Transcriptional regulation of TacL-mediated lipoteichoic acids biosynthesis by ComE during competence impacts pneumococcal transformation.

Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of teichoic acid biosynthesis is tightly controlled during pneumococcal competence; however, the mechanism governing this regulation and its impact on transformation remains poorly understood. We demonstrated that a defect in lipoteichoic acid ligase (TacL)-mediated lipoteichoic acids (LTAs) biosynthesis was associated with impaired pneumococcal transformation. Using a fragment of tacL regulatory probe as bait in a DNA pulldown assay, we successfully identified several regulatory proteins, including ComE. Electrophoretic mobility shift assays revealed that phosphomimetic ComE, but not wild-type ComE, exhibited specific binding to the probe. DNase I footprinting assays revealed the specific binding sequences encompassing around 30 base pairs located 31 base pairs upstream from the start codon of tacL. Expression of tacL was found to be upregulated in the ΔcomE strain, and the addition of exogenous competence-stimulating peptide repressed the tacL transcription in the wild-type strain but not the ΔcomE mutant, indicating that ComE exerted a negative regulatory effect on the transcription of tacL. Mutation in the JH2 region of tacL upstream regulatory sequence led to increased LTAs abundance and displayed higher transformation efficiency. Collectively, our work identified the regulatory mechanisms that control LTAs biosynthesis during competence and thereby unveiled a repression mechanism underlying pneumococcal transformation.

Novel protein ligase based on dual split intein.

Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans-splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans-splicing-based protein engineering and provide new clues for intein based protein ligase.

Complex genetic interaction between glucose sensor HXK1 and E3 SUMO ligase SIZ1 in regulating plant morphogenesis.

Sugar signaling forms the basis of metabolic activities crucial for an organism to perform essential life activities. In plants, sugars like glucose, mediate a wide range of physiological responses ranging from seed germination to cell senescence. This has led to the elucidation of cell signaling pathways involving glucose and its counterparts and the mechanism of how these sugars take control over major hormonal pathways such as auxin, ethylene, abscisic acid and cytokinin in Arabidopsis. Plants use HXK1(Hexokinase) as a glucose sensor to modulate changes in photosynthetic gene expression in response to high glucose levels. Other proteins such as SIZ1, a major SUMO E3 ligase have recently been implicated in controlling sugar responses via transcriptional and translational regulation of a wide array of sugar metabolic genes. Here, we show that these two genes work antagonistically and are epistatic in controlling responsiveness toward high glucose conditions.

Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A.

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5'-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. KEY POINTS: • Optimizing 5'-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.

METTL3 Promotes Osteosarcoma Metastasis via an m6A-dependent Epigenetic Activity of CBX4.

BACKGROUND: Osteosarcoma cells are prone to metastasis, and the mechanism of N6-methyladenosine (m6A) methylation modification in this process is still unclear. Methylation modification of m6A plays an important role in the development of osteosarcoma, which is mainly due to abnormal expression of enzymes related to methylation modification of m6A, which in turn leads to changes in the methylation level of downstream target genes messenger RNA (mRNA) leading to tumor development. METHODS: We analyzed the expression levels of m6A methylation modification-related enzyme genes in GSE12865 whole-genome sequencing data. And we used shRNA (short hairpin RNA) lentiviral interference to interfere with METTL3 (Methyltransferase 3) expression in osteosarcoma cells. We studied the cytological function of METTL3 by Cell Counting Kit-8 (CCK8), flow cytometry, migration and other experiments, and the molecular mechanism of METTL3 by RIP (RNA binding protein immunoprecipitation), Western blot and other experiments. RESULTS: We found that METTL3 is abnormally highly expressed in osteosarcoma and interferes with METTL3 expression in osteosarcoma cells to inhibit metastasis, proliferation, and apoptosis of osteosarcoma cells. We subsequently found that METTL3 binds to the mRNA of CBX4 (chromobox homolog 4), a very important regulatory protein in osteosarcoma metastasis, and METTL3 regulates the mRNA and protein expression of CBX4. Further studies revealed that METTL3 inhibited metastasis of osteosarcoma cells by regulating CBX4. METTL3 has been found to be involved in osteosarcoma cells metastasis by CBX4 affecting the protein expression of matrix metalloproteinase 2 (MMP2), MMP9, E-Cadherin and N-Cadherin associated with osteosarcoma cells metastasis. CONCLUSIONS: These results suggest that the combined action of METTL3 and CBX4 plays an important role in the regulation of metastasis of osteosarcoma, and therefore, the METTL3-CBX4 axis pathway may be a new potential therapeutic target for osteosarcoma.

