Expression Patterns Of Transcription Factors Research Articles

8342 A Rare Case of a Corticotropic and Gonadotropic Pituitary Double Adenoma

Abstract Disclosure: S. Gruber: None. D. Kirschenbaum: None. T. Hortobagyi: None. C. Serra: None. A. Bink: None. F. Beuschlein: None. Z. Erlic: None. Background: Pituitary adenomas account for about 15% of all intracranial neoplasms and typically arise from monoclonal proliferation of anterior pituitary cells. While lactotropic and clinically hormone-inactive adenomas make up by far the largest proportion, somatotropic and corticotropic adenomas have a significantly lower prevalence. This case describes the very rare finding of a corticotropic and gonadotropic double adenoma. Clinical Case: A 63-year-old Caucasian man (BMI 36.7 kg/m2) presented in the obesity clinic. Hypercortisolism was suspected by repeated lack of cortisol suppression in low-dose dexamethasone suppression tests [minimal 87 nmol/l] and disrupted circadian rhythm of cortisol secretion according to salivary cortisol profile. Cortisol levels in a 24-hour sample of urine were normal. ACTH levels were elevated [73 ng/l; n<61 ng/l] leading to suspicion of Morbus Cushing. Concomitant diseases include sleep apnea, arterial hypertension and newly diagnosed type 2 diabetes mellitus. Medical history was notable for deep vein thrombosis and pulmonary embolism. Other stigmata of hypercortisolism were not evident. The rest of endocrine work up confirmed partial secondary hypothyroidism and hypogondotropic hypogonadism. An MRI of the pituitary gland revealed a pituitary macroadenoma with a maximum dimension of 20 mm. The patient underwent an endoscopic transnasal adenomectomy with post-operative corticotropic insufficiency. Contrary to our expectations, the histological examination of the surgical resection revealed a gonadotrophic pituitary adenoma with SF1 and focal FSH expression. The discrepancy between clinical, biochemical and histological findings led to subsequent examinations of the specimen with detection of an small fragment accounting for less than 5% of the total sample with expression of T-Pit and ACTH but neither SF1 nor Pit-1, corresponding to an additional corticotropic adenoma with densely granulated pattern. The tumor cells did not show overlapping expression patterns of transcription factors or hormones, which indicates the coincidence of two distinct adenomas. Three months postoperatively, the patient is biochemically cured. Partial anterior pituitary insufficiency remained. Conclusion: Pituitary double adenomas are found in 0.5-1.5% of all surgical specimens. They are characterized by their origin from different cell lines, which distinguishes them from plurihormonal adenomas, which originate from one single cell line. Here we demonstrate a SF1-T-Pit lineage combination resulting in a neighboring ACTH and FSH-adenoma. In this particular case, the clinically relevant corticotropic adenoma was comparatively tiny, making it easy to miss. This illustrates well the importance of careful correlation of clinical, laboratory and histologic findings as correct diagnosis is crucial for postoperative patient management. Presentation: 6/2/2024

Delayed Blastocyst Formation Reduces the Quality and Hatching Ability of Porcine Parthenogenetic Blastocysts by Increasing DNA Damage, Decreasing Cell Proliferation, and Altering Transcription Factor Expression Patterns.

The purpose of this study was to investigate the influence of blastocyst formation timing on the quality of porcine embryos derived from parthenogenetic activation. Newly formed blastocysts at days 6, 7, and 8 of culture [termed formation 6, 7, and 8 blastocysts (F6, F7, and F8 blastocysts)] were obtained, and a series of parameters related to the quality of blastocysts, including apoptosis incidents, DNA replication, pluripotent factors, and blastocyst hatching capacity, were assessed. Delayed blastocyst formation (F7 and/or F8 blastocysts) led to increased levels of ROS, DNA damage, and apoptosis while decreasing the mitochondrial membrane potential, DNA replication, Oct4 levels, and numbers of Sox2-positive cells. F7 blastocysts showed a significantly reduced hatching rate compared to F6 blastocysts; however, F8 blastocysts were unable to develop to the hatching stage. Collectively, our findings suggest a negative correlation between delayed blastocyst formation and blastocyst quality.

