Levels Of Transcription Factors Research Articles

Engineered Transcription Factor Binding Arrays for DNA-based Gene Expression Control in Mammalian Cells.

Manipulating gene expression in mammalian cells is critical for cell engineering applications. Here we explore the potential of transcription factor (TF) recognition element arrays as DNA tools for modifying free TF levels in cells and thereby controlling gene expression. We first demonstrate proof-of-concept, showing that Tet TF-binding recognition element (RE) arrays of different lengths can tune gene expression and alter gene circuit performance in a predictable manner. We then open-up the approach to interface with any TF with a known binding site by developing a new method called Cloning Troublesome Repeats in Loops (CTRL) that can assemble plasmids with up to 256 repeats of any RE sequence. Transfection of RE array plasmids assembled by CTRL into mammalian cells show potential to modify host cell gene regulation at longer array sizes by sequestration of the TF of interest. RE array plasmids built using CTRL were demonstrated to target both synthetic and native mammalian TFs, illustrating the ability to use these tools to modulate genetic circuits and instruct cell fate. Together this work advances our ability to assemble repetitive DNA arrays and showcases the use of TF-binding RE arrays as a method for manipulating mammalian gene expression, thus expanding the possibilities for mammalian cell engineering.

Transcriptome-wide Characterization of Novel WRKY Family Genes Engaged in Crocin Biosynthesis in Crocus sativus

Crocins comprise a family of hydrophilic carotenoids with pharmacological properties that are produced in significant quantities in stigma of Crocus sativus. Although the biosynthesis pathway of crocins has been sufficiently elucidated, there is a paucity of information regarding how transcription factors (TFs) regulate crocin biosynthesis in various stigma developmental stages. WRKY TFs play a role in modulating carotenoid/apocarotenoid metabolism. To provide an overview of the WRKY family in Crocus sativus (CsWRKY) and characterize candidate CsWRKY TFs involved in the biosynthesis of crocins, CsWRKY genes were identified from RNA-sequenced stigma at different developmental stages. A phylogenetic analysis was performed to characterize their evolutionary interrelatedness. A coexpression analysis of CsWRKY genes and crocin biosynthesis-related genes was performed. A quantitative real-time polymerase chain reaction was used to corroborate the expression level of CsWRKY TFs in various tissue and at different developmental stages. A total of 34 CsWRKY TFs were identified from the stigma of C. sativus. The CsWRKY TFs, together with their orthologs from Arabidopsis, were clustered into group I, II, or III following phylogenetic analysis. A correlation analysis revealed that the expressions of the TFs CsWRKY1, CsWRKY2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28 were strongly related to the expression of crocin biosynthesis-related genes CsBCH, CsCCD2L, CsALDH, and CsUGT. CsWRKY2, CsWRKY15, and CsWRKY28 exhibited identical motifs and were stratified into group IId. Transcript levels of candidate CsWRKY genes were higher in stigma than in other tissues and were proportional to the crocin content.

Single-cell multi-omics analysis reveals cooperative transcription factors for gene regulation in oligodendrocytes.

Oligodendrocytes are the myelinating cells within the central nervous system. Many oligodendrocyte genes have been associated with brain disorders. However, how transcription factors (TFs) cooperate for gene regulation in oligodendrocytes remains largely uncharacterized. To address this, we integrated scRNA-seq and scATAC-seq data to identify the cooperative TFs that co-regulate the target gene (TG) expression in oligodendrocytes. First, we identified co- binding TF pairs whose binding sites overlapped in oligodendrocyte-specific regulatory regions. Second, we trained a deep learning model to predict the expression level of each TG using the expression levels of co-binding TFs. Third, using the trained models, we computed the TF importance and TF-TF interaction scores for predicting TG expression by the Shapley interaction scores. We found that the co-binding TF pairs involving known important TF pairs for oligodendrocyte differentiation, such as SOX10-TCF12, SOX10-MYRF, and SOX10-OLIG2, exhibited significantly higher Shapley scores than others (t-test, p-value < 1e-4). Furthermore, we identified 153 oligodendrocyte-associated eQTLs that reside in oligodendrocyte-specific enhancers or promoters where their eGenes (TGs) are regulated by cooperative TFs, suggesting potential novel regulatory roles from genetic variants. We also experimentally validated some identified TF pairs such as SOX10-OLIG2 and SOX10-NKX2.2 by co-enrichment analysis, using ChIP-seq data from rat peripheral nerve.

