Frank P. Buxton

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells

This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.

Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers

Significance Mammalian SWI/SNF (mSWI/SNF) alterations are highly prevalent, now estimated to occur in 20% of cancers. The inactivating nature of mSWI/SNF mutations presents a challenge for devising strategies to target these epigenetic lesions. By performing a comprehensive pooled shRNA screen of the epigenome using a unique deep coverage design shRNA (DECODER) library across a large cancer cell line panel, we identified that BRG1/SMARCA4 mutant cancer cells are highly sensitive to BRM/SMARCA2 depletion. Our study provides important mechanistic insight into the BRM/BRG1 synthetic lethal relationship, shows this finding translates in vivo, and highlights BRM as a promising therapeutic target for the treatment BRG1-mutant cancers. Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1-mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in ∼10–15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of “cancer-selective paralog dependency” may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.

Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans

A mutant of Aspergillus niger defective in ornithine transcarbamylase function was transformed with plasmids carrying a functional copy of the argB gene of Aspergillus nidulans after treatment of spheroplasts in the presence of polyethylene glycol and calcium ions. The plasmid pDG3 gave stable transformants at a frequency of 4 per microgram of input DNA. Southern blot analysis of DNA from transformants showed that pDG3 DNA had integrated into the A. niger chromosomes at a variety of locations. The transformants were phenotypically stable for many mitotic divisions. This procedure may potentially be used to insert any gene into the genome of A. niger. A cosmid shuttle vector, pDG1, for cloning in Aspergillus was also constructed.

Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II

SummaryThe structure and expression of the polygalacturonase-encoding pgaII genes of two recently recognized species, Aspergillus niger and Aspergillus tubigensis, was investigated. While the structure of the pgaII genes is very similar, showing 83% DNA sequence identity and 94% identity at the amino acid level, they have diverged significantly. The NH2-terminal sequence suggests that these PGs are made as pre pro-proteins and the secretory propeptide of the PGII precursors shows sequence homology with some other fungal pro-peptides. The expression of the pgaII genes is strongly regulated by the carbon source and the A. tubigensis gene is expressed and regulated in A. niger transformants. The low similarity of the fungal PGs with those of bacterial and plant origin is discussed in relation to the possible functional role of specific amino acids.

Role of the cysteine protease cathepsin S in neuropathic hyperalgesia

Abstract Using a gene expression analysis approach we found that the mRNA encoding the lysosomal cysteine protease cathepsin S (CatS) was up‐regulated in rat dorsal root ganglia (DRG) following peripheral nerve injury. CatS protein was expressed in infiltrating macrophages in DRG and near the site of injury. At both sites CatS expression progressively increased from day 3 to day 14 after injury. In naïve rats, intraplantar injection of activated rat recombinant (rr) CatS (0.3, 1 μg/rat) induced a mechanical hyperalgesia that developed within half‐an‐hour, diminished by 3 h and was absent after 24 h. Activated rrCathepsin B (CatB) and non‐activated rrCatS injected intraplantarly at the same or higher doses than activated rrCatS had no effect on rat nociceptive thresholds. In nerve‐injured rats, mechanical hyperalgesia, but not allodynia, was significantly reversed for up to 3 h by systemic administration of a non‐brain penetrant, irreversible CatS inhibitor (LHVS, 3–30 mg/kg s.c.). Depletion of peripheral macrophages by intravenous injection of liposome encapsulate clodronate (1 ml, 5 mg/ml) partially reduced established mechanical hyperalgesia but not allodynia, and abolished the anti‐hyperalgesic effect of LHVS. Our results demonstrate a pro‐nociceptive effect of CatS and indicate that endogenous CatS released by peripheral macrophages contributes to the maintenance of neuropathic hyperalgesia following nerve injury.

