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Mami Oikawa, Angela Simeone, Eva Hormanseder, Marta Teperek, Vincent Gaggioli, Alan O’Doherty, Emma Falk, Matthieu Sporniak, Clive D’Santos, Valar Nila Roamio Franklin, Kamal Kishore, Charles R. Bradshaw, Declan Keane, Thomas Freour, Laurent David, Adrian T. Grzybowski, Alexander J. Ruthenburg, John Gurdon, and Jerome Jullien
Nature Communications, eISSN: 20411723, Published: 1 December 2020 Springer Science and Business Media LLC
Sperm contributes genetic and epigenetic information to the embryo to efficiently support development. However, the mechanism underlying such developmental competence remains elusive. Here, we investigated whether all sperm cells have a common epigenetic configuration that primes transcriptional program for embryonic development. Using calibrated ChIP-seq, we show that remodelling of histones during spermiogenesis results in the retention of methylated histone H3 at the same genomic location in most sperm cell. This homogeneously methylated fraction of histone H3 in the sperm genome is maintained during early embryonic replication. Such methylated histone fraction resisting post-fertilisation reprogramming marks developmental genes whose expression is perturbed upon experimental reduction of histone methylation. A similar homogeneously methylated histone H3 fraction is detected in human sperm. Altogether, we uncover a conserved mechanism of paternal epigenetic information transmission to the embryo through the homogeneous retention of methylated histone in a sperm cells population. In addition to paternal genetic material, sperm contributes epigenetic information to the embryo to efficiently support development. Here, the authors demonstrate a homogeneous paternal contribution to epigenetic information via sperm-derived modified histone transmission to the developing vertebrate embryo.
Adrian T. Grzybowski, Rohan N. Shah, William F. Richter, and Alexander J. Ruthenburg
Nature Protocols, ISSN: 17542189, eISSN: 17502799, Pages: 3275-3302, Published: 1 December 2019 Springer Science and Business Media LLC
Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly. ICeChIP (internally calibrated ChIP) uses spiked-in, defined nucleosomal standards to overcome the pitfalls of traditional ChIP experiments, enabling the measurement of antibody specificity and the absolute measurement of histone modification density at genomic loci.
Richard L. Bennett, Aditya Bele, Eliza C. Small, Christine M. Will, Behnam Nabet, Jon A. Oyer, Xiaoxiao Huang, Rajarshi P. Ghosh, Adrian T. Grzybowski, Tao Yu, Qiao Zhang, Alberto Riva, Tanmay P. Lele, George C. Schatz, Neil L. Kelleher, Alexander J. Ruthenburg, Jan Liphardt, and Jonathan D. Licht
Cancer Discovery, ISSN: 21598274, eISSN: 21598290, Pages: 1438-1451, Published: October 2019 American Association for Cancer Research (AACR)
By examination of the cancer genomics database we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K) found particularly in bladder and head and neck cancer that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 in vitro and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with PIK3CA to promote colony formation, caused a significant drift in gene expression and fundamental changes in chromatin accessibility particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression.
