TATA Box Binding Research Articles

STAT1 mediated long non-coding RNA LINC00504 influences radio-sensitivity of breast cancer via binding to TAF15 and stabilizing CPEB2 expression

ABSTRACT Radiotherapy plays important roles in the treatment of breast cancer (BC), which develops from malignant cells in the breast. Long non-coding RNAs (lncRNAs) have been reported to be implicated in radio-resistance or radio-sensitivity of human cancer, which includes breast cancer. Nevertheless, long intergenic non-protein coding RNA 0504 (LINC00504) has not been investigated in BC. In our study, from RT-qPCR analysis, LINC00504 was found to be up-regulated in BC cells. By conducting in vitro assays, it was confirmed that the knockdown of LINC00504 could enhance the radio-sensitivity of BC cells. The regulatory mechanism of LINC00504 in BC was also verified by chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) and luciferase reporter assays. From the experimental results, we knew that the up-regulation of LINC00504 was mediated by signal transducer and activator of transcription 1 (STAT1). Moreover, LINC00504 stabilized the expression of cytoplasmic polyadenylation element-binding protein 2 (CPEB2) via binding to TATA-box binding protein associated factor 15 (TAF15). Furthermore, rescue assays validated that LINC00504 participated in regulating the radio-sensitivity of BC cells via up-regulating CPEB2. In summary, our study disclosed that STAT1 could mediate LINC00504 and weaken the radio-sensitivity of BC cells via binding to TAF15 and stabilizing CPEB2 expression.

TAF1 inhibitor Bay-299 induces cell death in acute myeloid leukemia.

BackgroundAcute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. The cure rate of currently intensive chemotherapy in AML was only 40% or less, and there is an urgent need to develop novel effective therapeutic targets or drugs. The TATA-box binding protein associated factor 1 (TAF1) plays important roles in transcriptional regulation and leukemogenesis. However, the potential of TAF1 as a therapeutic target for AML remains unclear. The present study examined the effects of the TAF1 inhibitor Bay-299 on AML cells and the underlying molecular mechanisms.MethodsThe expression of TAF1 in various types of tumors was analyzed using The Cancer Genome Atlas (TCGA) and the UALCAN database. The effects of Bay-299 on cell proliferation were evaluated using the Cell Counting Kit-8 (CCK-8) assay. Cell death, EdU incorporation, and cell differentiation were detected using flow cytometry. Western blot analysis was utilized to confirm the activation of the apoptotic pathway. Expression of cell cycle and cell death-related genes was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsAnalysis of the public databases showed that TAF1 expression was elevated in multiple types of tumors. Treatment of AML cells with the TAF1 inhibitor Bay-299 resulted in a remarkable inhibition of cell growth, increased cell death, reduced Edu incorporation, and increased cell differentiation. The apoptosis inhibitor Z-VAD and the receptor-interacting protein kinase 1 (RIPK1) inhibitor Nec-2 could rescue cell death induced by Bay-299. Bay-299 treatment increased the cleavage of key pro-apoptotic proteins, and this effect was ameliorated by administration of Z-VAD and Nec-2. Moreover, Bay-299 treatment was associated with increased expression of cell cycle inhibitor genes and multiple pyroptosis-promoting genes, contributing to the phenotypes observed in AML cell lines.ConclusionsThe TAF1 inhibitor Bay-299 induced AML cell death through multiple mechanisms and may be a promising candidate for the treatment of patients with AML.

The analysis of reference genes expression stability in susceptible and resistant Apera spica-venti populations under herbicide treatment

Weed resistance to herbicides constitutes a serious problem to world crop production. One of the weeds that are significantly threatening the crops’ yield and quality is Apera spica-venti. The target-site resistance (TSR) mechanism of A. spica-venti has been widely studied, though, little is known about its non-target-site resistance (NTSR) mechanisms at the molecular level. Molecular examination of NTSR is, to a great extent, based on the expression profiles of selected genes, e.g. those participating in detoxification. However, to obtain reliable results of gene expression analysis, the use of a normalizer is required. The aim of this study was to select the best reference genes in A. spica-venti plants of both populations, susceptible and resistant to ALS inhibitor, under treatment with herbicide. Eleven housekeeping genes were chosen for their expression stability assessment. The efficiency correction of raw quantification cycles (Cq) was included in the gene expression stability analyses, which resulted in indicating the TATA-box binding protein (TBP), glyceraldehyde-3-phosphate dehydrogenase, cytosolic (GAPC), and peptidyl-prolyl cis–trans isomerase CYP28 (CYP28) genes as the most stably expressed reference genes. The obtained results are of vital importance for future studies on the expression of genes associated with the non-target-site resistance mechanisms in the A. spica-venti populations susceptible and resistant to herbicides.

