Structural Paradigms in the Recognition of the Nucleosome Core Particle by Histone Lysine Methyltransferases.
Frontiers in Cell and Developmental Biology. 2020. 8:600.PMID: 32850785
Post-translational modifications (PTMs) of histone proteins play essential functions in shaping chromatin environment. Alone or in combination, these PTMs create templates recognized by dedicated proteins or change the chemistry of chromatin, enabling a myriad of nuclear processes to occur. Referred to as cross-talk, the positive or negative impact of a PTM on another PTM has rapidly emerged as a mechanism controlling nuclear transactions. One of those includes the stimulatory functions of histone H2B ubiquitylation on the methylation of histone H3 on K79 and K4 by Dot1L and COMPASS, respectively. While these findings were established early on, the structural determinants underlying the positive impact of H2B ubiquitylation on H3K79 and H3K4 methylation were resolved only recently. We will also review the molecular features controlling these cross-talks and the impact of H3K27 tri-methylation on EZH2 activity when embedded in the PRC2 complex.
Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication.
Science. 2014. 343(6176):1249-53.PMID: 24626927
Histone variants have been proposed to act as determinants for posttranslational modifications with widespread regulatory functions. We identify a histone-modifying enzyme that selectively methylates the replication-dependent histone H3 variant H3.1. The crystal structure of the SET domain of the histone H3 lysine-27 (H3K27) methyltransferase ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) in complex with a H3.1 peptide shows that ATXR5 contains a bipartite catalytic domain that specifically “reads” alanine-31 of H3.1. Variation at position 31 between H3.1 and replication-independent H3.3 is conserved in plants and animals, and threonine-31 in H3.3 is responsible for inhibiting the activity of ATXR5 and its paralog, ATXR6. Our results suggest a simple model for the mitotic inheritance of the heterochromatic mark H3K27me1 and the protection of H3.3-enriched genes against heterochromatization during DNA replication.
A Complex of C9ORF72 and p62 Uses Arginine Methylation to Eliminate Stress Granules by Autophagy.
Nature Communications. 2018. 9(1):2794.PMID: 30022074
Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.
Molecular Basis for the Methylation Specificity of ATXR5 for Histone H3.
Nucleic Acids Research. 2017. 45(11):6375-6387.PMID: 28383693
In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELATED PROTEIN 5 and 6 (ATXR5/6) regulate heterochromatic DNA replication and genome stability. Our initial studies showed that ATXR5/6 discriminate between histone H3 variants and preferentially methylate K27 on H3.1. In this study, we report three regulatory mechanisms contributing to the specificity of ATXR5/6. First, we show that ATXR5 preferentially methylates the R/F-K*-S/C-G/A-P/C motif with striking preference for hydrophobic and aromatic residues in positions flanking this core of five amino acids. Second, we demonstrate that post-transcriptional modifications of residues neighboring K27 that are typically associated with actively transcribed chromatin are detrimental to ATXR5 activity. Third, we show that ATXR5 PHD domain employs a narrow binding pocket to selectively recognize unmethylated K4 of histone H3. Finally, we demonstrate that deletion or mutation of the PHD domain reduces the catalytic efficiency (kcat/Km of AdoMet) of ATXR5 up to 58-fold, highlighting the multifunctional nature of ATXR5 PHD domain. Overall, our results suggest that several molecular determinants regulate ATXR5/6 methyltransferase activity and epigenetic inheritance of H3.1 K27me1 mark in plants.
Acetylation of PCNA Sliding Surface by Eco1 Promotes Genome Stability Through Homologous Recombination.
Molecular Cell. 2017. 65(1):78-90.PMID: 27916662
During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.
MFG-E8 Is Critical for Embryonic Stem Cell-Mediated T Cell Immunomodulation.
Stem Cell Reports. 2015. 5(5):741-752.PMID: 26455415
The molecules and mechanisms pertinent to the low immunogenicity of undifferentiated embryonic stem cells (ESCs) remain poorly understood. Here, we provide evidence that milk fat globule epidermal growth factor 8 (MFG-E8) is a vital mediator in this phenomenon and directly suppresses T cell immune responses. MFG-E8 is enriched in undifferentiated ESCs but diminished in differentiated ESCs. Upregulation of MFG-E8 in ESCs increases the successful engraftment of both undifferentiated and differentiated ESCs across major histocompatibility complex barriers. MFG-E8 suppresses T cell activation/proliferation and inhibits Th1, Th2, and Th17 subpopulations while increasing regulatory T cell subsets. Neutralizing MFG-E8 substantially abrogates these effects, whereas addition of recombinant MFG-E8 to differentiated ESCs restores immunosuppression. Furthermore, we provide the evidence that MFG-E8 suppresses T cell activation and regulates T cell polarization by inhibiting PKCθ phosphorylation through the α3/5βV integrin receptor. Our findings offer an approach to facilitate transplantation acceptance.
