SOD1 inhibition enhances sorafenib efficacy in HBV-related hepatocellular carcinoma by modulating PI3K/Akt/mTOR pathway and ROS-mediated cell death.

Hepatitis B Virus (HBV) infection significantly elevates the risk of hepatocellular carcinoma (HCC), with the HBV X protein (HBx) playing a crucial role in cancer progression. Sorafenib, the primary therapy for advanced HCC, shows limited effectiveness in HBV-infected patients due to HBx-related resistance. Numerous studies have explored combination therapies to overcome this resistance. Sodium diethyldithiocarbamate (DDC), known for its anticancer effects and its inhibition of superoxide dismutase 1 (SOD1), is hypothesized to counteract sorafenib (SF) resistance in HBV-positive HCCs. Our research demonstrates that combining DDC with SF significantly reduces HBx and SOD1 expressions in HBV-positive HCC cells and human tissues. This combination therapy disrupts the PI3K/Akt/mTOR signalling pathway and promotes apoptosis by increasing reactive oxygen species (ROS) levels. These cellular changes lead to reduced tumour viability and enhanced sensitivity to SF, as evidenced by the synergistic suppression of tumour growth in xenograft models. Additionally, DDC-mediated suppression of SOD1 further enhances SF sensitivity in HBV-positive HCC cells and xenografted animals, thereby inhibiting cancer progression more effectively. These findings suggest that the DDC-SF combination could serve as a promising strategy for overcoming SF resistance in HBV-related HCC, potentially optimizing therapy outcomes.

E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.

Dietary Restriction Impacts Peripheral Circadian Clock Output Important for Longevity in Drosophila.

Circadian clocks may mediate lifespan extension by caloric or dietary restriction (DR). We find that the core clock transcription factor Clock is crucial for a robust longevity and fecundity response to DR in Drosophila. To identify clock-controlled mediators, we performed RNA-sequencing from abdominal fat bodies across the 24 h day after just 5 days under control or DR diets. In contrast to more chronic DR regimens, we did not detect significant changes in the rhythmic expression of core clock genes. Yet we discovered that DR induced de novo rhythmicity or increased expression of rhythmic clock output genes. Network analysis revealed that DR increased network connectivity in one module comprised of genes encoding proteasome subunits. Adult, fat body specific RNAi knockdown demonstrated that proteasome subunits contribute to DR-mediated lifespan extension. Thus, clock control of output links DR-mediated changes in rhythmic transcription to lifespan extension.

Antitumor Effect of Metformin in Combination with Binimetinib on Melanoma Cells.

Cutaneous melanoma is a fatal disease for patients with distant metastasis. Metformin is the most widely used anti-diabetic drug, and proved to suppress cell proliferation and metastasis in diverse cancers including melanoma. We previously reported that MEK inhibitor trametinib increases the expression of epithelial-mesenchymal transition (EMT) regulators and melanoma cell motility, which are suppressed by addition of metformin in A375 melanoma cells. To confirm our findings further, we first evaluated the effect of metformin in combination with another MEK inhibitor binimetinib on cell viability in G361 melanoma cells. We then investigated whether binimetinib affects the expression of EMT regulators and cell motility. We finally monitored the effect of metformin on binimetinib-induced cell migration. Cell viability assay showed that combination index (CI) value at ED50 is 0.80, suggesting synergy for the combination of metformin with binimetinib. Our results also revealed that binimetinib increased the expression of EMT regulators such as integrin αV, fibronectin and slug, which correlate well with the enhanced cell migration in wound healing assay. Metformin, on the contrary, suppressed the expression of sparc, integrin αV, fibronectin and N-cadherin with the reduced cell motility. The combination treatment showed that metformin counteracts the binimetinib-induced increase of cell motility. Overall, these results suggest that metformin with binimetinib might be useful as a potential therapeutic adjuvant against cell survival and metastatic activity in melanoma patients.

A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila.

Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.

QuBiT: a quantitative tool for analyzing epithelial tubes reveals unexpected patterns of organization in the Drosophila trachea.

