Biology subjects

Pu, S.

Publications and source records attributed to Pu, S..

17 recordsLinked to original sources

bioRxiv2026

Integrative analysis reveals extensive interactions among C2H2 zinc finger proteins at chromatin loop anchors

Cys2-His2 -zinc-finger proteins (C2H2-ZFPs) form the largest class of human transcription factors, yet their potential role (s) in higher-order genome organization has remained largely poorly defined. To date, only a small number of family members, e.g., CTCF and YY1, have been shown to function in chromatin organization. Here, we examined the global relationship between C2H2-ZFPs and long-range chromatin interactions (LRIs). By integrating ChIP-seq datasets for 216 C2H2-ZFPs and genome-wide maps of chromatin looping, we observe that more than 40% of the human C2H2-ZFPs are significantly enriched at LRI anchors. We found that depletion of a subset of such C2H2-ZFPs is associated with altered expression of genes linked by the LRIs they occupy. To investigate whether protein-protein interactions (PPIs) contribute to this pattern, we generated a large-scale interactome using affinity purification followed by mass spectrometry experiments, encompassing 345 C2H2-ZFPs, and LUMIER binary interaction assays for 204 C2H2-ZFPs. We identified 1,732 binary interactions, suggesting extensive connectivity among the C2H2-ZFP family members. Integrative analysis of PPI, ChIP-seq, and chromatin interaction datasets revealed that interacting C2H2-ZFP pairs are significantly co-enriched at LRIs and frequently localize to either the same or opposing loop anchors. Finally, by correlating ChIP-seq with cancer mutational datasets, we observe that DNA-binding sites of [~]35% of LRI-associated C2H2-ZFPs overlap somatic mutations in cancer genomes. Together, our results reveal a widespread network of C2H2-ZFP interactions associated with chromatin loop anchors, providing an important resource for elucidating mechanisms regulating chromatin organization.

genomics↗

bioRxiv2026

ZNF121 recruits YTHDF2 to modulate mRNA stability

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and has been shown to regulate gene expression through the binding of specific reader proteins, such as YTHDF2, which promotes mRNA decay. Previous studies indicate that YTHDF2 has relatively weak intrinsic RNA-binding affinity, suggesting that additional factors may facilitate its association with target transcripts. Here, we show that the C2H2-Zinc Finger protein, ZNF121, binds mRNA in cells and physically interacts with YTHDF2 in the cytoplasm. We demonstrate that approximately 80% of ZNF121-bound mRNAs are also YTHDF2 targets, and that their binding sites highly correlate with each other. Loss of ZNF121 impairs YTHDF2 binding to shared targets and increases their stability, independent of the presence of m6A modifications. Moreover, co-regulated transcripts are enriched for cell-cycle-related pathways, and ZNF121 depletion leads to elevated expression of the oncogene MDM2, implicating ZNF121 in growth control and the DNA damage response. Our findings identify ZNF121 as a cofactor that enhances YTHDF2-mediated mRNA regulation and reveal a previously unknown layer of control in mRNA decay.

molecular biology↗

bioRxiv2024

CircAge: A Comprehensive Resource for Aging-Associated Circular RNAs Across Species and Tissues

Circular RNAs (circRNAs) represent a novel class of RNA molecules characterized by a circular structure and enhanced stability. Emerging evidence indicates that circRNAs play pivotal regulatory roles in the aging process. Despite this, there is a lack of a systematic resource that integrates aging-associated circRNA data. Therefore, we developed a comprehensive database named circAge, which encompasses 689 aging-related samples from 7 species and 21 tissue types. We also generated 47 new tissue samples from mice and rhesus monkeys through high-throughput sequencing. Integrating predictions from multiple bioinformatics tools, we identified over 413,378 unique circRNAs. Our data analysis revealed a general increase in circRNA expression levels with age, with approximately 22% of circRNAs demonstrating sequence conservation across species. The circAge database systematically predicts potential interactions between circRNAs and miRNAs, RNA-binding proteins, and assesses the coding potential of circRNAs. This resource lays a foundation for elucidating the regulatory mechanisms of circRNAs in aging. As a comprehensive repository of aging-associated circRNAs, circAge will significantly accelerate research in this field, facilitating the discovery of novel biomarkers and therapeutic targets for aging biology and developing diagnostic and therapeutic strategies for aging and age-related diseases. CircAge is publicly available at https://circage.kiz.ac.cn.