Exploring the Influence of T Cell Marker Gene Expression on the Pathobiology and Clinical Prognostic Outcomes in Intestinal-Type Gastric Carcinoma.

Gastric cancer (GC) poses a significant global health challenge. This study is aimed at elucidating the role of the immune system, particularly T cells and their subtypes, in the pathogenesis and progression of intestinal-type gastric carcinoma (GC), and at evaluating the predictive utility of a T cell marker gene-based risk score for overall survival. We performed an extensive analysis using single-cell RNA sequencing data to map the diversity of immune cells and identify specific T cell marker genes within GC. Pseudotime trajectory analysis was employed to observe the expression patterns of tumor-related pathways and transcription factors (TFs) at various disease stages. We developed a risk score using data from The Cancer Genome Atlas (TCGA) as a training set and validated it with the GSE15459 dataset. Our analysis revealed distinct patterns of T cell marker gene expression associated with different stages of GC. The risk score, based on these markers, successfully stratified patients into high-risk and low-risk groups with significantly different overall survival prospects. High-risk patients exhibited poorer survival outcomes compared to low-risk patients (p < 0.05). Additionally, the risk score was capable of identifying patients across a spectrum from chronic atrophic gastritis to early GC. The findings enhance the understanding of the tumor immune microenvironment in GC and propose new immunotherapeutic targets. The T cell marker gene-based risk score offers a potential tool for gastroenterologists to tailor treatment plans more precisely according to the cancer's severity.

Physiological and molecular responses of 'Hamlin' sweet orange trees expressing the VvmybA1 gene under cold stress conditions.

Overexpression of VvmybA1 transcription factor in 'Hamlin' citrus enhances cold tolerance by increasing anthocyanin accumulation. This results in improved ROS scavenging, altered gene expression, and stomatal regulation, highlighting anthocyanins' essential role in citrus cold acclimation. Cold stress is a significant threat to citrus cultivation, impacting tree health and productivity. Anthocyanins are known for their role as pigments and have emerged as key mediators of plant defense mechanisms against environmental stressors. This study investigated the potential of anthocyanin overexpression regulated by grape (Vitis vinifera) VvmybA1 transcription factor to enhance cold stress tolerance in citrus trees. Transgenic 'Hamlin' citrus trees overexpressing VvmybA1 were exposed to a 30-day cold stress period at 4°C along with the control wild-type trees. Our findings reveal that anthocyanin accumulation significantly influences chlorophyll content and their fluorescence parameters, affecting leaf responses to cold stress. Additionally, we recorded enhanced ROS scavenging capacity and distinct expression patterns of key transcription factors and antioxidant-related genes inthe transgenic leaves. Furthermore, VvmybA1 overexpression affected stomatal aperture regulation by moderating ABA biosynthesis, resulting in differential responses in a stomatal opening between transgenic and wild-type trees under cold stress. Transgenic trees exhibited reduced hydrogen peroxide levels, enhanced flavonoids, radical scavenging activity, and altered phytohormonal profiles. These findings highlighted the role of VvmybA1-mediated anthocyanin accumulation in enhancing cold tolerance. The current study alsounderlines the potential of anthocyanin overexpression as a critical regulator of the cold acclimation process by scavenging ROS in plant tissues.

Genome-wide profiling of WRKY genes involved in flavonoid biosynthesis in Erigeron breviscapus.

