General Transcription Factor Research Articles

The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity.

Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

Comparative Transcriptome Analysis Provides Novel Molecular Events for the Differentiation and Maturation of Hepatocytes during the Liver Development of Zebrafish.

The liver plays an essential role in multiple biological functions including metabolism, detoxification, digestion, coagulation, and homeostasis in vertebrates. The specification and differentiation of embryonic hepatoblasts, the proliferation of hepatocytes, and the hepatic tissue architecture are well documented, but molecular events governing the maturation of hepatocytes during liver development remain largely unclear. In this study, we performed a comparative transcriptome analysis of hepatocytes that were sorted by flow cytometry from developing zebrafish embryos at 60, 72, and 96 hpf. We identified 667 up-regulated and 3640 down-regulated genes in hepatocytes between 60 and 72 hpf, 606 up-regulated and 3924 down-regulated genes between 60 and 96 hpf, and 1693 up-regulated genes and 1508 down-regulated genes between 72 and 96 hpf. GO enrichment analysis revealed that key biological processes, cellular components, and molecular functions in hepatocytes between 60 to 72 hpf, such as cell cycle, DNA replication, DNA repair, RNA processing, and transcription regulation, are mainly associated with the proliferation of hepatocytes. In addition to biological processes, cellular components, and molecular functions for cell proliferation, molecular functions for carbohydrate metabolism were enriched in hepatocytes during 72 to 96 hpf. KEGG enrichment analysis identified key signaling pathways, such as cell cycle, RNA degradation, ubiquitin-mediated proteolysis, ErbB and Hedgehog signaling, basal transcription factors, Wnt signaling, and glycan degradation, which are closely associated with cell proliferation or carbohydrate metabolism in hepatocytes between 60 to 72 hpf. Newly enriched signaling pathways in hepatocytes during 72 to 96 hpf include metabolisms of pyrimidine, purine, nicotinate and nicotinamide, caffeine, glycine, serine and threonine, ABC transporters, and p53 signaling that function in metabolisms of lipid, protein and energy, cellular secretion, or detoxification, indicating the functional maturation of hepatocytes between 72 to 96 hpf. These findings provide novel clues for further understanding the functional differentiation and maturation of hepatocytes during liver development.

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

Exosome-derived GTF2H2 from Huh7 cells can inhibit endothelial cell viability, migration, tube formation, and permeability

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related morbidity and mortality worldwide. Given that HCC is an extraordinarily heterogeneous malignant disease, finding an effective therapeutic strategy for treating it has been difficult. Because of the importance of angiogenesis in tumorigenesis, targeting the more homogenous HCC endothelial cells may be a better therapeutic strategy. In a unpublished manuscript, we found that the expression levels of vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase 2/9 (MMP2/9) were reduced in human HCC tissues that overexpressed DNA damage repair gene general transcription factor II subunit H2 (GTF2H2). This suggested that GTF2H2 may have an inhibitory effect on angiogenesis. Therefore, we hypothesized that GTF2H2 acts as an anti-angiogenesis gene. However, our results showed that GTF2H2 overexpression had no effect on endothelial cell viability, migration, or permeability. To our surprise, treating human umbilical vein endothelial cells (HUVECs) with the culture medium of Huh 7 cells overexpressing GTF2H2 could inhibit their viability, migration, and permeability. We then isolated the culture medium into exosomes and other components from the culture medium. Only GTF2H2-enriched exosomes could inhibit the viability, migration, tube formation, and permeability of HUVECs. Our results suggest that overexpressing GTF2H2 had no effect on HUVECs, while GTF2H2 enriched exosomes from Huh7 cells could inhibit HUVEC phenotypes such as proliferation and migration. Therefore, GTF2H2-enriched exosomes can possibly be utilized as a novel drug for treating HCC and also serve as a potential molecular target for inhibiting tumor angiogenesis.