Discovery of Substrates for a SET Domain Lysine Methyltransferase Predicted by Multistate Computational Protein Design.
Structure. 2015. 23(1):206-215.PMID: 25533488
Characterization of lysine methylation has proven challenging despite its importance in biological processes such as gene transcription, protein turnover, and cytoskeletal organization. In contrast to other key posttranslational modifications, current proteomics techniques have thus far shown limited success at characterizing methyl-lysine residues across the cellular landscape. To complement current biochemical characterization methods, we developed a multistate computational protein design procedure to probe the substrate specificity of the protein lysine methyltransferase SMYD2. Modeling of substrate-bound SMYD2 identified residues important for substrate recognition and predicted amino acids necessary for methylation. Peptide- and protein- based substrate libraries confirmed that SMYD2 activity is dictated by the motif [LFM]-1-K(∗)-[AFYMSHRK]+1-[LYK]+2 around the target lysine K(∗). Comprehensive motif-based searches and mutational analysis further established four additional substrates of SMYD2. Our methodology paves the way to systematically predict and validate posttranslational modification sites while simultaneously pairing them with their associated enzymes.
The Functional Diversity of Protein Lysine Methylation.
Molecular Systems Biology. 2014. 10(4):724.PMID: 24714364
Large-scale characterization of post-translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination, has highlighted their importance in the regulation of a myriad of signaling events. While high-throughput technologies have tremendously helped cataloguing the proteins modified by these PTMs, the identification of lysine-methylated proteins, a PTM involving the transfer of one, two or three methyl groups to the ε-amine of a lysine side chain, has lagged behind. While the initial findings were focused on the methylation of histone proteins, several studies have recently identified novel non-histone lysine-methylated proteins. This review provides a compilation of all lysine methylation sites reported to date. We also present key examples showing the impact of lysine methylation and discuss the circuitries wired by this important PTM.
A non-canonical monovalent zinc finger stabilizes the integration of Cfp1 into the H3K4 methyltransferase complex COMPASS.
Nucleic Acids Research. 2020. 48(1):421-431.PMID: 31724694
COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS’s subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.
Structure and Conformational Dynamics of a COMPASS Histone H3K4 Methyltransferase Complex.
Cell. 2018. 174(5):1117-1126.PMID: 30100186
The methylation of histone 3 lysine 4 (H3K4) is carried out by an evolutionarily conserved family of methyltransferases referred to as complex of proteins associated with Set1 (COMPASS). The activity of the catalytic SET domain (su(var)3-9, enhancer-of-zeste, and trithorax) is endowed through forming a complex with a set of core proteins that are widely shared from yeast to humans. We obtained cryo-electron microscopy (cryo-EM) maps of the yeast Set1/COMPASS core complex at overall 4.0- to 4.4-Å resolution, providing insights into its structural organization and conformational dynamics. The Cps50 C-terminal tail weaves within the complex to provide a central scaffold for assembly. The SET domain, snugly positioned at the junction of the Y-shaped complex, is extensively contacted by Cps60 (Bre2), Cps50 (Swd1), and Cps30 (Swd3). The mobile SET-I motif of the SET domain is engaged by Cps30, explaining its key role in COMPASS catalytic activity toward higher H3K4 methylation states.
ATXR5/6 Forms Alternative Protein Complexes With PCNA and the Nucleosome Core Particle.