AMPK Reprograms Gene Expression and Promotes Adaptation/Survival in Response to Metabolic Stress through Binding to a Chromatin-Associated Transcription Complex in Acute Lymphoblastic Leukemia

Survival rates for relapsed/refractory acute lymphoblastic leukemia (ALL), the most common cancer in children and adolescents, remain dismal. We and others have reported that ALL cells are vulnerable to conditions inducing energy/ER-stress mediated by AMP-activated protein kinase (AMPK) activation. AMPK has been reported to interact with chromatin-associated proteins (e.g., histone H2B) in MEF cells to epigenetically regulate gene expression in response to environmental/cellular stress (Bungard et al. Science, 2010; 329:1201). To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we first constructed Bp-ALL (NALM6, REH) and T-ALL (CCRF-CEM, KE-37) stable cell lines expressing HA-AMPKα1 or HA-AMPKα2. Next, using HA and RNA pol II antibodies, we performed ChIP-seq assays in CCRF-CEM/HA-AMPKα2 (CN2) grown in glucose-free RPMI for 24 h. ChIP-seq differentially identified 171 candidate genes in CN2 treated with no-glucose vs. 431 genes in untreated controls. Data analysis using the Encode and ChEA database identified the TATA-Box Binding Protein Associated Factor, CCAAT Enhancer Binding Protein Delta (CEBPD), the negative elongation factor complex member E (NELFE), and the Promyelocytic leukemia protein (PML) among highly ranked transcription factors (TFs) may associated with AMPKα2 on chromatin. To correlate the level of gene mRNA expression and recruitment of AMPKα2 to chromatin gene loci regulated in response to energy/metabolic stress, we performed RNA-seq assays in CN2 cells treated with or without glucose deprivation for 24 h. RNA-seq data analysis indicated that of the 3497 genes altered by AMPK activation, two thirds were downregulated whereas the remaining were upregulated. Kyoto Encyclopedia of Genes and Genomes gene set and BioPlanet 2019 gene set analysis identified metabolic pathways, DNA replication/metabolism, and cell cycle as the main biological processes altered in CN2 cells in response to metabolic stress. Among downregulated genes in response to metabolic stress, we uncovered a cluster of histone genes. To confirm and validate our data, we used RT-qPCR and ChIP-qPCR assays on selected histone gene candidates (H1-2/ HIST1H1C, H1-3/HIST1H1D, H4C4/HIST1H4D) which exhibited both decreased recruitment of HA-AMPKα2 to chromatin and mRNA downregulation in response to metabolic stress. Further ChIP-qPCR assays using an AMPKα2 antibody confirmed these data in KASUMI-2 cells (Bp-ALL). Similar data were also observed in CN2 cells treated with AICAR, another AMPK activator. Additional experiments were conducted using the allosteric AMPK activators compound 991 and PF-06409577. Using Co-IP experiments, we uncovered that AMPKα2 interacted with putative members of an AMPK/chromatin-associated transcription factor complex which included the TATA-Box Binding Protein Associated Factor (TAF), integrator (INT), and RNA polymerase II. To investigate the role of AMPK kinase activity on AMPKα2-associated chromatin regulated gene targets, we determined the effect of genetic constructs encoding a constitutively active (CA) form of AMPKα2 on histone gene mRNA expression in CCRF-CEM and MEF AMPKα1/AMPKα2 double knockout (DKO) cells, and found that in both cell types the expression of the full-length CA-AMPKα2 (T172D) lead to decreased mRNA expression of the histone genes examined, suggesting AMPK kinase activity is required to regulate histone genes in response to energy/metabolic stress. In conclusion, our data show that in response to metabolic stress, AMPK binds directly to a multi-protein complex on chromatin to reprogram gene expression to promote cellular adaptation/survival in ALL. Further elucidation of AMPK's interactions with members of the putative AMPK/chromatin-associated transcription complex may lead to unique opportunities for epigenetic-based therapeutic interventions and combination strategies to exploit synthetic lethality in relapse/refractory ALL and other hematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