Genome-wide identification analysis of the 4-Coumarate: CoA ligase (4CL) gene family expression profiles in Juglans regia and its wild relatives J. Mandshurica resistance and salt stress.

Persian walnut (Juglans regia) and Manchurian walnut (Juglans mandshurica) belong to Juglandaceae, which are vulnerable, temperate deciduous perennial trees with high economical, ecological, and industrial values. 4-Coumarate: CoA ligase (4CL) plays an essential function in plant development, growth, and stress. Walnut production is challenged by diverse stresses, such as salinity, drought, and diseases. However, the characteristics and expression levels of 4CL gene family in Juglans species resistance and under salt stress are unknown. Here, we identified 36 Jr4CL genes and 31 Jm4CL genes, respectively. Based on phylogenetic relationship analysis, all 4CL genes were divided into three branches. WGD was the major duplication mode for 4CLs in two Juglans species. The phylogenic and collinearity analyses showed that the 4CLs were relatively conserved during evolution, but the gene structures varied widely. 4CLs promoter region contained multiply cis-acting elements related to phytohormones and stress responses. We found that Jr4CLs may be participated in the regulation of resistance to anthracnose. The expression level and some physiological of 4CLs were changed significantly after salt treatment. According to qRT-PCR results, positive regulation was found to be the main mode of regulation of 4CL genes after salt stress. Overall, J. mandshurica outperformed J. regia. Therefore, J. mandshurica can be used as a walnut rootstock to improve salt tolerance. Our results provide new understanding the potential functions of 4CL genes in stress tolerance, offer the theoretical genetic basis of walnut varieties adapted to salt stress, and provide an important reference for breeding cultivated walnuts for stress tolerance.

E3 SUMO ligase SIZ1 splicing variants localize and function according to external conditions.

SIZ1 (SAP and MIZ1) is a member of the Siz/PIAS-type RING family of E3 SUMO (small ubiquitin-related modifier) ligases that play key roles in growth, development, and stress responses in plant and animal systems. Nevertheless, splicing variants of SIZ1 have not yet been characterized. Here, we identified four splicing variants of Arabidopsis (Arabidopsis thaliana) SIZ1, which encode three different protein isoforms. The SIZ1 gene encodes an 873-amino acid (aa) protein. Among the four SIZ1 splicing variants (SSVs), SSV1 and SSV4 encode identical 885 aa proteins; SSV2 encodes an 832 aa protein; and SSV3 encodes an 884 aa protein. SSV2 mainly localized to the plasma membrane, whereas SIZ1, SSV1/SSV4, and SSV3 localized to the nucleus. Interestingly, SIZ1 and all SSVs exhibited similar E3 SUMO ligase activities and preferred SUMO1 and SUMO2 for their E3 ligase activity. Transcript levels of SSV2 were substantially increased by heat treatment, while those of SSV1, SSV3, and SSV4 transcripts were unaffected by various abiotic stresses. SSV2 directly interacted with and sumoylated cyclic nucleotide-gated ion channel 6 (CNGC6), a positive thermotolerance regulator, enhancing the stability of CNGC6. Notably, transgenic siz1-2 mutants expressing SSV2 exhibited greater heat stress tolerance than wild-type plants, whereas those expressing SIZ1 were sensitive to heat stress. Furthermore, transgenic cngc6 plants overaccumulating a mutated mCNGC6 protein (K347R, a mutation at the sumoylation site) were sensitive to heat stress, similar to the cngc6 mutants, while transgenic cngc6 plants overaccumulating CNGC6 exhibited restored heat tolerance. Together, we propose that alternative splicing is an important mechanism that regulates the function of SSVs during development or under adverse conditions, including heat stress.