The transcription factors of WRKY genes play essential roles in plant growth, stress responses, and metabolite biosynthesis. Erigeron breviscapus, a traditional Chinese herb, is abundant in flavonoids and has been used for centuries to treat cardiovascular and cerebrovascular diseases. However, the WRKY transcription factors that regulate flavonoid biosynthesis in E. breviscapus remain unknown. In this study, a total of 75 EbWRKY transcription factors were predicted through comprehensive genome-wide characterization of E. breviscapus and the chromosomal localization of each EbWRKY gene was investigated. RNA sequencing revealed transient responses of 74 predicted EbWRKY genes to exogenous abscisic acid (ABA), salicylic acid (SA), and gibberellin 3 (GA3) after 4h of treatment. In contrast, the expression of key structural genes involved in flavonoid biosynthesis increased after 4h in GA3 treatment. However, the content of flavonoid metabolites in leaves significantly increased at 12h. The qRT-PCR results showed that the expression patterns of EbWRKY11, EbWRKY30, EbWRKY31, EbWRKY36, and EbWRKY44 transcription factors exhibited a high degree of similarity to the 11 structural genes involved in flavonoid biosynthesis. Protein-DNA interactions were performed between the key genes involved in scutellarin biosynthesis and candidate WRKYs. The result showed that F7GAT interacts with EbWRKY11, EbWRKY36, and EbWRKY44, while EbF6H has a self-activation function. This study provides comprehensive information on the regulatory control network of flavonoid accumulation mechanisms, offering valuable insights for breeding E. breviscapus varieties with enhanced scutellarin content.

Changes in characteristics of spermatogonial stem cells in response to heat stress in stallions

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions.

Differential Gene Expression Profiling during Avian Immune Organ Development: Insights from Thymus and Bursa of Fabricius Transcriptome Analysis

Pluripotent hematopoietic stem cells undergo maturation to regenerate blood cells. T-cell and B-cell maturation from common lymphoid progenitors (CLPs) is regulated by distinct transcription factors and signaling pathways. Notch signaling, GATA3, and TCF1 drive T-cell fate, while EBF1, PU.1, E2A, Pax5, TCF1, and Foxo1 regulate B-cell differentiation. Alterations in these factors can cause lineage deviations. Gene expression profiling of the chick thymus and bursa reveals dynamic expression patterns of transcription factors, cytokines, and signal molecules. Notch1 and Dll1 expression that increase during later stages indicate the ongoing role of Notch signaling in T-cell lineage maintenance. The current study aimed to identify differentially expressed genes during the development of the avian immune organs, focusing on the thymus and bursa using 24 Ross 308 avian breed. The mRNA libraries from these organs were analyzed using quantitative Real-time PCR analysis at six time points spanning the embryonic ages (days 15 and 18) and post-hatch age (days 3, 7, 14, and 28). The data for the gene expression indicated significant variations across different stages of immune organ development. Differential gene expression was observed between sorted T and B-cells, with GATA3, CD3e, CD4, and Ptprc showing higher expression in the T-cell population, and Pax5 and CD81 exhibiting higher expression in the B-cell population. Notably, ENO1 and IRF4 showed higher expression in T-cells at E15 and B-cells at E18. The study highlights the importance of regulatory factors and genes in maintaining cellular identity, furthers the understanding of avian immunology, and has the potential for improving poultry health and studying immune-related diseases in humans. These findings pave the way for further research on the role of biochemical components under important disease conditions in avian immunology and their potential applications for poultry health. Keywords: Avian lymphocyte, B-cell maturation, Gene expression, Regulatory elements, T-cell

Abstract 6602: Targeting the SWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies

Abstract Background: Small cell lung cancer (SCLC) is a rapidly progressing subtype of lung cancer with a high growth rate and early metastasis propensity, often resulting in a more advanced disease stage at diagnosis. A recent transcriptome analysis of human SCLC tumors revealed that SCLC could be characterized by the expression pattern of certain transcription factors (TFs), including ASCL1 (achaete-scute family bHLH transcription factor 1) and POU2F3 (POU domain class 2 transcription factor 3), exemplifying SCLC as a TF-driven malignancy. ASCL1-driven SCLC (SCLC-A) manifests a neuroendocrine phenotype, while POU2F3-driven SCLC (SCLC-P) is characterized as a tuft-cell-like variant. Recent studies revealed that POU domain class 2 transcription factors uniquely rely on coactivators to achieve their lineage-defining functions in the tuft cell and B cell lineages. In tuft-cell-like SCLC cells, the coactivators of POU2F3 (POU2AF2 and POU2AF3) endow POU2F3 with a critical transactivation domain by forming a master regulator complex, which supports enhancer-mediated cancer-promoting gene activation in SCLC-P cells. This indicates a potential therapeutic vulnerability of tuft-cell-like SCLC whereby strategies aimed at blocking POU2F3-POU2AF2/3 function may lead to clinical benefit. Methods: We conducted a domain-targeted CRISPR screen to identify druggable targets for SCLC-P, followed by pharmacological validation in preclinical SCLC models. Multi-omics techniques, including ATAC-seq, ChIP-seq, RNA-seq, and RIME, were employed to elucidate the SWI/SNF complex's regulatory influence on the POU2F-POU2AF axis. Results: Here we identified that the POU2F3 molecular subtype of SCLC (SCLC-P) exhibits an exquisite dependence on the activity of the SWI/SNF epigenetic complex. SCLC-P cell lines were sensitive to low nanomolar levels of a SWI/SNF ATPase degrader when compared to other molecular subtypes of SCLC. Co-factors of POU2F were found to interact with components of the SWI/SNF complex. The POU2F3 transcription factor complex is evicted from chromatin upon SWI/SNF ATPase degradation. A novel, orally bioavailable SWI/SNF ATPase PROTAC degrader demonstrated preferential efficacy in SCLC-P relative to the SCLC-A subtype. The SWI/SNF ATPase PROTAC degrader did not alter normal tuft cell numbers in lung or colon, nor did it exhibit toxicity in mice. B cell malignancies which displayed a dependency on the POU2F co-factor, POU2AF1, were also remarkably sensitive to SWI/SNF ATPase degradation. In a POU2AF1-dependent, disseminated murine model of multiple myeloma, AU-24118 had an enhanced survival benefit as compared to pomalidomide, an approved treatment for multiple myeloma. Conclusions: Taken together, our studies suggest that POU2F-POU2AF driven malignancies have an intrinsic dependence on the SWI/SNF complex representing a striking therapeutic vulnerability. Citation Format: Lanbo Xiao, Tongchen He, Olaf Klingbeil, Young Eleanor, Xiaoli Wu, Yuanyuan Qiao, Abhijit Parolia, Sanjana Eyunni, Rahul Mannan, Somnath Mahapatra, NamHoon Kim, Heng Zheng, Fengyun Su, Xuhong Cao, Susanta Samajdar, Murali Ramachandra, Christopher R. Vakoc, Arul M. Chinnaiyan. Targeting the SWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies [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 6602.

High expression of UBE2S promotes progression of hepatocellular carcinoma by increasing cancer cell stemness

To investigate the expression of the ubiquitination enzyme UBE2S in different cell types in hepatocellular carcinoma (HCC) microenvironment and its impact on proliferation and stemness of HCC cells. TCGA and CPTAC database were used to analyze the transcriptional and promoter methylation levels and protein expressions of UBE2S in HCC. Specific expression patterns of UBE2S, intercellular communication and key transcription factors in different cell types were analyzed based on single-cell sequencing data from TISCH website. We further examined UBE2S expressions in clinical samples of HCC tissues, HCC cells and T cells using immunohistochemistry and immunofluorescence staining. We also tested the effects of UBE2S knockdown on stemness of HCC-LM3 and HepG2 cells using clone formation experiments and sphere formation assay. Analysis based on TCGA database suggested significant overexpression of UBE2S in both paired and non-paired tumor tissues (P < 0.001), and its transcriptional level increased with tumor grades. The methylation level of UBE2S promoter was significantly decreased in HCC (P < 0.001), and its transcription level increased obviously in HCC with TP53 mutation (P < 0.001). Analysis of CPTAC database also demonstrated overexpression of UBE2S protein in HCC tissues (P < 0.001). Three prognostic models suggested that HCC patients with high UBE2S expression had poorer prognosis (P < 0.001). Single-cell sequencing data analysis revealed high expressions of UBE2S in T cells and high intensities of interaction between endothelial cells, epithelial cells and fibroblasts in HCC microenvironment. Immunohistochemistry and immunofluorescence staining demonstrated high UBE2S expressions in clinical samples of HCC tissues, HCC cells and T cells. In HCC-LM3 and HepG2 cells, UBE2S knockdown significantly inhibited cell clone formation and tumor sphere formation (P < 0.05). UBE2S is highly expressed in T cells in HCC microenvironment in close correlation with a poor prognosis. High UBE2S expression promotes the stemness of HCC cells.

