LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase–dependent proteolysis of the stem-cell protein SOX2

Zhang, Chunxiao; Hoang, Nam V.; Feng, Lu; Saxena, Lovely; Lee, Logan; Alejo, Salvador; Qi, Dandan; Khal, Anthony; Sun, Hong; Lu, Fei; Zhang, Hui

doi:10.1074/jbc.ra117.000342

Cited by 35 publications

(108 citation statements)

References 45 publications

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“…PCNA also reciprocally associated with CDT2. Our studies further revealed that treatment of cells with MG132, an inhibitor of the 26S proteosome (22), significantly enhanced the interaction between CDT2 and PCNA (Fig. 1C).…”

Section: Cdt2 Interacts With Pcna To Target Cdt1 For Degradationsupporting

confidence: 62%

“…Human PCNA cDNA was cloned into the pET vector and expressed in E. coli BL21 strain. The site-directed mutagenesis was conducted as previously described (22). For binding assays, 1 g of GST fusion proteins were mixed with bacterial lysates containing the expressed PCNA and incubated at 4°C for 2 h. The protein complexes were isolated by 30 l of GSH-Sepharose and detected by Western blotting with anti-GST or anti-PCNA antibodies.…”

Section: Recombinant Proteins and Binding Assaysmentioning

confidence: 99%

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Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis–induced degradation of CDT1

Feng

Saxena

Hoang

et al. 2018

Journal of Biological Chemistry

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Edited by Xiao-Fan Wang During DNA replication or repair, the DNA polymerase cofactor, proliferating cell nuclear antigen (PCNA), homotrimerizes and encircles the replicating DNA, thereby acting as a DNA clamp that promotes DNA polymerase processivity. The formation of the PCNA trimer is also essential for targeting the replication-licensing protein, chromatin-licensing, and DNA replication factor 1 (CDT1), for ubiquitin-dependent proteolysis to prevent chromosomal DNA re-replication. CDT1 uses its PCNA-interacting peptide box (PIP box) to interact with PCNA, and the CRL4 E3 ubiquitin ligase subunit CDT2 is recruited through the formation of PCNA-CDT1 complexes. However, it remains unclear how CDT1 and many other PIP box-containing proteins are marked for degradation by the CRL4 CDT2 ubiquitin ligase during DNA replication or damage. Here, using recombinant protein expression coupled with site-directed mutagenesis, we report that CDT2 and PCNA directly interact and this interaction depends on the presence of a highly conserved, C-terminal PIP box-like region in CDT2. Deletion or mutation of this region abolished the CDT2-PCNA interaction between CDT2 and PCNA both in vitro and in vivo. Moreover, PCNA-dependent CDT1 degradation in response to DNA damage and replication during the cell cycle requires an intact PIP box in CDT2. The requirement of the PIP boxes in both CDT2 and its substrate CDT1 suggests that the formation of the PCNA trimeric clamp around DNA during DNA replication and repair may bring together CDT1 and CRL4 CDT2 ubiquitin E3 ligase to target CDT1 for proteolysis in a DNA synthesis-dependent manner. Proliferating cell nuclear antigen (PCNA) 2 is central to DNA replication and DNA repair (1, 2). It normally exists in a mono-This work was supported by National Institutes of Health Grants R15 NS096694 (to H. S.), R15GM116087 and R15GM131255 (to H. Z.

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Section: Cdt2 Interacts With Pcna To Target Cdt1 For Degradationsupporting

confidence: 62%

Section: Recombinant Proteins and Binding Assaysmentioning

confidence: 99%

Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis–induced degradation of CDT1

Feng

Saxena

Hoang

et al. 2018

Journal of Biological Chemistry

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“…LSD1 is a flavin-dependent monoamine oxidase that demethylates mono-and dimethylated histone 3 lysines 4 and 9 (H3K4me1/2 and H3K9me1/2) to impact chromatin structure and gene expression (48,49). LSD1 also demethylates nonhistone targets, including p53, STAT3, estrogen receptor ␣ (ER␣), MYPT1, hypoxia-inducible factor 1␣ (HIF1␣), DNMT1, E2F, and SOX2, suggesting a broad influence over cellular homeostasis (50)(51)(52)(53)(54)(55)(56)(57). LSD1 operates within multiple assemblies that regulate transcription, including nucleosome remodeling and deacetylase (NuRD), C-terminal binding protein (CtBP), mixed-lineage leukemia (MLL) coactivator, and BRAF-histone deacetylase (BHC)/CoREST complexes (58)(59)(60)(61)(62).…”

