects.

Epigenetic targets and drug discovery Part 1: Histone methylation

Dynamic chromatin structure is modulated by post-translational modifications on histones, such as acetylation, phosphorylation and methylation. Research on histone methylation has become the most flourishing area of epigenetics in the past fourteen years, and a large amount of data has been accumulated regarding its biology and disease implications. Correspondingly, a lot of efforts have been made to develop small molecule compounds that can specifically modulate histone methyltransferases and methylation reader proteins, aiming for potential therapeutic drugs. Here, we summarize recent progress in chemical probe and drug discovery of histone methyltransferases and methylation reader proteins. For each target, we will review their biological/biochemical functions first, and then focus on their disease implications and drug discovery. We can also see that structure-based compound design and optimization plays a critical role in facilitating the development of highly potent and selective chemical probes and inhibitors for these targets.

Tudor domains of the PRC2 components PHF1 and PHF19 selectively bind to histone H3K36me3

PRC2 is the major H3K27 methyltransferase and is responsible for maintaining repressed gene expression patterns throughout development. It contains four core components: EZH2, EED, SUZ12 and RbAp46/48 and some cell-type specific components. In this study, we focused on characterizing the histone binding domains of PHF1 and PHF19, and found that the Tudor domains of PHF1 and PHF19 selectively bind to histone H3K36me3. Structural analysis of these Tudor domains also shed light on how these Tudor domains selectively bind to histone H3K36me3. The histone H3K36me3 binding by the Tudor domains of PHF1, PHF19 and likely MTF2 provide another recruitment and regulatory mechanism for the PRC2 complex. In addition, we found that the first PHD domains of PHF1 and PHF19 do not exhibit histone H3K4 binding ability, nor do they affect the Tudor domain binding to histones.

Associate editor: B. TeicherEpigenetic targets and drug discovery: Part 1: Histone methylation

Dynamic chromatin structure is modulated by post-translational modifications on histones, such as acetylation, phosphorylation and methylation. Research on histone methylation has become the most flourishing area of epigenetics in the past fourteen years, and a large amount of data has been accumulated regarding its biology and disease implications. Correspondingly, a lot of efforts have been made to develop small molecule compounds that can specifically modulate histone methyltransferases and methylation reader proteins, aiming for potential therapeutic drugs. Here, we summarize recent progress in chemical probe and drug discovery of histone methyltransferases and methylation reader proteins. For each target, we will review their biological/biochemical functions first, and then focus on their disease implications and drug discovery. We can also see that structure-based compound design and optimization plays a critical role in facilitating the development of highly potent and selective chemical probes and inhibitors for these targets.

Family-wide characterization of histone binding abilities of human CW domain containing proteins

Covalent modifications of histone N-terminal tails play a critical role in regulating chromatin structure and controlling gene expression. These modifications are controlled by histone-modifying enzymes and read out by histone-binding proteins. Numerous proteins have been identified as histone modification readers. Here we report the family-wide characterization of histone binding abilities of human CW domain-containing proteins. We demonstrate that the CW domains in ZCWPW2 and MORC3/4 selectively recognize histone H3 trimethylated at Lys-4, similar to ZCWPW1 reported previously, while the MORC1/2 and LSD2 lack histone H3 Lys-4 binding ability. Our crystal structures of the CW domains of ZCWPW2 and MORC3 in complex with the histone H3 trimethylated at Lys-4 peptide reveal the molecular basis of this interaction. In each complex, two tryptophan residues in the CW domain form the “floor” and “right wall,” respectively, of the methyllysine recognition cage. Our mutation results based on ZCWPW2 reveal that the right wall tryptophan residue is essential for binding, and the floor tryptophan residue enhances binding affinity. Our structural and mutational analysis highlights the conserved roles of the cage residues of CW domain across the histone methyllysine binders but also suggests why some CW domains lack histone binding ability.

Structural basis for substrate recognition by the human N-terminal methyltransferase 1

α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides, respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.

Structural basis for the regulatory role of the PPxY motifs in the thioredoxin-interacting protein TXNIP.

