Publications by authors named "Brianna J Klein"

26 Publications

  • Page 1 of 1

Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis.

J Mol Cell Cardiol 2020 Dec 24;153:44-59. Epub 2020 Dec 24.

Division of Cardiology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; The Consortium for Fibrosis Research & Translation, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Pharmacology Graduate Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

Direct reprogramming of fibroblasts into cardiomyocytes (CMs) represents a promising strategy to regenerate CMs lost after ischemic heart injury. Overexpression of GATA4, HAND2, MEF2C, TBX5, miR-1, and miR-133 (GHMT2m) along with transforming growth factor beta (TGF-β) inhibition efficiently promote reprogramming. However, the mechanisms by which TGF-β blockade promotes cardiac reprogramming remain unknown. Here, we identify interactions between the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3, the SWI/SNF remodeling complex subunit BRG1, and cardiac transcription factors. Furthermore, canonical TGF-β signaling regulates the interaction between GATA4 and JMJD3. TGF-β activation impairs the ability of GATA4 to bind target genes and prevents demethylation of H3K27 at cardiac gene promoters during cardiac reprogramming. Finally, a mutation in GATA4 (V267M) that is associated with congenital heart disease exhibits reduced binding to JMJD3 and impairs cardiomyogenesis. Thus, we have identified an epigenetic mechanism wherein canonical TGF-β pathway activation impairs cardiac gene programming, in part by interfering with GATA4-JMJD3 interactions.
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http://dx.doi.org/10.1016/j.yjmcc.2020.12.005DOI Listing
December 2020

Molecular mechanism of the MORC4 ATPase activation.

Nat Commun 2020 10 29;11(1):5466. Epub 2020 Oct 29.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, 80045, USA.

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.
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http://dx.doi.org/10.1038/s41467-020-19278-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596504PMC
October 2020

TCF19 Promotes Cell Proliferation through Binding to the Histone H3K4me3 Mark.

Biochemistry 2020 02 2;59(4):389-399. Epub 2019 Dec 2.

Biophysics and Structural Genomics Division , Saha Institute of Nuclear Physics , 1/AF Bidhannagar , Kolkata 700064 , India.

Transcription factor 19 (TCF19) plays critical roles in type 1 diabetes and the maintenance of pancreatic β cells. Recent studies have also implicated TCF19 in cell proliferation of hepatic carcinoma and non-small cell lung carcinoma; however, the mechanism underlying this regulation remains elusive. At the molecular level, TCF19 contains two modules, the plant homeodomain (PHD) finger and the forkhead-associated (FHA) domain, of unclear function. Here, we show that TCF19 mediates hepatocellular carcinoma HepG2 cell proliferation through its PHD finger that recognizes trimethylated lysine 4 of histone 3 (H3K4me3). W316 of the PHD finger of TCF19 is one of the critical residues eliciting this function. Whole genome microarray analysis and orthogonal cell-based assays identified a large subset of genes involved in cell survival and proliferation that depend on TCF19. Our data suggest that TCF19 acts as a pro-oncogene in hepatocellular carcinoma cells and that its functional PHD finger is critical in cell proliferation.
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http://dx.doi.org/10.1021/acs.biochem.9b00771DOI Listing
February 2020

Molecular Basis for the PZP Domain of BRPF1 Association with Chromatin.

Structure 2020 01 8;28(1):105-110.e3. Epub 2019 Nov 8.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

The assembly of human histone acetyltransferase MOZ/MORF complexes relies on the scaffolding bromodomain plant homeodomain (PHD) finger 1 (BRPF1) subunit. The PHD-zinc-knuckle-PHD module of BRPF1 (BRPF1) has been shown to associate with the histone H3 tail and DNA; however, the molecular mechanism underlying recognition of H3 and the relationship between the histone and DNA-binding activities remain unclear. In this study, we report the crystal structure of BRPF1 bound to the H3 tail and characterize the role of the bipartite interaction in the engagement of BRPF1 with the nucleosome core particle (NCP). We find that although both interactions of BRPF1 with the H3 tail and DNA are required for tight binding to NCP and for acetyltransferase function of the BRPF1-MORF-ING5-MEAF6 complex, binding to extranucleosomal DNA dominates. Our findings suggest that functionally active BRPF1 might be important in stabilization of the MOZ/MORF complexes at chromatin with accessible DNA.
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http://dx.doi.org/10.1016/j.str.2019.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949404PMC
January 2020

E2F1 acetylation directs p300/CBP-mediated histone acetylation at DNA double-strand breaks to facilitate repair.