Biological tubes are essential for animal survival, and their functions are dependent on tube shape. Analyzing the contributions of cell shape and organization to the morphogenesis of small tubes has been hampered by the limitations of existing programs in quantifying cell geometry on highly curved tubular surfaces and calculating tube-specific parameters. We therefore developed QuBiT (Quantitative Tool for Biological Tubes) and used it to analyze morphogenesis of the embryonic Drosophila trachea (airway). In the main tube, we find previously unknown anterior-to-posterior (A-P) gradients of cell apical orientation and aspect ratio, and periodicity in the organization of apical cell surfaces. Inferred cell intercalation during development dampens an A-P gradient of the number of cells per cross-section of the tube, but does not change the patterns of cell connectivity. Computationally 'unrolling' the apical surface of wild-type trachea and the hindgut reveals previously unrecognized spatial patterns of the apical marker Uninflatable and a non-redundant role for the Na+/K+ ATPase in apical marker organization. These unexpected findings demonstrate the importance of a computational tool for analyzing small diameter biological tubes.

Chronic Nonatherosclerotic Occlusive Popliteal Artery Disease.

BACKGROUND: The outcomes of revascularization of nonatherosclerotic occlusive popliteal artery disease are unknown. Therefore, the objective of this study was to analyze the outcomes of recanalization of nonatherosclerotic occlusive popliteal artery disease, the results of which would be useful in creating surgical strategies for such cases. METHODS: From January 2000 to December 2015, a total of 22 patients with nonatherosclerotic occlusive popliteal artery disease underwent open surgical repair. We retrospectively analyzed the data of these patients. Thrombectomy with angioplasty on the occluded popliteal artery, graft interposition, and bypass surgery were conducted for revascularization of the affected region. We analyzed the overall primary patency rate, type of graft, and surgical approach. Furthermore, we compared the primary patency rate after surgical treatment. RESULTS: Of 22 patients, 3 (13.6%) had cystic adventitial disease, 16 (72.7%) had popliteal artery entrapment syndrome, and 3 (13.6%) were diagnosed as having thromboangiitis obliterans. Five patients (22.7%) underwent thrombectomy with patch angioplasty, 8 (36.3%) underwent bypass surgery, and 9 (40.9%) underwent graft interposition of the popliteal artery. All graft interpositions and thrombectomies with patch angioplasty were performed through a posterior approach, whereas all bypass surgeries were performed through a medial approach except in 1 case. The mean follow-up period was 54.95 ± 42.99 months. The overall primary patency rate at 1, 3, and 10 years was 100%, 86.9%, and 69.5%, respectively. In the bypass group, the primary patency rate at 1, 3, and 10 years was 100%, 66.7%, and 44.4%, respectively. In the other groups, the primary patency rate was 100% during the follow-up period. The difference in primary patency rate was statistically significant (P ≤ 0.05). CONCLUSIONS: The result of surgical treatment for nonatherosclerotic occlusive popliteal artery disease was better than that of atherosclerotic popliteal artery disease. Direct popliteal artery reconstruction, such as graft interposition or thrombectomy with patch angioplasty, showed better short- and long-term patency than did bypass surgery.

Identification of Drosophila Zfh2 as a Mediator of Hypercapnic Immune Regulation by a Genome-Wide RNA Interference Screen.

Hypercapnia, elevated partial pressure of CO2 in blood and tissue, develops in many patients with chronic severe obstructive pulmonary disease and other advanced lung disorders. Patients with advanced disease frequently develop bacterial lung infections, and hypercapnia is a risk factor for mortality in such individuals. We previously demonstrated that hypercapnia suppresses induction of NF-κB-regulated innate immune response genes required for host defense in human, mouse, and Drosophila cells, and it increases mortality from bacterial infections in both mice and Drosophila. However, the molecular mediators of hypercapnic immune suppression are undefined. In this study, we report a genome-wide RNA interference screen in Drosophila S2* cells stimulated with bacterial peptidoglycan. The screen identified 16 genes with human orthologs whose knockdown reduced hypercapnic suppression of the gene encoding the antimicrobial peptide Diptericin (Dipt), but did not increase Dipt mRNA levels in air. In vivo tests of one of the strongest screen hits, zinc finger homeodomain 2 (Zfh2; mammalian orthologs ZFHX3/ATBF1 and ZFHX4), demonstrate that reducing zfh2 function using a mutation or RNA interference improves survival of flies exposed to elevated CO2 and infected with Staphylococcus aureus. Tissue-specific knockdown of zfh2 in the fat body, the major immune and metabolic organ of the fly, mitigates hypercapnia-induced reductions in Dipt and other antimicrobial peptides and improves resistance of CO2-exposed flies to infection. Zfh2 mutations also partially rescue hypercapnia-induced delays in egg hatching, suggesting that Zfh2's role in mediating responses to hypercapnia extends beyond the immune system. Taken together, to our knowledge, these results identify Zfh2 as the first in vivo mediator of hypercapnic immune suppression.