Harinad B. Maganti, Hani Jrade, Christopher Cafariello, Janet L. Manias Rothberg, Christopher J. Porter, Julien Yockell-Lelièvre, Hannah L. Battaion, Safwat T. Khan, Joel P. Howard, Yuefeng Li, Adrian T. Grzybowski, Elham Sabri, Alexander J. Ruthenburg, F. Jeffrey Dilworth, Theodore J. Perkins, Mitchell Sabloff, Caryn Y. Ito, and William L. Stanford
Cancer Discovery, ISSN: 21598274, eISSN: 21598290, Pages: 1376-1389, Published: November 2018 American Association for Cancer Research (AACR)
Deep sequencing has revealed that epigenetic modifiers are the most mutated genes in acute myeloid leukemia (AML). Thus, elucidating epigenetic dysregulation in AML is crucial to understand disease mechanisms. Here, we demonstrate that metal response element binding transcription factor 2/polycomblike 2 (MTF2/PCL2) plays a fundamental role in the polycomb repressive complex 2 (PRC2) and that its loss elicits an altered epigenetic state underlying refractory AML. Unbiased systems analyses identified the loss of MTF2-PRC2 repression of MDM2 as central to, and therefore a biomarker for, refractory AML. Thus, immature MTF2-deficient CD34+CD38- cells overexpress MDM2, thereby inhibiting p53 that leads to chemoresistance due to defects in cell-cycle regulation and apoptosis. Targeting this dysregulated signaling pathway by MTF2 overexpression or MDM2 inhibitors sensitized refractory patient leukemic cells to induction chemotherapeutics and prevented relapse in AML patient-derived xenograft mice. Therefore, we have uncovered a direct epigenetic mechanism by which MTF2 functions as a tumor suppressor required for AML chemotherapeutic sensitivity and identified a potential therapeutic strategy to treat refractory AML.Significance: MTF2 deficiency predicts refractory AML at diagnosis. MTF2 represses MDM2 in hematopoietic cells and its loss in AML results in chemoresistance. Inhibiting p53 degradation by overexpressing MTF2 in vitro or by using MDM2 inhibitors in vivo sensitizes MTF2-deficient refractory AML cells to a standard induction-chemotherapy regimen. Cancer Discov; 8(11); 1376-89. ©2018 AACR. See related commentary by Duy and Melnick, p. 1348 This article is highlighted in the In This Issue feature, p. 1333.
Rohan N. Shah, Adrian T. Grzybowski, Evan M. Cornett, Andrea L. Johnstone, Bradley M. Dickson, Brandon A. Boone, Marcus A. Cheek, Martis W. Cowles, Danielle Maryanski, Matthew J. Meiners, Rochelle L. Tiedemann, Robert M. Vaughan, Neha Arora, Zu-Wen Sun, Scott B. Rothbart, Michael-Christopher Keogh, and Alexander J. Ruthenburg
Molecular Cell, ISSN: 10972765, eISSN: 10974164, Pages: 162-177.e7, Published: 4 October 2018 Elsevier BV
Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments has not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the "reproducibility crisis," demonstrating the need for rigorous, platform-appropriate validation.
Sophiya Karki, Domenick E. Kennedy, Kaitlin Mclean, Adrian T. Grzybowski, Mark Maienschein-Cline, Shiladitya Banerjee, Heping Xu, Elizabeth Davis, Malay Mandal, Christine Labno, Sarah E. Powers, Michelle M. Le Beau, Aaron R. Dinner, Harinder Singh, Alexander J. Ruthenburg, and Marcus R. Clark
Cell Reports, eISSN: 22111247, Pages: 2443-2456, Published: 28 August 2018 Elsevier BV
SUMMARY Expression of vast repertoires of antigen receptors by lymphocytes, with each cell expressing a single receptor, requires stochastic activation of individual variable (V) genes for transcription and recombination. How this occurs remains unknown. Using single-cell RNA sequencing (scRNA-seq) and allelic variation, we show that individual pre-B cells monoallelically transcribe divergent arrays of Vκ genes, thereby opening stochastic repertoires for subsequent Vκ-Jκ recombination. Transcription occurs upon translocation of Vκ genes to RNA polymerase II arrayed on the nuclear matrix in transcription factories. Transcription is anchored by CTCF-bound sites or E2A-loaded Vκ promotors and continues over large genomic distances delimited only by topological associating domains (TADs). Prior to their monoallelic activation, Vκ loci are transcriptionally repressed by cyclin D3, which prevents capture of Vκ gene containing TADs by transcription factories. Cyclin D3 also represses protocadherin, olfactory, and other monoallelically expressed genes, suggesting a widely deployed mechanism for coupling monoallelic gene activation with cell cycle exit.