Nat Commun 2019 10 30;10(1):4951. Epub 2019 Oct 30.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park, Smithville, TX, 78957, USA.

E2F1 and retinoblastoma (RB) tumor-suppressor protein not only regulate the periodic expression of genes important for cell proliferation, but also localize to DNA double-strand breaks (DSBs) to promote repair. E2F1 is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here we demonstrate that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases and that this interaction is required for the recruitment of p300 and CBP to DSBs and the induction of histone acetylation at sites of damage. A knock-in mutation that blocks E2F1 acetylation abolishes the recruitment of p300 and CBP to DSBs and also the accumulation of other chromatin modifying activities and repair factors, including Tip60, BRG1 and NBS1, and renders mice hypersensitive to ionizing radiation (IR). These findings reveal an important role for E2F1 acetylation in orchestrating the remodeling of chromatin structure at DSBs to facilitate repair.
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http://dx.doi.org/10.1038/s41467-019-12861-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821830PMC
October 2019

Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation.

Nat Commun 2019 10 17;10(1):4724. Epub 2019 Oct 17.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, 80045, USA.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORF). The crystal structure of MORF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.
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http://dx.doi.org/10.1038/s41467-019-12551-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797804PMC
October 2019

Selective binding of the PHD6 finger of MLL4 to histone H4K16ac links MLL4 and MOF.

Nat Commun 2019 05 24;10(1):2314. Epub 2019 May 24.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, 80045, USA.

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation.
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http://dx.doi.org/10.1038/s41467-019-10324-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534582PMC
May 2019

Mechanism for autoinhibition and activation of the MORC3 ATPase.

Proc Natl Acad Sci U S A 2019 03 8;116(13):6111-6119. Epub 2019 Mar 8.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045;

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW.
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http://dx.doi.org/10.1073/pnas.1819524116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442546PMC
March 2019

Structural insights into the π-π-π stacking mechanism and DNA-binding activity of the YEATS domain.

Nat Commun 2018 11 1;9(1):4574. Epub 2018 Nov 1.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, 80045, USA.

The YEATS domain has been identified as a reader of histone acylation and more recently emerged as a promising anti-cancer therapeutic target. Here, we detail the structural mechanisms for π-π-π stacking involving the YEATS domains of yeast Taf14 and human AF9 and acylated histone H3 peptides and explore DNA-binding activities of these domains. Taf14-YEATS selects for crotonyllysine, forming π stacking with both the crotonyl amide and the alkene moiety, whereas AF9-YEATS exhibits comparable affinities to saturated and unsaturated acyllysines, engaging them through π stacking with the acyl amide. Importantly, AF9-YEATS is capable of binding to DNA, whereas Taf14-YEATS is not. Using a structure-guided approach, we engineered a mutant of Taf14-YEATS that engages crotonyllysine through the aromatic-aliphatic-aromatic π stacking and shows high selectivity for the crotonyl H3K9 modification. Our findings shed light on the molecular principles underlying recognition of acyllysine marks and reveal a previously unidentified DNA-binding activity of AF9-YEATS.
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http://dx.doi.org/10.1038/s41467-018-07072-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212594PMC
November 2018

The ZZ domain of p300 mediates specificity of the adjacent HAT domain for histone H3.

Nat Struct Mol Biol 2018 09 27;25(9):841-849. Epub 2018 Aug 27.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA.