A continuum of zinc finger transcription factor retention on native chromatin underlies dynamic genome organization

Transcription factor (TF) residence on chromatin translates into quantitative transcriptional or structural outcomes on genome. Commonly used formaldehyde crosslinking fixes TF-DNA interactions cumulatively and compromises the measured occupancy level. Here we mapped the occupancy level of global or individual zinc finger TFs like CTCF and MAZ, in the form of highly resolved footprints, on native chromatin. By incorporating reinforcing perturbation conditions, we established S-score, a quantitative metric to proxy the continuum of CTCF or MAZ retention across different motifs on native chromatin. The native chromatin-retained CTCF sites harbor sequence features within CTCF motifs better explained by S-score than the metrics obtained from other crosslinking or native assays. CTCF retention on native chromatin correlates with local SUMOylation level, and anti-correlates with transcriptional activity. The S-score successfully delineates the otherwise-masked differential stability of chromatin structures mediated by CTCF, or by MAZ independent of CTCF. Overall, our study established a paradigm continuum of TF retention across binding sites on native chromatin, explaining the dynamic genome organization.

Genome-wide identification and expression pattern analysis of WRKY transcription factors in response to biotic and abiotic stresses in tea plants (Camellia sinensis)

Plants would encounter various biotic and abiotic stresses during the growth and development. WRKY transcription factors (TFs) as plant-specific TFs, play an important role in responding to various adverse circumstances. Despite some advances were achieved in functional studies of WRKY TFs in tea plants, systematic analysis of the involvement of CsWRKY TFs when facing cold, salt, drought stresses and pathogen and insect attack was lacked. In present study, a total of 78 CsWRKY TFs were identified following the genomic and transcript databases. The expression patterns of CsWRKYs in various organs of tea plants and the expression profiles in response to biotic and abiotic stresses were investigated by examining representative RNA-seq data. Moreover, the effects of hormone treatments (SA and MeJA) on the transcription levels of WRKY TFs were also investigated. The phylogenetic tree of CsWRKY TFs from different species indicated the functional diversity of WRKY TFs was not closely related to their protein classification. Concurrently, CsWRKY70-2 TF was identified as a positive regulator in response to drought stress. This study provided solid and valuable information, helping us better understand the functional diversity of CsWRKY TFs, and laid the foundation for further research on the function of key WRKY genes in tea plants.

Abstract 1694: Using high-throughput screening to identify DNA response elements that sense cancer dysregulated pathways

Abstract Earli is developing a highly sensitive, orthogonal approach that is designed to be delivered via IV injection and uses a genetic construct to usurp dysregulated pathways, and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. The key element of our cancer-activated construct that drives specificity and sensitivity of expression is a promoter sequence, synthetically engineered to leverage transcription factors (TFs) activated in cancer pathways. Acting as a molecular sensor for dysregulated TF levels, the DNA construct expresses a measurable biomarker in cancer cells, while remaining transcriptionally silent in normal adjacent tissues and benign lesions. Previously, we described a multiomics factor analysis (MOFA) to interrogate 200 non-small cell lung cancer (NSCLC) and matched normal adjacent tissue samples comprised of RNA-seq based transcriptional profiling, and MS-based proteomics and phospho-proteomics from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset, and generated a list of top TFs dysregulated in NSCLC. In this study, we show the capability to design, engineer, and test cancer specific activity of combinations of response elements using a Massively Parallel Reporter Assay (MPRA) high-throughput pooled screening (HTS) method. More than 2,000 unique sequences of homotypic or heterotypic TF binding sites corresponding to the top 100 TFs were arrayed with diverse spacing and individually barcoded for the high-throughput screens in NSCLC cell lines. To validate our top hits, we individually tested 10 sequences from the top 25% and 8 sequences from the bottom 25%. Nine of the top sequences produced signals 5-10X higher than the background, whereas only one of the eight bottom sequences produced signal higher than the background. We have individually validated 30 different response elements and found that 28 of 30 drove the expected expression, with the majority of response elements (n=20) showing expression 5-10X higher than background. Remarkably, two of the engineered response elements were able to drive expression 20-30X higher in a specific cell line activated by WNT-signaling, suggesting that these response elements can sense specific cancer dysregulated pathways. Our lead response elements are being tested across a broad range of in vitro primary tumor lines with diverse genetic backgrounds and transcriptional profiles prior to in vivo testing using our imaging platform to distinguish malignant lung nodules. In summary, we have established a robust screening platform to identify response elements that can be activated by cancer dysregulated pathways. We are currently expanding this platform to other cancer models and indications, and leveraging this to explore more complex response elements that use combinations of TFs and natural spacing. Citation Format: Elizabeth Stroebele, Yue (Wendy) Zhang, Ishan Podar, Chloe Xia, Ajda Rojc, Dariusz Wodziak, Maggie Louie, David Suhy. Using high-throughput screening to identify DNA response elements that sense cancer dysregulated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1694.

