Publications by authors named "Christopher B Mulholland"

9 Publications

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Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals.

Nat Commun 2020 11 24;11(1):5972. Epub 2020 Nov 24.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
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http://dx.doi.org/10.1038/s41467-020-19603-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686362PMC
November 2020

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency.

Sci Rep 2020 07 21;10(1):12066. Epub 2020 Jul 21.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.
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http://dx.doi.org/10.1038/s41598-020-68600-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374584PMC
July 2020

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Nat Commun 2020 03 6;11(1):1222. Epub 2020 Mar 6.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
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http://dx.doi.org/10.1038/s41467-020-15006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060239PMC
March 2020

Direct modulation of the bone marrow mesenchymal stromal cell compartment by azacitidine enhances healthy hematopoiesis.

Blood Adv 2018 12;2(23):3447-3461

Department of Medicine III, Technische Universität München, Munich, Germany.

Mesenchymal stromal cells (MSCs) are crucial components of the bone marrow (BM) microenvironment essential for regulating self-renewal, survival, and differentiation of hematopoietic stem/progenitor cells (HSPCs) in the stem cell niche. MSCs are functionally altered in myelodysplastic syndromes (MDS) and exhibit an altered methylome compared with MSCs from healthy controls, thus contributing to disease progression. To determine whether MSCs are amenable to epigenetic therapy and if this affects their function, we examined growth, differentiation, and HSPC-supporting capacity of ex vivo-expanded MSCs from MDS patients in comparison with age-matched healthy controls after direct treatment in vitro with the hypomethylating agent azacitidine (AZA). Strikingly, we find that AZA exerts a direct effect on healthy as well as MDS-derived MSCs such that they favor support of healthy over malignant clonal HSPC expansion in coculture experiments. RNA-sequencing analyses of MSCs identified stromal networks regulated by AZA. Notably, these comprise distinct molecular pathways crucial for HSPC support, foremost extracellular matrix molecules (including collagens) and interferon pathway components. Our study demonstrates that the hypomethylating agent AZA exerts its antileukemic activity in part through a direct effect on the HSPC-supporting BM niche and provides proof of concept for the therapeutic potential of epigenetic treatment of diseased MSCs. In addition, our comprehensive data set of AZA-sensitive gene networks represents a valuable framework to guide future development of targeted epigenetic niche therapy in myeloid malignancies such as MDS and acute myeloid leukemia.
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http://dx.doi.org/10.1182/bloodadvances.2018022053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290099PMC
December 2018

Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin.

Mol Cell 2018 11 1;72(4):739-752.e9. Epub 2018 Nov 1.

Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany; MRC London Institute of Medical Sciences (LMS), Du Cane Road, London W12 0NN, UK; Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. Electronic address:

The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the "backside" of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.
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http://dx.doi.org/10.1016/j.molcel.2018.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242706PMC
November 2018

DNA methylation analysis on purified neurons and glia dissects age and Alzheimer's disease-specific changes in the human cortex.

Epigenetics Chromatin 2018 07 25;11(1):41. Epub 2018 Jul 25.

Department of Genetics, University of Saarland (UdS), Campus, 66123, Saarbrücken, Germany.

Background: Epigenome-wide association studies (EWAS) based on human brain samples allow a deep and direct understanding of epigenetic dysregulation in Alzheimer's disease (AD). However, strong variation of cell-type proportions across brain tissue samples represents a significant source of data noise. Here, we report the first EWAS based on sorted neuronal and non-neuronal (mostly glia) nuclei from postmortem human brain tissues.

Results: We show that cell sorting strongly enhances the robust detection of disease-related DNA methylation changes even in a relatively small cohort. We identify numerous genes with cell-type-specific methylation signatures and document differential methylation dynamics associated with aging specifically in neurons such as CLU, SYNJ2 and NCOR2 or in glia RAI1,CXXC5 and INPP5A. Further, we found neuron or glia-specific associations with AD Braak stage progression at genes such as MCF2L, ANK1, MAP2, LRRC8B, STK32C and S100B. A comparison of our study with previous tissue-based EWAS validates multiple AD-associated DNA methylation signals and additionally specifies their origin to neuron, e.g., HOXA3 or glia (ANK1). In a meta-analysis, we reveal two novel previously unrecognized methylation changes at the key AD risk genes APP and ADAM17.

Conclusions: Our data highlight the complex interplay between disease, age and cell-type-specific methylation changes in AD risk genes thus offering new perspectives for the validation and interpretation of large EWAS results.
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http://dx.doi.org/10.1186/s13072-018-0211-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058387PMC
July 2018

Ubiquitome Analysis Reveals PCNA-Associated Factor 15 (PAF15) as a Specific Ubiquitination Target of UHRF1 in Embryonic Stem Cells.

J Mol Biol 2017 12 18;429(24):3814-3824. Epub 2017 Oct 18.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany. Electronic address:

Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNA methyltransferase 1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation, we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15; also known as Pclaf, Ns5atp9, KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response.
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http://dx.doi.org/10.1016/j.jmb.2017.10.014DOI Listing
December 2017

A modular open platform for systematic functional studies under physiological conditions.

Nucleic Acids Res 2015 Sep 24;43(17):e112. Epub 2015 May 24.

Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.
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http://dx.doi.org/10.1093/nar/gkv550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787826PMC
September 2015