Human p300 is a transcriptional co-activator and a major acetyltransferase that acetylates histones and other proteins facilitating gene transcription. The activity of p300 relies on the fine-tuned interactome that involves a dozen p300 domains and hundreds of binding partners and links p300 to a wide range of vital signaling events. Here, we report a novel function of the ZZ-type zinc finger (ZZ) of p300 as a reader of histone H3. We show that the ZZ domain and acetyllysine-recognizing bromodomain of p300 play critical roles in modulating p300 enzymatic activity and its association with chromatin. The acetyllysine binding function of bromodomain is essential for acetylation of histones H3 and H4, whereas interaction of the ZZ domain with H3 promotes selective acetylation of the histone H3K27 and H3K18 sites.
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http://dx.doi.org/10.1038/s41594-018-0114-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482957PMC
September 2018

Recognition of cancer mutations in histone H3K36 by epigenetic writers and readers.

Epigenetics 2018 23;13(7):683-692. Epub 2018 Aug 23.

a Department of Pharmacology , University of Colorado School of Medicine , Aurora , CO , USA.

Histone posttranslational modifications control the organization and function of chromatin. In particular, methylation of lysine 36 in histone H3 (H3K36me) has been shown to mediate gene transcription, DNA repair, cell cycle regulation, and pre-mRNA splicing. Notably, mutations at or near this residue have been causally linked to the development of several human cancers. These observations have helped to illuminate the role of histones themselves in disease and to clarify the mechanisms by which they acquire oncogenic properties. This perspective focuses on recent advances in discovery and characterization of histone H3 mutations that impact H3K36 methylation. We also highlight findings that the common cancer-related substitution of H3K36 to methionine (H3K36M) disturbs functions of not only H3K36me-writing enzymes but also H3K36me-specific readers. The latter case suggests that the oncogenic effects could also be linked to the inability of readers to engage H3K36M.
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http://dx.doi.org/10.1080/15592294.2018.1503491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224213PMC
March 2019

Yaf9 subunit of the NuA4 and SWR1 complexes targets histone H3K27ac through its YEATS domain.

Nucleic Acids Res 2018 01;46(1):421-430

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Yaf9 is an integral part of the NuA4 acetyltransferase and the SWR1 chromatin remodeling complexes. Here, we show that Yaf9 associates with acetylated histone H3 with high preference for H3K27ac. The crystal structure of the Yaf9 YEATS domain bound to the H3K27ac peptide reveals that the sequence C-terminal to K27ac stabilizes the complex. The side chain of K27ac inserts between two aromatic residues, mutation of which abrogates the interaction in vitro and leads in vivo to phenotypes similar to YAF9 deletion, including loss of SWR1-dependent incorporation of variant histone H2A.Z. Our findings reveal the molecular basis for the recognition of H3K27ac by a YEATS reader and underscore the importance of this interaction in mediating Yaf9 function within the NuA4 and SWR1 complexes.
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http://dx.doi.org/10.1093/nar/gkx1151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758897PMC
January 2018

Characterization of histone acylations links chromatin modifications with metabolism.

Nat Commun 2017 10 26;8(1):1141. Epub 2017 Oct 26.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Over the last decade, numerous histone acyl post-translational modifications (acyl-PTMs) have been discovered, of which the functional significance is still under intense study. Here, we use high-resolution mass spectrometry to accurately quantify eight acyl-PTMs in vivo and after in vitro enzymatic assays. We assess the ability of seven histone acetyltransferases (HATs) to catalyze acylations on histones in vitro using short-chain acyl-CoA donors, proving that they are less efficient towards larger acyl-CoAs. We also observe that acyl-CoAs can acylate histones through non-enzymatic mechanisms. Using integrated metabolomic and proteomic approaches, we achieve high correlation (R  > 0.99) between the abundance of acyl-CoAs and their corresponding acyl-PTMs. Moreover, we observe a dose-dependent increase in histone acyl-PTM abundances in response to acyl-CoA supplementation in in nucleo reactions. This study represents a comprehensive profiling of scarcely investigated low-abundance histone marks, revealing that concentrations of acyl-CoAs affect histone acyl-PTM abundances by both enzymatic and non-enzymatic mechanisms.
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http://dx.doi.org/10.1038/s41467-017-01384-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656686PMC
October 2017

A Unique pH-Dependent Recognition of Methylated Histone H3K4 by PPS and DIDO.