Mechanism and Effect of HNF4α Decrease in a Rat Model of Cirrhosis and Liver Failure

Background & AimsHNF4α, a master regulator of liver development and the mature hepatocyte phenotype, is downregulated in chronic and inflammatory liver disease. We used contemporary transcriptomics and epigenomics to study the cause and effects of this downregulation, and characterized a multicellular etiology. MethodsProgressive changes in the rat CCl4-model were studied by deep RNA sequencing and genome-wide ChIP-seq analysis of transcription factor (TF) binding and chromatin modification. Studies compared decompensated cirrhosis with liver failure after 26 weeks of treatment to earlier compensated cirrhosis, and to additional rat models of chronic fibrosis. Finally, to resolve cell-specific responses and intercellular signaling, we compared transcriptomes of liver, non-parenchymal, and inflammatory cells. ResultsHNF4α was significantly lower in 26-week cirrhosis, part of a general reduction of TF that regulate metabolism. Nevertheless, increased binding of HNF4α contributed to strong activation of major phenotypic genes, while reduced binding to other genes had a moderate phenotypic effect. Decreased Hnf4a expression was the combined effect of STAT3 and NFκB activation, which similarly reduced expression of other metabolic TF. STAT/NFκB also induced de novo expression of Osmr by hepatocytes, to complement induced expression of Osm by nonparenchymal cells. ConclusionsLiver decompensation by inflammatory STAT3 and NFκB signaling was not a direct consequence of progressive cirrhosis. Despite significant reduction of Hnf4a expression, residual levels of this abundant TF still stimulated strong new gene expression. Reduction of HNF4α was part of a broad hepatocyte transcriptional response to inflammation.

In-organoid single-cell CRISPR screening reveals determinants of hepatocyte differentiation and maturation

BackgroundHarnessing hepatocytes for basic research and regenerative medicine demands a complete understanding of the genetic determinants underlying hepatocyte differentiation and maturation. Single-cell CRISPR screens in organoids could link genetic perturbations with parallel transcriptomic readout in single cells, providing a powerful method to delineate roles of cell fate regulators. However, a big challenge for identifying key regulators during data analysis is the low expression levels of transcription factors (TFs), which are difficult to accurately estimate due to noise and dropouts in single-cell sequencing. Also, it is often the changes in TF activities in the transcriptional cascade rather than the expression levels of TFs that are relevant to the cell fate transition.ResultsHere, we develop Organoid-based Single-cell CRISPR screening Analyzed with Regulons (OSCAR), a framework using regulon activities as readouts to dissect gene knockout effects in organoids. In adult-stem-cell-derived liver organoids, we map transcriptomes in 80,576 cells upon 246 perturbations associated with transcriptional regulation of hepatocyte formation. Using OSCAR, we identify known and novel positive and negative regulators, among which Fos and Ubr5 are the top-ranked ones. Further single-gene loss-of-function assays demonstrate that Fos depletion in mouse and human liver organoids promote hepatocyte differentiation by specific upregulation of liver metabolic genes and pathways, and conditional knockout of Ubr5 in mouse liver delays hepatocyte maturation.ConclusionsAltogether, we provide a framework to explore lineage specifiers in a rapid and systematic manner, and identify hepatocyte determinators with potential clinical applications.

Epigenomic profiling nominates master transcription factors (TFs) driving sarcomatoid differentiation (SD) of renal cell carcinoma (RCC)

Abstract Background Sarcomatoid differentiation of RCC (sRCC) is associated with poor survival. Recent studies showed marked response of sRCC to immune checkpoint blockade (ICB). While distinctive patterns of gene expression in sRCC have been identified, the gene regulatory programs and TFs that drive SD remain unknown. The aim of this study is to nominate TFs responsible for SD and to investigate their association with the clinical outcomes of patients with RCC. Methods Chromatin immunoprecipitation and sequencing (ChIP-seq) for H3K27ac – a histone modification associated with active regulatory elements – was performed on pathologically reviewed sRCC and non-sRCC samples collected at the Dana-Farber Cancer Institute. Regulatory elements that were differentially active between the two groups were identified based on levels of H3K27ac (Benjamini-Hochberg q&amp;lt;0.01, log-fold change (LFC) threshold=1). Enrichment of specific TF binding motifs at activated regulatory elements in sRCC was assessed using HOMER. Differential gene expression analysis of TFs was performed using DESeq2 on RNA-seq data from TCGA. A Mann-Whitney U test was performed on RNA-seq data from the IMmotion151 and Javelin Renal 101 clinical trials to compare mean expression level of TFs in transcript per million (TPM) in these trials. Patients with non-sRCC enrolled in the IMmotion151 trials were divided into quartiles based on gene expression levels of candidate TFs. Progression-free survival (PFS) was compared between non-sRCC patients stratified by expression quartiles as well as patients with sRCC using a multivariable Cox proportional-hazards model accounting for age and IMDC risk score. To validate these findings, a similar analysis was performed in the Javelin Renal 101 trial. Results We obtained high-quality H3K27ac ChIP-seq profiles for 9 sRCC and 17 non-sRCC samples. We identified 278 candidate regulatory elements with increased H3K27ac levels in sRCC vs. non-sRCC. These regulatory elements were enriched for nucleotide motifs bound by the TFs FOSL1 and E2F7. Differential expression analysis between 48 sRCC vs. 493 non-sRCC samples showed that FOSL1 and E2F7 were significantly upregulated in sRCC vs. non-sRCC (LFC=1.7, q=5e-11; LFC=1.8, q=1.3e-20; resp.). Mean TPMs of FOSL1 and E2F7 were significantly increased in sRCC vs. non-sRCC in IMmotion151 cohort and Javelin Renal 101 (all p&amp;lt;0.001). Among patients who received sunitinib, those with the highest quartile of FOSL1 and E2F7 expression showed significantly shorter PFS in IMmotion151 patients compared to patients with the lowest quartile of expression (HR=1.6, 95%CI=1.3-2.2, p=0.008 &amp; HR=2.6, 95%CI=1.8-3.7, p&amp;lt;0.001; resp.). Furthermore, patients with highest quartile of expression showed similar PFS compared to patients with sRCC (p=0.56 and p=0.64; resp.). These results were validated in the sunitinib arm of the Javelin Renal 101 cohort (Figure). Figure: Kaplan-Meier curves of progression-free survival (PFS) in patients with in the sunitinib arm of Javelin Renal 101 by sarcomatoid differentiation and FOSL1 expression levels. Conclusions This is the first study to characterize the epigenomic landscape of sRCC by integrating ChIP-seq and RNA-seq data. Our findings implicated FOSL1 and E2F7 as transcriptional regulators of SD with prognostic relevance. These TFs seems to be associated with aggressive behavior in non-sRCC as well. Further studies are underway to functionally validate these results.