An Analysis of Structure-function Co-relation between GLI Oncoprotein and HLA Immune-gene Transcriptional Regulation through Molecular Docking

Background:GLI proteins play a significant role in the transduction of the Hedgehog (Hh) signaling pathway. A variety of human cancers, including the brain, gastrointestinal, lung, breast, and prostate cancers, demonstrate inappropriate activation of this pathway. GLI helps in proliferation and has an inhibitory role in the differentiation of hematopoietic stem cells. Malignancies may have a defect in differentiation. Different types of malignancies and undifferentiated cells have a low level of HLA expression on their cell surface.Objective:Human Leukocytic Antigen (HLA) downregulation is frequently observed in cancer cells. This work is aimed to hypothesize whether this downregulation of HLA molecules is GLI oncoprotein mediated or not. To understand the roles of different types of GLI oncoproteins on different classes of HLA transcriptional machinery was carried out through structure-based modeling and molecular docking studies.Methods:To investigate the role of GLI in HLA expression /downregulation is Hh-GLI mediated or not, molecular docking based computational interaction studies were performed between different GLI proteins (GLI1, GLI2, and GLI3) with TATA box binding protein (TBP) and compare the binding efficiencies of different HLA gene (both HLA class I and –II) regulating transcription factors (RelA, RFX5, RFXAP, RFXANK, CIITA, CREB1, and their combinations) with TBP. Due to unavailability of 3D protein structures of GLI2 and cyclin D2 (a natural ligand of GLI1) were modelled followed by structural validation by Ramachandran plot analysis.Results:GLI proteins especially, GLI1 and GLI2, have almost similar binding energy of RFX5-RFXANK- RFXAP and CIITA multi-protein complex to TBP but has lower binding energy between RelA to TBP.Conclusion:This study suggests that HLA class I may not be downregulated by GLI; however, over-expression of GLI1 is may be responsible for HLA class II downregulation. Thus this protein may be responsible for the maintenance of the undifferentiated state of malignant cells. This study also suggests the implicative role of GLI1 in the early definitive stage of hematopoiesis.

Genomic organization and hypoxia inducible factor responsive regulation of teleost hsp90β gene during hypoxia stress.

The physiological significance of a large family of heat-shock proteins (HSPs), comprised of the cytosolic HSP90A and the endoplasmic reticulum component of HSPB, is evident in prokaryotes and eukaryotes. The HSP90A is believed to play critical roles in diverse physiological functions of cell viability and chromosomal stability including stress management. Heightened abundance of hsp90β transcript was documented in Channa striatus, a freshwater fish, which is capable of surviving within an extremely hypoxic environment. To better understand the mechanism of hsp90β gene expression, we investigated its genomic organization. Eleven exons were identified, including a long upstream intron with a remarkable similarity with human, but not with chicken counterpart. Dual-luciferase assays identified promoter activity in a 1366bp 5'-flanking segment beyond the transcription initiation site. Examination detected a minimal promoter of 754bp containing a TATA-box, CAAT-enhancer in addition to providing clues regarding other enhancer and repressor elements. The driving capability of this minimal promoter was further validated by its binding ability with TATA-box binding protein and the generation of GFP expressing transgenic zebrafish (F2). Further, deletion of an inverted HIF (hypoxia inducible factor) motif RCGTG (upstream of the TATA-box) dramatically reduced luciferase expression in a hypoxic environment (CoCl2 treated cultivable cells) and was identified as a cis-acting HIF responsive element, necessary for the hypoxia-induced expression. The results obtained herein provide an insight regarding how hsp90β gene expression is controlled by HIF responsive element in teleost both during hypoxia stress management and normal physiological functions, and suggested that the hsp90β gene promoter could be used as a potential candidate for generating ornamental and food-fish transgenics.

Striatal Cholinergic Dysregulation after Neonatal Decrease in X-Linked Dystonia Parkinsonism-Related TAF1 Isoforms.

