Biology subjects

Fang, H.

Publications and source records attributed to Fang, H..

At least 73 records · Page 4Linked to original sources

bioRxiv2020

Single-cell landscape of nuclear configuration and gene expression during stem cell differentiation and X inactivation

Mammalian development is associated with extensive changes in gene expression, chromatin accessibility, and nuclear structure. Here, we follow such changes associated with mouse embryonic stem cell differentiation and X inactivation by integrating, for the first time, allele-specific data obtained by high-throughput single-cell RNA-seq, ATAC-seq, and Hi-C. In differentiated cells, contact decay profiles, which clearly distinguish the active and inactive X chromosomes, reveal loss of the inactive X-specific structure at mitosis followed by a rapid reappearance, suggesting a bookkeeping mechanism. In differentiating embryonic stem cells, changes in contact decay profiles are detected in parallel on both the X chromosomes and autosomes, suggesting profound simultaneous reorganization. The onset of the inactive X-specific structure in single cells is notably delayed relative to that of gene silencing, consistent with the idea that chromatin compaction is a late event of X inactivation. Novel computational approaches to effectively align single-cell gene expression, chromatin accessibility, and 3D chromosome structure reveal that long-range structural changes to chromosomes appear as discrete events, unlike progressive changes in gene expression and chromatin accessibility.

genomics↗

bioRxiv2020

Deficiency in DNA mismatch repair of methylation damage is a major mutational process in cancer

DNA mismatch repair (MMR) is essential for maintaining genome integrity with its deficiency predisposing to cancer1. MMR is well known for its role in the post-replicative repair of mismatched base pairs that escape proofreading by DNA polymerases following cell division2. Yet, cancer genome sequencing has revealed that MMR deficient cancers not only have high mutation burden but also harbour multiple mutational signatures3, suggesting that MMR has pleotropic effects on DNA repair. The mechanisms underlying these mutational signatures have remained unclear despite studies using a range of in vitro4,5 and in vivo6 models of MMR deficiency. Here, using mutation data from cancer genomes, we identify a previously unknown function of MMR, showing that the loss of non-canonical replication-independent MMR activity is a major mutational process in human cancers. MMR is comprised of the MutS (MSH2/MSH6) and MutL (MLH1/PMS2) complexes7. Cancers with deficiency of MutS exhibit mutational signature contributions distinct from those deficient of MutL. This disparity is attributed to mutations arising from the unrepaired deamination of 5-methylcytosine (5mC), i.e. methylation damage, as opposed to replicative errors by DNA polymerases induced mismatches. Repair of methylation damage is strongly associated with H3K36me3 chromatin but independent of binding of MBD4, a DNA glycosylase that recognise 5mC and can repair methylation damage. As H3K36me3 recruits MutS, our results suggest that MutS is the essential factor in mediating the repair of methylation damage. Cell line models of MMR deficiency display little evidence of 5mC deamination-induced mutations as their rapid rate of proliferation limits for the opportunity for methylation damage. We thus uncover a non-canonical role of MMR in the protection against methylation damage in non-dividing cells.

genomics↗

bioRxiv2020

Allele-specific gene regulation by KDM6A

KDM6A demethylates the repressive histone mark H3K27me3 and thus plays an important role in developmental gene regulation. KDM6A expression is female-biased due to escape from X inactivation, suggesting that this protein may play a role in sex differences. Here, we report that maternal and paternal alleles of a subset of mouse genes are differentially regulated by KDM6A. Knockouts of Kdm6a in male and female embryonic stem cells derived from F1 hybrid mice from reciprocal interspecific crosses resulted in preferential downregulation of maternal alleles of a number of genes implicated in development. Moreover, the majority of these genes exhibited a maternal allele expression bias, which was observed in both reciprocal crosses. Promoters of genes downregulated on maternal but not paternal alleles demonstrated a loss of chromatin accessibility, while the expected increase in H3K27me3 levels occurred only at promoters of genes downregulated on paternal but not maternal alleles. These results illustrate parent-of-origin mechanisms of gene regulation by KDM6A, consistent with histone demethylation-dependent and -independent activities.