CUL4A Ubiquitin Ligase Is an Independent Predictor of Overall Survival in Pancreatic Adenocarcinoma.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with dismal prognosis. Genomic instability due to defects in cell-cycle regulation/mitosis or deficient DNA-damage repair is a major driver of PDAC progression with clinical relevance. Deregulation of licensing of DNA replication leads to DNA damage and genomic instability, predisposing cells to malignant transformation. While overexpression of DNA replication-licensing factors has been reported in several human cancer types, their role in PDAC remains largely unknown. We aimed here to examine the expression and prognostic significance of the DNA replication-licensing factors chromatin licensing and DNA replication factor 1 (CDT1), cell-division cycle 6 (CDC6), minichromosome maintenance complex component 7 (MCM7) and also of the ubiquitin ligase regulator of CDT1, cullin 4A (CUL4A), in PDAC. MATERIALS AND METHODS: Expression levels of CUL4, CDT1, CDC6 and MCM7 were evaluated by immunohistochemistry in 76 formalin-fixed paraffin-embedded specimens of PDAC patients in relation to DNA-damage response marker H2AX, clinicopathological parameters and survival. We also conducted bioinformatics analysis of data from online available databases to corroborate our findings. RESULTS: CUL4A and DNA replication-licensing factors were overexpressed in patients with PDAC and expression of CDT1 positively correlated with H2AX. Expression of CUL4A and CDT1 positively correlated with lymph node metastasis. Importantly, elevated CUL4A expression was associated with reduced overall survival and was an independent indicator of poor prognosis on multivariate analysis. CONCLUSION: Our findings implicate CUL4A, CDT1, CDC6 and MCM7 in PDAC progression and identify CUL4A as an independent prognostic factor for this disease.

Ubiquitylation of RUNX3 by RNA-binding ubiquitin ligase MEX3C promotes tumorigenesis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common pathological type of lung cancer, but the early diagnosis rate is low. The RNA-binding ubiquitin ligase MEX3C promotes tumorigenesis in several cancers but its mechanism of action in LUAD is unclear. In this study, the biological activity of MEX3C was assessed in LUAD. MEX3C and RUNX3 mRNA levels in the tissues of LUAD patients were determined using reverse transcription­quantitative PCR. The involvement of MEX3C in the growth and metastasis of LUAD cells was measured by EdU assay, CCK-8, colony formation, Transwell assay, TUNEL, and flow cytometry. Expression of apoptosis and epithelial-mesenchymal transition related proteins were determined using western blotting analysis. LUAD cells transfected with si-MEX3C were administered to mice subcutaneously to monitor tumor progression and metastasis. We found that MEX3C is strongly upregulated in LUAD tissue sections, and involved in proliferation and migration. A549 and H1299 cells had significantly higher levels of MEX3C expression compared to control HBE cells. Knockdown of MEX3C dramatically decreased cell proliferation, migration, and invasion, and accelerated apoptosis. Mechanistically, we demonstrate MEX3C induces ubiquitylation and degradation of tumor suppressor RUNX3. Moreover, RUNX3 transcriptionally represses Suv39H1, as revealed by RNA pull-down and chromatin immunoprecipitation assays. The in vivo mice model demonstrated that knockdown of MEX3C reduced LUAD growth and metastasis significantly. Collectively, we reveal a novel MEX3C-RUNX3-Suv39H1 signaling axis driving LUAD pathogenesis. Targeting MEX3C may represent a promising therapeutic strategy against LUAD.

[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

Transcriptomic and metabolomic analysis unveils a negative effect of glutathione metabolism on laccase activity in Cerrena unicolor 87613.

The white rot fungus Cerrena unicolor 87613 has been previously shown to be a promising resource in laccase production, an enzyme with significant biotechnological applications. Conventional methods face technical challenges in improving laccase activity. Attempts are still being made to develop novel approaches for further enhancing laccase activity. This study aimed to understand the regulation of laccase activity in C. unicolor 87613 for a better exploration of the novel approach. Transcriptomic and metabolomic analyses were performed to identify key genes and metabolites involved in extracellular laccase activity. The findings indicated a strong correlation between the glutathione metabolism pathway and laccase activity. Subsequently, experimental verifications were conducted by manipulating the pathway using chemical approaches. The additive reduced glutathione (GSH) dose-dependently repressed laccase activity, while the GSH inhibitors (APR-246) and reactive oxygen species (ROS) inducer (H2O2) enhanced laccase activity. Changes in GSH levels could determine the intracellular redox homeostasis in interaction with ROS and partially affect the expression level of laccase genes in C. unicolor 87613 in turn. In addition, GSH synthetase was found to mediate GSH abundance in a feedback loop. This study suggests that laccase activity is negatively influenced by GSH metabolism and provides a theoretical basis for a novel strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.IMPORTANCEThe production of laccase activity is limited by various conventional approaches, such as heterologous expression, strain screening, and optimization of incubation conditions. There is an urgent need for a new strategy to meet industrial requirements more effectively. In this study, we conducted a comprehensive analysis of the transcriptome and metabolome of Cerrena unicolor 87613. For the first time, we discovered a negative role played by reduced glutathione (GSH) and its metabolic pathway in influencing extracellular laccase activity. Furthermore, we identified a feedback loop involving GSH, GSH synthetase gene, and GSH synthetase within this metabolic pathway. These deductions were confirmed through experimental investigations. These findings not only advanced our understanding of laccase activity regulation in its natural producer but also provide a theoretical foundation for a strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.