X-linked dystonia parkinsonism is a generalized, progressive dystonia followed by parkinsonism with onset in adulthood and accompanied by striatal neurodegeneration. Causative mutations are located in a noncoding region of the TATA-box binding protein-associated factor 1 (TAF1) gene and result in aberrant splicing. There are 2 major TAF1 isoforms that may be decreased in symptomatic patients, including the ubiquitously expressed canonical cTAF1 and the neuronal-specific nTAF1. The objective of this study was to determine the behavioral and transcriptomic effects of decreased cTAF1 and/or nTAF1 in vivo. We generated adeno-associated viral (AAV) vectors encoding microRNAs targeting Taf1 in a splice-isoform selective manner. We performed intracerebroventricular viral injections in newborn mice and rats and intrastriatal infusions in 3-week-old rats. The effects of Taf1 knockdown were assayed at 4 months of age with evaluation of motor function, histology, and RNA sequencing of the striatum, followed by its validation. We report motor deficits in all cohorts, more pronounced in animals injected at P0, in which we also identified transcriptomic alterations in multiple neuronal pathways, including the cholinergic synapse. In both species, we show a reduced number of striatal cholinergic interneurons and their marker mRNAs after Taf1 knockdown in the newborn. This study provides novel information regarding the requirement for TAF1 in the postnatal maintenance of striatal cholinergic neurons, the dysfunction of which is involved in other inherited forms of dystonia. © 2021 International Parkinson and Movement Disorder Society.

LncRNA APOA1-AS facilitates proliferation and migration and represses apoptosis of VSMCs through TAF15-mediated SMAD3 mRNA stabilization

ABSTRACT Coronary atherosclerosis (CAS) is a major cause of cardiovascular disease. Long non-coding RNAs (lncRNAs) have been implicated as novel biomarkers in coronary artery disease (CAD). APOA1 antisense RNA (APOA1-AS) was proven to show high expression during atherosclerotic development, but no report has uncovered the detailed mechanism of APOA1-AS in CAS. Thus, this paper aims to explore the role of APOA1-AS in CAS. Vascular smooth muscle cells (VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL) to mimic atherosclerosis-like injury. Quantitative real-time PCR (RT-qPCR) and western blot analysis analyzed gene expression. Cell counting kit-8 (CCK-8), wound healing assay, and flow cytometry were implemented to assess the function of APOA1-AS in modulating pathological phenotype of VSMCs. Results demonstrated that APOA1-AS was notably up-regulated in ox-LDL treated VSMCs (ox-LDL-VSMCs). The deficiency of APOA1-AS hindered proliferation and migration and stimulated apoptosis in ox-LDL-VSMCs. Mechanistically, APOA1-AS recruited TATA-box binding protein associated factor 15 (TAF15) protein to stabilized SMAD family member 3 (SMAD3) mRNA and activate the TGF-β/SMAD3 signaling pathway. In conclusion, APOA1-AS contributed to proliferation and migration and repressed apoptosis of VSMCs through TAF15-mediated SMAD3 mRNA stabilization, indicating that APOA1-AS could be a promising target for CAS.

Profile of Patrick Cramer

Patrick Cramer’s interest in the scientific method may have been sparked by a childhood gift. While growing up, his parents gave him a kit to try little experiments at home that inspired him to become a scientist. Patrick Cramer. Image credit: Patrick Cramer. Cramer studied chemistry, first at the University of Stuttgart and later at Heidelberg University in Germany. “I was in love with the beauty of biological molecules, so I moved into biochemistry and then into structural biology,” says Cramer. This move was facilitated by stints at the University of Bristol and Cambridge University. “At Cambridge I really fell in love with structural biology, and so I chose that as a subject for my PhD studies at the European Molecular Biology Laboratory,” he says. At the European Molecular Biology Laboratory in Grenoble, France, Cramer began to study gene transcription, launching a career unraveling the mysteries of this fundamental biological process. Cramer has determined the three-dimensional (3D) structure of transcription complexes, providing numerous insights into the mechanisms of transcription initiation, elongation, and regulation. He has also contributed to the development of functional genomics and computational biology to study how the genome is transcribed and regulated in living cells and understand how gene activity is controlled. Now director at the Max Planck Institute for Biophysical Chemistry in Gottingen, Germany, Cramer was elected to the National Academy of Sciences in 2020. In his Inaugural Article (1), his team describes the first structures of the highly conserved transcription factor TATA-box binding protein (TBP) in complex with a nucleosome. When Cramer received his doctorate in 1998, a major question about transcription was the structure of RNA polymerase II, the central enzyme that makes messenger RNA. To work on this problem, he joined Roger Kornberg’s laboratory at Stanford University as a postdoctoral researcher, funded by …

Synergistic AHR Binding Pathway with EMT Effects on Serous Ovarian Tumors Recognized by Multidisciplinary Integrated Analysis.