bioinformatics↗

bioRxiv2024

Unraveling the impact of hyperleptinemia on female reproduction: Insights from transgenic pig model

BackgroundInfertility is a growing global health concern affecting millions of couples worldwide. Among several factors, an extreme body weight adversely affects reproductive functions. Leptin is a well-known adipokine that serves as an endocrine signal between adiposity and fertility. However, the exact mechanisms underlying the effects of high leptin on female reproduction remain unclear. MethodsTransgenic pig overexpressing leptin (11) were produced by back cross and screened for leptin overexpression, and the growth curve, fat deposition, reproductive performance, apoptosis, serum hormones and cholesterol production, RNA sequencing, and single nucleus RNA sequencing of the leptin-overexpressed pigs and control group were evaluated. ResultsTransgenic pig overexpressing leptin (11) were obtained, which exhibited significantly reduced body weight, body size, and back fat thickness. These pigs manifested late onset of puberty (327{+/-}48.5 vs 150{+/-}6.5 days), irregular estrous behavior characterized by increased inter-estrous interval (28.1{+/-}4.2 vs 21.3 {+/-} 0.9 days), and more numbers of mating until pregnancy (at least 3 times). This reproductive impairment in leptin pigs was related to hormonal imbalances characterized by increased levels of FSH, LH, prolactin, E2, P4, and TSH, altered steroidogenesis such as increased levels of serum CE along with steroidogenic markers (STAR, CYP19A), and ovarian dysfunctions manifested by neutrophilic infiltration and low expression of caspase-3 positive cells on leptin pigs ovary. Meanwhile, bulk RNA sequencing of the ovaries also revealed neutrophilic infiltration followed by upregulation of inflammation-related genes. Further, leptin overexpression triggered immune response, suppressed follicle development and luteinization, imposing metabolism dysfunction and hormone imbalance in the ovary by single-nucleus RNA sequencing (snRNA-seq). ConclusionLow body weight in leptin overexpression pigs adversely affects reproductive performance, causing delayed puberty, irregular estrous cycles, and reduced breeding efficiency. This is linked to metabolic imbalances, increased immune response, and altered ovarian functions. This study provided a theoretical basis for the complex mechanisms underlying leptin, and infertility by employing leptin-overexpressed female pigs.

genetics↗

bioRxiv2024

Recruitment of the m6A/Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48

N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2'-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity. Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO- dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA- binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

molecular biology↗

bioRxiv2023

Cryo-EM structure of the Spo11 core complex bound to DNA

The DNA double-strand breaks that initiate meiotic recombination are formed by topoisomerase relative Spo11, supported by conserved auxiliary factors. Because high-resolution structural data are lacking, many questions remain about the architecture of Spo11 and its partners and how they engage with DNA. We report cryo-EM structures at up to 3.3 [A] resolution of DNA-bound core complexes of Saccharomyces cerevisiae Spo11 with Rec102, Rec104, and Ski8. In these structures, monomeric core complexes make extensive contacts with the DNA backbone and with the recessed 3-OH and first 5 overhanging nucleotide, definitively establishing the molecular determinants of DNA end-binding specificity and providing insight into DNA cleavage preferences in vivo. The structures of individual subunits and their interfaces, supported by functional data in yeast, provide insight into the role of metal ions in DNA binding and uncover unexpected structural variation in homologs of the Top6BL component of the core complex.

biochemistry↗

bioRxiv2023

N-6-methyladenosine (m6A) Promotes the Nuclear Retention of mRNAs with Intact 5' Splice Site Motifs