Journal of Molecular Biology. 2019. 431(7):1370-79.PMID: 30826376
The proliferating cell nuclear antigen (PCNA) is a sliding clamp associated with DNA polymerases and serves as a binding platform for the recruitment of regulatory proteins linked to DNA damage repair, cell cycle regulation, and epigenetic signaling. The histone H3 lysine-27 (H3K27) mono-methyltransferase Arabidopsis trithorax-related protein 5/6 (ATXR5/6) associates with PCNA, and this interaction has been proposed to act as a key determinant controlling the reestablishment of H3K27 mono-methylation following replication. In this study, we provide biochemical evidence showing that PCNA inhibits ATXR6 enzymatic activity. The structure of the ATXR6 PCNA-interacting peptide (PIP) in complex with PCNA indicates that a trio of hydrophobic residues contributes to the binding of the enzyme to the sliding clamp. Finally, despite the presence of three PIP binding clefts, only two molecules of ATXR6 bind to PCNA likely enabling the recruitment of a third protein to the sliding clamp. Collectively, these results rule out the model wherein PCNA-bound ATXR6 actively reestablishes H3K27 mono-methylation following DNA replication and provides insights into the role of ATXR6 PIP motif in its interaction with PCNA.
Crystal Structure of Campylobacter Jejuni Peroxide Regulator.
FEBS Letters. 2018. 592(13):2351-2360.PMID: 29856899
In Campylobacter jejuni (Cj), the metal-cofactored peroxide response regulator (PerR) transcription factor allows C. jejuni to respond to oxidative stresses. The crystal structure of the metalated form of CjPerR shows that the protein folds as an asymmetric dimer displaying structural differences in the orientation of its DNA-binding domain. Comparative analysis shows that such asymmetry is a conserved feature among crystallized PerR proteins, and mutational analysis reveals that residues found in the first α-helix of CjPerR contribute to DNA binding. These studies present the structure of CjPerR protein and highlight structural heterogeneity in the orientation of the metalated PerR DNA-binding domain which may underlie the ability of PerR to recognize DNA, control gene expression, and contribute to bacterial pathogenesis.
Lysine Methylation of FEN1 by SET7 Is Essential for Its Cellular Response to Replicative Stress.
Oncotarget. 2017. 8(39):64918-64931.PMID: 29029401
The DNA damage response (DDR) is central to the cell survival and it requires post-translational modifications, in part, to sense the damage, amplify the signaling response and recruit and regulate DNA repair enzymes. Lysine methylation of histones such as H4K20 and non-histone proteins including p53 has been shown to be essential for the mounting of the DDR. It is well-known that the lysine methyltransferase SET7 regulates the DDR, as cells lacking this enzyme are hypersensitive to chemotherapeutic drugs. To define addition substrates of SET7 involved in the DDR, we screened a peptide array encompassing potential lysine methylation sites from >100 key DDR proteins and identified peptides from 58 proteins to be lysine methylated defining a methylation consensus sequence of [S>K-2; S>R-1; K0] consistent with previous findings. We focused on K377 methylation of the Flap endonuclease 1 (FEN1), a structure specific endonuclease with important functions in Okazaki fragment processing during DNA replication as a substrate of SET7. FEN1 was monomethylated by SET7 in vivo in a cell cycle dependent manner with levels increasing as cells progressed through S phase and decreasing as they exited S phase, as detected using K377me1 specific antibodies. Although K377me1 did not affect the enzymatic activity of FEN1, it was required for the cellular response to replicative stress by FEN1. These finding define FEN1 as a new substrate of SET7 required for the DDR.
Improvement of the Reverse Tetracycline Transactivator by Single Amino Acid Substitutions That Reduce Leaky Target Gene Expression to Undetectable Levels.
Scientific Reports. 2016. 6:27697.PMID: 27323850
Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount.
Brighter Red Fluorescent Proteins by Rational Design of Triple-Decker Motif.
ACS Chemical Biology. 2016. 11(2):508-17.PMID: 26697759
Red fluorescent proteins (RFPs) are used extensively in chemical biology research as fluorophores for live cell imaging, as partners in FRET pairs, and as signal transducers in biosensors. For all of these applications, brighter RFP variants are desired. Here, we used rational design to increase the quantum yield of monomeric RFPs in order to improve their brightness. We postulated that we could increase quantum yield by restricting the conformational degrees of freedom of the RFP chromophore. To test our hypothesis, we introduced aromatic residues above the chromophore of mRojoA, a dim RFP containing a π-stacked Tyr residue directly beneath the chromophore, in order to reduce chromophore conformational flexibility via improved packing and steric complementarity. The best mutant identified displayed an absolute quantum yield increase of 0.07, representing an over 3-fold improvement relative to mRojoA. Remarkably, this variant was isolated following the screening of only 48 mutants, a library size that is several orders of magnitude smaller than those previously used to achieve equivalent gains in quantum yield in other RFPs. The crystal structure of the highest quantum yield mutant showed that the chromophore is sandwiched between two Tyr residues in a triple-decker motif of aromatic rings. Presence of this motif increases chromophore rigidity, as evidenced by the significantly reduced temperature factors compared to dim RFPs. Overall, the approach presented here paves the way for the rapid development of fluorescent proteins with higher quantum yield and overall brightness.