CDK7/GRP78 signaling axis contributes to tumor growth and metastasis in osteosarcoma.

Osteosarcoma derives from primitive bone-forming mesenchymal cells and is the most common primary bone malignancy. Therapeutic targeting of osteosarcoma has been unsuccessful; therefore, identifying novel osteosarcoma pathogenesis could offer new therapeutic options. CDK7 is a subunit within the general transcription factor TFIIH. We aim to explore the new mechanism by which CDK7 regulates osteosarcoma and our studies may provide new theoretical support for the use of CDK7 inhibitors in the treatment of osteosarcoma. Here, we investigate the molecular mechanism underlying the association between CDK7 and GRP78 in osteosarcoma. Specifically, we find that an E3 ubiquitin ligase TRIM21 binds and targets GRP78 for ubiquitination and degradation, whereas CDK7 phosphorylates GRP78 at T69 to inhibit TRIM21 recruitment, leading to GRP78 stabilization. Notably, a CDK7-specific inhibitor, THZ1, blunts osteosarcoma growth and metastasis. Combination treatment with CDK7 and GRP78 inhibitors yield additive effects on osteosarcoma growth and progression inhibition. Thus, simultaneous suppression of CDK7 and GRP78 activity represents a potential new approach for the treatment of osteosarcoma. In conclusion, the discovery of this previously unknown CDK7/GRP78 signaling axis provides the molecular basis and the rationale to target human osteosarcoma.

Comprehensive characterization of tumor microenvironment and m6A RNA methylation regulators and its effects on PD-L1 and immune infiltrates in cervical cancer.

Understanding the role of N6-adenosine methylation (m6A) in the tumor microenvironment (TME) is important since it can contribute to tumor development. However, the research investigating the association between m6A and TME and cervical cancer is still in its early stages. The aim of this study was to discover the possible relationship between m6A RNA methylation regulators, TME, PD-L1 expression levels, and immune infiltration in cervical cancer. We gathered RNA-seq transcriptome data and clinical information from cervical cancer patients using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. To begin, researchers assessed the differences in m6A regulatory factor expression levels between cervical cancer and normal tissues. Clustering analysis was adapted to assess PD-L1 expression, immunological score, immune cell infiltration, TME, and probable pathways in cervical cancer samples. The majority of m6A regulators were found to be considerably overexpressed in cervical cancer tissues. Using consensus clustering of 21 m6A regulators, we identified two subtypes (clusters 1/2) of cervical cancer, and we found that WHO stage and grade were associated with the subtypes. PD-L1 expression increased dramatically in cervical cancer tissues and was significantly linked to ALKBH5, FTO, METTL3, RBM15B, YTHDF1, YTHDF3, and ZC3H13 expression levels. Plasma cells and regulatory T cells (Tregs) were considerably elevated in cluster 2. Cluster 1 is involved in numerous signature pathways, including basal transcription factors, cell cycle, RNA degradation, and the spliceosome. The prognostic signature-based riskscore (METTL16, YTHDF1, and ZC3H13) was found to be an independent prognostic indicator of cervical cancer. The tumor immune microenvironment (TIME) was linked to m6A methylation regulators, and changes in their copy number will affect the quantity of tumor-infiltrating immune cells dynamically. Overall, our research discovered a powerful predictive signature based on m6A RNA methylation regulators. This signature correctly predicted the prognosis of cervical cancer patients. The m6A methylation regulator could be a critical mediator of PD-L1 expression and immune cell infiltration, and it could have a significant impact on the TIME of cervical cancer.