Structure 2017 10 14;25(10):1530-1539.e3. Epub 2017 Sep 14.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

The protein partner of Sans-fille (PPS) and its human homolog DIDO mediate diverse chromatin activities, including the regulation of stemness genes in embryonic stem cells and splicing in Drosophila. Here, we show that the PHD fingers of PPS and DIDO recognize the histone mark H3K4me3 in a pH-dependent manner: the binding is enhanced at high pH values but is decreased at low pH. Structural analysis reveals that the pH dependency is due to the presence of a histidine residue in the K4me3-binding aromatic cage of PPS. The pH-dependent mechanism is conserved in DIDO but is lost in yeast Bye1. Acidification of cells leads to the accelerated efflux of endogenous DIDO, indicating the pH-dependent sensing of H3K4me3 in vivo. This novel mode for the recognition of H3K4me3 establishes the PHD fingers of PPS and DIDO as unique epigenetic readers and high pH sensors and suggests a role for the histidine switch during mitosis.
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http://dx.doi.org/10.1016/j.str.2017.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679713PMC
October 2017

Recognition of Histone H3K14 Acylation by MORF.

Structure 2017 04 9;25(4):650-654.e2. Epub 2017 Mar 9.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

The monocytic leukemia zinc-finger protein-related factor (MORF) is a transcriptional coactivator and a catalytic subunit of the lysine acetyltransferase complex implicated in cancer and developmental diseases. We have previously shown that the double plant homeodomain finger (DPF) of MORF is capable of binding to acetylated histone H3. Here we demonstrate that the DPF of MORF recognizes many newly identified acylation marks. The mass spectrometry study provides comprehensive analysis of H3K14 acylation states in vitro and in vivo. The crystal structure of the MORF DPF-H3K14butyryl complex offers insight into the selectivity of this reader toward lipophilic acyllysine substrates. Together, our findings support the mechanism by which the acetyltransferase MORF promotes spreading of histone acylation.
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http://dx.doi.org/10.1016/j.str.2017.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415407PMC
April 2017

PHF20 Readers Link Methylation of Histone H3K4 and p53 with H4K16 Acetylation.

Cell Rep 2016 10;17(4):1158-1170

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

PHF20 is a core component of the lysine acetyltransferase complex MOF (male absent on the first)-NSL (non-specific lethal) that generates the major epigenetic mark H4K16ac and is necessary for transcriptional regulation and DNA repair. The role of PHF20 in the complex remains elusive. Here, we report on functional coupling between methylation readers in PHF20. We show that the plant homeodomain (PHD) finger of PHF20 recognizes dimethylated lysine 4 of histone H3 (H3K4me2) and represents an example of a native reader that selects for this modification. Biochemical and structural analyses help to explain this selectivity and the preference of Tudor2, another reader in PHF20, for dimethylated p53. Binding of the PHD finger to H3K4me2 is required for histone acetylation, accumulation of PHF20 at target genes, and transcriptional activation. Together, our findings establish a unique PHF20-mediated link between MOF histone acetyltransferase (HAT), p53, and H3K4me2, and suggest a model for rapid spreading of H4K16ac-enriched open chromatin.
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http://dx.doi.org/10.1016/j.celrep.2016.09.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125728PMC
October 2016

ASH1L Links Histone H3 Lysine 36 Dimethylation to MLL Leukemia.

Cancer Discov 2016 07 6;6(7):770-83. Epub 2016 May 6.

Department of Pathology, Stanford University School of Medicine, Stanford, California.

Unlabelled: Numerous studies in multiple systems support that histone H3 lysine 36 dimethylation (H3K36me2) is associated with transcriptional activation; however, the underlying mechanisms are not well defined. Here, we show that the H3K36me2 chromatin mark written by the ASH1L histone methyltransferase is preferentially bound in vivo by LEDGF, a mixed-lineage leukemia (MLL)-associated protein that colocalizes with MLL, ASH1L, and H3K36me2 on chromatin genome wide. Furthermore, ASH1L facilitates recruitment of LEDGF and wild-type MLL proteins to chromatin at key leukemia target genes and is a crucial regulator of MLL-dependent transcription and leukemic transformation. Conversely, KDM2A, an H3K36me2 demethylase and Polycomb group silencing protein, antagonizes MLL-associated leukemogenesis. Our studies are the first to provide a basic mechanistic insight into epigenetic interactions wherein placement, interpretation, and removal of H3K36me2 contribute to the regulation of gene expression and MLL leukemia, and suggest ASH1L as a novel target for therapeutic intervention.