Molecular Basis for DPY-30 Association to COMPASS-like and NURF Complexes.
Structure. 2014. 22(12):1821-1830.PMID: 25456412
DPY-30 is a subunit of mammalian COMPASS-like complexes (complex of proteins associated with Set1) and regulates global histone H3 Lys-4 trimethylation. Here we report structural evidence showing that the incorporation of DPY-30 into COMPASS-like complexes is mediated by several hydrophobic interactions between an amphipathic α helix located on the C terminus of COMPASS subunit ASH2L and the inner surface of the DPY-30 dimerization/docking (D/D) module. Mutations impairing the interaction between ASH2L and DPY-30 result in a loss of histone H3K4me3 at the β locus control region and cause a delay in erythroid cell terminal differentiation. Using overlay assays, we defined a consensus sequence for DPY-30 binding proteins and found that DPY-30 interacts with BAP18, a subunit of the nucleosome remodeling factor complex. Overall, our results indicate that the ASH2L/DPY-30 complex is important for cell differentiation and provide insights into the ability of DPY-30 to associate with functionally divergent multisubunit complexes.
Context Dependency of Set1/COMPASS-mediated Histone H3 Lys4 Trimethylation.
Genes & Development. 2014. 28(2):115-20.PMID: 24402317
The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS’s H3K4me3 activity at promoter-proximal regions in a context-dependent manner.
Nanoparticle Concentration vs Surface Area in the Interaction of Thiol-Containing Molecules: Toward a Rational Nanoarchitectural Design of Hybrid Materials.
ACS Applied Materials & Interfaces. 2019. 11(19):17697-17705.PMID: 31013043
The effect of accounting for the total surface in the association of thiol-containing molecules to nanosilver was assessed using isothermal titration calorimetry, along with a new open access algorithm that calculates the total surface area for samples of different polydispersity. Further, we used advanced molecular dynamic calculations to explore the underlying mechanisms for the interaction of the studied molecules in the presence of a nanosilver surface in the form of flat surfaces or as three-dimensional pseudospherical nanostructures. Our data indicate that not only is the total surface area available for binding but also the supramolecular arrangements of the molecules in the near proximity of the nanosilver surface strongly affects the affinity of thiol-containing molecules to nanosilver surfaces.
Structural Analysis of the Ash2L/Dpy-30 Complex Reveals a Heterogeneity in H3K4 Methylation.
Structure. 2018. 26(12):1594-1603.PMID: 30270175
Dpy-30 is a regulatory subunit controlling the histone methyltransferase activity of the KMT2 enzymes in vivo. Paradoxically, in vitro methyltransferase assays revealed that Dpy-30 only modestly participates in the positive heterotypic allosteric regulation of these methyltransferases. Detailed genome-wide, molecular and structural studies reveal that an extensive network of interactions taking place at the interface between Dpy-30 and Ash2L are critical for the correct placement, genome-wide, of H3K4me2 and H3K4me3 but marginally contribute to the methyltransferase activity of KMT2 enzymes in vitro. Moreover, we show that H3K4me2 peaks persisting following the loss of Dpy-30 are found in regions of highly transcribed genes, highlighting an interplay between Complex of Proteins Associated with SET1 (COMPASS) kinetics and the cycling of RNA polymerase to control H3K4 methylation. Overall, our data suggest that Dpy-30 couples its modest positive heterotypic allosteric regulation of KMT2 methyltransferase activity with its ability to help the positioning of SET1/COMPASS to control epigenetic signaling.
Functional Insights Into the Interplay Between DNA Interaction and Metal Coordination in Ferric Uptake Regulators.