Molecular biology of the cell

Molecular biology of the cell

Transcriptional regulator Taf14 binds DNA and is required for the function of transcription factor TFIID in the absence of histone H2A.Z

The transcriptional regulator Taf14 is a component of multiple protein complexes involved in transcription initiation and chromatin remodeling in yeast cells. Although Taf14 is not required for cell viability, it becomes essential in conditions where the formation of the transcription preinitiation complex is hampered. The specific role of Taf14 in mediating transcription initiation and preinitiation complex formation is unclear. Here, we explored its role in the general transcription factor IID by mapping Taf14 genetic and proteomic interactions and found that it was needed for the function of the complex if Htz1, the yeast homolog of histone H2A.Z, was absent from chromatin. Dissecting the functional domains of Taf14 revealed that the linker region between the YEATS and ET domains was required for cell viability in the absence of Htz1 protein. We further show that the linker region of Taf14 interacts with DNA. We propose that providing additional DNA binding capacity might be a general role of Taf14 in the recruitment of protein complexes to DNA and chromatin.

M6A modification-mediated BATF2 suppresses metastasis and angiogenesis of tongue squamous cell carcinoma through inhibiting VEGFA

ABSTRACT The aim is to explore the underlying mechanism of basic leucine zipper ATF-like transcription factor 2 (BATF2) in tongue squamous cell carcinoma (TSCC). The expression of BATF2 in TSCC tissues and corresponding adjacent normal TSCC tissues, human TSCC cell lines (SCC-15 and CAL-27) and human normal tongue epithelial cells NTEC was detected. Then, SCC-15 cells with stable BATF2 knockdown and CAL-27 cells with BATF2 overexpression were established to investigate the functional effect of BATF2 on TSCC. Thereafter, the effect of BATF2 on TSCC angiogenesis and BATF2 m6A methylation was also examined. BATF2 was significantly downregulated in TSCC tissues and cell lines, and BATF2 overexpression could suppress growth, metastasis and angiogenesis of TSCC. Mechanistically, vascular endothelial growth factor A (VEGFA) was identified as a downstream gene of BATF2, and it was confirmed that BATF2 suppressed growth, metastasis and angiogenesis of TSCC via inhibiting VEGFA. In addition, the N6-methyladenosine (m6A) modification of BATF2 mRNA mediated by METTL14 suppressed its expression in TSCC. METTL14/BATF2 axis could serve as a novel promising therapeutic candidate against angiogenesis for TSCC.

Differential dependencies of human RNA polymerase II promoters on TBP, TAF1, TFIIBand XPB.

The effects of rapid acute depletion of components of RNA polymerase II (Pol II) general transcription factors (GTFs) that are thought to be critical for formation of preinitiation complexes (PICs) and initiation in vitro were quantified in HAP1 cells using precision nuclear run-on sequencing (PRO-Seq). The average dependencies for each factor across >70 000 promoters varied widely even though levels of depletions were similar. Some of the effects could be attributed to the presence or absence of core promoter elements such as the upstream TBP-specificity motif or downstream G-rich sequences, but some dependencies anti-correlated with such sequences. While depletion of TBP had a large effect on most Pol III promoters only a small fraction of Pol II promoters were similarly affected. TFIIB depletion had the largest general effect on Pol II and also correlated with apparent termination defects downstream of genes. Our results demonstrate that promoter activity is combinatorially influenced by recruitment of TFIID and sequence-specific transcription factors. They also suggest that interaction of the preinitiation complex (PIC) with nucleosomes can affect activity and that recruitment of TFIID containing TBP only plays a positive role at a subset of promoters.

Binary organization of epidermal basal domains highlights robustness to environmental exposure.

Adulte interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here, we elucidate the molecular and cellular states of basal IFE domains by marker expression and single-cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that in part reflect the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Furthermore, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains in adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge preferred basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis.

Unspliced XBP1 contributes to cholesterol biosynthesis and tumorigenesis by stabilizing SREBP2 in hepatocellular carcinoma.