molecular biology↗

bioRxiv2020

RNF219 interacts with CCR4-NOT in regulating stem cell differentiation

Regulation of RNA stability plays a crucial role in gene expression control. Deadenylation is the initial rate-limiting step for the majority of RNA decay events. Here, we show that RING Finger Protein 219 (RNF219) interacts with the CCR4-NOT deadenylase complex. RNF219-CCR4-NOT exhibits deadenylation activity in vitro. RNA-seq analyses identify some of the 2-cell specific genes and the neuronal genes significantly down-regulated upon RNF219 knockdown, while up-regulated after depletion of the CCR4-NOT subunit CNOT10 in mouse embryonic stem (ES) cells. RNF219 depletion leads to impaired neuronal lineage commitment during ES cell differentiation. Our study suggests that RNF219 is a novel interacting partner of CCR4-NOT, and required for maintenance of ES cell pluripotency.View Full Text

molecular biology↗

bioRxiv2020

Unsupervised manifold alignment for single-cell multi-omics data

Integrating single-cell measurements that capture different properties of the genome is vital to extending our understanding of genome biology. This task is challenging due to the lack of a shared axis across datasets obtained from different types of single-cell experiments. For most such datasets, we lack corresponding information among the cells (samples) and the measurements (features). In this scenario, unsupervised algorithms that are capable of aligning single-cell experiments are critical to learning an in silico co-assay that can help draw correspondences among the cells. Maximum mean discrepancy-based manifold alignment (MMD-MA) is such an unsupervised algorithm. Without requiring correspondence information, it can align single-cell datasets from different modalities in a common shared latent space, showing promising results on simulations and a small-scale single-cell experiment with 61 cells. However, it is essential to explore the applicability of this method to larger single-cell experiments with thousands of cells so that it can be of practical interest to the community. In this paper, we apply MMD-MA to two recent datasets that measure transcriptome and chromatin accessibility in ~2000 single cells. To scale the runtime of MMD-MA to a more substantial number of cells, we extend the original implementation to run on GPUs. We also introduce a method to automatically select one of the user-defined parameters, thus reducing the hyperparameter search space. We demonstrate that the proposed extensions allow MMD-MA to accurately align state-of-the-art single-cell experiments.

bioinformatics↗

bioRxiv2020

Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium

The biomanufacturing of D-glucaric acid has been attracted increasing interest and the industrial yeast Saccharomyces cerevisiae is regarded as an excellent host for D-glucaric acid production. Here we constructed the biosynthetic pathway of D-glucaric acid in S. cerevisiae INVSc1 whose opi1 was knocked out and obtained two engineered strains, LGA-1 and LGA-C, producing record breaking titers of D-glucaric acid, 9.53 {+/-} 0.46 g/L and 11.21 {+/-} 0.63 g/L D-glucaric acid from 30 g/L glucose and 10.8 g/L myo-inositol in the mode of fed-batch fermentation, respectively. Due to the genetic stability and the outperformance in subsequent applications, however, LGA-1 was a preferable strain. As one of the top chemicals from biomass, there have been no reports on D-glucaric acid production from lignocellulose, which is the most abundant renewable on earth. Therefore, the biorefinery processes of lignocellulose for D-glucaric acid production including separated hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP) were investigated in this work and CBP by an artificial microbial consortium composed of Trichoderma reesei Rut-C30 and S. cerevisiae LGA-1 was found to have relatively high D-glucaric acid titers and yields after 7 d fermentation, 0.54 {+/-} 0.12 g/L D-glucaric acid from 15 g/L Avicel, and 0.45 {+/-} 0.06 g/L D-glucaric acid from 15 g/L steam exploded corn stover (SECS), respectively. In attempts to design the microbial consortium for more efficient CBP the team consisted of the two members, T. reesei Rut-C30 and S. cerevisiae LGA-1, was found to be the best with excellent work distribution and collaboration. This desirable and promising approach for direction production of D-glucaric acid from lignocellulose deserves extensive and in-depth research.

synthetic biology↗

bioRxiv2020

Deletion of FUNDC2 and CMC4 on chromosome Xq28 is sufficient to cause hypergonadotropic hypogonadism in men