Abstract A029: Epigenomic profiling nominates master transcription factors (TFs) driving sarcomatoid differentiation of renal cell carcinoma (RCC)

Abstract Introduction Sarcomatoid RCC (sRCC) is associated with poor prognosis. Recent studies showed promising response rates of sRCC to immune checkpoint blockade (ICB). Although distinct patterns of gene expression in sRCC have been reported, the gene regulatory programs that drive SD remain unknown. This study aims to nominate TFs driving SD and to evaluate their impact on the clinical outcomes of patients (pts) with RCC. Methods Chromatin immunoprecipitation and sequencing (ChIP-seq) for H3K27ac was performed on histologically annotated areas of sRCC and non-sRCC fresh frozen tissue samples collected at Dana-Farber Cancer Institute. Regulatory elements that were differentially active between the two groups were identified (cutoff p&amp;lt;0.01, log-fold change (LFC) threshold=1). Enrichment of specific TF binding motifs at activated regulatory elements in sRCC was assessed using HOMER. Differential gene expression analysis of TFs was performed using DESeq2 on RNA-seq data from TCGA (Benjamini-Hochberg q &amp;lt;0.01, LFC threshold=1). An unpaired student’s t-test was performed to compare mean gene expression levels in transcript per million (TPM) on baseline sRCC and non-sRCC tissue samples from the IMmotion151 trial (sunitinib vs. atezolizumab-bevacizumab in metastatic RCC). Pts enrolled in the IMmotion151 trial were divided into quartiles based on gene expression levels of candidate TFs. Progression-free survival (PFS) was compared between the quartiles using a multivariable Cox proportional-hazards model accounting for age and IMDC risk score. The association of TF expression with the objective radiographic response (ORR) was analyzed using a logistic regression model. Results ChIP-seq was performed on 9 sRCC &amp; 17 non-sRCC samples. We identified 278 candidate regulatory elements with increased H3K27ac levels in sRCC vs. non-sRCC that were enriched for nucleotide motifs bound by the TFs FOSL1 and E2F7. Differential expression analysis between 48 sRCC vs. 493 non-sRCC samples showed that FOSL1 &amp; E2F7 were significantly upregulated in sRCC vs. non-sRCC (LFC=1.7, q=5e-11; LFC=1.8, q=1.3e-20; resp.). Mean TPMs of FOSL1 &amp; E2F7 were significantly increased in sRCC vs. non-sRCC in IMmotion151 cohort (2.5 vs. 1.5 TPM for FOSL1, and 2.9 vs. 1.9 TPM for E2F7, all p&amp;lt;0.001). Pts in the highest quartile of FOSL1 and E2F7 expression showed significantly shorter PFS in IMmotion151 pts receiving sunitinib alone (HR=1.6, 95%CI=1.3-2.2, p=0.008 &amp; HR=2.6, 95%CI=1.8-3.7, p&amp;lt;0.001; resp.). We observed similar results even after controlling for the presence of SD. Increased expression of E2F7 was associated with worse ORR in IMmotion151 pts who received sunitinib alone, even after controlling for the presence of SD (OR=0.24±0.4, p&amp;lt;0.001). Conclusion This is the first study to analyze the epigenomic landscape of sRCC by integrating ChIP-seq and RNA-seq data. Our findings implicated FOSL1 &amp; E2F7 as transcriptional regulators of SD with prognostic relevance. Further studies are underway to validate these results. Citation Format: Karl Semaan, Talal El Zarif, Marc Eid, Valisha Shah, Brad Fortunato, Renee Maria Saliby, Amin H. Nassar, Sarah Abou Alaiwi, Ji-Heui Seo, Chris Labaki, Ziad Bakouny, Sayed Matar, Nourhan El Ahmar, Yasmin Nabil Laimon, Gwo-Shu Mary Lee, Mark Pomerantz, Jacob Berchuck, Sabina Signoretti, Eliezer Van Allen, Toni Choueiri, David Braun, Matthew Freedman, Sylvan Baca. Epigenomic profiling nominates master transcription factors (TFs) driving sarcomatoid differentiation of renal cell carcinoma (RCC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A029.

