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No description available.

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal and cardiac myocytes from undifferentiated stem cells. The present study aims to understand and compare the transcriptional regulation of skeletal and/or cardiac muscle development in the absence of Sox7 or in the presence of a collagen-based matrix in P19 embyonal carcinoma (EC) and mouse embryonic stem (ES) cells. First, knock-down of Sox7 , by shRNA, in muscle inducing conditions (+DMSO) and in the absence of RA (-RA), decreased muscle progenitor transcription factor and myogenic regulatory factor (MRF) levels, suggesting that Sox7 is necessary for myogenesis. However, knock-down of Sox7 in the presence of RA (+RA) and DMSO increased expression of muscle progenitor markers and MRFs, suggesting that Sox7 is inhibitory for myogenesis +RA. Furthermore, Sox7 overexpression enhanced myogenesis -RA, but inhibited myogenesis and enhanced neurogenesis +RA. These results suggest an important interplay between RA signalling and Sox7 function during P19 differentiation. Second, Q-PCR analysis showed that compared to the mouse ES cells differentiated on the regular TC plates, differentiation on the collagen matrices had a higher expression of skeletal and cardiac precursors, MRFs and terminal differentiation markers. Collagen alone enhanced myotube formation. The enhanced collagen matrix, containing the oligosaccharide sialyl LewisX (sLeX), specifically enhanced cardiomyogenesis. These studies have added to our understanding of the transcriptional regulation of premyogenic mesoderm factors and the role of Sox7 in this process. In addition these studies provide a vision for possible use of biomaterials in directed differentiation of stem cells for the purpose of cell therapy.

Triacylglycerols (TAG) play vital roles in plant development and are also utilized by humans for consumption, chemical and industrial feed stocks, and production of biofuels. Their primary function in plants is as an energy reserve stored in seeds to allow for the growth of the developing plant after germination. While many plants accumulate significant quantities of TAGs in the developing embryo or other parts of the seed, significant quantities of TAG accumulation also occurs in nonseed tissues such as the mesocarp of avocado and oil palm, the roots of nutsedge, and the fruits of bay berry. In Arabidopsis the transcription factor Wrinkled 1 (WRI1) was shown to be responsible for regulating the accumulation of TAG in seeds, and homologues of WRI1 have been shown to have similar functions in other plants and plant tissues. However, WRI1 likely does not act alone to control the synthesis and accumulation of TAG in plants. This project aims to identify additional transcription factors that are responsible for controlling the synthesis and accumulation of TAG in nonseed tissues by utilizing transcriptome data from a variety of plant species. The transcriptome data also revealed three homologues of WRI1 that are highly expressed in Avocado mesocarp. Their function in Avocado will be determined by complementing Arabidopsis wri1 mutants and examining TAG accumulation in the resulting transgenic plants. This study is aimed at identifying genes that play a role in the accumulation of high levels of TAG in various plant tissues and confirming the function of the WRI homologues in Avocado. Future studies will be able to use this information to better understand regulation of TAG accumulation in plants which will allow increased oil accumulation and yield in plants for various commercial and industrial applications.

All sequenced archaeal genomes encode a general transcription factor, TFE, which is highly conserved and homologous to the alpha subunit of the eukaryotic transcription factor TFIIE. TFE functions to increase promoter opening efficiency during transcription initiation, although the mechanism for this is unclear. The N-terminus of TFE contains a common DNA binding motif, a winged helix. At the tip of this winged helix is a highly conserved region of aromatic amino acids that is close to DNA during initiation. TFE activation can compensate for mutations in another transcription factor, TFB2, which is homologous to TFIIB. P. furiosus encodes two paralogs of the eukaryotic RNA polymerase II transcription factor TFIIB: TFB1 and TFB2. TFB2 lacks a portion of the highly conserved N-terminus, and functions in transcription complexes at a lower efficiency than TFB1. It has been demonstrated that the presence of TFE is able to assist in transcription with TFB2 in vitro bringing its efficiency to almost TFB1 levels. Thus, TFB2 provides a unique opportunity to evaluate the function of the TFE winged helix in transcription. In this study the aromatic patch of the TFE winged helix was mutated to test its role in activation of TFB1 and TFB2-containing transcription complexes, because this aromatic patch is required for full TFE activity especially when NTP concentrations are low.