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confidence: 99%

Growth Factor Independence 1B-Mediated Transcriptional Repression and Lineage Allocation Require Lysine-Specific Demethylase 1-Dependent Recruitment of the BHC Complex

McClellan

Casey

Bareyan

et al. 2019

Molecular and Cellular Biology

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Growth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B’s obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.

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“…Recently, a novel function of non-histone protein methylation is to trigger the proteolysis of methylated proteins. In human ovarian teratocarcinoma PA-1 cells, PHF20L1 and the lysine demethylase LSD1 coordinate to protect SOX2 from methylation-dependent proteolysis [8,9]. The first Tudor domain (Tudor1) of PHF20L1 is ascribed to read not only the mono-methylated lysine K4 of histone H3 and K20 of histone H4 but also mono-methylated K142 (K142 me1 ) of non-histone protein DNA (cytosine-5) methyltransferase 1 (DNMT1) [10,11].…”

Section: Introductionmentioning

confidence: 99%

Conformational Selection in Ligand Recognition by the First Tudor Domain of PHF20L1

Gao

et al. 2020

Preprint

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The first Tudor domain of PHF20L1 (PHF20L1 Tudor1) recognizes both histone methylation and non-histone methylation to play versatile roles, e.g., PHF20L1 Tudor1 binds to the oncogenic target DNA (cytosine-5) methyltransferase 1 (DNMT1) to prevent it from degradation. However, the crystal structure of the PHF20 Tudor domain, a homolog of PHF20L1, reveals a closed aromatic cage of the Tudor domain. It is thus highly desirable to interrogate the ligand-recognition mechanism of PHF20L1 Tudor1, which will in turn validate the potential druggability of this target. Here, we solved the crystal structure of the free form PHF20L1 Tudor1, which adopts the closed conformation similar to PHF20. NMR relaxation dispersion and molecular dynamics (MD) simulations suggest a pre-existing low-population conformation with a remarkable rearrangement of aromatic cage residues. Such structural rearrangement is further revealed by the crystal structures of PHF20L1 Tudor1 in complex with the lysine 142 methylated (K142 me1 ) DNMT1, and a small molecule cosolvent 2-(N-morpholino)ethanesulfonic acid (MES), respectively. This result thus ignites interest in the discovery of small molecule inhibitors against PHF20L1 Tudor1. The hit identified from NMR fragment-based screening protrudes into the same open form aromatic cage of PHF20L1 Tudor1, and blocks the interaction between PHF20L1 Tudor1 and methylated DNMT1. Further free form crystal structures of key mutants reveal one open form and one closed form aromatic cage, which is energetically trapped observed in the NMR relaxation dispersion and MD simulations. The binding of DNMT1 with PHF20L1 Tudor1 mutants was also recapitulated in cancer cells. The mutagenesis thus alters the structure, dynamics and eventually the function of PHF20L1 Tudor1. Our results demonstrate that PHF20L1 Tudor1 utilizes the same conformational selection mechanism to recognize ligands, regardless of whether it is a natural substrate or a small molecule identified from fragment-based screening. Albeit at a low population, the pre-existing ligand-binding conformation shall shift the paradigm in the druggability assessment of a dynamic protein, even though it may lack a small molecule binding pocket in its free form structure. The inhibition of PHF20L1 paves an alternative way to target DNMT1 degradation.

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LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase–dependent proteolysis of the stem-cell protein SOX2

Cited by 35 publications

References 45 publications

Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis–induced degradation of CDT1

Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis–induced degradation of CDT1

Growth Factor Independence 1B-Mediated Transcriptional Repression and Lineage Allocation Require Lysine-Specific Demethylase 1-Dependent Recruitment of the BHC Complex

Conformational Selection in Ligand Recognition by the First Tudor Domain of PHF20L1

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