Cholesterol biosynthesis plays a critical role in rapidly proliferating tumor cells. X-box binding protein 1 (XBP1), which was first characterized as a basic leucine zipper-type transcription factor, exists in an unspliced (XBP1-u) and spliced (XBP1-s) form. Recent studies showed that unspliced XBP1 (XBP1-u) has unique biological functions independent from XBP1-s and could promote tumorigenesis; however, whether it is involved in tumor metabolic reprogramming remains unknown. Herein, we found that XBP1-u promotes tumor growth by enhancing cholesterol biosynthesis in hepatocellular carcinoma (HCC) cells. Specifically, XBP1-u colocalizes with sterol regulatory element-binding protein 2 (SREBP2) and inhibits its ubiquitination/proteasomal degradation. The ensuing stabilization of SREBP2 activates the transcription of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol biosynthesis. We subsequently show that the XBP1-u/SREBP2/HMGCR axis is crucial for enhancing cholesterol biosynthesis and lipid accumulation as well as tumorigenesis in HCC cells. Taken together, these findings reveal a novel function of XBP1-u in promoting tumorigenesis through increased cholesterol biosynthesis in hepatocarcinoma cells. Hence, XBP1-u might be a potential target for anti-tumor therapeutic strategies that focus on cholesterol metabolism in HCC.

Myelin oligodendrocyte glycoprotein-associated disease is associated with BANK1, RNASET2 and TNIP1 polymorphisms

AimHere we aimed to compare association of common immune-related genetic variants with three autoimmune central nervous system (CNS) demyelinating diseases, namely myelin oligodendrocyte glycoprotein-associated disease (MOGAD), multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). MethodsIn this retrospective cross-sectional study, 26 common immune-related single nucleotide polymorphisms were genotyped in 102 patients with MOGAD, 100 patients with MS, 198 patients with NMOSD and 541 healthy control subjects recruited from Guangzhou, China. ResultsAmong all tested genetic variations, one polymorphism, B cell scaffold protein with ankyrin repeats 1 (BANK1) rs4522865 was associated with multiple disorders, namely MOGAD (OR = 1.94, 95% CI:1.19–3.17, P = 0.0059) and NMOSD (OR = 1.69, 95% CI:1.17–2.45). Besides BANK1 rs4522865, two other non-HLA loci, ribonuclease T2 (RNASET2) rs9355610 (OR = 0.47, 95% CI: 0.26–0.85) and TNFAIP3 interacting protein 1 (TNIP1) rs10036748 (OR = 1.76, 95% CI: 1.16–2.71), were associated with MOGAD. In addition, NMOSD was associated with signal transducer and activator of transcription 4 (STAT4) rs7574865 (OR = 1.58, 95% CI: 1.12–2.24) and general transcription factor Iii (GTF2I) rs73366469 (OR = 1.60, 95% CI:1.12–2.29), while MS was associated with a killer cell lectin like receptor G1 (KLRG1) rs1805673 (OR = 0.61, 95% CI: 0.40–0.94) and T-box transcription factor 21 (TBX21) rs17244587 (OR = 2.25, 95% CI: 1.25–4.06). ConclusionThe current study suggests for the first time three non-HLA susceptibility loci for MOGAD. In addition, comparison of association of 26 immune-related polymorphisms with three autoimmune CNS demyelinating diseases demonstrates substantial difference in genetic basis of those disorders.

BHLHDB: A next generation database of basic helix loop helix transcription factors based on deep learning model.

The basic helix loop helix (bHLH) superfamily is a large and diverse protein family that plays a role in various vital functions in nearly all animals and plants. The bHLH proteins form one of the largest families of transcription factors found in plants that act as homo- or heterodimers to regulate the expression of their target genes. The bHLH transcription factor is involved in many aspects of plant development and metabolism, including photomorphogenesis, light signal transduction, secondary metabolism, and stress response. The amount of molecular data has increased dramatically with the development of high-throughput techniques and wide use of bioinformatics techniques. The most efficient way to use this information is to store and analyze the data in a well-organized manner. In this study, all members of the bHLH superfamily in the plant kingdom were used to develop and implement a relational database. We have created a database called bHLHDB (www.bhlhdb.org) for the bHLH family members on which queries can be conducted based on the family or sequences information. The Hidden Markov Model (HMM), which is frequently used by researchers for the analysis of sequences, and the BLAST query were integrated into the database. In addition, the deep learning model was developed to predict the type of TF with only the protein sequence quickly, efficiently, and with 97.54% accuracy and 97.76% precision. We created a unique and next-generation database for bHLH transcription factors and made this database available to the world of science. We believe that the database will be a valuable tool in future studies of the bHLH family.