Epithelial ovarian cancers (EOCs) are fatal and obstinate among gynecological malignancies in advanced stage or relapsed status, with serous carcinomas accounting for the vast majority. Unlike EOCs, borderline ovarian tumors (BOTs), including serous BOTs, maintain a semimalignant appearance. Using gene ontology (GO)-based integrative analysis, we analyzed gene set databases of serous BOTs and serous ovarian carcinomas for dysregulated GO terms and pathways and identified multiple differentially expressed genes (DEGs) in various aspects. The SRC (SRC proto-oncogene, non-receptor tyrosine kinase) gene and dysfunctional aryl hydrocarbon receptor (AHR) binding pathway consistently influenced progression-free survival and overall survival, and immunohistochemical staining revealed elevated expression of related biomarkers (SRC, ARNT, and TBP) in serous BOT and ovarian carcinoma samples. Epithelial–mesenchymal transition (EMT) is important during tumorigenesis, and we confirmed the SNAI2 (Snail family transcriptional repressor 2, SLUG) gene showing significantly high performance by immunohistochemistry. During serous ovarian tumor formation, activated AHR in the cytoplasm could cooperate with SRC, enter cell nuclei, bind to AHR nuclear translocator (ARNT) together with TATA-Box Binding Protein (TBP), and act on DNA to initiate AHR-responsive genes to cause tumor or cancer initiation. Additionally, SNAI2 in the tumor microenvironment can facilitate EMT accompanied by tumorigenesis. Although it has not been possible to classify serous BOTs and serous ovarian carcinomas as the same EOC subtype, the key determinants of relevant DEGs (SRC, ARNT, TBP, and SNAI2) found here had a crucial role in the pathogenetic mechanism of both tumor types, implying gradual evolutionary tendencies from serous BOTs to ovarian carcinomas. In the future, targeted therapy could focus on these revealed targets together with precise detection to improve therapeutic effects and patient survival rates.

Current updates and future perspectives in the evaluation of azoospermia: A systematic review

ABSTRACT Objectives: To provide a summary of the current evaluation of azoospermia and insights into future perspectives in the evaluation and counselling of men with azoospermia. Methods: A search of PubMed, Cochrane Reviews and Web of Science databases was performed for full-text English-language articles published between 1943 and 2020 focussing on ‘future perspectives’, ‘azoospermia’ and ‘evaluation’. Results: Azoospermia represents a severe form of male infertility characterised by sperm production so impaired that there are no sperm present in the ejaculate. The current evaluation of azoospermia focusses on patient history and physical examination with selected adjunctive laboratory investigations including serum hormones, a karyotype and screening for Y chromosome microdeletions. Future diagnostics are focussed on identifying the underlying genetic aetiologies for azoospermia, as well as a greater emphasis on screening for systemic illness that men with severe infertility may be predisposed to develop. Conclusion: Azoospermia represents an extreme form of male infertility, and evaluation relies heavily on history and physical examination, as genetic evaluations for these individuals remain limited. Future evaluation will focus on next-generation sequencing and more rigorous evaluation for possible co-existing and future risk of systemic disease. ABBREVIATIONS: ADGRG2, adhesion G protein-coupled receptor G2; ASRM: American Society of Reproductive Medicine; AZF: azoospermia factor; CBAVD: congenital bilateral absence of the vas deferens; CFTR: cystic fibrosis transmembrane conductance regulator; CRKL: CRK-like proto-oncogene; E2F1: E2F transcription factor 1; HAUS7: HAUS augmin-like complex subunit 7; HR: hazard ratio; KS: Klinefelter syndrome; MAZ, MYC-associated zinc finger protein; NGS: next-generation sequencing; NOA: non-obstructive azoospermia; OA: obstructive azoospermia; RHOX: reproductive homeobox on the X chromosome; SH2: SRC homology 2; TAF7L: TATA-box binding protein associated factor 7-like; TEX11: testis-expressed 11; WES: whole-exome sequencing

EWSR1-The Most Common Rearranged Gene in Soft Tissue Lesions, Which Also Occurs in Different Bone Lesions: An Updated Review.