In vitro immune cell monitoring as a guide for long-term immunosuppression in adult liver transplant recipients.

BACKGROUNDS/AIMS: We evaluated the clinical usability of immune cell monitoring in adult liver transplantation (LT) recipients. METHODS: This study was composed of two parts as using calcineurin phosphatase (CNP) activity assay and ImmuKnow assay independently as in vitro monitoring tools of immune cell function in adult LT recipients. RESULTS: There was a rough correlation between CNP activity and tacrolimus concentration in 33 patients. This association was evident in patients who were only administered tacrolimus, but disappeared after the co-administration of mycophenolate. In 118 healthy individuals, the mean proportion of helper T-cells was 37.4±8.1%. According to ImmuKnow assay, their immune responses were strong in 12 patients (10.2%), moderate in 92 patients (78.0%), and low in 14 patients (11.9%). In 85 patients waiting for LT, there was a rough correlation between the ImmuKnow ATP level and age. Their immune responses were strong in 0 patients (0%), moderate in 8 patients (9.4%), and low in 77 patients (90.6%). There was a difference in the ImmuKnow ATP levels between healthy individuals and patients with liver disease. In 137 LT recipients, there was no correlation between the ImmuKnow ATP levels and tacrolimus concentration. This trend did not change after grouping the patients according to co-administration with mycophenolate. Eight recipients experienced acute rejection, but none showed strong immune response. CONCLUSIONS: We think that both CNP activity assay and ImmuKnow assay are too limited to objectively determine the level of immunosuppression. Further studies should be performed to identify other methods for immune function monitoring.

Anatomical Popliteal Artery Entrapment Syndrome Caused by an Aberrant Plantaris Muscle.

PURPOSE: We report on cases of anatomical popliteal artery entrapment syndrome (PAES) caused by an aberrant plantaris muscle and highlight the involvement of this muscle in PAES. MATERIALS AND METHODS: Seven symptomatic PAES legs in six patients treated at The Division of Vascular Surgery, Asan Medical Center, Seoul, Korea, between 1995 and 2011 were included in this study. We retrospectively analyzed patient records, magnetic resonance imaging (MRI) and/or computed tomography (CT) scans of the knee joint, Doppler pressure studies, CT angiographies, and conventional femoral arteriographies. RESULTS: Five males and one female patient with a median age of 32 (18-53) years old were enrolled in the study. All patients complained of intermittent claudication of the affected leg. All aberrant plantaris muscles were higher and more medially located than normal plantaris muscles, causing occlusion of the popliteal artery upon forced plantar flexion of the ankle. For arterial lesions, five occlusions of the popliteal artery and two patent popliteal arteries with positive provocation were noted. As for treatment, myotomy of the aberrant plantaris muscle was done for two non-occlusive PAES legs. For occlusive PAES legs, one thrombectomy, one saphenous vein graft interposition of the popliteal artery followed by myotomy, and two below-knee femoro-popliteal bypasses were performed. The median follow-up period was 88 (7-148) months. CONCLUSION: An aberrant plantaris muscle can cause anatomical PAES. Classification or diagnosis of PAES should be based on axial studies using CT scans or MRI using various reconstruction methods. Treatment, including myotomy of the plantaris muscle, should be individualized.

