TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.

Epigenetic Silencing of OR and TAS2R Genes Expression in Human Orbitofrontal Cortex At Early Stages of Sporadic Alzheimer’s Disease

Modulation of brain olfactory (OR) and taste receptors (TASR) expression was recently reported in neurological diseases. We explored the possible expression and regulation of selected OR and TASR genes in human orbitofrontal cortex of sporadic Alzheimer’s disease (AD) and found that these are expressed and markedly downregulated at early stages. The expression pattern did not follow disease progression suggesting regulation through epigenetic mechanisms. We found an increase of global H3K9me3 levels and substantial enrichment of this repressive signature at ORs and TAS2Rs proximal promoter at early stages, ultimately lost at advanced stages. By mass spectrometry-based proteomic and further validation, we found that H3K9me3 interacts with MeCP2 at early stages and that this protein is increased in sporadic AD. Findings suggest MeCP2 might be implicated in OR and TAS2R genes expression regulation through interaction with H3K9me3, and as an early event, it may uncover a novel etiopathogenetic mechanism of sporadic AD.

Concordant Androgen-Regulated Expression of Divergent Rhox5 Promoters in Sertoli Cells

Concordant transcriptional regulation can generate multiple gene products that collaborate to achieve a common goal. Here we report a case of concordant transcriptional regulation that instead drives a single protein to be produced in the same cell type from divergent promoters. This gene product—the RHOX5 homeobox transcription factor—is translated from 2 different mRNAs with different 5′ untranslated regions (UTRs) transcribed from alternative promoters. Despite the fact that these 2 promoters—the proximal promoter (Pp) and the distal promoter (Pd)—exhibit different patterns of tissue-specific activity, share no obvious sequence identity, and depend on distinct transcription factors for expression, they exhibit a remarkably similar expression pattern in the testes. In particular, both depend on androgen signaling for expression in the testes, where they are specifically expressed in Sertoli cells and have a similar stage-specific expression pattern during the seminiferous epithelial cycle. We report evidence for 3 mechanisms that collaborate to drive concordant Pp/Pd expression. First, both promoters have an intrinsic ability to respond to androgen receptor and androgen. Second, the Pp acts as an enhancer to promote androgen-dependent transcription from the Pd. Third, Pd transcription is positively autoregulated by the RHOX5 protein, which is first produced developmentally from the Pp. Together, our data support a model in which the Rhox5 homeobox gene evolved multiple mechanisms to activate both of its promoters in Sertoli cells to produce Rhox5 in an androgen-dependent manner during different phases of spermatogenesis.

Over-representation of potential SP4 target genes within schizophrenia-risk genes

Reduction of Sp4 expression causes age-dependent hippocampal vacuolization and many other intermediate phenotypes of schizophrenia in Sp4 hypomorphic mice. Recent human genetic studies from both the Schizophrenia Exome Sequencing Meta-Analysis (SCHEMA) and the Genome-Wide Association Study (GWAS) validated SP4 as a schizophrenia-risk gene over the exome-wide or the genome-wide significance. Truncation of the human SP4 gene has an odds ratio of 9.37 (3.38–29.7) for schizophrenia. Despite successful identification of many schizophrenia-risk genes, it is unknown whether and how these risk genes may interact with each other in the development of schizophrenia. By taking advantage of the specific localization of the GC-boxes bound by SP4 transcription factors, I analyzed the relative abundance of these GC-boxes in the proximal promoter regions of schizophrenia-risk genes. I found that the GC-box containing genes are significantly over-represented within schizophrenia-risk genes, suggesting that SP4 is not only a high-risk gene for schizophrenia, but may also act as a hub of network in the regulation of many other schizophrenia-risk genes via these GC-boxes in the pathogenesis of schizophrenia.

Molecular Characterization and Expression of the LAP3 Gene and Its Association with Growth Traits in the Blood Clam Tegillarca granosa