EWSR1 belongs to the FET family of RNA-binding proteins including also Fused in Sarcoma (FUS), and TATA-box binding protein Associated Factor 15 (TAF15). As consequence of the multifunctional role of EWSR1 leading to a high frequency of transcription of the chromosomal region where the gene is located, EWSR1 is exposed to aberrations such as rearrangements. Consecutive binding to other genes leads to chimeric proteins inducing oncogenesis. The other TET family members are homologous. With the advent of widely used modern molecular techniques during the last decades, it has become obvious that EWSR1 is involved in the development of diverse benign and malignant tumors with mesenchymal, neuroectodermal, and epithelial/myoepithelial features. As oncogenic transformation mediated by EWSR1-fusion proteins leads to such diverse tumor types, there must be a selection on the multipotent stem cell level. In this review, we will focus on the wide variety of soft tissue and bone entities, including benign and malignant lesions, harboring EWSR1 rearrangement. Fusion gene analysis is the diagnostic gold standard in most of these tumors. We present clinicopathologic, immunohistochemical, and molecular features and discuss differential diagnoses.

BVES-AS1 inhibits the malignant behaviors of colon adenocarcinoma cells via regulating BVES.

The underexpression of the long noncoding RNA blood vessel epicardial substance antisense RNA 1 (BVES-AS1) has been shown in colon adenocarcinoma (COAD) patients. However, its role in COAD remains to be explored. This study aimed to investigate the function and potential mechanism of BVES-AS1 in COAD. Colony formation, Cell Counting Kit-8, JC-1 mitochondrial membrane potential assay, wound healing, transwell, and western blot analyses were used to measure cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) in COAD cells. RNA pull-down, luciferase reporter, and RNA binding protein immunoprecipitation assays were used to detect the interaction of BVES-AS1 and downstream genes. BVES-AS1 was expressed at low levels in COAD cells. Overexpressed BVES-AS1 inhibited COAD cell proliferation, migration, invasion, and EMT while elevating cell apoptosis. Mechanistically, BVES-AS1 functioned as a competing endogenous RNA sponging miR-522-3p to regulate the expression of nearby gene blood vessel epicardial substance (BVES). Besides this, BVES-AS1 recruited TATA-box binding protein associated factor 15 (TAF15) to promote BVES messenger RNA stability. Taken together, our study confirmed that BVES-AS1 inhibited COAD progression via interacting with miR-522-3p and TAF15 to regulate BVES expression, which might offer a perspective for COAD treatment.

Small-expanded allele spinocerebellar ataxia 17: imaging and phenotypic variability.

Spinocerebellar ataxia 17 (SCA17) is a rare genetic cause of adult-onset ataxia caused by an abnormal expansion of the CAG/CAA sequence in the TATA-box Binding Protein (TBP) gene. A number of repeats higher than 49 are full penetrance-expanded. The range between 41 and 49 repeats is characterized by decreased penetrance, and it is usually referred to as "small." Here, we describe two patients with the SCA17 phenotype and with 43 and 44 CAG repeats in the TBP gene, and review all the previously reported cases of SCA17 with a small range of expansions. We focus on both clinical features and imaging findings, which, in the case of small-expanded alleles, can resemble those of atypical parkinsonisms. Thus, we suggest to consider the small-expanded allele SCA17 as a possible diagnosis in patients with adult-onset ataxia, even when both clinical and imaging characteristics are suggestive for other non-genetic neurodegenerative diseases.

Screening of Reference Genes for RT-qPCR in Chicken Adipose Tissue and Adipocytes.