Customized left-sided hepatectomy and bile duct resection for perihilar cholangiocarcinoma in a patient with left-sided gallbladder and multiple combined anomalies.

Left-sided gallbladder (LSGB) is a rare anomaly, but it is often associated with multiple combined variations of the liver anatomy. We present the case of a patient with LSGB who underwent successful resection of perihilar cholangiocarcinoma. The patient was a 67-year-old male who presented with upper abdominal pain and obstructive jaundice. Initial imaging studies led to the diagnosis of Bismuth-Corlette type IIIB perihilar cholangiocarcinoma. Due to the unique location of the gallbladder and combined multiple hepatic anomalies, LSGB was highly suspected. During surgery after hilar dissection, we recognized that the tumor was located at the imaginary hilar bile duct bifurcation, but its actual location was corresponding to the biliary confluence of the left median and lateral sections. The extent of resection included extended left lateral sectionectomy, caudate lobe resection, and bile duct resection. Since some of the umbilical portion of the portal vein was invaded, it was resected and repaired with a portal vein branch patch. Due to anatomical variation of the biliary system, only one right-sided duct was reconstructed. The patient recovered uneventfully without any complication. LSGB should be recognized as a constellation of multiple hepatic anomalies, and therefore, thorough investigations are necessary to enable the performance of safe hepatic and biliary resections.

Balloon dilation of jejunal afferent loop functional stenosis following left hepatectomy and hepaticojejunostomy long time after pylorus-preserving pancreaticoduodenectomy: a case report.

We present a rare case of functional stenosis of the jejunal loop following left hepatectomy and hepaticojejunostomy long after pylorus-preserving pancreaticoduodenectomy (PPPD), which was successfully managed by balloon dilation. A 70-year-old Korean man had undergone PPPD 6 years before due to 1.8 cm-sized distal bile duct cancer. Sudden onset of obstructive jaundice led to diagnosis of recurrent bile duct cancer mimicking perihilar cholangiocarcinoma of type IIIb. After left portal vein embolization, the patient underwent resection of the left liver and caudate lobe and remnant extrahepatic bile duct. The pre-existing jejunal loop and choledochojejunostomy site were used again for new hepaticojejunostomy. The patient recovered uneventfully, but clamping of the percutaneous transhepatic biliary drainage (PTBD) tube resulted in cholangitis. Biliary imaging studies revealed that biliary passage into the afferent jejunal limb was significantly impaired. We performed balloon dilation of the afferent jejunal loop by using a 20 mm-wide balloon. Follow-up hepatobiliary scintigraphy showed gradual improvement in biliary excretion and the PTBD tube was removed at 1 month after balloon dilation. This very unusual condition was regarded as disuse atrophy of the jejunal loop, which was successfully managed by balloon dilation and intraluminal keeping of a large-bore PTBD tube for 1 month.

Degradation of multi-DNAPLs by a UV/persulphate/ethanol system with the additional injection of a base solution.

This study was conducted to investigate the inhibited influences on and solution to the degradation of four types of dense non-aqueous phase liquids (DNAPLs) (i.e. perchloroethylene [PCE], trichloroethylene [TCE], chloroform [CF], and carbon tetrachloride [CT]) all at the same instance in groundwater (GW). Degradations of DNAPLs in de-ionized water (DW) and GW were carried out by applying an ultraviolet radiation-activated persulphate (UV/PS) system. PCE and TCE were degraded by over 90% and CT was only degraded by 25% in both DW and GW. However, CF was degraded by over 90% in DW, while it was only degraded by 50% in GW. First of all, degradations with an inorganic anion (either Cl- or HCO3-) indicated that the lower degradation of CF in GW was caused by the existence of the chloride ion. Moreover, the low CF degradation in GW was overcome by the additional injection of a base solution (sodium hydroxide [NaOH]) into the UV/PS system. The results showed that PCE, TCE, and CF were degraded by over 90%, respectively, when a molar ratio of [base]0:[PS]0 was larger than 0.5:1, but CT was still not effectively degraded in the UV/PS system. To achieve effective CT degradation, UV/PS with the ethanol (EtOH) system was evaluated and it was found that it degraded CT over 90%. However, at this time, CF was not effectively degraded in the UV/PS/EtOH system. Finally, degradations of DNAPLs in the UV/PS/EtOH system with the additional injection of a base solution were conducted and it showed that multi-DNAPLs were degraded by over 90%, respectively, when the molar ratio of [PS]0:[EtOH]0:[base]0 was 1:1:3.