In humans, misprocessed mRNAs containing intact 5' Splice Site (5'SS) motifs are nuclear retained and targeted for decay by ZFC3H1, a component of the Poly(A) Exosome Targeting complex, and U1-70K, a component of the U1 snRNP. In S. pombe, the ZFC3H1 homolog, Red1, binds to the YTH-domain containing protein Mmi1 to target certain RNA transcripts to nuclear foci for nuclear retention and decay. Here we show that YTHDC1 and YTHDC2, two YTH domain-containing proteins that bind to N-6-methyladenosine (m6A) modified RNAs, interact with ZFC3H1 and U1-70K, and are required for the nuclear retention of mRNAs with intact 5'SS motifs. Disruption of m6A deposition inhibits both the nuclear retention of these transcripts and their accumulation in YTHDC1-enriched foci that are adjacent to nuclear speckles. Endogenous RNAs with intact 5'SS motifs, such as intronic polyadenylated transcripts, tend to be m6A-modified at low levels. Thus, m6A modification acts in a conserved quality control mechanism that targets misprocessed mRNAs for nuclear retention and decay.

molecular biology↗

bioRxiv2023

Structure and DNA bridging activity of the essential Rec114-Mei4 trimer interface

The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo, these proteins form large immunostaining foci that are integrated with higher order chromosome structures. In vitro, they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C-terminus of Rec114 with the N-terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.

biochemistry↗

bioRxiv2023

Omicron Spike Protein Is Vulnerable to Reduction

SARS-CoV-2 virus spike (S) protein is an envelope protein responsible for binding to the ACE2 receptor, driving subsequent entry into host cells. The existence of multiple disulfide bonds in the S protein makes it potentially susceptible to reductive cleavage. Using a tri-part split luciferase-based binding assay, we evaluated the impacts of chemical reduction on S proteins from different virus variants and found that those from the Omicron family are highly vulnerable to reduction. Through manipulation of different Omicron mutations, we found that alterations in the receptor binding module (RBM) are the major determinants of this vulnerability. Specifically we discovered that Omicron mutations facilitate the cleavage of C480-C488 and C379-C432 disulfides, which consequently impairs binding activity and protein stability. The vulnerability of Omicron S proteins suggests a mechanism that can be harnessed to treat specific SARS-CoV-2 strains.

biochemistry↗

bioRxiv2022

Prefrontal neuronal dynamics in the absence of task execution

Prefrontal cortical activity represents stimuli in working memory tasks in a low-dimensional manifold that transforms over the course of a trial. Such transformations reflect specific cognitive operations, so that, for example, the rotation of stimulus representations is thought to reduce interference by distractor stimuli. Here we show that rotations occur in the low-dimensional activity space of prefrontal neurons in naive monkeys, while passively viewing familiar stimuli. Moreover, some aspects of these rotations remain remarkably unchanged after training to perform working memory tasks. Significant training effects are still present in population dynamics, which further distinguish correct and error trials during task execution. Our results reveal automatic functions of prefrontal neural circuits, allow transformations that may aid cognitive flexibility.

neuroscience↗

bioRxiv2021

KRAB Zinc Finger protein Znf684 interacts with Nxf1 to regulate mRNA export

Cys2His2 (C2H2) type zinc finger (ZnF) proteins constitute a large class of proteins that are generally considered to be DNA-binding transcription factors. Using affinity purification followed by mass spectrometry, as well as reciprocal co-immunoprecipitation experiments, we determined that the C2H2-ZnF protein Znf684 interacts physically with several proteins involved in mRNA export, including Nxf1 and Alyref. We utilized individual nucleotide resolution cross-linking immunoprecipitation followed by high throughput sequencing (iCLIP-seq) experiments to show that Znf684 binds directly to specific mRNAs in vivo and has an RNA-binding profile similar to those of Nxf1 and Alyref, suggesting a role in mRNA export regulation. Immunofluorescence microscopy (IF) experiments revealed that Znf684 localizes to both the nucleus and cytoplasm. Using cellular fractionation experiments, we demonstrate that overexpression of Znf684 negatively impacts the export of SMAD3 and other target mRNAs. Taken together, our results suggest that Znf684 regulates the export of a subset of transcripts through physical interactions with Nxf1 and specific target mRNAs.