Reverse transcription quantitative real-time PCR is the most commonly used method to detect gene expression levels. In experiments, it is often necessary to correct and standardize the expression level of target genes with reference genes. Therefore, it is very important to select stable reference genes to obtain accurate quantitative results. Although application examples of reference genes in mammals have been reported, no studies have investigated the use of reference genes in studying the growth and development of adipose tissue and the proliferation and differentiation of preadipocytes in chickens. In this study, GeNorm, a reference gene stability statistical algorithm, was used to analyze the expression stability of 14 candidate reference genes in the abdominal adipose tissue of broilers at 1, 4, and 7 weeks of age, the proliferation and differentiation of primary preadipocytes, as well as directly isolated preadipocytes and mature adipocytes. The results showed that the expression of the TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) genes was most stable during the growth and development of abdominal adipose tissue of broilers, the expression of the peptidylprolyl isomerase A (PPIA) and HMBS genes was most stable during the proliferation of primary preadipocytes, the expression of the TBP and RPL13 genes was most stable during the differentiation of primary preadipocytes, and the expression of the TBP and HMBS genes was most stable in directly isolated preadipocytes and mature adipocytes. These results provide reference bases for accurately detecting the mRNA expression of functional genes in adipose tissue and adipocytes of chickens.

Assembling for transcription initiation

Transcription Eukaryotic transcription initiation by RNA polymerase II (Pol II) requires the assembly of a preinitiation complex (PIC) on core promoters. The binding of TATA box–binding protein (TBP) to the TATA box promoter has been thought to be a general rule in PIC assembly and transcription initiation. However, most coding genes lack a TATA box, and nearly all Pol II–mediated gene transcription requires the TBP-containing multisubunit complex transcription factor IID (TFIID). Chen et al. determined the structures of human TFIID-based PIC in sequential assembly states and revealed that TFIID supports distinct PIC assembly on TATA-containing and TATA-lacking promoters. The finding resolves the long-standing mystery of how one set of general transcription machinery initiates transcription on diverse promoters. Science , this issue p. [eaba8490][1] [1]: /lookup/doi/10.1126/science.aba8490

A homozygous variant in TBPL2 was identified in women with oocyte maturation defects and infertility.

What are the genetic causes of oocyte maturation defects? A homozygous splicing variant (c.788 + 3A>G) in TATA-box binding protein like 2 (TBPL2) was identified as a contributory genetic factor in oocyte maturation defects. TBPL2, a vertebrate oocyte-specific general transcription factor, is essential for oocyte development. TBPL2 variants have not been studied in human oocyte maturation defects. Two infertile families characterized by oocyte maturation defects were recruited for whole-exome sequencing (WES). Genomic DNA was extracted from peripheral blood for WES analysis. Sanger sequencing was performed for data validation. Pathogenicity of variants was predicted by in silico analysis. Minigene assay and single-oocyte RNA sequencing were performed to investigate the effects of the variant on mRNA integrity and oocyte transcriptome, respectively. A homozygous splicing variant (c.788 + 3A>G) in TBPL2 was identified in two unrelated families characterized by oocyte maturation defects. Haplotype analysis indicated that the disease allele of Families 1 and 2 was independent. The variant disrupted the integrity of TBPL2 mRNA. Transcriptome sequencing of affected oocytes showed that vital genes for oocyte maturation and fertilization were widely and markedly downregulated, suggesting that a mutation in the transcriptional factor, TBPL2, led to global gene alterations in oocytes. Limitations include the lack of direct functional evidence. Owing to the scarcity of human oocyte samples, only two immature MI oocytes were obtained from the patients, and we could only investigate the effect of the mutation at the transcriptional level by high-throughput sequencing technology. No extra oocytes were obtained to assess the transcriptional activity of the mutant oocytes by immunofluorescence, or investigate the effects on the binding of TBPL2 caused by the mutation. Our findings highlight a critical role of TBPL2 in female reproduction and identify a homozygous splicing mutation in TBPL2 that might be related to defects in human oocyte maturation. This information will facilitate the genetic diagnosis of infertile individuals with repeated failures of IVF, providing a basis for genetic counseling. This study was supported by the National Key Research and Development Program of China (2018YFC1004000, 2017YFC1001504 and 2017YFC1001600), the National Natural Science Foundation of China (81871168, 31900409 and 31871509), the Foundation for Distinguished Young Scholars of Shandong Province (JQ201816), the Innovative Research Team of High-Level Local Universities in Shanghai (SSMU-ZLCX20180401) and the Fundamental Research Funds of Shandong University. The authors have no competing interests to declare. N/A.

Structural insights into preinitiation complex assembly on core promoters.