BackgroundHypergonadotropic hypogonadism (HH) is characterized by low sex steroid levels and secondarily elevated gonadotropin levels with either congenital or acquired etiology. Genetic factors leading to HH have yet to be fully elucidated. MethodsHere, we report on genome and transcriptome data analyses from a male patient with HH and history of growth delay who has an inherited deletion of chromosome Xq28. Furthermore, expression analyses were done for this patient and his unaffected family members and compared to normal controls to identify dysregulated genes due to this deletion. ResultsOur patients Xq28 deletion is 44,806bp and contains only two genes FUNDC2 and CMC4. Expression of both FUNDC2 and CMC4 are completely abolished in the patient. Gene ontology analyses of differentially expressed genes in the patient in comparison to controls show that significantly up-regulated genes in the patient are enriched in Sertoli cell barrier regulation, apoptosis, inflammatory response and gonadotropin-releasing regulation. Indeed, our patient has an elevated FSH level, which regulates Sertoli cell proliferation and spermatogenesis. In his mother and sister, who are heterozygous for this deletion, X-chromosome inactivation is skewed towards the deleted X, suggesting a mechanism to avoid FSH dysregulation. ConclusionCompared to the previously reported men with variable sized Xq28 deletions, our study suggests that loss of function of FUNDC2 and/or CMC4 results in dysregulation of apoptosis, inflammation and FSH, and is sufficient to cause Xq28-associated HH.

genomics↗

bioRxiv2019

A Quantitative Proteome Map of the Human Body

Determining protein levels in each tissue and how they compare with RNA levels is important for understanding human biology and disease as well as regulatory processes that control protein levels. We quantified the relative protein levels from 12,627 genes across 32 normal human tissue types prepared by the GTEx project. Known and new tissue specific or enriched proteins (5,499) were identified and compared to transcriptome data. Many ubiquitous transcripts are found to encode highly tissue specific proteins. Discordance in the sites of RNA expression and protein detection also revealed potential sites of synthesis and action of protein signaling molecules. Overall, these results provide an extraordinary resource, and demonstrate that understanding protein levels can provide insights into metabolism, regulation, secretome, and human diseases.\n\nSummaryQuantitative proteome study of 32 human tissues and integrated analysis with transcriptome data revealed that understanding protein levels could provide in-depth knowledge to post transcriptional or translational regulations, human metabolism, secretome, and diseases.

systems biology↗

bioRxiv2019

Architectural RNA is required for heterochromatin organization

In addition to its known roles in protein synthesis and enzyme catalysis, RNA has been proposed to stabilize higher-order chromatin structure. To distinguish presumed architectural roles of RNA from other functions, we applied a ribonuclease digestion strategy to our CUT&RUN in situ chromatin profiling method (CUT&RUN.RNase). We find that depletion of RNA compromises association of the murine nucleolar protein Nucleophosmin with pericentric heterochromatin and alters the chromatin environment of CCCTC-binding factor (CTCF) bound regions. Strikingly, we find that RNA maintains the integrity of both constitutive (H3K9me3 marked) and facultative (H3K27me3 marked) heterochromatic regions as compact domains, but only moderately stabilizes euchromatin. To establish the specificity of heterochromatin stabilization by RNA, we performed CUT&RUN on cells deleted for the Firre long non-coding RNA and observed disruption of H3K27me3 domains on several chromosomes. We conclude that RNA maintains local and global chromatin organization by acting as a structural scaffold for heterochromatic domains.

genomics↗

bioRxiv2019

Mutational processes of distinct POLE exonuclease domain mutants drive an enrichment of a specific TP53 mutation in colorectal cancer

Cancer genomes with mutations in the exonuclease domain of Polymerase Epsilon (POLE) present with an extraordinarily high somatic mutation burden. In vitro studies have shown that distinct POLE mutants exhibit different polymerase activity and yet, how these POLE mutants generate mutations across cancer genomes and influence driver events remains poorly understood. Here we analyzed 7,345 colorectal cancer samples, including nine whole genome sequenced samples harboring POLE mutations. Our analysis identified differential mutation spectra across the mutants including methylation-independent enrichment of C>T mutations in POLE V411L. In contrast, analysis of other genomic regions showed similar mutation profiles across the different POLE mutants. Notably, we found that POLE mutants with the TP53 R213* mutation, caused by a TT[C>T]GA substitution, have significantly higher relative frequency of this mutational context compared with samples without this mutation. This finding demonstrates that variations in underlying mutation spectra can increase the likelihood of specific driver mutation formation.