Investigation of autism-related transcription factors underlying sex differences in the effects of bisphenol A on transcriptome profiles and synaptogenesis in the offspring hippocampus

BackgroundBisphenol A (BPA) has been linked to susceptibility to autism spectrum disorder (ASD). Our recent studies have shown that prenatal BPA exposure disrupted ASD-related gene expression in the hippocampus, neurological functions, and behaviors associated with ASD in a sex-specific pattern. However, the molecular mechanisms underlying the effects of BPA are still unclear.MethodsTranscriptome data mining and molecular docking analyses were performed to identify ASD-related transcription factors (TFs) and their target genes underlying the sex-specific effects of prenatal BPA exposure. Gene ontology analysis was conducted to predict biological functions associated with these genes. The expression levels of ASD-related TFs and targets in the hippocampus of rat pups prenatally exposed to BPA were measured using qRT-PCR analysis. The role of the androgen receptor (AR) in BPA-mediated regulation of ASD candidate genes was investigated using a human neuronal cell line stably transfected with AR-expression or control plasmid. Synaptogenesis, which is a function associated with genes transcriptionally regulated by ASD-related TFs, was assessed using primary hippocampal neurons isolated from male and female rat pups prenatally exposed to BPA.ResultsWe found that there was a sex difference in ASD-related TFs underlying the effects of prenatal BPA exposure on the transcriptome profiles of the offspring hippocampus. In addition to the known BPA targets AR and ESR1, BPA could directly interact with novel targets (i.e., KDM5B, SMAD4, and TCF7L2). The targets of these TFs were also associated with ASD. Prenatal BPA exposure disrupted the expression of ASD-related TFs and targets in the offspring hippocampus in a sex-dependent manner. Moreover, AR was involved in the BPA-mediated dysregulation of AUTS2, KMT2C, and SMARCC2. Prenatal BPA exposure altered synaptogenesis by increasing synaptic protein levels in males but not in females, but the number of excitatory synapses was increased in female primary neurons only.ConclusionsOur findings suggest that AR and other ASD-related TFs are involved in sex differences in the effects of prenatal BPA exposure on transcriptome profiles and synaptogenesis in the offspring hippocampus. These TFs may play an essential role in an increased ASD susceptibility associated with endocrine-disrupting chemicals, particularly BPA, and the male bias of ASD.

Genome-wide analysis of the XTH gene family and functional analysis of DlXTH23.5/25 during early longan somatic embryogenesis.

Xyloglucan endotransglucosylase (XET)/hydrolase (XTH) is a cell wall-modifying protein that affects cell expansion and loosening of the cell wall. This study focused on the regulatory mechanism of DlXTH genes during early somatic embryogenesis (SE) and the heat stress response in longan. Mining of the available D. longan genome sequence yielded 25 putative XTH genes. Transcript profiles based on RNA sequencing (RNA-seq) data showed that most of the 17 detected DlXTH genes were highly expressed in the embryogenic callus (EC) (8) and globular embryo (GE) (8), and 13 of them responded significantly to heat stress. The assay for transposase-accessible chromatin sequencing (ATAC-seq) data analysis showed that in terms of chromatin accessibility, 22 of the 25 DlXTH genes were open during early SE, and most of the peak DlXTH genes with transcription differences during early SE were associated with high levels of H3K4me1. The most differentially expressed genes, DlXTH23.5 and DlXTH25, were selected for analysis. According to subcellular localization and quantitative real-time PCR (qRT-PCR) analysis, DlXTH23.5/25, which encode cell membrane-localized proteins, were expressed at the highest level in the GE and significantly responded to heat stress. Dual-luciferase assays and transient transformation showed that the transcription factors (TFs) DlWRKY31, DlERF1, and DlERF5 might bind to the DlXTH23.5/25 promoters to activate gene transcription. Transient overexpression of TFs and DlXTH23.5/25 induced XET activity in Nicotiana benthamiana leaves. Under heat stress in the longan EC, the XET activities and expression levels of TFs and DlXTH23.5/25 were significantly increased, and a high concentration of XET might inhibit longan SE. Thus, the regulatory network composed of DlXTH23.5/25 and its related TFs may regulate early longan SE and participate in the regulatory pathway of longan under heat stress via cell wall repair through the action of XET.