Genome-wide analysis of DNA methylation and the gene expression change in lung cancer.

INTRODUCTION: The recent DNA methylation studies on cancers have revealed the necessity of profiling an entire human genome and not to restrict the profiling to specific regions of the human genome. It has been suggested that genome-wide DNA methylation analysis enables us to identify the genes that are regulated by DNA methylation in carcinogenesis. METHODS: So, we performed whole-genome DNA methylation analysis for human lung squamous cell carcinoma (SCC), which is strongly related with smoking. We also performed microarrays using 21 pairs of normal lung tissues and tumors from patients with SCC. By combining these data, 30 hypermethylated and down-regulated genes, and 22 hypomethylated and up-regulated genes were selected. The gene expression level and DNA methylation pattern were confirmed by semiquantitative reverse-transcriptase polymerase chain reaction and pyrosequencing, respectively. RESULTS: By these validations, we selected five hypermethylated and down-regulated genes and one hypomethylated and up-regulated gene. Moreover, these six genes were proven to be actually regulated by DNA methylation by confirming the recovery of their DNA methylation pattern and gene expression level using a demethylating agent. The DNA methylation pattern of the CYTL1 promoter region was significantly different between early and advanced stages of SCC. CONCLUSION: In conclusion, by combining the whole-genome DNA methylation pattern and the gene expression profile, we identified the six genes (CCDC37, CYTL1, CDO1, SLIT2, LMO3, and SERPINB5) that are regulated by DNA methylation, and we suggest their value as target molecules for further study of SCC.

Feasibility study on an oxidant-injected permeable reactive barrier to treat BTEX contamination: adsorptive and catalytic characteristics of waste-reclaimed adsorbent.

The adsorptive and catalytic characteristics of waste-reclaimed adsorbent (WR), which is a calcined mixture of bottom-ash and dredged-soil, was investigated for its application to treating BTEX contamination. BTEX adsorption in WR was 54%, 64%, 62%, and 65%, respectively, for a 72 h reaction time. Moreover, the catalytic characteristics of WR were observed when three types of oxidation systems (i.e., H(2)O(2), persulfate (PS), and H(2)O(2)/Fe(III)/oxalate) were tested, and these catalytic roles of WR could be due to iron oxide on its surface. In PS/WR system, large amounts of metal ions from WR were released because of large drops of solution pH, and the surface area of WR was also greatly reduced. Moreover, the BTEX that was removed per consumed oxidant (ΔC(rem)/ΔOx) increased with increasing PS. In H(2)O(2)/Fe(III)/oxalate with WR system, the highest BTEX degradation rate constants (k(deg)) were calculated as 0.338, 0.365, 0.500 and 0.716 h(-1), respectively, when 500 mM of H(2)O(2) was used, and the sorbed BTEX on the surface of WR was also degraded, which suggests the regeneration of WR. Therefore, the oxidant-injected permeable reactive barrier filled in WR could be an alternative to treating BTEX with both adsorption and catalytic degradation.

Effect of metal oxides on the reactivity of persulfate/Fe(II) in the remediation of diesel-contaminated soil and sand.

The effect of metal oxides on the ability of persulfate (PS) with Fe(II) to remediate diesel-contaminated soil was investigated. In both natural soil and purchased sand, the highest diesel degradation occurred at pH 3 and the optimum molar ratio of PS/Fe(II) was 100:1 (i.e. 500 mM PS to 5 mM Fe(II)). Moreover, adding Fe(II) increased PS reactivity more in soil than it did in sand, indicating the involvement of metal oxides in the soil matrix. Evaluating the effects of metal oxides (i.e. goethite, hematite, magnetite, and manganese oxide) on the reactivity of PS with/without Fe(II) in a system containing diesel-contaminated sand revealed that manganese oxide increased PS activity the most and that the highest diesel degradation by PS occurred when both manganese oxide and Fe(II) were used as activators. XRD did not show the transformation of manganese oxide in the presence of Fe(II). SEM-EDS showed the association of Fe(II) on the surface of manganese oxide, and ICP analysis revealed that almost all the added Fe(II) adsorbed to manganese oxide but almost none adsorbed to iron oxides under acidic conditions. Therefore, the high reactivity of PS could be due to the high density of Fe(II) over the surface of manganese oxide.

