Publications by authors named "Christopher J Petell"

9 Publications

  • Page 1 of 1

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

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation.

Proc Natl Acad Sci U S A 2020 08 16;117(31):18439-18447. Epub 2020 Jul 16.

Department of Biochemistry, University of California, Riverside, CA 92521;

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.
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http://dx.doi.org/10.1073/pnas.2009316117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414182PMC
August 2020

Engineered Reader Proteins for Enhanced Detection of Methylated Lysine on Histones.

ACS Chem Biol 2020 01 1;15(1):103-111. Epub 2019 Nov 1.

Department of Chemistry , University of North Carolina at Chapel Hill , CB 3290, Chapel Hill , North Carolina 27599 , United States.

Histone post-translational modifications (PTMs) are crucial for many cellular processes including mitosis, transcription, and DNA repair. The cellular readout of histone PTMs is dependent on both the chemical modification and histone site, and the array of histone PTMs on chromatin is dynamic throughout the eukaryotic life cycle. Accordingly, methods that report on the presence of PTMs are essential tools for resolving open questions about epigenetic processes and for developing therapeutic diagnostics. Reader domains that recognize histone PTMs have shown potential as advantageous substitutes for anti-PTM antibodies, and engineering efforts aimed at enhancing reader domain affinities would advance their efficacy as antibody alternatives. Here we describe engineered chromodomains from and humans that bind more tightly to H3K9 methylation (H3K9me) marks and result in the tightest reported reader-H3K9me interaction to date. Point mutations near the binding interface of the HP1 chromodomain were screened in a combinatorial fashion, and a triple mutant was found that binds 20-fold tighter than the native scaffold without any loss in PTM-site selectivity. The beneficial mutations were then translated to a human homologue, CBX1, resulting in an even tighter interaction with H3K9me3. Furthermore, we show that these engineered readers (Readers) increase detection of H3K9me marks in several biochemical assays and outperform a commercial anti-H3K9me antibody in detecting H3K9me-containing nucleosomes , demonstrating the utility of Readers to complement antibodies in epigenetics research.
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http://dx.doi.org/10.1021/acschembio.9b00651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365037PMC
January 2020

Improved methods for the detection of histone interactions with peptide microarrays.

Sci Rep 2019 04 18;9(1):6265. Epub 2019 Apr 18.

Department of Biochemistry and Biophysics, 120 Mason Farm Rd, University of North Carolina at Chapel Hill, NC, Chapel Hill, 27599, USA.

Histone post-translational modifications contribute to chromatin function largely through the recruitment of effector proteins that contain specialized "reader" domains. While a significant number of reader domains have been characterized for their histone binding specificities, many of these domains remain poorly characterized. Peptide microarrays have been widely employed for the characterization of histone readers, as well as modifying enzymes and histone antibodies. While powerful, this platform has limitations in terms of its sensitivity and they frequently miss low affinity reader domain interactions. Here, we provide several technical changes that improve reader domain detection of low-affinity interactions. We show that 1% non-fat milk in 1X PBST as the blocking reagent during incubation improved reader-domain interaction results. Further, coupling this with post-binding high-salt washes and a brief, low-percentage formaldehyde cross-linking step prior to the high-salt washes provided the optimal balance between resolving specific low-affinity interactions and minimizing background or spurious signals. We expect this improved methodology will lead to the elucidation of previously unreported reader-histone interactions that will be important for chromatin function.
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http://dx.doi.org/10.1038/s41598-019-42711-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472351PMC
April 2019

A refined DNA methylation detection method using MspJI coupled quantitative PCR.

Anal Biochem 2017 09 15;533:1-9. Epub 2017 Jun 15.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, United States; Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, United States. Electronic address:

DNA methylation is a highly conserved epigenetic modification with critical roles ranging from protection against phage infection in bacteria to the regulation of gene expression in mammals. DNA methylation at specific sequences can be measured by using methylation dependent or sensitive restriction enzymes coupled to semi- or quantitative PCR (MD-qPCR). This study reports a refined MD-qPCR method for detecting gain or loss of DNA methylation at specific sites through the specific use of MspJI or HpaII, respectively. By employing varying concentrations of DNA with methylation ranging from 0 to 100%, our data provide evidence that compared to HpaII, MspJI increases the sensitivity and accuracy of detecting relative DNA methylation gains by MD-qPCR. We also show that the MspJI-coupled MD-qPCR can accurately determine the percent gain in DNA methylation at the Sall4 enhancer and is more sensitive than HpaII in detecting relative gains in DNA methylation at the Oct4 proximal enhancer during embryonic stem cell (ESC) differentiation. The high specificity and sensitivity of this targeted approach increases its potential as a diagnostic tool to detect relatively smaller gains in DNA methylation at specific sites from limited amounts of sample.
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http://dx.doi.org/10.1016/j.ab.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972016PMC
September 2017

Dnmt3b Methylates DNA by a Noncooperative Mechanism, and Its Activity Is Unaffected by Manipulations at the Predicted Dimer Interface.

Biochemistry 2018 07 4;57(29):4312-4324. Epub 2016 Nov 4.

Department of Biochemistry, Purdue University Center for Cancer Research , Purdue University , West Lafayette , Indiana 47907 , United States.