The single-cell expression profile of transposable elements and transcription factors in human early biparental and uniparental embryonic development.

Transposable elements (TEs) and transcription factors (TFs) are involved in the precise regulation of gene expression during the preimplantation stage. Activation of TEs is a key event for mammalian embryonic genome activation and preimplantation early embryonic development. TFs are involved in the regulation of drastic changes in gene expression patterns, but an inventory of the interplay between TEs and TFs during normal/abnormal human embryonic development is still lacking. Here we used single-cell RNA sequencing data generated from biparental and uniparental embryos to perform an integrative analysis of TE and TF expression. Our results showed that endogenous retroviruses (ERVs) are mainly expressed during the minor embryonic genome activation (EGA) process of early embryos, while Alu is gradually expressed in the middle and later stages. Some important ERVs (e.g., LTR5_Hs, MLT2A1) and Alu TEs are expressed at significantly lower levels in androgenic embryos. Integrative analysis revealed that the expression of the transcription factors CTCF and POU5F1 is correlated with the differential expression of ERV TEs. Comparative coexpression network analysis further showed distinct expression levels of important TFs (e.g., LEUTX and ZSCAN5A) in dizygotic embryos vs. parthenogenetic and androgenic embryos. This systematic investigation of TE and TF expression in human early embryonic development by single-cell RNA sequencing provides valuable insights into mammalian embryonic development.

Growth and flavonol accumulation of Ginkgo biloba leaves affected by red and blue light

Ginkgo biloba L. is an economically important tree species in China rich in resources. Flavonoids in their leaves have a high medicinal value. Research on improving the flavonoid yield of G. biloba leaves by regulating environmental factors has been widely conducted. However, the effect of light quality on G. biloba growth and flavonoid accumulation is not clear, and its molecular mechanism has not been reported. In this study, one-year-old ginkgo seedlings were used as experimental material. Three treatments of red, mixed (red:blue = 1:1), and blue light were set up using LED lamps. Using white light as the control, differences of G. biloba growth and flavonol accumulation under different light qualities were explored. The results showed that under red, mixed, and blue light, the petiole of G. biloba elongated, the leaf area decreased, and the leaves tended to be slender, which was not conducive to the accumulation of leaves. The blue light significantly increased the height of G. biloba seedlings. The net photosynthetic rate under blue light was relatively lower, the transpiration rate was lower in the treatment groups, the stomatal conductance decreased significantly under mixed light, and the change in intercellular CO2 concentration was not significant. RNA sequencing (RNA-seq) combined with quantitative real-time polymerase chain reaction (qRT-PCR) showed that the transcription factors (TFs) HY5 (Gb_12012) and MYB (Gb_39081) and structural genes CHS (Gb_20355), F3H (Gb_29563), and FLS (Gb_14028, Gb_14029, and Gb_14030) positively regulated the biosynthesis of flavonols, whereas MYB-related TFs (Gb_27262 and Gb_34639) played a negative regulatory role. Combined with the existing research and the results of this experiment, we preliminarily speculated the mechanism of G. biloba response to red and blue light quality. After G. biloba leaves were stimulated by red and blue light, the expression of Gb_12012 changed the abundance of HY5 and affected the expression levels of downstream MYB TFs. MYB directly binds to the promoters of the structural genes CHS, F3H, and FLS to regulate its expression to promote (or inhibit) the biosynthesis of flavonols. Finally, Mixed and blue light significantly increased the flavonol content in G. biloba leaves, while red light was the opposite. Blue light significantly increased the flavone yield of G. biloba leaves per plant (increased by 0.76 times) and its antioxidant capacity. This lays a foundation for applying light quality in the cultivation of G. biloba for leaf use.

Differential nuclear import sets the timing of protein access to the embryonic genome

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels stay constant, suggesting that alternative mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter the nucleus sequentially. Moreover, we find that the timing of nuclear proteins’ access to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities.

Expression Pattern of Inflammatory and Anti-Inflammatory Cytokines and Key Differential Transcription Factors in Peripheral Blood Mononuclear Cells of Iranian Coronavirus Disease 2019 Patients with Different Disease Severity.