Genome-wide identification and analysis of expression pattern of REVEILLE transcription factors in castor (Ricinus communis L.)

Genome-wide identification and analysis of expression pattern of REVEILLE transcription factors in castor (Ricinus communis L.)

Expression of FOXI1 and POU2F3 varies among different salivary gland neoplasms and is higher in Warthin tumor

PurposeSalivary gland tumors are histologically diverse. Ionocytes and tuft cells, rare epithelial cells found in normal salivary glands, might be associated with salivary tumors. Here, we explored the expression of FOXI1 and POU2F3, master regulators of ionocytes and tuft cells, respectively, for common salivary neoplasms using immunohistochemistry.MethodsWe analyzed normal salivary tissues and nine salivary gland tumors; Warthin tumors (WT), pleomorphic adenomas (PA), basal cell adenomas, and oncocytomas were benign, whereas mucoepidermoid, adenoid cystic, acinic cell, salivary duct carcinomas, and polymorphous adenocarcinomas were malignant.ResultsNormal salivary glands contained a few FOXI1- and POU2F3-positive cells in the ducts instead of the acini, consistent with ionocytes and tuft cells, respectively. Among the benign tumors, only WTs and PAs consistently expressed FOXI1 (10/10 and 9/10, respectively). The median H-score of WTs was significantly higher than that of PAs (17.5 vs. 4, P = 0.01). While WTs and PAs harbored POU2F3-positive cells (10/10 and 9/10, respectively), the median H-score was higher in WTs than in PAs (10.5 vs 4, respectively). Furthermore, WTs exhibited a unique staining pattern of FOXI1- and POU2F3-positive cells, which were present in luminal and abluminal locations, respectively. Whereas none of the malignant tumors expressed FOXI1, only adenoid cystic carcinoma consistently expressed POU2F3 (5/5), with a median H-score of 4.ConclusionThe expression patterns of the characteristic transcription factors found in ionocytes and tuft cells vary among salivary gland tumor types and are higher in WT, which might be relevant for understanding and diagnosing salivary gland neoplasms.

Identification and Expression Patterns of WOX Transcription Factors under Abiotic Stresses in Pinus massoniana.

WUSCHEL-related homeobox (WOX) transcription factors (TFs) play a crucial role in regulating plant development and responding to various abiotic stresses. However, the members and functions of WOX proteins in Pinus massoniana remain unclear. In this study, a total of 11 WOX genes were identified, and bioinformatics methods were used for preliminary identification and analysis. The phylogenetic tree revealed that most PmWOXs were distributed in ancient and WUS clades, with only one member found in the intermediate clade. We selected four highly conserved WOX genes within plants for further expression analysis. These genes exhibited expressions across almost all tissues, while PmWOX2, PmWOX3, and PmWOX4 showed high expression levels in the callus, suggesting their potential involvement in specific functions during callus development. Expression patterns under different abiotic stresses indicated that PmWOXs could participate in resisting multiple stresses in P. massoniana. The identification and preliminary analysis of PmWOXs lay the foundation for further research on analyzing the resistance molecular mechanism of P. massoniana to abiotic stresses.

A full-body transcription factor expression atlas with completely resolved cell identities in C. elegans

Invariant cell lineage in C. elegans enables spatiotemporal resolution of transcriptional regulatory mechanisms controlling the fate of each cell. Here, we develop RAPCAT (Robust-point-matching- And Piecewise-affine-based Cell Annotation Tool) to automate cell identity assignment in three-dimensional image stacks of L1 larvae and profile reporter expression of 620 transcription factors in every cell. Transcription factor profile-based clustering analysis defines 80 cell types distinct from conventional phenotypic cell types and identifies three general phenotypic modalities related to these classifications. First, transcription factors are broadly downregulated in quiescent stage Hermaphrodite Specific Neurons, suggesting stage- and cell type-specific variation in transcriptome size. Second, transcription factor expression is more closely associated with morphology than other phenotypic modalities in different pre- and post-differentiation developmental stages. Finally, embryonic cell lineages can be associated with specific transcription factor expression patterns and functions that persist throughout postembryonic life. This study presents a comprehensive transcription factor atlas for investigation of intra-cell type heterogeneity.