Transcription factor IID (TFIID) recognizes core promoters and supports preinitiation complex (PIC) assembly for RNA polymerase II (Pol II)-mediated eukaryotic transcription. We determined the structures of human TFIID-based PIC in three stepwise assembly states and revealed two-track PIC assembly: stepwise promoter deposition to Pol II and extensive modular reorganization on track I (on TATA-TFIID-binding element promoters) versus direct promoter deposition on track II (on TATA-only and TATA-less promoters). The two tracks converge at an ~50-subunit holo PIC in identical conformation, whereby TFIID stabilizes PIC organization and supports loading of cyclin-dependent kinase (CDK)-activating kinase (CAK) onto Pol II and CAK-mediated phosphorylation of the Pol II carboxyl-terminal domain. Unexpectedly, TBP of TFIID similarly bends TATA box and TATA-less promoters in PIC. Our study provides structural visualization of stepwise PIC assembly on highly diversified promoters.

O-GlcNAcylation and O-GlcNAc Cycling Regulate Gene Transcription: Emerging Roles in Cancer

Simple SummaryO-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification (PTM) linking nutrient flux through the hexosamine biosynthetic pathway (HBP) to gene transcription. Mounting experimental and clinical data implicates aberrant O-GlcNAcylation in the development and progression of cancer. Herein, we discuss how alteration of O-GlcNAc-regulated transcriptional mechanisms leads to atypical gene expression in cancer. We discuss the challenges associated with studying O-GlcNAc function and present several new approaches for studies of O-GlcNAc-regulated transcription.O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar post-translational modification (PTM) of intracellular proteins linking nutrient flux through the Hexosamine Biosynthetic Pathway (HBP) to the control of cis-regulatory elements in the genome. Aberrant O-GlcNAcylation is associated with the development, progression, and alterations in gene expression in cancer. O-GlcNAc cycling is defined as the addition and subsequent removal of the modification by O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) provides a novel method for cells to regulate various aspects of gene expression, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. We will focus on the complex relationship between phosphorylation and O-GlcNAcylation in the regulation of the RNA Polymerase II (RNAP II) pre-initiation complex and the regulation of the carboxyl-terminal domain of RNAP II via the synchronous actions of OGT, OGA, and kinases. Additionally, we discuss how O-GlcNAcylation of TATA-box binding protein (TBP) alters cellular metabolism. Next, in a non-exhaustive manner, we will discuss the current literature on how O-GlcNAcylation drives gene transcription in cancer through changes in transcription factor or chromatin remodeling complex functions. We conclude with a discussion of the challenges associated with studying O-GlcNAcylation and present several new approaches for studying O-GlcNAc regulated transcription that will advance our understanding of the role of O-GlcNAc in cancer.

Downregulation of TAF9B by miR-7-5p Inhibits the Progression of Osteosarcoma.

BackgroundOsteosarcoma (OS) is a malignant bone tumor with high metastatic potential. As a regulatory factor of apoptosis, TATA-box binding protein (TBP) associated factor 9B (TAF9B) is rarely studied in tumors.MethodsWe investigated the role and mechanism of TAF9B in OS cells by overexpression and knockdown. CCK8, colony formation, transwell, and flow cytometry analysis were performed to detect proliferation, migration, invasion, and apoptosis.ResultsTAF9B overexpression promotes the proliferation, migration, and invasion of OS cells, while TAF9B knockdown gives the opposite result. TAF9B inhibits apoptosis by upregulating Bcl-2 and downregulating Bax and Cleaved-caspase-3. Through starBase analysis, it was found that miR-7-5p can bind to the 3ʹUTR region of TAF9B, which is further confirmed by the dual luciferase reporter system assay. MiR-7-5p downregulates the expression of TAF9B in MG63 and U2OS cells. The proliferation and invasion of OS cells are inhibited after miR-7-5p mimics transfection and are promoted after miR-7-5p inhibitor transfection. TAF9B rescues the inhibitory effect of miR-7-5p on OS cells. TAF9B also activates the AKT/mTOR signaling pathway.ConclusionAccording to our results, miR-7-5p inhibits the translation of TAF9B and then suppresses growth and metastasis through the AKT/mTOR signaling pathway in OS cells, thereby indicating the potential value of miR-7-5p and TAF9B as therapeutic targets for human OS.