Significance: Epigenetic regulators play vital roles in cancer pathogenesis and represent a new frontier in therapeutic targeting. Our studies provide basic mechanistic insight into the role of H3K36me2 in transcription activation and MLL leukemia pathogenesis and implicate ASH1L histone methyltransferase as a promising target for novel molecular therapy. Cancer Discov; 6(7); 770-83. ©2016 AACR.See related commentary by Balbach and Orkin, p. 700This article is highlighted in the In This Issue feature, p. 681.
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http://dx.doi.org/10.1158/2159-8290.CD-16-0058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4930721PMC
July 2016

Chromatin condensation and recruitment of PHD finger proteins to histone H3K4me3 are mutually exclusive.

Nucleic Acids Res 2016 07 25;44(13):6102-12. Epub 2016 Mar 25.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA

Histone post-translational modifications, and specific combinations they create, mediate a wide range of nuclear events. However, the mechanistic bases for recognition of these combinations have not been elucidated. Here, we characterize crosstalk between H3T3 and H3T6 phosphorylation, occurring in mitosis, and H3K4me3, a mark associated with active transcription. We detail the molecular mechanisms by which H3T3ph/K4me3/T6ph switches mediate activities of H3K4me3-binding proteins, including those containing plant homeodomain (PHD) and double Tudor reader domains. Our results derived from nuclear magnetic resonance chemical shift perturbation analysis, orthogonal binding assays and cell fluorescence microscopy studies reveal a strong anti-correlation between histone H3T3/T6 phosphorylation and retention of PHD finger proteins in chromatin during mitosis. Together, our findings uncover the mechanistic rules of chromatin engagement for H3K4me3-specific readers during cell division.
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http://dx.doi.org/10.1093/nar/gkw193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291243PMC
July 2016

G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression.

Nat Commun 2016 Mar 10;7:10810. Epub 2016 Mar 10.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression.
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http://dx.doi.org/10.1038/ncomms10810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792926PMC
March 2016

Bivalent interaction of the PZP domain of BRPF1 with the nucleosome impacts chromatin dynamics and acetylation.

Nucleic Acids Res 2016 Jan 30;44(1):472-84. Epub 2015 Nov 30.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA

BRPF1 (bromodomain PHD finger 1) is a core subunit of the MOZ histone acetyltransferase (HAT) complex, critical for normal developmental programs and implicated in acute leukemias. BRPF1 contains a unique assembly of zinc fingers, termed a PZP domain, the physiological role of which remains unclear. Here, we elucidate the structure-function relationship of this novel epigenetic reader and detail the biological and mechanistic consequences of its interaction with nucleosomes. PZP has a globular architecture and forms a 2:1 stoichiometry complex with the nucleosome, bivalently interacting with histone H3 and DNA. This binding impacts the nucleosome dynamics, shifting the DNA unwrapping/rewrapping equilibrium toward the unwrapped state and increasing DNA accessibility. We demonstrate that the DNA-binding function of the BRPF1 PZP domain is required for the MOZ-BRPF1-ING5-hEaf6 HAT complex to be recruited to chromatin and to acetylate nucleosomal histones. Our findings reveal a novel link between chromatin dynamics and MOZ-mediated acetylation.
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http://dx.doi.org/10.1093/nar/gkv1321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705663PMC
January 2016

An acetyl-methyl switch drives a conformational change in p53.