genomics↗

bioRxiv2019

Ribosomal DNA methylation as stable biomarkers for detection of cancer in plasma

BackgroundRecently, liquid biopsy for cancer detection has pursued great progress. However, there are still a lack of high quality markers. It is a challenge to detect cancer stably and accurately in plasma cell free DNA (cfDNA), when the ratio of cancer signal is low. Repetitive genes or elements may improve the robustness of signals. In this study, we focused on ribosomal DNA which repeats hundreds of times in human diploid genome and investigated performances for cancer detection in plasma.\n\nResultsWe collected bisulfite sequencing samples including normal tissues and 4 cancer types and found that intergenic spacer (IGS) of rDNA has high methylation levels and low variation in normal tissues and plasma. Strikingly, IGS of rDNA shows significant hypo-methylation in tumors compared with normal tissues. Further, we collected plasma bisulfite sequencing data from 224 healthy subjects and cancer patients. Means of AUC in testing set were 0.96 (liver cancer), 0.94 (lung cancer and), 0.92 (colon cancer) with classifiers using only 10 CpG sites. Due to the feature of high copy number, when liver cancer plasma WGBS was down-sampled to 10 million raw reads (0.25x whole genome coverage), the prediction performance decreased only a bit (mean AUC=0.93). Finally, methylation of rDNA could also be used for monitor cancer progression and treatment.\n\nConclusionTaken together, we provided the high-resolution map of rDNA methylation in tumors and supported that methylation of rDNA was a competitive and robust marker for detecting cancer and monitoring cancer progression in plasma.

genomics↗

bioRxiv2019

C1orf109L promote R-loop accumulation induced DNA damage to inhibit cell growth

As a function unknown gene, C1orf109 is lower expression in various cells. Here, we reported that C1orf109L, the longest variant of C1orf109, which interacted with R-loop-regulating proteins to trigger R-loop, a three-stranded nucleic acid structure frequently mediated genome instability, accumulation. C1orf109L induce chronic DNA damage to promote P21 upregulation and strongly inhibits cell growth in vitro and in vivo by arresting the cell cycle in the G2 phase. With camptothecin (CPT), an R-loop activator, treatment, C1orf109L further triggers R-loop accumulation-induced DNA damage and promotes cell death by activating cell-death pathway. Furthermore, CPT treatment increases C1orf109L ubiquitination and turnover, which inhibits cell death and promotes the G0/G1 phase of the cell cycle. Therefore, our data illustrated the mechanisms underlying C1orf109L-related cell growth inhibition and provide feasibility and limitations for C1orf109L as a potential target for cancer therapy.

cell biology↗

bioRxiv2019

Exploring the influence of surface proteins on probiotic activity of Lactobacillus pentosus HC-2 in Litopenaeus vannamei midgut via Label-free quantitative proteomic analysis

Our previously work showed that Lactobacillus pentosus HC-2 as probiotic could improve the growth performance, immune response, gut bacteria diversity and disease resistance of Litopenaeus vannamei. However, the probiotic mechanism of was not fully characterized. In the present study, histology and proteomic analysis was performed to explore the influence of HC-2 surface protein on its probiotic effects to L. vannamei after feeding the intact surface proteins or the probiotic treated with lithium chloride (LiCl) to remove non-covalently bound surface proteins or no probiotic for four weeks. Histological observation found that feeding with normal HC-2 obviously improved the intestinal histology and enhanced the protective effect against pathogens damages, but fed with LiCl-treated HC-2 didnt improve the intestinal environment. A total over 2,764 Peptides and 1,118 uniproteins were identified from L. vannamei midgut, 211 proteins were significant differentially expressed normal HC-2 group compared with control, 510 proteins were significant differentially expressed in LiCl-tread HC-2 group compared with control, and 458 proteins were significant differentially expressed in LiCl-tread HC-2 group compared with normal HC-2 group. GO/KEGG enrichment analysis of the significantly different proteins demonstrated that fed with normal HC-2 mainly induced immune response, metabolic, cell adhesion and cell-cell signaling related proteins up-regulation, which were contributed to bacteria adhesion and colonization in midgut to improve shrimp immune system and growth, but these proteins were suppressed after feeding with deprived surface proteins bacteria. Taken together, these results indicating that the surface proteins were indispensable for HC-2 to execute probiotic effects in midgut of shrimp.

microbiology↗

bioRxiv2019

Transcriptome Analysis of juvenile Pacific White Shrimp (Litopenaeus vannamei) with symptom of stunted growth

