PH domain-only protein PHLDA3 is a p53-regulated repressor of Akt.

p53 And Akt are critical players regulating tumorigenesis with opposite effects: whereas p53 transactivates target genes to exert its function as a tumor suppressor, Akt phosphorylates its substrates and transduces downstream survival signals. In addition, p53 and Akt negatively regulate each other to balance survival and death signals within a cell. We now identify PHLDA3 as a p53 target gene that encodes a PH domain-only protein. We find that PHLDA3 competes with the PH domain of Akt for binding of membrane lipids, thereby inhibiting Akt translocation to the cellular membrane and activation. Ablation of endogenous PHLDA3 results in enhanced Akt activity and decrease of p53-dependent apoptosis. We also demonstrate the suppression of anchorage-independent cell growth by PHLDA3. Loss of the PHLDA3 genomic locus was frequently observed in primary lung cancers, suggesting a role of PHLDA3 in tumor suppression. Our results reveal a new mode of coordination between the p53 and Akt pathways.

Stimulation of interferon-ß responses by aberrant SARS-CoV-2 small viral RNAs acting as retinoic acid-inducible gene-I agonists.

Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines. Despite this, the interferon (IFN) response is delayed, contributing to disease progression. Here, we report that SARS-CoV-2 excessively generates small viral RNAs (svRNAs) encoding exact 5' ends of positive-sense genes in human cells in vitro and ex vivo, whereas endemic human coronaviruses (OC43 and 229E) produce significantly fewer similar svRNAs. SARS-CoV-2 5' end svRNAs are RIG-I agonists and induce the IFN-ß response in the later stages of infection. The first 60-nt ends bearing duplex structures and 5'-triphosphates are responsible for immune-stimulation. We propose that RIG-I activation by accumulated SARS-CoV-2 5' end svRNAs may contribute to later drive over-exuberant IFN production. Additionally, the differences in the amounts of svRNAs produced and the corresponding IFN response among CoV strains suggest that lower svRNA production during replication may correlate with the weaker immune response seen in less pathogenic CoVs.

Cell type-specific target selection by combinatorial binding of Smad2/3 proteins and hepatocyte nuclear factor 4alpha in HepG2 cells.

Specific regulation of target genes by transforming growth factor-ß (TGF-ß) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In this study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and we compared them with those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-ß. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4α (HNF4α) is expressed in HepG2 cells but not in HaCaT cells, and the HNF4α-binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. Chromatin immunoprecipitation sequencing analysis of HNF4α binding regions under TGF-ß stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4α bindings. MIXL1 was identified as a new combinatorial target of HNF4α and Smad2/3, and both the HNF4α protein and its binding motif were required for the induction of MIXL1 by TGF-ß in HepG2 cells. These findings generalize the importance of binding of HNF4α on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-ß and suggest that certain transcription factors expressed in a cell type-specific manner play important roles in the transcription regulated by the TGF-ß-Smad signaling pathway.

Allelic imbalances and homozygous deletion on 8p23.2 for stepwise progression of hepatocarcinogenesis.

UNLABELLED: Early hepatocellular carcinoma (eHCC) originates from the hepatocytes of chronic liver disease and develops into classical hepatocellular carcinoma (HCC). To identify sequential genetic changes in multistep hepatocarcinogenesis, we analyzed molecular karyotypes using oligonucleotide genotyping 50K arrays. First, 1q21.3-44 gain and loss of heterozygosity (LOH) on 1p36.21-36.32 and 17p13.1-13.3 were frequently observed in eHCC, but not in chronic liver diseases, suggesting that such chromosomal aberrations are early, possibly causative events in liver cancer. Next, we detected 25 chromosomal loci associated with liver cancer progression in five HCCs with nodule-in-nodule appearance, in which the inner nodule develops within eHCC lesion. Using these chromosomal regions as independent variables, decision tree analysis was applied on 14 early and 25 overt HCCs, and extracted combination of chromosomal gains on 5q11.1-35.3 and 8q11.1-24.3 and LOH on 4q11-34.3 and 8p11.21-23.3 as distinctive attributes, which can classify early and overt HCCs recursively. In these four altered regions identified as late events of hepatocarcinogenesis, two tumors in 32 overt HCCs analyzed in the present study and one in a set of independent samples of 36 overt HCCs in our previous study harbored a homozygous deletion near the CSMD1 locus on 8p23.2. CSMD1 messenger RNA expression was decreased in HCC without 8p23.2 deletion, possibly due to hypermethylation of the CpG islands in its promoter region. CONCLUSION: 1q gain and 1p and 17p LOH are early molecular events, whereas gains in 5q and 8q and LOH on 4q and 8p only occur in advanced HCC, and inactivation of the putative suppressor gene, CSMD1, may be the key event in progression of liver cancer.

Promoter-wide analysis of Smad4 binding sites in human epithelial cells.