The Role of Pyrazolopyridine Derivatives on Different Steps of Herpes Simplex Virus Type-1 In Vitro Replicative Cycle.

Herpes simplex virus type-1 (HSV-1) infection causes several disorders, and acyclovir is used as a reference compound. However, resistant strains are commonly observed. Herein, we investigate the effects of N-heterocyclic compounds (pyrazolopyridine derivatives), named ARA-04, ARA-05, and AM-57, on HSV-1 in vitro replication. We show that the 50% effective concentration (EC50) values of the compounds ARA-04, ARA-05, and AM-57 were 1.00 ± 0.10, 1.00 ± 0.05, and 0.70 ± 0.10 µM, respectively. These compounds presented high 50% cytotoxic concentration (CC50) values, which resulted in a selective index (SI) of 1000, 1000, and 857.1 for ARA-04, ARA-05, and AM-57, respectively. To gain insight into which step of the HSV-1 replication cycle these molecules would impair, we performed adsorption and penetration inhibition assays and time-of-addition experiments. Our results indicated that ARA-04 and ARA-05 affected viral adsorption, while AM-57 interfered with the virus replication during its α- and γ-phases and decreased ICP27 content during initial and late events of HSV-1 replication. In addition, we also observed that AM-57 caused a strong decrease in viral gD content, which was reinforced by in silico calculations that suggested AM-57 interacts preferentially with the viral complex between a general transcription factor and virion protein (TFIIBc-VP16). In contrast, ARA-04 and ARA-05 interact preferentially in the proteins responsible for the viral adsorption process (nectin-1 and glycoprotein). Thus, our results suggest that the 1H-pyrazolo[3,4-b]pyridine derivatives inhibit the HSV-1 replicative cycle with a novel mechanism of action, and its scaffold can be used as a template for the synthesis of promising new molecules with antiviral effects, including to reinforce the presented data herein for a limited number of molecules.

Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL.

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients. Significance: This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

Identifying Biomarkers of Cisplatin Sensitivity in Non-Small Cell Lung Cancer via Comprehensive Integrative Analysis

Background: Only 30-40% of non-small cell lung cancer (NSCLC) patients are clinically sensitive to cisplatin-based chemotherapy. Thus, it is necessary to identify biomarkers for personalized cisplatin chemotherapy in NSCLC. However, data heterogeneity and low-value density make it challenging to detect reliable cisplatin efficacy biomarkers using traditional analysis methods. Objective: This paper aims to find reliable cisplatin efficacy biomarkers for NSCLC patients using comprehensive integrative analysis. Method: We searched online resources and collected six NSCLC transcriptomics data sets with responses to cisplatin. The six data sets are divided into two groups: the learning group for biomarker identification and the test group for independent validation. We performed comprehensive integrative analysis under two kinds of frameworks, i.e., one-level and two-level, with three integrative models. Pathway analysis was performed to estimate the biological significance of the resulting biomarkers. For independent validation, logrank statistic was employed to test how significant the difference of Kaplan- Meier (KM) curves between two patient groups is, and the Cox proportional-hazards model was used to test how the expression of a gene is associated with patients’ survival time. Especially, a permutation test was performed to verify the predictive power of a biomarker panel on cisplatin efficacy. For comparison, we also analyzed each learning data set individually, in which three popular differential expression models, Limma, SAM, and RankSum, were used. Results: A total of 318 genes were identified as a core panel of cisplatin efficacy markers for NSCLC patients, exhibiting consistent differential expression between cisplatin-sensitive and –resistant groups across studies. A total of 129 of 344 KEGG pathways were found to be enriched in the core panel, reflecting a picture of the molecular mechanism of cisplatin resistance in NSCLC. By mapping onto the KEGG pathway tree, we found that a KEGG pathway-level I module, genetic information processing, is most active in the core panel with the highest activity ratio in response to cisplatin in NSCLC as expected. Related pathways include mismatch repair, nucleotide excision repair, aminoacyl-tRNA biosynthesis, and basal transcription factors, most of which respond to DNA double-strand damage in patients. Evaluation on two independent data sets demonstrated the predictive power of the core marker panel for cisplatin sensitivity in NSCLC. Also, some single markers, e.g., MST1R, were observed to be remarkably predictive of cisplatin resistance in NSCLC. Conclusion: Integrative analysis is more powerful in detecting biomarkers for cisplatin efficacy by overcoming data heterogeneity and low-value density in data sets, and the identified core panel (318 genes) can help develop personalized medicine of cisplatin chemotherapy for NSCLC patients.