molecular biology↗

bioRxiv2021

Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2

SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies. IN BRIEFSARS-CoV-2, depends on host cell components for infection and replication. Genome-wide CRISPR screens were performed in multiple human cell lines to elucidate common host dependencies required for SARS-CoV-2 infection. Only ACE2, the cognate SARS-CoV-2 entry receptor, was common amongst cell lines, while all other host genes identified were cell line specific, several of which converged on pathways involved in cell signalling, lipid metabolism, immune pathways, and chromatin modulation. Overall, a network of host factors was identified that will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate productive targeting of host genes and pathways. HIGHLIGHTS- Genome-wide CRISPR screens for SARS-CoV-2 in multiple human cell lines - Identification of wide-ranging cell-type dependent genetic dependencies for SARS-CoV-2 infection - ACE2 is the only common host factor identified across different cell types

systems biology↗

bioRxiv2020

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links associated with Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion decreased cellular viability and altered the expression of selected targets. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Nucleocapsid protein attenuates stress granule formation and alters gene expression via direct interaction with host mRNAs

The COVID-19 pandemic has caused over one million deaths thus far. There is an urgent need for the development of specific viral therapeutics and a vaccine. SARS-CoV-2 nucleocapsid (N) protein is highly expressed upon infection and is essential for viral replication, making it a promising target for both antiviral drug and vaccine development. Here, starting from a functional proteomics workflow, we initially catalogued the protein-protein interactions of 21 SARS-CoV-2 proteins in HEK293 cells, finding that the stress granule resident proteins G3BP1 and G3BP2 copurify with N with high specificity. We demonstrate that N protein expression in human cells sequesters G3BP1 and G3BP2 through its physical interaction with these proteins, attenuating stress granule (SG) formation. The ectopic expression of G3BP1 in N-expressing cells was sufficient to reverse this phenotype. Since N is an RNA-binding protein, we performed iCLIP-sequencing experiments in cells, with or without exposure to oxidative stress, to identify the host RNAs targeted by N. Our results indicate that SARS-CoV-2 N protein binds directly to thousands of mRNAs under both conditions. Like the G3BPs stress granule proteins, N was found to predominantly bind its target mRNAs in their 3UTRs. RNA sequencing experiments indicated that expression of N results in wide-spread gene expression changes in both unstressed and oxidatively stressed cells. We suggest that N regulates host gene expression by both attenuating stress granules and binding directly to target mRNAs.

molecular biology↗

bioRxiv2020

Structural and functional characterization of the Spo11 core complex

Spo11, which makes DNA double-strand breaks (DSBs) essential for meiotic recombination, is poorly understood mechanistically because it has been recalcitrant to biochemical study. Here, we provide a molecular analysis of S. cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal Topoisomerase VI, with Rec104 similar to a GHKL domain but without conserved ATPase motifs. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), indicating that dimerization may control DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex-duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing, and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.

molecular biology↗

bioRxiv2020

DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

Formation of meiotic DNA double-strand breaks (DSBs) by Spo11 is tightly regulated and tied to chromosome structure, but the higher-order assemblies that execute and control DNA breakage are poorly understood. We address this question through molecular characterization of Saccharomyces cerevisiae RMM proteins (Rec114, Mei4 and Mer2)--essential, conserved components of the DSB machinery. Each subcomplex of Rec114-Mei4 (2:1 heterotrimer) or Mer2 (homotetrameric coiled coil) is monodisperse in solution, but they independently condense with DNA into dynamic, reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation, which correlates with DSB formation in vivo. Condensates fuse into mixed Rec114-Mei4-Mer2 clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from recruitment of regulatory components and modulation of biophysical properties of the condensates.

molecular biology↗

bioRxiv2019

Regulation of transcription termination by FUS and TDP-43

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAPII) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. We previously found that symmetrical dimethylation (me2s) of a CTD Arginine residue (R1810 in human) causes recruitment of the Tudor domain of SMN, which interacts with Senataxin. SMN is mutated in spinal muscular atrophy (SMA), and Senataxin is sometimes mutated in Amyotrophic Lateral Sclerosis (ALS). R1810me2s and SMN, like Senataxin, are important for resolving R-loops (DNA:RNA hybrids) at transcription terminators. FUS and TDP-43 (TARDBP) are DNA/RNA binding proteins that are sometimes mutated in ALS and FTD (Frontotemporal dementia). Here we show that TDP-43 and, to some extent, FUS are recruited by the R1810me2s-SMN pathway. Defects in FUS and TDP-43 recruitment influence RNAPII termination and R-loop accumulation, leading to elevated DNA damage at terminators that may contribute to neurodegenerative disorders like ALS and FTD.

molecular biology↗