The purpose of this research was to investigate the gene expression levels of inflammatory cytokines interferon (IFN)γ, tumor necrosis factor (TNF)α, interleukin (IL)1β, IL2, IL6, IL8, and IL17, and anti-inflammatory cytokines IL4, IL10, IFNα, and IFNβ, as well as relevant key transcription factors (TFs), including GATA3, PU1, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), IRF3 (interferon regulatory factor 3), BCL6 (B cell lymphoma 6 protein), FOXP3 (forkhead box P3), RORγt, and T-bet (T-box expressed in T cell) in Iranian patients with moderate and severe coronavirus disease 2019 (COVID-19). Fifty-six patients with COVID-19, and 25 healthy controls (HCs) age and sex matched were investigated. Based on the interim guidance of COVID-19 from the World Health Organization, the patients were classified into 33 moderate and 23 severe patients with COVID-19. The gene expression levels of cytokines and relevant TFs were quantified in peripheral blood mononuclear cells by quantitative real-time polymerase chain reaction (qRT-PCR). The gene expression levels of TFs RoRγ (RAR-related orphan nuclear receptor γ), NF-κB, and T-bet were significantly higher in patients with COVID-19 compared with HCs. Furthermore, the gene expression levels of cytokines, including IL2, IFNγ, IL6, TNFα, IL1β, IL8, and IL17, were significantly higher in patients with COVID-19 than HCs. However, there was a significant increase for IL6, TNFα, and IL17 in severe compared with moderate patients with COVID-19. Finally, The Spearman correlation analysis revealed a significantly positive correlation for IL6 and TNFα, IL6 and IL2, IL6, IFNγ, and IL2 and IFNγ. These data suggest that expression of IL6, TNFα, and IL17 as well as the synergic effect of elevated values of IL2 and IFNγ should be considered in the treatment and management of patients with severe COVID-19.

Identification of prognostic stemness biomarkers in colon adenocarcinoma drug resistance

BackgroundColon adenocarcinoma (COAD) is one of the leading causes of death worldwide. Cancer stem cells (CSCs) are vital for COAD chemoresistance and recurrence, however little is known about stem cell-related biomarkers in drug resistance and COAD prognosis prediction.MethodsTo uncover the roles of CSC in COAD tumorigenesis, chemoresistance, and prognosis, we retrieved COAD patients’ RNAseq data from TCGA (The Cancer Genome Atlas). We further performed analysis of differentially expressed genes (DEGs) and mRNA expression-based stemness index (mRNAsi) to identify stemness-related COAD biomarkers. We then evaluated the roles of mRNAsi in tumorigenesis, clinical-stage, overall survival (OS), and chemoresistance. Afterward, we used identified prognostic stemness-related genes (PSRGs) to construct a prediction model. After constructing the prediction model, we used elastic Net regression and area under the curve (AUC) to explore the prediction value of PSRGs based on risk scores and the receiver operator characteristic (ROC) curve. To elucidate the underlying interconnected systems, we examined relationships between the levels of TFs, PSRGs, and 50 cancer hallmarks by a Pearson correlation analysis.ResultsTwelve thousand one hundred eight DEGs were identified by comparing 456 primary COADs and 41 normal solid tissue samples. Furthermore, we identified 4351 clinical stage-related DEGs, 16,516 stemness-associated DEGs, and 54 chemoresistance-related DEGs from cancer stages: mRNAsi, and COAD chemoresistance. Compared to normal tissue samples, mRNAsi in COAD patients were marked on an elevation and involved in prognosis (p = 0.027), stemness-related DEGs based on chemoresistance (OR = 3.28, p ≤ 0.001) and AJCC clinical stage relating (OR = 4.02, p ≤ 0.001) to COAD patients. The prediction model of prognosis were constructed using the 6 PSRGs with high accuracy (AUC: 0.659). The model identified universal correlation between NRIP2 and FDFT1 (key PRSGs), and some cancer related transcription factors (TFs) and trademarks of cancer gene were in the regulatory network.ConclusionWe found that mRNAsi is a reliable predictive biomarker of tumorigenesis and COAD prognosis. Our established prediction model of COAD chemoresistance, which includes the six PSRGs, is effective, as the model provides promising therapeutic targets in the COAD.

Identification of Serum Exosome-Derived circRNA-miRNA-TF-mRNA Regulatory Network in Postmenopausal Osteoporosis Using Bioinformatics Analysis and Validation in Peripheral Blood-Derived Mononuclear Cells.