Michael S Werner, Matthew A Sullivan, Rohan N Shah, Rangarajan D Nadadur, Adrian T Grzybowski, Vasiliy Galat, Ivan P Moskowitz, and Alexander J Ruthenburg
Nature Structural and Molecular Biology, ISSN: 15459993, eISSN: 15459985, Pages: 596-603, Published: 1 July 2017 Springer Science and Business Media LLC
We recently described a new class of long noncoding RNAs (lncRNAs) that are distinguished by especially tight chromatin association and whose presence is strongly correlated to expression of nearby genes. Here, we examine the cis-enhancer mechanism of this class of chromatin-enriched RNA (cheRNA) across multiple human cell lines. cheRNAs are largely cell type specific and provide the most reliable chromatin signature to predict cis-gene transcription in every human cell type examined. Targeted depletion of three cheRNAs decreases expression of their neighboring genes, indicating potential co-activator function, and single-molecule fluorescence in situ hybridization (smFISH) of one cheRNA-distal target gene pair suggests a spatial overlap consistent with a role in chromosome looping. Additionally, the cheRNA HIDALGO stimulates the fetal hemoglobin subunit gamma 1 (HBG1) gene during erythroid differentiation by promoting contacts to a downstream enhancer. Our results suggest that multiple cheRNAs activate proximal lineage-specific gene transcription.
Takamitsu Hattori, Darson Lai, Irina S. Dementieva, Sherwin P. Montaño, Kohei Kurosawa, Yupeng Zheng, Louesa R. Akin, Kalina M. Świst-Rosowska, Adrian T. Grzybowski, Akiko Koide, Krzysztof Krajewski, Brian D. Strahl, Neil L. Kelleher, Alexander J. Ruthenburg, and Shohei Koide
Proceedings of the National Academy of Sciences of the United States of America, ISSN: 00278424, eISSN: 10916490, Volume: 113, Pages: 2092-2097, Published: 23 February 2016 Proceedings of the National Academy of Sciences
Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.
Scott B. Rothbart, Bradley M. Dickson, Jesse R. Raab, Adrian T. Grzybowski, Krzysztof Krajewski, Angela H. Guo, Erin K. Shanle, Steven Z. Josefowicz, Stephen M. Fuchs, C. David Allis, Terry R. Magnuson, Alexander J. Ruthenburg, and Brian D. Strahl
Molecular Cell, ISSN: 10972765, eISSN: 10974164, Pages: 502-511, Published: 6 August 2015 Elsevier BV
Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.
Adrian T. Grzybowski, Zhonglei Chen, and Alexander J. Ruthenburg
Molecular Cell, ISSN: 10972765, eISSN: 10974164, Pages: 886-899, Published: June 04, 2015 Elsevier BV
Chromatin immunoprecipitation (ChIP) serves as a central experimental technique in epigenetics research, yet there are serious drawbacks: it is a relative measurement, which untethered to any external scale obscures fair comparison among experiments; it employs antibody reagents that have differing affinities and specificities for target epitopes that vary in abundance; and it is frequently not reproducible. To address these problems, we developed Internal Standard Calibrated ChIP (ICeChIP), wherein a native chromatin sample is spiked with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons, and reveals unique insight into the nature of bivalent domains. This technology provides in situ assessment of the immunoprecipitation step, accommodating for many experimental pitfalls as well as providing a critical examination of untested assumptions inherent to conventional ChIP.
Zhonglei Chen, Adrian T. Grzybowski, and Alexander J. Ruthenburg
ChemBioChem, ISSN: 14394227, eISSN: 14397633, Pages: 2071-2075, Published: 2015 Wiley
Considerable mechanistic insight into the function of histone post‐translational modifications and the enzymes that install and remove them derives from in vitro experiments with modified histones, often embedded in nucleosomes. We report the first semisyntheses of native‐like histone 3 (H3) bearing tri‐ and dimethyllysines at position 79 and trimethyllysine at position 36, as well as more facile and traceless semisyntheses of K9 and K27 trimethylated species. These semisyntheses are practical on a multi‐milligram scale and can also generate H3 with combinations of marks. Each of these modifications has distinct functional consequences, although the pathways by which H3K36me3 and H3K79me2/3 act have not been entirely mapped. To this end, we demonstrated that our semisynthetic histones, when reconstituted into nucleosomes, are valuable affinity reagents for unbiased binding partner discovery and compare them to their methyllysine analogue (MLA) counterparts at the nucleosome level.