Structure 2015 Feb;23(2):322-31

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

Individual posttranslational modifications (PTMs) of p53 mediate diverse p53-dependent responses; however, much less is known about the combinatorial action of adjacent modifications. Here, we describe crosstalk between the early DNA damage response mark p53K382me2 and the surrounding PTMs that modulate binding of p53 cofactors, including 53BP1 and p300. The 1.8 Å resolution crystal structure of the tandem Tudor domain (TTD) of 53BP1 in complex with p53 peptide acetylated at K381 and dimethylated at K382 (p53K381acK382me2) reveals that the dual PTM induces a conformational change in p53. The α-helical fold of p53K381acK382me2 positions the side chains of R379, K381ac, and K382me2 to interact with TTD concurrently, reinforcing a modular design of double PTM mimetics. Biochemical and nuclear magnetic resonance analyses show that other surrounding PTMs, including phosphorylation of serine/threonine residues of p53, affect association with TTD. Our findings suggest a novel PTM-driven conformation switch-like mechanism that may regulate p53 interactions with binding partners.
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http://dx.doi.org/10.1016/j.str.2014.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317571PMC
February 2015

The histone-H3K4-specific demethylase KDM5B binds to its substrate and product through distinct PHD fingers.

Cell Rep 2014 Jan 9;6(2):325-35. Epub 2014 Jan 9.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA; Molecular Biology Program, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation and is implicated in carcinogenesis. It contains multiple conserved chromatin-associated domains, including three PHD fingers of unknown function. Here, we show that the first and third, but not the second, PHD fingers of KDM5B possess histone binding activities. The PHD1 finger is highly specific for unmodified histone H3 (H3K4me0), whereas the PHD3 finger shows preference for the trimethylated histone mark H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for genes involved in inflammatory responses, cell proliferation, adhesion, and migration. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with the histone deacetylase 1 (HDAC1) in gene repression. KDM5B is downregulated in triple-negative breast cancer relative to estrogen-receptor-positive breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion, and the PHD1-H3K4me0 interaction is essential for inhibiting migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a multivalent mechanism for KDM5B-mediated transcriptional regulation.
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http://dx.doi.org/10.1016/j.celrep.2013.12.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918441PMC
January 2014

Crosstalk between epigenetic readers regulates the MOZ/MORF HAT complexes.

Epigenetics 2014 Feb 29;9(2):186-93. Epub 2013 Oct 29.

Department of Pharmacology; University of Colorado School of Medicine; Aurora, CO USA.

The MOZ/MORF complexes represent an example of a chromatin-binding assembly whose recruitment to specific genomic regions and activity can be fine-tuned by posttranslational modifications of histones. Here we detail the structures and biological functions of epigenetic readers present in the four core subunits of the MOZ/MORF complexes, highlight the imperative role of combinatorial readout by the multiple readers, and discuss new research directions to advance our understanding of histone acetylation.
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http://dx.doi.org/10.4161/epi.26792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962528PMC
February 2014

Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity.

Genes Dev 2013 Sep;27(18):2009-24

Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Québec G1R 2J6, Canada;

Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.
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http://dx.doi.org/10.1101/gad.223396.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792477PMC
September 2013

RNA polymerase and transcription elongation factor Spt4/5 complex structure.

Proc Natl Acad Sci U S A 2011 Jan 27;108(2):546-50. Epub 2010 Dec 27.

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.

Spt4/5 in archaea and eukaryote and its bacterial homolog NusG is the only elongation factor conserved in all three domains of life and plays many key roles in cotranscriptional regulation and in recruiting other factors to the elongating RNA polymerase. Here, we present the crystal structure of Spt4/5 as well as the structure of RNA polymerase-Spt4/5 complex using cryoelectron microscopy reconstruction and single particle analysis. The Spt4/5 binds in the middle of RNA polymerase claw and encloses the DNA, reminiscent of the DNA polymerase clamp and ring helicases. The transcription elongation complex model reveals that the Spt4/5 is an upstream DNA holder and contacts the nontemplate DNA in the transcription bubble. These structures reveal that the cellular RNA polymerases also use a strategy of encircling DNA to enhance its processivity as commonly observed for many nucleic acid processing enzymes including DNA polymerases and helicases.
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http://dx.doi.org/10.1073/pnas.1013828108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3021056PMC
January 2011

The X-ray crystal structure of RNA polymerase from Archaea.

Nature 2008 Feb 30;451(7180):851-4. Epub 2008 Jan 30.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising an RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB orthologue TFB. It has been widely understood that precise comparisons of cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 A resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 A) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulphur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.
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http://dx.doi.org/10.1038/nature06530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805805PMC
February 2008