BackgroundOsteoporosis is one of the most common systemic metabolic bone diseases, especially in postmenopausal women. Circular RNA (circRNA) has been implicated in various human diseases. However, the potential role of circRNAs in postmenopausal osteoporosis (PMOP) remains largely unknown. The study aims to identify potential biomarkers and further understand the mechanism of PMOP by constructing a circRNA-associated ceRNA network.MethodsThe PMOP-related datasets GSE161361, GSE64433, and GSE56116 were downloaded from the Gene Expression Omnibus (GEO) database and were used to obtain differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied to determine possible relevant functions of differentially expressed messenger RNAs (mRNAs). The TRRUST database was used to predict differential transcription factor (TF)-mRNA regulatory pairs. Afterwards, combined CircBank and miRTarBase, circRNA-miRNA as well as miRNA-TF pairs were constructed. Then, a circRNA-miRNA-TF-mRNA network was established. Next, the correlation of mRNAs, TFs, and PMOP was verified by the Comparative Toxicogenomics Database. And expression levels of key genes, including circRNAs, miRNAs, TFs, and mRNAs in the ceRNA network were further validated by quantitative real-time PCR (qRT-PCR). Furthermore, to screen out signaling pathways related to key mRNAs of the ceRNA network, Gene Set Enrichment Analysis (GSEA) was performed.ResultsA total of 1201 DE mRNAs, 44 DE miRNAs, and 1613 DE circRNAs associated with PMOP were obtained. GO function annotation showed DE mRNAs were mainly related to inflammatory responses. KEGG analysis revealed DE mRNAs were mainly enriched in osteoclast differentiation, rheumatoid arthritis, hematopoietic cell lineage, and cytokine-cytokine receptor interaction pathways. We first identified 26 TFs and their target mRNAs. Combining DE miRNAs, miRNA-TF/mRNA pairs were obtained. Combining DE circRNAs, we constructed the ceRNA network contained 6 circRNAs, 4 miRNAs, 4 TFs, and 12 mRNAs. The expression levels of most genes detected by qRT-PCR were generally consistent with the microarray results. Combined with the qRT-PCR validation results, we eventually identified the ceRNA network that contained 4 circRNAs, 3 miRNAs, 3 TFs, and 9 mRNAs. The GSEA revealed that 9 mRNAs participate in many important signaling pathways, such as “olfactory transduction”, “T cell receptor signaling pathway”, and “neuroactive ligand-receptor interaction”. These pathways have been reported to the occurrence and development of PMOP. To sum up, key mRNAs in the ceRNA network may participate in the development of osteoporosis by regulating related signal pathways.ConclusionsA circRNA-associated ceRNA network containing TFs was established for PMOP. The study may help further explore the molecular mechanisms and may serve as potential biomarkers or therapeutic targets for PMOP.

YY1 and NFYA: Potential tr-KIT Specific Transcription Factors in Prostate Cancer

Objective: Via the use of an alternative promoter, a truncated c-KIT protein (tr-KIT) of 30-50 kDa is generated, lacking extracellular and transmembrane domains. Moreover, over-expression of tr-KIT, a stronger activator than c-KIT, appears to be specific to prostate cancer (PCa). Also, Imatinib, a tyrosine kinase inhibitor, blocks the activity of full-length c-KIT but has no effect on tr-KIT in PCa. Tr-KIT has its own nuclear factor binding site. However, the transcription factors (TFs) binding to this region specific to tr-KIT are not known yet. This study was conducted to define the most potential TFs specific for tr-KIT via in silico analysis.Methods: Tr-KIT potential TF binding sequence was uploaded into Tfsitescan database. Five TFs with the highest potential binding to this sequence were selected. Transcriptomic data of LNCaP (PCa expressing tr-KIT), PC3 (PCa not expressing tr-KIT) and RWPE-1 (normal prostate) cell lines (GSM1647378, GSE36022 and GSM738189, respectively) from Gene Expression Omnibus (GEO) database were compared for gene expression levels of pre-defined potential tr-KIT specific TFs using DESeq package of R-program. Finally, two TFs having higher expression levels in both LNCaP and PC3 compared to RWPE-1 and higher expression levels in LNCaP compared to PC3 were detected.Results: Five TFs having the highest potential were selected as: YY1, c-MYB, IL8, NFYA and TCF3. Via in silico analysis performed, it was found that YY1 and NFYA have the highest potential to be tr-KIT specific TFs in PCa, among them.Conclusion: YY1 and NFYA TFs may take a role in formation of tr-KIT in PCa.

Transcription factor regulation of eQTL activity across individuals and tissues.

Tens of thousands of genetic variants associated with gene expression (cis-eQTLs) have been discovered in the human population. These eQTLs are active in various tissues and contexts, but the molecular mechanisms of eQTL variability are poorly understood, hindering our understanding of genetic regulation across biological contexts. Since many eQTLs are believed to act by altering transcription factor (TF) binding affinity, we hypothesized that analyzing eQTL effect size as a function of TF level may allow discovery of mechanisms of eQTL variability. Using GTEx Consortium eQTL data from 49 tissues, we analyzed the interaction between eQTL effect size and TF level across tissues and across individuals within specific tissues and generated a list of 10,098 TF-eQTL interactions across 2,136 genes that are supported by at least two lines of evidence. These TF-eQTLs were enriched for various TF binding measures, supporting with orthogonal evidence that these eQTLs are regulated by the implicated TFs. We also found that our TF-eQTLs tend to overlap genes with gene-by-environment regulatory effects and to colocalize with GWAS loci, implying that our approach can help to elucidate mechanisms of context-specificity and trait associations. Finally, we highlight an interesting example of IKZF1 TF regulation of an APBB1IP gene eQTL that colocalizes with a GWAS signal for blood cell traits. Together, our findings provide candidate TF mechanisms for a large number of eQTLs and offer a generalizable approach for researchers to discover TF regulators of genetic variant effects in additional QTL datasets.