Biotin protein ligase as you like it: Either extraordinarily specific or promiscuous protein biotinylation.

Biotin (vitamin H or B7) is a coenzyme essential for all forms of life. Biotin has biological activity only when covalently attached to a few key metabolic enzyme proteins. Most organisms have only one attachment enzyme, biotin protein ligase (BPL), which attaches biotin to all target proteins. The sequences of these proteins and their substrate proteins are strongly conserved throughout biology. Structures of both the biotin ligase- and biotin-acceptor domains of mammals, plants, several bacterial species, and archaea have been determined. These, together with mutational analyses of ligases and their protein substrates, illustrate the exceptional specificity of this protein modification. For example, the Escherichia coli BPL biotinylates only one of the >4000 cellular proteins. Several bifunctional bacterial biotin ligases transcriptionally regulate biotin synthesis and/or transport in concert with biotinylation. The human BPL has been demonstrated to play an important role in that mutations in the BPL encoding gene cause one form of the disease, biotin-responsive multiple carboxylase deficiency. Promiscuous mutant versions of several BPL enzymes release biotinoyl-AMP, the active intermediate of the ligase reaction, to solvent. The released biotinoyl-AMP acts as a chemical biotinylation reagent that modifies lysine residues of neighboring proteins in vivo. This proximity-dependent biotinylation (called BioID) approach has been heavily utilized in cell biology.

Small-molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states.

Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small-molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase and demonstrated that CFTR-F508del ERAD is robust. Gene-drug interaction experiments illustrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.

miRNA-211 maintains metabolic homeostasis in medulloblastoma through its target gene long-chain acyl-CoA synthetase 4.

The prognosis of childhood medulloblastoma (MB) is often poor, and it usually requires aggressive therapy that adversely affects quality of life. microRNA-211 (miR-211) was previously identified as an important regulator of cells that descend from neural cells. Since medulloblastomas primarily affect cells with similar ontogeny, we investigated the role and mechanism of miR-211 in MB. Here we showed that miR-211 expression was highly downregulated in cell lines, PDXs, and clinical samples of different MB subgroups (SHH, Group 3, and Group 4) compared to normal cerebellum. miR-211 gene was ectopically expressed in transgenic cells from MB subgroups, and they were subjected to molecular and phenotypic investigations. Monoclonal cells stably expressing miR-211 were injected into the mouse cerebellum. miR-211 forced expression acts as a tumor suppressor in MB both in vitro and in vivo, attenuating growth, promoting apoptosis, and inhibiting invasion. In support of emerging regulatory roles of metabolism in various forms of cancer, we identified the acyl-CoA synthetase long-chain family member (ACSL4) as a direct miR-211 target. Furthermore, lipid nanoparticle-coated, dendrimer-coated, and cerium oxide-coated miR-211 nanoparticles were applied to deliver synthetic miR-211 into MB cell lines and cellular responses were assayed. Synthesizing nanoparticle-miR-211 conjugates can suppress MB cell viability and invasion in vitro. Our findings reveal miR-211 as a tumor suppressor and a potential therapeutic agent in MB. This proof-of-concept paves the way for further pre-clinical and clinical development.

The YmgB-SpoT interaction triggers the stringent response in Escherichia coli.

Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent of its proposed role in the two-component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.

Deficiency of ligase IV leads to reduced NHEJ, accumulation of DNA damage, and can sensitize cells to cancer therapeutics.

Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient mouse cells, which are embryonic lethal, Ligase IV deficient human cells, including pre-B cells, are viable. Using CRISPR-Cas9 mediated genome editing, we have generated six different LIG4 mutants in cervical cancer and normal kidney epithelial cell lines. While the LIG4 mutant cells showed a significant reduction in NHEJ, joining mediated through microhomology-mediated end joining (MMEJ) and homologous recombination (HR) were significantly high. The reduced NHEJ joining activity was restored by adding purified Ligase IV/XRCC4. Accumulation of DSBs and reduced cell viability were observed in LIG4 mutant cells. LIG4 mutant cells exhibited enhanced sensitivity towards DSB-inducing agents such as ionizing radiation (IR) and etoposide. More importantly, the LIG4 mutant of cervical cancer cells showed increased sensitivity towards FDA approved drugs such as Carboplatin, Cisplatin, Paclitaxel, Doxorubicin, and Bleomycin used for cervical cancer treatment. These drugs, in combination with IR showed enhanced cancer cell death in the background of LIG4 gene mutation. Thus, our study reveals that mutation in LIG4 results in compromised NHEJ, leading to sensitization of cervical cancer cells towards currently used cancer therapeutics.

Systematic profiling of dominant ubiquitin variants reveals key functional nodes contributing to evolutionary selection.

Dominant-negative mutations can help to investigate the biological mechanisms and to understand the selective pressures for multifunctional proteins. However, most studies have focused on recessive mutant effects that occur in the absence of a second functional gene copy, which overlooks the fact that most eukaryotic genomes contain more than one copy of many genes. We have identified dominant effects on yeast growth rate among all possible point mutations in ubiquitin expressed alongside a wild-type allele. Our results reveal more than 400 dominant-negative mutations, indicating that dominant-negative effects make a sizable contribution to selection acting on ubiquitin. Cellular and biochemical analyses of individual ubiquitin variants show that dominant-negative effects are explained by varied accumulation of polyubiquitinated cellular proteins and/or defects in conjugation of ubiquitin variants to ubiquitin ligases. Our approach to identify dominant-negative mutations is general and can be applied to other proteins of interest.

GID complex regulates the differentiation of neural stem cells by destabilizing TET2.

Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.

CBX4 deletion promotes tumorigenesis under KrasG12D background by inducing genomic instability.

Chromobox protein homolog 4 (CBX4) is a component of the Polycomb group (PcG) multiprotein Polycomb repressive complexes 1 (PRC1), which is participated in several processes including growth, senescence, immunity, and tissue repair. CBX4 has been shown to have diverse, even opposite functions in different types of tissue and malignancy in previous studies. In this study, we found that CBX4 deletion promoted lung adenocarcinoma (LUAD) proliferation and progression in KrasG12D mutated background. In vitro, over 50% Cbx4L/L, KrasG12D mouse embryonic fibroblasts (MEFs) underwent apoptosis in the initial period after Adeno-Cre virus treatment, while a small portion of survival cells got increased proliferation and transformation abilities, which we called selected Cbx4-/-, KrasG12D cells. Karyotype analysis and RNA-seq data revealed chromosome instability and genome changes in selected Cbx4-/-, KrasG12D cells compared with KrasG12D cells. Further study showed that P15, P16 and other apoptosis-related genes were upregulated in the primary Cbx4-/-, KrasG12D cells due to chromosome instability, which led to the large population of cell apoptosis. In addition, multiple pathways including Hippo pathway and basal cell cancer-related signatures were altered in selected Cbx4-/-, KrasG12D cells, ultimately leading to cancer. We also found that low expression of CBX4 in LUAD was associated with poorer prognosis under Kras mutation background from the human clinical data. To sum up, CBX4 deletion causes genomic instability to induce tumorigenesis under KrasG12D background. Our study demonstrates that CBX4 plays an emerging role in tumorigenesis, which is of great importance in guiding the clinical treatment of lung adenocarcinoma.

Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation.

Polycomb repressive complex 1 (PRC1) undergoes phase separation to form Polycomb condensates that are multi-component hubs for silencing Polycomb target genes. In this study, we demonstrate that formation and regulation of PRC1 condensates are consistent with the scaffold-client model, where the Chromobox 2 (CBX2) protein behaves as the scaffold while the other PRC1 proteins are clients. Such clients induce a re-entrant phase transition of CBX2 condensates. The composition of the multi-component PRC1 condensates (1) determines the dynamic properties of the scaffold protein; (2) selectively promotes the formation of CBX4-PRC1 condensates while dissolving condensates of CBX6-, CBX7-, and CBX8-PRC1; and (3) controls the enrichment of CBX4-, CBX7-, and CBX8-PRC1 in CBX2-PRC1 condensates and the exclusion of CBX6-PRC1 from CBX2-PRC1 condensates. Our findings uncover how multi-component PRC1 condensates are assembled via an intricate scaffold-client mechanism whereby the properties of the PRC1 condensates are sensitively regulated by its composition and stoichiometry.