Crystallization of amorphous Fe90Zr10 under ball milling.

The present study deals with structural transformations induced by high-energy ball-milling of an amorphous Fe90Zr10 alloy prepared by melt-spinning. The amorphous melt-spun ribbons were found to undergo crystallization into BCC alpha-Fe(Zr) nanocrystallites under high-energy ball milling. The decomposition degree of the amorphous phase increased with increasing milling time and intensity. Our results suggest that the observed crystallization is a deformation-induced process rather than a thermally induced one.

Isolating bronchial epithelial cell preparations from gross lung specimens.

Tissue microdissection is appropriate for separating pure cells from heterogeneous tissues. Recently, we have focused on whole genome DNA methylation patterns of lung squamous cell carcinoma (SCC) and needed to obtain the appropriate counterpart cells of lung SCC. However, in some regions of human tissues, such as in bronchial epithelium, it is difficult to apply tissue microdissection as a means to isolate pure cells from a heterogenous mixture of cells. Accordingly, we developed the pop brush method to retrieve sufficient amounts of pure bronchial epithelium from gross lung specimens, and this method enables us to study epigenetic variations of lung SCC.

Expression patterns of aurora kinase B, heat shock protein 47, and periostin in esophageal squamous cell carcinoma.

To elucidate the proteins related to the development and progress of esophageal squamous cell carcinoma (ESCC), we performed gene expression profile analysis of 14 ESCC tissues. We identified 182 genes that were commonly upregulated (p < 10(-5)) and 54 genes that were downregulated in ESCC tissues (p < 10(-6)). In order to validate the gene expression profiles, we did semiquantitative RT-PCR analysis and found 11 genes upregulated in greater than 70% of 19 tissue samples. We further examined the protein expression level and the distribution patterns of four genes: aurora kinase B (AURKB), periostin (POSTN), heat shock protein 47 (HSP47), and matrix metalloprotease 1 (MMP1). Ultimately, three genes (AURKB, HSP47, POSTN) were verified to be increased at both the mRNA and protein levels. Among them, AURKB and HSP47 were found to increase in the early development stage of ESCC and POSTN might be considered to function in the cell-cell interactions between cancer cell and adjacent stromal cells. Accordingly, these studies may provide valuable information for the identification of the genes that are necessary to study further their functions. Moreover, these genes might be used as potential diagnostic or therapeutic target molecules for ESCC patients.

MED16 and MED23 of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced transcriptional activators.

Transcriptional activators interact with diverse proteins and recruit transcriptional machinery to the activated promoter. Recruitment of the Mediator complex by transcriptional activators is usually the key step in transcriptional activation. However, it is unclear how Mediator recognizes different types of activator proteins. To systematically identify the subunits responsible for the signal- and activator-specific functions of Mediator in Drosophila melanogaster, each Mediator subunit was depleted by RNA interference, and its effect on transcriptional activation of endogenous as well as synthetic promoters was examined. The depletion of some Mediator gene products caused general transcriptional defects, whereas depletion of others caused defects specifically related to activation. In particular, MED16 and MED23 were required for lipopolysaccharide- and heat-shock-specific gene expression, respectively, and their activator-specific functions appeared to result from interaction with specific activators. The corequirement of MED16 for other forms of differentiation-inducing factor-induced transcription was confirmed by microarray analysis of differentiation-inducing factor (DIF)- and MED16-depleted cells individually. These results suggest that distinct Mediator subunits interact with specific activators to coordinate and transfer activator-specific signals to the transcriptional machinery.