Genome-Wide Identification and Expression Pattern of MYB Family Transcription Factors in Erianthus fulvus.

MYB family genes have many functions and are widely involved in plant abiotic-stress responses. Erianthus fulvus is an important donor material for stress-resistance genes in sugarcane breeding. However, the MYB family genes in E. fulvus have not been systematically investigated. In this study, 133 EfMYB genes, including 48 Ef1R-MYB, 84 EfR2R3-MYB and 1 Ef3R-MYB genes, were identified in the E. fulvus genome. Among them, the EfR2R3-MYB genes were classified into 20 subgroups. In addition, these EfMYB genes were unevenly distributed across 10 chromosomes. A total of 4 pairs of tandemly duplicated EfMYB genes and 21 pairs of segmentally duplicated EfMYB genes were identified in the E. fulvus genome. Protein-interaction analysis predicted that 24 EfMYB proteins had potential interactions with 14 other family proteins. The EfMYB promoter mainly contains cis-acting elements related to the hormone response, stress response, and light response. Expression analysis showed that EfMYB39, EfMYB84, and EfMYB124 could be significantly induced using low-temperature stress. EfMYB30, EfMYB70, EfMYB81, and EfMYB101 responded positively to drought stress. ABA treatment significantly induced EfMYB1, EfMYB30, EfMYB39, EfMYB84, and EfMYB130. All nine genes were induced using MeJA treatment. These results provide comprehensive information on EfMYB genes and can serve as a reference for further studies of gene function.

Screening for Intracellular Phosphorylation Cascades That Positively and Negatively Regulate the Self-Renewing Proliferation of Immature HPCs

For the purpose of studying hematological diseases involving complex lineages of cells, hematopoietic differentiation using patient-derived iPS cells has already proved to be powerful in the analysis of various diseases. However, in terms of sourcing for the expansion of its use to larger scale studies, the time-consuming and inefficient differentiation process still presents a significant obstacle. In this context, the development of a method that allows the proliferation of progenitor cells (HPCs) which maintains their ability to differentiate into multiple lineages is an important issue. In vivo, immature multipotent hematopoietic stem cells (HSCs) reside in the bone marrow, where intracellular signals via phosphorylation cascades co-ordinately regulate the expression pattern of transcription factors (TFs), leading to fate decisions within the microenvironment between self-renewal and one-way differentiation into HPCs. In contrast, iPS cell-derived hematopoietic differentiation does not involve HSCs and the microenvironment differs from that of bona fide cells. Therefore, the signal combinations required to amplify iPS cell-derived HPCs without further differentiation should naturally differ from those observed in HSCs in vivo. Based on this assumption, we constructed a CRISPRa and CRISPRi screen for phosphorylation-related genes using iPS cell-derived differentiation to search for factors that enable long-term maintenance of multipotent HPCs. First, Doxycycline-inducible dCas9-VPR (CRISPRa) or dCas9-KRAB-MecP2 (CRISPRi) expression cassettes were introduced into human iPS cells before, and then acted in combination with the phosphorylation-focused sgRNA libraries at the time when HPCs begin to be produced from mesodermal progenitors during their hematopoietic differentiation. Then, to identify the sgRNAs of interest with single-cell accuracy, colony formation assays were performed by isolating HPCs one by one by single-cell sorting. 2 weeks later, cells were collected only from wells that had formed CFU-Mix. Amplicon-sequences were performed using genomic DNA extracted from those cells and the sgRNAs that significantly contributed to the phenotype were identified by calculating the difference in enrichment with control sgRNAs. As a result, the CRISPRa screen identified a group of intracellular signals related with eight candidate genes known to be specifically activated in immature cells in the normal hematopoiesis. On the other hand, the results of CRISPRi showed that suppression of necroptosis-related signalling probably promotes long-term culture of HPCs in the stroma-free, artificial culture circumstances. Our results showed the potential to obtain multipotent HPCs from iPS cells over a long period of time and in large quantities, simply by adjusting the intracellular signals without artificial adding or removing of individual genes.