Smad4, the common partner Smad, is a key molecule in transforming growth factor-beta (TGF-beta) family signaling. Loss of Smad4 expression is found in several types of cancer, including pancreatic cancer and colon cancer, and is related to carcinogenesis. Here we identified Smad4 binding sites in the promoter regions of over 25 500 known genes by chromatin immunoprecipitation on a microarray (ChIP-chip) in HaCaT human keratinocytes. We identified 925 significant Smad4 binding sites. Approximately half of the identified sites overlapped the binding regions of Smad2 and Smad3 (Smad2/3, receptor-regulated Smads in TGF-beta signaling), while the rest of the regions appeared dominantly occupied by Smad4 even when a different identification threshold for Smad2/3 binding regions was used. Distribution analysis showed that Smad4 was found in the regions relatively distant from the transcription start sites, while Smad2/3 binding regions were more often present near the transcription start sites. Motif analysis also revealed that activator protein 1 (AP-1) sites were especially enriched in the sites common to Smad2/3 and Smad4 binding regions. In contrast, GC-rich motifs were enriched in Smad4-dominant binding regions. We further determined putative target genes of Smad4 whose expression was regulated by TGF-beta. Our findings revealed some general characteristics of Smad4 binding regions, and provide resources for examining the role of Smad4 in epithelial cells and cancer pathogenesis.

Distinct chromosomal bias of gene expression signatures in the progression of hepatocellular carcinoma.

To identify the chromosomal aberrations associated with the progression of liver cancer, we applied expression imbalance map analysis to gene expression data from 31 hepatocellular carcinomas and 19 noncancerous tissues. Expression imbalance map analysis, which detects mRNA expression imbalance correlated with chromosomal regions, showed that expression gains of 1q21-23 (74%), 8q13-21 (48%), 12q23-24 (41%), 17q12-21(48%), 17q25 (25%), and 20q11 (22%) and losses of 4q13 (48%), 8p12-21 (32%), 13q14 (32%), and 17p13 (29%) were significantly associated with hepatocellular carcinoma. Most regions with altered expression identified by expression imbalance map were also identified in previous reports using comparative genomic hybridization. We demonstrated chromosomal copy number gain in 1q21-23 and loss in 17p13 by genomic quantitative PCR, suggesting that gene expression profiles reflect chromosomal alterations. Furthermore, expression imbalance map analysis revealed that more poorly differentiated hepatocellular carcinoma contain more chromosomal alterations, which are accumulated in a stepwise manner in the course of hepatocellular carcinoma progression: expression imbalance of 1q, 8p, 8q, and 17p occur as early events in hepatocarcinogenesis, and 12q, 17q25 and 20q occur as later events. In particular, expression gain of 17q12-21 and loss of 4q were seen to accumulate constantly through the dedifferentiation process. Our data suggest that gene expression profiles are subject to chromosomal bias and that expression imbalance map can correlate gene expression to gene loci with high resolution and sensitivity.

Genome-wide profiling of the core clock protein BMAL1 targets reveals a strict relationship with metabolism.

Circadian rhythms are common to most organisms and govern much of homeostasis and physiology. Since a significant fraction of the mammalian genome is controlled by the clock machinery, understanding the genome-wide signaling and epigenetic basis of circadian gene expression is essential. BMAL1 is a critical circadian transcription factor that regulates genes via E-box elements in their promoters. We used multiple high-throughput approaches, including chromatin immunoprecipitation-based systematic analyses and DNA microarrays combined with bioinformatics, to generate genome-wide profiles of BMAL1 target genes. We reveal that, in addition to E-boxes, the CCAATG element contributes to elicit robust circadian expression. BMAL1 occupancy is found in more than 150 sites, including all known clock genes. Importantly, a significant proportion of BMAL1 targets include genes that encode central regulators of metabolic processes. The database generated in this study constitutes a useful resource to decipher the network of circadian gene control and its intimate links with several fundamental physiological functions.

Chromatin immunoprecipitation on microarray analysis of Smad2/3 binding sites reveals roles of ETS1 and TFAP2A in transforming growth factor beta signaling.

The Smad2 and Smad3 (Smad2/3) proteins are principally involved in the transmission of transforming growth factor beta (TGF-beta) signaling from the plasma membrane to the nucleus. Many transcription factors have been shown to cooperate with the Smad2/3 proteins in regulating the transcription of target genes, enabling appropriate gene expression by cells. Here we identified 1,787 Smad2/3 binding sites in the promoter regions of over 25,500 genes by chromatin immunoprecipitation on microarray in HaCaT keratinocytes. Binding elements for the v-ets erythroblastosis virus E26 oncogene homolog (ETS) and transcription factor AP-2 (TFAP2) were significantly enriched in Smad2/3 binding sites, and knockdown of either ETS1 or TFAP2A resulted in overall alteration of TGF-beta-induced transcription, suggesting general roles for ETS1 and TFAP2A in the transcription induced by TGF-beta-Smad pathways. We identified novel Smad binding sites in the CDKN1A gene where Smad2/3 binding was regulated by ETS1 and TFAP2A. Moreover, we showed that small interfering RNAs for ETS1 and TFAP2A affected TGF-beta-induced cytostasis. We also analyzed Smad2- or Smad3-specific target genes regulated by TGF-beta and found that their specificity did not appear to be solely determined by the amounts of the Smad2/3 proteins bound to the promoters. These findings reveal novel regulatory mechanisms of Smad2/3-induced transcription and provide an essential resource for understanding their roles.