Pujuan Deng

All Publications

• Protein-templated synthesis of dinucleotide repeat DNA by an antiphage reverse transcriptase. Science (New York, N.Y.) Deng, P., Lee, H., Armijo, C., Wang, H., Gao, A. 2026: eaed1656

  Abstract

  Defense-associated reverse transcriptases (DRTs) are widespread bacterial anti-phage systems that use unconventional mechanisms of polynucleotide synthesis. We show that DRT3, which comprises two distinct RTs (Drt3a and Drt3b) and a noncoding RNA (ncRNA), synthesizes alternating poly(GT/AC) double-stranded DNA. Cryo-electron microscopy structures at 2.6 Å resolution reveal a D3-symmetric 6:6:6 complex of Drt3a, Drt3b, and ncRNA. Drt3a produces the poly(GT) strand using a conserved ACACAC template within the ncRNA. Notably, Drt3b synthesizes a complementary, protein-primed poly(AC) strand in the complete absence of a nucleic acid template, using conserved active site residues specific to Drt3b to enforce precise base alternation. These findings expand the functional landscape of nucleic acid polymerases, revealing a protein-templated mechanism for sequence-specific DNA synthesis.

  View details for DOI 10.1126/science.aed1656

  View details for PubMedID 41990131

• Systematic discovery of DNA-binding tandem repeat proteins. Nucleic acids research Hu, X., Zhang, X., Sun, W., Liu, C., Deng, P., Cao, Y., Zhang, C., Xu, N., Zhang, T., Zhang, Y. E., Liu, J. G., Wang, H. 2024; 52 (17): 10464-10489

  Abstract

  Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools.

  View details for DOI 10.1093/nar/gkae710

  View details for PubMedID 39189466

  View details for PubMedCentralID PMC11417379

• Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon. Cell Deng, P., Tan, S. Q., Yang, Q. Y., Fu, L., Wu, Y., Zhu, H. Z., Sun, L., Bao, Z., Lin, Y., Zhang, Q. C., Wang, H., Wang, J., Liu, J. G. 2023; 186 (13): 2865-2879.e20

  Abstract

  Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.

  View details for DOI 10.1016/j.cell.2023.05.032

  View details for PubMedID 37301196

• Structural Basis of the Transcriptional Elongation Factor Paf1 Core Complex from Saccharomyces eubayanus. International journal of molecular sciences Qin, Y., Zhou, Y., Cao, Y., Ren, Y., Deng, P., Jiang, J., Wang, Z. 2023; 24 (10)

  Abstract

  The multicomponent polymerase associated factor 1 (Paf1) complex (PAF1C) is an important transcription elongation factor that upregulates RNA polymerase II-mediated genome-wide transcription. PAF1C can regulate transcription through direct association with the polymerase or by impacting the chromatin structure epigenetically. In recent years, significant progress has been made in understanding the molecular mechanisms of PAF1C. However, high-resolution structures that can clarify the interaction details among the components of the complex are still needed. In this study, we evaluated the structural core of the yeast PAF1C containing the four components Ctr9, Paf1, Cdc73 and Rtf1 at high resolution. We observed the interaction details among these components. In particular, we identified a new binding surface of Rtf1 on PAF1C and found that the C-terminal sequence of Rtf1 dramatically changed during evolution, which may account for its different binding affinities to PAF1C among species. Our work presents a precise model of PAF1C, which will facilitate our understanding of the molecular mechanism and the in vivo function of the yeast PAF1C.

  View details for DOI 10.3390/ijms24108730

  View details for PubMedID 37240075

  View details for PubMedCentralID PMC10217977

• LncRNA-Smad7 mediates cross-talk between Nodal/TGF-β and BMP signaling to regulate cell fate determination of pluripotent and multipotent cells. Nucleic acids research Kong, X., Yan, K., Deng, P., Fu, H., Sun, H., Huang, W., Jiang, S., Dai, J., Zhang, Q. C., Liu, J. G., Xi, Q. 2022; 50 (18): 10526-10543

  Abstract

  Transforming growth factor β (TGF-β) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-β and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-β and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.