In search for viruses associated with Litopenaeus vannamei with symptom of stunted growth, we have undertaken a comparative transcriptome analysis of total RNA sequences using Illumina based high-throughput sequencing (HTS). We obtain 89000242 and 95126160 high-quality clean reads from cDNA libraries of L.vannamei with stunted growth symptom (sick group) and health shrimp (health group control), respectively. Totally, 108221 unigenes with an average length of 716 bp were obtained from RNA-seq data. The unigenes were annotated against NR, NT, KO, KOG, SwissProt, PFAM and GO databases. 3975 (3.67%) showed significant matches in all the above databases and 39812 (36.78%) unigenes were annotated in at least one database. Comparative transcriptomic analysis revealed that 807 significantly differentially expressed unigenes were identified, including 282 down-regulated genes and 525 up-regulated genes. Thirteen up-regulated virus-related genes were only discovered in the sick shrimp groups, but not in health ones. Five of them were closely related to virus family Picornaviridae. From phylogenetic tree, we could find five viral genes were closely related to genus Ampvirus, Falcovirus, Aquamavirus (Seal picornavirus). Some up-regulated genes in the Sick groups mainly included genes involved in virus infecting processes concerning antigen processing and presenting, apoptosis, lysosome, phagosome and inflammation. While, many down-regulated genes in the Sick groups mainly involved in the process of starch and sucrose metabolism, and protein digestion and absorption. Our research provided some useful information about pathogenic factor and mechanism on the stunted growth symptom of L.vannamei, and was significant for the control and prevention of this disease.

pathology↗

bioRxiv2018

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

The only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea, and the only unknown step in its biosynthesis is the production of the intermediate adenosylcobinamide phosphate. Here, using genetic and metabolic engineering, we generated an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. Excitingly, the BluE and CobC enzymes from Rhodobacter capsulatus transform L-threonine into (R)-1-Amino-2-propanol O-2-Phosphate, which is then condensed with adenosylcobyric acid to yield adenosylcobinamide phosphate by either CobD from the aeroic R. capsulatus or CbiB from the anerobic Salmonella typhimurium. These findings suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increased the vitamin B12 yield of a recombinant E. coli strain by more than [~]250-fold to 307.00 {micro}g/g DCW via metabolic engineering and optimization of fermentation conditions. Beyond our scientific insights about the aerobic and anaerobic pathways and our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

synthetic biology↗

bioRxiv2018

WDR45 contributes to neurodegeneration through regulation of ER homeostasis and neuronal death

Mutations in the autophagy gene WDR45 cause {beta}-propeller protein-associated neurodegeneration (BPAN); however the molecular and cellular mechanism of the disease process is largely unknown. Here we generated constitutive Wdr45 knockout (KO) mice that displayed cognitive impairments, abnormal synaptic transmission and lesions in hippocampus and basal ganglia. Immunohistochemistry analysis shows loss of neurons in prefrontal cortex and basal ganglion in aged mice, and increased apoptosis in these regions, recapitulating a hallmark of neurodegeneration. Quantitative proteomic analysis shows accumulation of endoplasmic reticulum (ER) proteins in KO mouse. Furthermore, we show that a defect in autophagy results in impaired ER turnover and ER stress. The unfolded protein response (UPR) is elevated through IRE1 and possibly other kinase signaling pathways, and eventually leads to neuronal apoptosis. Suppression of ER stress, or activation of autophagy through inhibition of mTOR pathway rescues neuronal death. Thus, our study not only provides mechanistic insights for BPAN, but also suggests that a defect in macroautophagy machinery leads to impairment in selective organelle autophagy.

cell biology↗

bioRxiv2017

Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences, and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available with nearly 20,000 variants present, most of which were missed by prior efforts. Surrounding the important HER2 locus, we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the transcriptome and sheds new light on the complexity of cancer progression.

genomics↗

bioRxiv2017

Accurate detection of complex structural variations using single molecule sequencing

Structural variations (SVs) are the largest source of genetic variation, but remain poorly understood because of limited genomics technology. Single molecule long read sequencing from Pacific Biosciences and Oxford Nanopore has the potential to dramatically advance the field, although their high error rates challenge existing methods. Addressing this need, we introduce open-source methods for long read alignment (NGMLR, https://github.com/philres/ngmlr) and SV identification (Sniffles, https://github.com/fritzsedlazeck/Sniffles) that enable unprecedented SV sensitivity and precision, including within repeat-rich regions and of complex nested events that can have significant impact on human disorders. Examining several datasets, including healthy and cancerous human genomes, we discover thousands of novel variants using long reads and categorize systematic errors in short-read approaches. NGMLR and Sniffles are further able to automatically filter false events and operate on low amounts of coverage to address the cost factor that has hindered the application of long reads in clinical and research settings.

bioinformatics↗