The catalytic domains of the de novo DNA methyltransferases Dnmt3a-C and Dnmt3b-C are highly homologous. However, their unique biochemical properties could potentially contribute to differences in the substrate preferences or biological functions of these enzymes. Dnmt3a-C forms tetramers through interactions at the dimer interface, which also promote multimerization on DNA and cooperativity. Similar to the case for processive enzymes, cooperativity allows Dnmt3a-C to methylate multiple sites on the same DNA molecule; however, it is unclear whether Dnmt3b-C methylates DNA by a cooperative or processive mechanism. The importance of the tetramer structure and cooperative mechanism is emphasized by the observation that the R882H mutation in the dimer interface of DNMT3A is highly prevalent in acute myeloid leukemia and leads to a substantial loss of its activity. Under conditions that distinguish between cooperativity and processivity, we show that in contrast to that of Dnmt3a-C, the activity of Dnmt3b-C is not cooperative and confirm the processivity of Dnmt3b-C and the full length Dnmt3b enzyme. Whereas the R878H mutation (mouse homologue of R882H) led to the loss of cooperativity of Dnmt3a-C, the activity and processivity of the analogous Dnmt3b-C R829H variant were comparable to those of the wild-type enzyme. Additionally, buffer acidification that attenuates the dimer interface interactions of Dnmt3a-C had no effect on Dnmt3b-C activity. Taken together, these results demonstrate an important mechanistic difference between Dnmt3b and Dnmt3a and suggest that interactions at the dimer interface may play a limited role in regulating Dnmt3b-C activity. These new insights have potential implications for the distinct biological roles of Dnmt3a and Dnmt3b.
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http://dx.doi.org/10.1021/acs.biochem.6b00964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992102PMC
July 2018

An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation.

Nucleic Acids Res 2016 09 13;44(16):7605-17. Epub 2016 May 13.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA

Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites.
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http://dx.doi.org/10.1093/nar/gkw426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027477PMC
September 2016

Regulation of glucose-dependent gene expression by the RNA helicase Dbp2 in Saccharomyces cerevisiae.

Genetics 2014 Nov 27;198(3):1001-14. Epub 2014 Aug 27.

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-2063 Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907-2064

Cellular homeostasis requires a fine balance between energy uptake, utilization, and growth. Dbp2 is a member of the DEAD-box protein family in Saccharomyces cerevisiae with characterized ATPase and helicase activity in vitro. DEAD-box RNA helicases are a class of enzymes that utilize ATP hydrolysis to remodel RNA and/or RNA-protein (RNP) composition. Dbp2 has been proposed to utilize its helicase activity in vivo to promote RNA-protein complex assembly of both messenger (m)RNAs and long noncoding (lnc)RNAs. Previous work from our laboratory demonstrated that loss of DBP2 enhances the lncRNA-dependent transcriptional induction of the GAL genes by abolishing glucose-dependent repression. Herein, we report that either a carbon source switch or glucose deprivation results in rapid export of Dbp2 to the cytoplasm. Genome-wide RNA sequencing identified a new class of antisense hexose transporter transcripts that are specifically upregulated upon loss of DBP2. Further investigation revealed that both sense and antisense hexose transporter (HXT) transcripts are aberrantly expressed in DBP2-deficient cells and that this expression pathway can be partially mimicked in wild-type cells by glucose depletion. We also find that Dbp2 promotes ribosome biogenesis and represses alternative ATP-producing pathways, as loss of DBP2 alters the transcript levels of ribosome biosynthesis (snoRNAs and associated proteins) and respiration gene products. This suggests that Dbp2 is a key integrator of nutritional status and gene expression programs required for energy homeostasis.
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http://dx.doi.org/10.1534/genetics.114.170019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224148PMC
November 2014

Long noncoding RNAs promote transcriptional poising of inducible genes.

PLoS Biol 2013 Nov 19;11(11):e1001715. Epub 2013 Nov 19.

Department of Biochemistry, Purdue University, West Lafayette, Indiana, United States of America.

Long noncoding RNAs (lncRNAs) are a class of molecules that impinge on the expression of protein-coding genes. Previous studies have suggested that the GAL cluster-associated lncRNAs of Saccharomyces cerevisiae repress expression of the protein-coding GAL genes. Herein, we demonstrate a previously unrecognized role for the GAL lncRNAs in activating gene expression. In yeast strains lacking the RNA helicase, DBP2, or the RNA decay enzyme, XRN1, we find that the GAL lncRNAs specifically accelerate gene expression from a prior repressive state. Furthermore, we provide evidence that the previously suggested repressive role is a result of specific mutant phenotypes, rather than a reflection of the normal, wild-type function of these noncoding RNAs. To shed light on the mechanism for lncRNA-dependent gene activation, we show that rapid induction of the protein-coding GAL genes is associated with faster recruitment of RNA polymerase II and reduced association of transcriptional repressors with GAL gene promoters. This suggests that the GAL lncRNAs enhance expression by derepressing the GAL genes. Consistently, the GAL lncRNAs enhance the kinetics of transcriptional induction, promoting faster expression of the protein-coding GAL genes upon the switch in carbon source. We suggest that the GAL lncRNAs poise inducible genes for rapid activation, enabling cells to more effectively trigger new transcriptional programs in response to cellular cues.
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http://dx.doi.org/10.1371/journal.pbio.1001715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3833879PMC
November 2013