  View details for DOI 10.1093/nar/gkac780

  View details for PubMedID 36134711

  View details for PubMedCentralID PMC9561265

• Diverse activation mechanisms of PI3Ks. Nature structural & molecular biology Deng, P., Liu, J. G. 2022; 29 (3): 185-187

  View details for DOI 10.1038/s41594-022-00744-4

  View details for PubMedID 35256803

• Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search. Chemical science Yang, M., Sun, R., Deng, P., Yang, Y., Wang, W., Liu, J. G., Chen, C. 2021; 12 (38): 12776-12784

  Abstract

  RNA-guided Streptococcus pyogenes Cas9 (SpCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how Cas9 locates its target among huge amounts of dsDNAs remains elusive. Here, combining biochemical and single-molecule fluorescence assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target with high efficiency. We further observed surprising apparent asymmetric target search regions flanking PAM sites on dsDNA under physiological salt conditions, which accelerates the target search efficiency of Cas9 by ∼10-fold. Illustrated by a cryo-EM structure of the Cas9/sgRNA/dsDNA dimer, non-specific interactions between DNA ∼8 bp downstream of the PAM site and lysines within residues 1151-1156 of Cas9, especially lys1153, are the key elements to mediate the one-dimensional diffusion of Cas9 and cause asymmetric target search regions flanking the PAM. Disrupting these non-specific interactions, such as mutating these lysines to alanines, diminishes the contribution of one-dimensional diffusion and reduces the target search rate by several times. In addition, low ionic concentrations or mutations on PAM recognition residues that modulate interactions between Cas9 and dsDNA alter apparent asymmetric target search behaviors. Together, our results reveal a unique searching mechanism of Cas9 under physiological salt conditions, and provide important guidance for both in vitro and in vivo applications of Cas9.

  View details for DOI 10.1039/d1sc02633j

  View details for PubMedID 34703564

  View details for PubMedCentralID PMC8494019

• Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases. Nature Li, W., Tian, W., Yuan, G., Deng, P., Sengupta, D., Cheng, Z., Cao, Y., Ren, J., Qin, Y., Zhou, Y., Jia, Y., Gozani, O., Patel, D. J., Wang, Z. 2020

  Abstract

  Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family, including NSD1, NSD2 and NSD3, have crucial roles in chromatin regulation and are implicated in oncogenesis1,2. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling dimethylation of histone H3 at Lys36 (H3K36)3-7. However, the molecular basis that underlies this mechanism is largely unknown. Here we solve the cryo-electron microscopy structures of NSD2 and NSD3 bound to mononucleosomes. We find that binding of NSD2 and NSD3 to mononucleosomes causes DNA near the linker region to unwrap, which facilitates insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between NSD2 or NSD3 and the nucleosome precisely defines the position of the enzyme on the nucleosome, explaining the specificity of methylation to H3K36. Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3. NSDs that contain these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes the proliferation of cancer cells and the growth of xenograft tumours. Together, our research provides molecular insights into the nucleosome-based recognition and histone-modification mechanisms of NSD2 and NSD3, which could lead to strategies for therapeutic targeting of proteins of the NSD family.

  View details for DOI 10.1038/s41586-020-03069-8

  View details for PubMedID 33361816

• Transcriptional elongation factor Paf1 core complex adopts a spirally wrapped solenoidal topology. Proceedings of the National Academy of Sciences of the United States of America Deng, P., Zhou, Y., Jiang, J., Li, H., Tian, W., Cao, Y., Qin, Y., Kim, J., Roeder, R. G., Patel, D. J., Wang, Z. 2018; 115 (40): 9998-10003

  Abstract

  The polymerase-associated factor 1 (Paf1) complex is a general transcription elongation factor of RNA polymerase II, which is composed of five core subunits, Paf1, Ctr9, Cdc73, Leo1, and Rtf1, and functions as a diverse platform that broadly affects gene expression genome-wide. In this study, we solved the 2.9-Å crystal structure of the core region composed of the Ctr9-Paf1-Cdc73 ternary complex from a thermophilic fungi, which provides a structural perspective of the molecular details of the organization and interactions involving the Paf1 subunits in the core complex. We find that Ctr9 is composed of 21 tetratricopeptide repeat (TPR) motifs that wrap three circular turns in a right-handed superhelical manner around the N-terminal region of an elongated single-polypeptide-chain scaffold of Paf1. The Cdc73 fragment is positioned within the surface groove of Ctr9, where it contacts mainly with Ctr9 and minimally with Paf1. We also identified that the Paf1 complex preferentially binds single-strand-containing DNAs. Our work provides structural insights into the overall architecture of the Paf1 complex and paves the road forward for understanding the molecular mechanisms of the Paf1 complex in transcriptional regulation.

  View details for DOI 10.1073/pnas.1812256115

  View details for PubMedID 30224485

  View details for PubMedCentralID PMC6176576

• Polycomb-like proteins link the PRC2 complex to CpG islands. Nature Li, H., Liefke, R., Jiang, J., Kurland, J. V., Tian, W., Deng, P., Zhang, W., He, Q., Patel, D. J., Bulyk, M. L., Shi, Y., Wang, Z. 2017; 549 (7671): 287-291

  Abstract

  The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo.

  View details for DOI 10.1038/nature23881

  View details for PubMedID 28869966

  View details for PubMedCentralID PMC5937281