Scientific Reports. 2018. 8(1):7140.PMID: 29739988
Ferric uptake regulators (Fur) are a family of transcription factors coupling gene regulatory events to metal concentration. Recent evidence has expanded the mechanistic repertoires employed by Fur to activate or repress gene expression in the presence or absence of regulatory metals. However, the mechanistic basis underlying this extended repertoire has remained largely unexplored. In this study, we used an extensive set of mutations to demonstrate that Campylobacter jejuni Fur (CjFur) employs the same surface to positively and negatively control gene expression regardless of the presence or absence of metals. Moreover, the crystal structure determination of a CjFur devoid of any regulatory metals shows that subtle reorientation of the transcription factor DNA binding domain negatively impacts DNA binding, gene expression and gut colonization in chickens. Overall, these results highlight the versatility of the CjFur DNA binding domain in mediating all gene regulatory events controlled by the metalloregulator and that the full metalation of CjFur is critical to the Campylobacter jejuni life cycle in vivo.
Method for the Successful Crystallization of the Ferric Uptake Regulator From Campylobacter Jejuni.
Methods in Molecular Biology. 2017. 1512:79-89.PMID: 27885600
The Ferric Uptake Regulator (FUR) is a transcription factor (TF) regulating the expression of several genes to control iron levels in prokaryotes. Members of this family of TFs share a common structural scaffold that typically comprises two regions that include a DNA binding and dimerization domains. While this structural organization is conserved, FUR proteins employ different mechanisms to bind divergent DNA binding elements and regulate gene expression in the absence or presence of regulatory metals. These findings, combined with the observations that FUR proteins display different geometries in regard to the relative orientation of the DNA binding and dimerization domains, have highlighted an expanding repertoire of molecular mechanisms controlling the activity of this family of TFs. In this chapter, we present an overview of the methods to purify, crystallize, and solve the structure of Campylobacter jejuni FUR.
A Charge-Suppressing Strategy for Probing Protein Methylation.
Chemical Communications (Cambridge). 2016. 52(31):5474-7.PMID: 27021271
Methylation of arginine and lysine (RK) residues play essential roles in epigenetics and the regulation of gene expression. However, research in this area is often hindered by the lack of effective tools for probing the protein methylation. Here, we present an antibody-free strategy to capture protein methylation on RK residues by using chemical reactions to eliminate the charges on un-modified RK residues and peptide N-termini. Peptides containing methylated RK residues remain positively charged and are then enriched by strong cation exchange chromatography, followed by high-resolution mass spectrometry identification.
UTX Inhibition as Selective Epigenetic Therapy Against TAL1-driven T-cell Acute Lymphoblastic Leukemia.
Genes & Development. 2016. 30(5):508-21.PMID: 26944678
T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.
A Phosphorylation Switch on RbBP5 Regulates Histone H3 Lys4 Methylation.
Genes & Development. 2015. 29(2):123-8.PMID: 25593305
The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.
Keeping Them All Together: β-Propeller Domains in Histone Methyltransferase Complexes
Journal of Molecular Biology. 2014. 426(20):3363-75.PMID: 24853063
Histone methyltransferases (HKMTs) residing in multi-subunit protein complexes frequently require the presence of β-propeller proteins to achieve their biological functions. Recent biochemical studies have highlighted the functional diversity of these scaffolding proteins in maintaining the integrity of the complexes, allosterically regulating HKMT enzymatic activity and acting as “histone tethering devices” to facilitate the interaction between HKMTs and their substrates. Structural studies have revealed that, while β-propeller domain proteins share structural similarity, they employ divergent mechanisms to achieve their functions. This review focuses on the progress made in the last decade to identify the biochemical determinants underlying the functions of these important proteins.
Binding of RNA by APOBEC3G Controls Deamination-Independent Restriction of Retroviruses
Nucleic Acid Research. 2013. 41(15):7438-52.PMID: 23761443
APOBEC3G (A3G) is a host-encoded protein that potently restricts the infectivity of a broad range of retroviruses. This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity. It is not yet known to what extent deamination-independent processes contribute to the overall restriction, how they exactly work or how they are regulated. Here, we show that alanine substitution of either tryptophan 94 (W94A) or 127 (W127A) in the non-catalytic N-terminal domain of A3G severely impedes RNA binding and alleviates deamination-independent restriction while still maintaining DNA mutator activity. Substitution of both tryptophans (W94A/W127A) produces a more severe phenotype in which RNA binding and RNA-dependent protein oligomerization are completely abrogated. We further demonstrate that RNA binding is specifically required for crippling late reverse transcript accumulation, preventing proviral DNA integration and, consequently, restricting viral particle release. We did not find that deaminase activity made a significant contribution to the restriction of any of these processes. In summary, this work reveals that there is a direct correlation between A3G’s capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.