Effects of curcumin on Wnt/β-catenin, Hedgehog pathways and expression of EMT transcription factors in basal conditions and upon HIF1A and RELA knockdown in HeLa cells

Targeting the HIF1A and NF-κB pathways with pharmacological inhibitors or small interfering RNAs is one of the most promising aspects of cancer stem cell therapy. However, the influence of HIF1A and NF-κB modulators on downstream targets such as components of the Wnt/β-catenin and Hedgehog pathways and EMT is still poorly understood.HeLa cells were treated with 10 and 50 μM curcumin for 24 h, and the function of the Wnt/β-catenin and Hedgehog pathways and EMT transcription factors were subsequently assessed using 14 RNAs expression qPCR-based analysis. We also applied siRNA-dependent HIF1A and RELA knockdown to investigate the influence of these pathways on the expression of the above RNAs along and in combination with curcumin treatment.At the higher concentration under basal conditions, curcumin suppressed most of the targets studied, while causing a significant increase in SNAI1 expression. Knockdown of HIF1A and RELA reduced the expression of Hedgehog pathway targets. In contrast to basal conditions, the expression of EMT transcription factors appeared to be increased after knockdown in response to 50 μM curcumin.Curcumin significantly inhibited Wnt/β-catenin, Hedgehog pathways and EMT transcription factor, while inducing an increase in SNAI1 gene expression, which may be part of cell adaptation systems. Knockdown of HIF1A and RELA revealed the strong dependence of Hedgehog target expression on HIF1A and NF-κB pathways. The activation status of HIF1A and NF-κB pathways determines the expression pattern of EMT transcription factors in HeLa cells.

Identification of Regulatory Elements in Primary Sensory Neurons Involved in Trauma-Induced Neuropathic Pain.

Chronic pain is a significant public health issue that is often refractory to existing therapies. Here we use a multiomic approach to identify cis-regulatory elements that show differential chromatin accessibility and reveal transcription factor (TF) binding motifs with functional regulation in the rat dorsal root ganglion (DRG), which contain cell bodies of primary sensory neurons, after nerve injury. We integrated RNA-seq to understand how differential chromatin accessibility after nerve injury may influence gene expression. Using TF protein arrays and chromatin immunoprecipitation-qPCR, we confirmed C/EBPγ binding to a differentially accessible sequence and used RNA-seq to identify processes in which C/EBPγ plays an important role. Our findings offer insights into TF motifs that are associated with chronic pain. These data show how interactions between chromatin landscapes and TF expression patterns may work together to determine gene expression programs in rat DRG neurons after nerve injury.

Mapping the splicing landscape of the human immune system.

Most human genes code for more than one transcript. Different ratios of transcripts of the same gene can be found in different cell types or states, indicating differential use of transcription start sites or differential splicing. Such differential transcript use (DTUs) events provide an additional layer of regulation and protein diversity. With the exceptions of PTPRC and CIITA, there are very few reported cases of DTU events in the immune system. To rigorously map DTUs between different human immune cell types, we leveraged four publicly available RNA sequencing datasets. We identified 282 DTU events between five human healthy immune cell types that appear in at least two datasets. The patterns of the DTU events were mostly cell-type-specific or lineage-specific, in the context of the five cell types tested. DTUs correlated with the expression pattern of potential regulators, namely, splicing factors and transcription factors. Of the several immune related conditions studied, only sepsis affected the splicing of more than a few genes and only in innate immune cells. Taken together, we map the DTUs landscape in human peripheral blood immune cell types, and present hundreds of genes whose transcript use changes between cell types or upon activation.