Leucine aminopeptidase 3 (LAP3) is a metallopeptidase that cleaves N-terminal residues and is involved in protein maturation and degradation. In this study, we characterized the leucine aminopeptidase 3 (LAP3) gene from Tegillarca granosa (Tg-LAP3 for short), which appeared to consist of 15,731 nucleotides encoding 530 amino acids. We identified 12 introns and 13 exons in the Tg-LAP3 gene, suggesting a highly conserved genomic structure. The proximal promoter sequence consists of 1922 bps with a typical TATA box structure, which is the general structural characteristic of core promoters in eukaryotes. We found two functional domains in the Tg-LAP3 protein, including an N-terminal domain (41–174aa) and a peptidase_M17 catalytic domain (209–522aa). Multiple alignment showed that Tg-LAP3 shares 73.4% identity with LAP3 of Mizuhopecten yessoensis and 55.2–70.7% identity with LAP3 of other species. Quantitative analysis of Tg-LAP3 in embryos/larvae and adult tissues indicated that the highest expression occurred in eyebot larva, with limited expression in other stages; among tissues, the highest expression was found in the liver (p < 0.05). Association analysis found that three single-nucleotide polymorphisms (SNPs) (g.-488A > G, g.-1123C > T, and g.-1304C > A) in the proximal promoter were successfully typed, but there was no significant difference in growth traits (body weight, shell length, shell width, and shell height) among these genotypes. The results of our study demonstrate the functional roles of the Tg-LAP3 gene and provide valuable information for molecular marker-assisted selection (MAS) of the blood clam.

Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance

BackgroundEya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown.MethodsWe engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo.ResultsEya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death–inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor–specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor–specific expression in response to Six2 activity.ConclusionsOur results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

Shaping modern human skull through epigenetic, transcriptional and post-transcriptional regulation of the RUNX2 master bone gene

RUNX2 encodes the master bone transcription factor driving skeletal development in vertebrates, and playing a specific role in craniofacial and skull morphogenesis. The anatomically modern human (AMH) features sequence changes in the RUNX2 locus compared with archaic hominins’ species. We aimed to understand how these changes may have contributed to human skull globularization occurred in recent evolution. We compared in silico AMH and archaic hominins’ genomes, and used mesenchymal stromal cells isolated from skull sutures of craniosynostosis patients for in vitro functional assays. We detected 459 and 470 nucleotide changes in noncoding regions of the AMH RUNX2 locus, compared with the Neandertal and Denisovan genomes, respectively. Three nucleotide changes in the proximal promoter were predicted to alter the binding of the zinc finger protein Znf263 and long-distance interactions with other cis-regulatory regions. By surface plasmon resonance, we selected nucleotide substitutions in the 3’UTRs able to affect miRNA binding affinity. Specifically, miR-3150a-3p and miR-6785-5p expression inversely correlated with RUNX2 expression during in vitro osteogenic differentiation. The expression of two long non-coding RNAs, AL096865.1 and RUNX2-AS1, within the same locus, was modulated during in vitro osteogenic differentiation and correlated with the expression of specific RUNX2 isoforms. Our data suggest that RUNX2 may have undergone adaptive phenotypic evolution caused by epigenetic and post-transcriptional regulatory mechanisms, which may explain the delayed suture fusion leading to the present-day globular skull shape.

Identification and validation of hub genes of synovial tissue for patients with osteoarthritis and rheumatoid arthritis

Background Osteoarthritis (OA) and rheumatoid arthritis (RA) were two major joint diseases with similar clinical phenotypes. This study aimed to determine the mechanistic similarities and differences between OA and RA by integrated analysis of multiple gene expression data sets. Methods Microarray data sets of OA and RA were obtained from the Gene Expression Omnibus (GEO). By integrating multiple gene data sets, specific differentially expressed genes (DEGs) were identified. The Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein–protein interaction (PPI) network analysis of DEGs were conducted to determine hub genes and pathways. The “Cell Type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT)” algorithm was employed to evaluate the immune infiltration cells (IICs) profiles in OA and RA. Moreover, mouse models of RA and OA were established, and selected hub genes were verified in synovial tissues with quantitative polymerase chain reaction (qPCR). Results A total of 1116 DEGs were identified between OA and RA. GO functional enrichment analysis showed that DEGs were enriched in regulation of cell morphogenesis involved in differentiation, positive regulation of neuron differentiation, nuclear speck, RNA polymerase II transcription factor complex, protein serine/threonine kinase activity and proximal promoter sequence-specific DNA binding. KEGG pathway analysis showed that DEGs were enriched in EGFR tyrosine kinase inhibitor resistance, ubiquitin mediated proteolysis, FoxO signaling pathway and TGF-beta signaling pathway. Immune cell infiltration analysis identified 9 IICs with significantly different distributions between OA and RA samples. qPCR results showed that the expression levels of the hub genes (RPS6, RPS14, RPS25, RPL11, RPL27, SNRPE, EEF2 and RPL19) were significantly increased in OA samples compared to their counterparts in RA samples (P < 0.05). Conclusion This large-scale gene analyses provided new insights for disease-associated genes, molecular mechanisms as well as IICs profiles in OA and RA, which may offer a new direction for distinguishing diagnosis and treatment between OA and RA.