Nitrogen, carbon, and pH regulation of extracellular acidic proteases of Aspergillus niger

Aspergillus niger secretes a number of enzymes, including proteases, into its culture fluid. The regulation of the two major acidic extracellular proteases, pepA and pepB, was investigated using Northern analyses. Our data suggest that the regulation of pepA and pepB expression occurs predominantly at the level of mRNA content and that, while they are regulated in a similar manner, differences are also clear in their expression. Both genes were found to be under complex regulatory control. The expression of the two genes could be turned off by the presence of good nitrogen or carbon sources in the media, and external protein sources did not induce expression of either gene under conditions of carbon and nitrogen repression. The pH of the medium also played a major role in their regulation as the expression of both genes was completely turned off under alkaline conditions, even when grown in media lacking good nitrogen and carbon sources but containing proteins. We isolated clones containing 5′ non-coding sequences of the pepA gene from a λ genomic library with a pepA specific probe. Analysis and comparison of the promoter sequences of the pepA and pepB genes revealed that both contain several putative AREA- and CREA-binding sites and they also share an 18-bp-long sequence which is 83% identical in these two genes.

A phosphate-repressible acid phosphatase gene from Aspergillus niger: its cloning, sequencing and transcriptional analysis

The cloning and sequencing of an Aspergillus niger gene encoding a secreted form of phosphate-repressible acid phosphatase by complementation of a pacA (phosphate-repressible acid phosphatase) mutant of Aspergillus nidulans is described. The gene contains two introns, 201 and 265 nt in length, and codes for a 1.6-kb transcript. Both phosphate concentration and pH of the growth medium affect the level of expression of the gene in A. niger. Similar regulation is observed in A. nidulans transformants. A putative signal peptide, resembling known signal sequences of yeast, is identified.

Cloning and characterisation ofpepC, a gene encoding a serine protease from Aspergillus niger

We have cloned a gene, pepC, encoding a serine proteinase, PEPC, from Aspergillus niger by screening a phage lambda genomic DNA library with a gene (PRB1) from Saccharomyces cerevisiae which codes for proteinase YscB. The nucleotide (nt) sequence of pepC revealed that the gene is composed of two exons of 369 nt and 1230 nt separated by a single 70-nt intron. The deduced protein of 533 amino acids (aa) has a putative signal sequence for transport into the endoplasmic reticulum. Based on the extensive homology shown with serine proteinases (SerP) of the subtilisin family, which includes the active site triad, we hypothesise that the protein is made as a larger precursor which is matured by the cleavage of 130-140 aa from its N terminus and possibly by the removal of approx. 70 aa from its C terminus.

Cloning of a new bidirectionally selectable marker for Aspergillus strains

Mutants that lack adenosine triphosphate sulfurylase (ATPsase; EC 2.7.7.4) are unable to use sulfate as sole source of sulfur and are also resistant to selenate. These mutants, denoted sC-, are readily obtained from any strain of Aspergillus niger or Aspergillus nidulans by the strong selection for selenate resistance. We have cloned the gene encoding ATPsase from A. nidulans by complementation of an sC mutant strain of A. nidulans with a gene library and show that plasmids containing this gene transform both A. niger and A. nidulans sC- strains, restoring their ability to grow on sulfate as sole sulfur source. The fact that strong selection for either sC+ or sC- can be applied provides a simple way of delivering genetically engineered constructs to any strain of A. niger including strains of industrial importance. In addition, this system is useful for gene replacements and other genomic DNA manipulations in Aspergillus species.

Cloning, characterization and expression of pepF, a gene encoding a serine carboxypeptidase from Aspergillus niger

We have cloned a gene (pepF) encoding a serine carboxypeptidase, proteinase F (PEPF), from Aspergillus niger. The sequences were identified in a phage lambda genomic DNA library using a synthetic probe based on the N-terminal sequence of PEPF. Nucleotide sequence data from pepF genomic and cDNA clones reveals that it is composed of four exons of 199, 283, 227 and 881 bp, interrupted by three introns of 53, 69 and 59 bp. The sequence of pepF codes for a polypeptide of 530 amino acids (aa), of which the first 52 aa are not present in the mature PEPF. This region may represent a prepro sequence that is removed by proteolytic cleavage as a monobasic cleavage site (Lys52). Northern blot analysis of total cellular RNA extracted from A. niger cells indicates that pepF is transcribed as a single 1.8-kb mRNA, which is regulated by nitrogen and carbon repression, specific induction and the pH of the culture medium.