BHLHE22 Expression Is Associated with a Proinflammatory Immune Microenvironment and Confers a Favorable Prognosis in Endometrial Cancer.

Endometrial cancer (EC) rates are rising annually. Additional prediction markers need to be evaluated because only 10–20% of EC cases show an objective response to immune-checkpoint inhibitors (ICIs). Our previous methylomic study found that BHLHE22 is hypermethylated in EC tissues and can be detected using a Pap-smear sample. BHLHE22, a basic helix loop helix transcription factor family member, is known as a transcriptional repressor and is involved in cell differentiation. However, the role of BHLHE22 in EC remains poorly understood. Herein, we analyzed BHLHE22 expression in 54 paired cancer and normal endometrial tissue samples, and confirmed with databases (TCGA, GTEx, and human protein atlas). We found that BHLHE22 protein expression was significantly downregulated in EC compared with normal endometrium. High BHLHE22 expression was associated with microsatellite-instable subtype, endometrioid type, grade, and age. It showed a significant favorable survival. BHLHE22 overexpression inhibited the proliferation and migration of EC cells. Functional enrichment analysis showed that BHLHE22 was significantly associated with immune-related pathways. Furthermore, BHLHE22 was positively correlated with proinflammatory leukocyte infiltration and expression of chemokine genes in EC. In conclusion, BHLHE22 regulates immune-related pathways and modulates the immune microenvironment of EC.

SIN-3 functions through multi-protein interaction to regulate apoptosis, autophagy, and longevity in Caenorhabditis elegans

SIN3/HDAC is a multi-protein complex that acts as a regulatory unit and functions as a co-repressor/co-activator and a general transcription factor. SIN3 acts as a scaffold in the complex, binding directly to HDAC1/2 and other proteins and plays crucial roles in regulating apoptosis, differentiation, cell proliferation, development, and cell cycle. However, its exact mechanism of action remains elusive. Using the Caenorhabditis elegans (C. elegans) model, we can surpass the challenges posed by the functional redundancy of SIN3 isoforms. In this regard, we have previously demonstrated the role of SIN-3 in uncoupling autophagy and longevity in C. elegans. In order to understand the mechanism of action of SIN3 in these processes, we carried out a comparative analysis of the SIN3 protein interactome from model organisms of different phyla. We identified conserved, expanded, and contracted gene classes. The C. elegans SIN-3 interactome -revealed the presence of well-known proteins, such as DAF-16, SIR-2.1, SGK-1, and AKT-1/2, involved in autophagy, apoptosis, and longevity. Overall, our analyses propose potential mechanisms by which SIN3 participates in multiple biological processes and their conservation across species and identifies candidate genes for further experimental analysis.