Genome-Wide Identification of R2R3-MYB Transcription Factor Family in Tartary Buckwheat (Fagopyrum tataricum) Identifies a Member Involved in Anthocyanin Biosynthesis

Tartary buckwheat (Fagopyrum tataricum Gaertn.) belongs to the family of Polygonaceae and is used as a multi-functional plant. R2R3-MYB transcription factors play a crucial part in plant growth and are involved in many biological processes where they regulate their internal environment. To date, there is no documented systematic research on the R2R3-MYB gene family in Tartary buckwheat. Here, domain features, chromosomal location, motif prediction, gene structure, cis-acting elements, as well as the expression pattern of R2R3-MYB transcription factors were analyzed comprehensively in Tartary buckwheat using a bioinformatic approach. Additionally, one R2R3-MYB gene was verified by transgenic Arabidopsis. Results indicate that a total of 152 R2R3-MYB genes were identified with special R2R3 domains and were distributed on 8 chromosomes of Tartary buckwheat. They were further classified into 25 sub-categories via phylogenetic analysis in terms of the R2R3-MYB gene family classification principles of Arabidopsis thaliana. This classification was further supported by analysis of exon–intron structure, motif, and cis-elements. Tandem and segmental duplication existed among the R2R3-MYB gene family of Tartary buckwheat, and there were 5, 8, 27, and 36 FtR2R3-MYB homologous genes, respectively, when comparing with Zea mays, Oryza sativa, Arabidopsis thaliana, and Solanum melongena by synteny analysis. The expression pattern of FtR2R3-MYB genes in different tissue and under salt stress and different light condition showed that members had tissue-specific expression levels and that these members may play diverse functions in plant growth and adaptation to varying environments. In addition, one of the FtR2R3-MYB gene families, FtMYB43, a homologue of AtTT2, clustered with R2R3-MYB from other plant species, which were reported to be involved in the regulation of anthocyanin or proanthocyanidin biosynthesis. This gene was located in the nucleus, and had transcriptional activation activity, indicating that FtMYB43 may be a positive transcript factor of anthocyanin or proanthocyadin biosynthesis. Moreover, the function of FtMYB43 was further verified to improve the production of anthocyanin in transgenic Arabidopsis by overexpression, and qRT-PCR assay implied that FtMYB43 may regulate the expression levels of most structural genes of the anthocyanin biosynthesis pathway in Arabidopsis seedlings. These results provide more insights into the structure and function of the R2R3-MYB gene family and may accelerate the breeding of ornamental buckwheat cultivars.

Transcriptome analysis during vernalization in wheat (Triticum aestivum L.)

BackgroundVernalization, as a vital process in the life cycle of winter cereal, has important effects on floral organ formation and flowering time. Many morphological changes together with molecular changes occur during the vernalization period. Here, we used transcriptome sequencing to analyze the transcriptomic changes in wheat leaves before, during and after vernalization using the winter wheat cultivar ‘Shiluan02-1’.ResultsA total of 16,370 differentially expressed genes were obtained across different vernalization periods. Gene Ontology enrichment analysis revealed that photoperiodism, photoprotection, photosynthesis, lipid transport and biosynthetic process, and chlorophyll metabolic process were closely related to vernalization. In addition, AP2/ERF, C2H2, bHLH, WRKY, MYB, MYB-related, and NAC transcription factors were significantly enriched during vernalization, and the transcription factor expression patterns suggested the intricate regulation of transcription factor modules in plant vernalization pathways. Analysis of gene expression patterns of the MADS-box transcription factor genes showed different expression patterns during vernalization phases, among which VERNALIZATION1 (VRN1) genes were found to gradually increase during vernalization periods from V0 to V35, while decline in the V42 phase, then increase after vernalization. The Tavrt-2 gene cooperated with Tavrn1 to regulate flowering induced by vernalization, and its expression level was rapidly increased by vernalization but declined in the V42 phase and then increased after vernalization. Some genes from the ICE-CBF-COR pathway were also identified, and additional analysis indicated that some key genes related to phytohormone biosynthesis and signal transduction were enriched during the vernalization period, such as gibberellic acid, ethylene, abscisic acid and jasmonic acid biosynthesis and signaling pathway genes.ConclusionsOur study provides valuable molecular information for future studies on wheat vernalization regulation and also serves as an excellent reference for future wheat breeding.