TXNIP (thioredoxin-interacting protein) negatively regulates the antioxidative activity of thioredoxin and participates in pleiotropic cellular processes. Its deregulation is linked to various human diseases, including diabetes, acute myeloid leukaemia and cardiovascular diseases. The E3 ubiquitin ligase Itch (Itchy homologue) polyubiquitinates TXNIP to promote its degradation via the ubiquitin-proteasome pathway, and this Itch-mediated polyubiquitination of TXNIP is dependent on the interaction of the four WW domains of Itch with the two PPxY motifs of TXNIP. However, the molecular mechanism of this interaction of TXNIP with Itch remains elusive. In the present study, we found that each of the four WW domains of Itch exhibited different binding affinities for TXNIP, whereas multivalent engagement between the four WW domains of Itch and the two PPxY motifs of TXNIP resulted in their strong binding avidity. Our structural analyses demonstrated that the third and fourth WW domains of Itch were able to recognize both PPxY motifs of TXNIP simultaneously, supporting a multivalent binding mode between Itch and TXNIP. Interestingly, the phosphorylation status on the tyrosine residue of the PPxY motifs of TXNIP serves as a molecular switch in its choice of binding partners and thereby downstream biological signalling outcomes. Phosphorylation of this tyrosine residue of TXNIP diminished the binding capability of PPxY motifs of TXNIP to Itch, whereas this phosphorylation is a prerequisite to the binding activity of TXNIP to SHP2 [SH2 (Src homology 2) domain-containing protein tyrosine phosphatase 2] and their roles in stabilizing the phosphorylation and activation of CSK (c-Src tyrosine kinase).

Peptide recognition by heterochromatin protein 1 (HP1) chromoshadow domains revisited: Plasticity in the pseudosymmetric histone binding site of human HP1.

Heterochromatin protein 1 (HP1), a highly conserved non-histone chromosomal protein in eukaryotes, plays important roles in the regulation of gene transcription. Each of the three human homologs of HP1 includes a chromoshadow domain (CSD). The CSD interacts with various proteins bearing the PXVXL motif but also with a region of histone H3 that bears the similar PXXVXL motif. The latter interaction has not yet been resolved in atomic detail. Here we demonstrate that the CSDs of all three human HP1 homologs have comparable affinities to the PXXVXL motif of histone H3. The HP1 C-terminal extension enhances the affinity, as does the increasing length of the H3 peptide. The crystal structure of the human HP1γ CSD (CSDγ) in complex with an H3 peptide suggests that recognition of H3 by CSDγ to some extent resembles CSD-PXVXL interaction. Nevertheless, the prolyl residue of the PXXVXL motif appears to play a role distinct from that of Pro in the known HP1β CSD-PXVXL complexes. We consequently generalize the historical CSD-PXVXL interaction model and expand the search scope for additional CSD binding partners.

Structural and functional analysis of the DOT1L-AF10 complex reveals mechanistic insights into MLL-AF10-associated leukemogenesis.

The mixed-lineage leukemia (MLL)-AF10 fusion oncoprotein recruits DOT1L to the homeobox A (HOXA) gene cluster through its octapeptide motif leucine zipper (OM-LZ), thereby inducing and maintaining the MLL-AF10-associated leukemogenesis. However, the recognition mechanism between DOT1L and MLL-AF10 is unclear. Here, we present the crystal structures of both apo AF10OM-LZ and its complex with the coiled-coil domain of DOT1L. Disruption of the DOT1L-AF10 interface abrogates MLL-AF10-associated leukemic transformation. We further show that zinc stabilizes the DOT1L-AF10 complex and may be involved in the regulation of the HOXA gene expression. Our studies may also pave the way for the rational design of therapeutic drugs against MLL-rearranged leukemia.

The Chromodomain of Polycomb: Methylation Reader and Beyond

Abstract Polycomb group (PcG) proteins are epigenetic repressors that are essential for cell differentiation and development. The Pc protein is a key member of the PcG family and a core component of the Polycomb repressive complex PRC1. In Drosophila, the chromodomain of Pc specifically recognizes the gene silencing mark trimethylated lysine 27 of histone H3 generated by the PRC2 complex, thus delivering the PRC1 complex to targeted chromatin sites. The mammalian Pc homologs, however, bind differentially to methylated histone H3. In addition, other mechanisms may also contribute to the chromatin targeting of Pc and its associated complexes. Here we summarize the current knowledge of the Pc chromodomain and discuss its role as a histone methylation reader from a structural point of view. The cross talk between lysine methylation and other posttranslational modifications on histones and other putative nonhistone targets of Pc protein is also discussed. Recent advancement in the chemical probe development against the chromodomain is also reviewed.Polycomb group (PcG) proteins are epigenetic repressors that are essential for cell differentiation and development. The Pc protein is a key member of the PcG family and a core component of the Polycomb repressive complex PRC1. In Drosophila, the chromodomain of Pc specifically recognizes the gene silencing mark trimethylated lysine 27 of histone H3 generated by the PRC2 complex, thus delivering the PRC1 complex to targeted chromatin sites. The mammalian Pc homologs, however, bind differentially to methylated histone H3. In addition, other mechanisms may also contribute to the chromatin targeting of Pc and its associated complexes. Here we summarize the current knowledge of the Pc chromodomain and discuss its role as a histone methylation reader from a structural point of view. The cross talk between lysine methylation and other posttranslational modifications on histones and other putative nonhistone targets of Pc protein is also discussed. Recent advancement in the chemical probe development against the chromodomain is also reviewed.