The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-reader (also called the B-finger) motif. TFB2 functions, though poorly, in promoter-dependent transcription initiation. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening. Archaeal RNA polymerase requires two transcription factors for initiation: TBP, which binds to TATA boxes, and TFB, which binds TBP and DNA, recruits RNAP and helps initiate transcription. Archaeal TFBs usually contain a conserved B-reader sequence homologous to the eukaryotic B-reader motif in their N-terminal domains. This region is involved in the assembly of the transcription complex, promoter melting and in transcription start site determination but its position and orientation relative to promoter DNA during initiation is not clear. In this study the positioning of the TFB B-reader relative to DNA was determined by cross-linking using TFB variants substituted with photoactivatable unnatural amino acids. The results demonstrate that the B-reader is in close proximity to the transcription start site on the template but not the non-template strand in transcription initiation complexes. Furthermore, the position of the B-reader varies between closed and open promoter complexes, and between open promoter and early initiation complexes. Thus the archaeal B-reader sequence is poised to interact with promoter DNA in a dynamic fashion, and is likely playing a role in positioning the template-strand in an open pre-initiation complex.

The hyperthermophile archaeon, Pyrococcus furiosus encodes two eukaryotic TFIIB family proteins, TFB1 and TFB2. TFB1 is very similar to TFIIB in terms of sequence homology and function, whereas TFB2 is unusual as it is missing highly conserved sequences in its N-terminal domain that are present in TFIIB and TFB1. Despite this, TFB2 is effective in transcription process, albeit with lower efficiency compared to TFB1. Other archaea also contain multiple TFBs, but unlike Pyrococcus furiosus TFB2, these multiple TFBs have higher sequence homology to each other and have similar transcription efficiencies. Photochemical cross-linking experiments have shown that the B-reader of TFB in archaea and TFIIB in eukaryotes is close to transcription start site and is very important in RNAP recruitment to promoter DNA and transcription start site selection. Thus the lack of the highly conserved B reader region in P. furiosus TFB2 presents the opportunity to further study the functional importance of this region. In this study several amino acids in N-terminal domain of TFB2 were mutated with photoactivable unnatural amino acid p-benzoyl L- phenylalanine (pBpa) and the proximity of TFB2 relative to DNA was determined by photochemical cross-linking experiments. The results showed that TFB2 interacts with DNA near the TATA box via its C-terminal domain, and interacts with both strands of DNA near the transcription start site via its divergent B-reader and the B-linker sequences. The B-reader loop region is close to transcription start site and interacts with the transcribed strand of promoter DNA while the B-linker strand cross-links with the non-transcribed strand. Some of the amino acids in between the B-reader loop and the B-linker strand region in TFB2 are seen to cross-link both the transcribed and the non-transcribed strand. Thus, despite the absence of strong homology to conserved B-reader and B-linker sequences, TFB2 is likely to interact with DNA in the transcription bubble and facilitate in transcription initiation.

Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 4 (STAT4) is an important transcription factor activated by IL-12 signaling. Activated STAT4 is essential for Th1 cell differentiation, a process characterized by increased potential for interferon (IFN)-γ production. Defective IFN-γ production due to STAT4 deficiency occurs after autologous stem cell transplantation for lymphoma. We have investigated the mechanisms of post-transplant STAT4 deficiency. The tumor-bearing state is ruled out to be the cause because STAT4 levels were not significantly different in peripheral blood mononuclear cells (PBMCs) obtained from lymphoma patients prior to treatment and healthy control subjects. The magnitude of the decrease in STAT4 levels corresponded with increasing intensity of chemotherapeutic treatment in vivo. Furthermore, treatment of normal PBMC cultures or a natural killer (NK) cell line with chemotherapy drugs in vitro also resulted in reduced STAT4 protein and reduced IL-12-induced IFN-γ production. Chemotherapy drugs are shown to have no impact on the stability of STAT4 mRNA, while steady-state levels of STAT4 transcripts are decreased in lymphoma patients. Our findings demonstrated that chemotherapeutic drugs up-regulate the ubiquitination rates of the STAT4 protein, which in turn promotes its degradation via the proteasome-mediated pathway. Treatment with the proteasome inhibitor bortezomib largely reversed the chemotherapy-induced STAT4 deficiency. Thus, acquired STAT4 deficiency in lymphoma patients is a consequence of treatment with chemotherapy. These results have important implications for design of optimal immunotherapy for lymphoma.

No description available.

With the critical role of aberrantly active Signal Transducer and Activator of Transcription (Stat) 3 protein in many human cancers, selective small-molecule inhibitors targeting the dimerization event which is required for stat3 activation, would be valuable as therapeutic agents. And the inhibitors will be useful chemical probes to clarify the complex biological functions of Stat3. By computational and structural analyses of the interaction between Stat3 and the lead dimerization disruptor, S3I-201, we have designed a diverse set of analogs. One of the most active analogs, S3I-201.1066 is derived to contain a cyclo-hexyl benzyl moiety on the amide nitrogen, which increases the binding to the Stat3 SH2 domain. Evidence is presented from in vitro biochemical and biophysical studies that S3I-201.1066 directly interacts with Stat3 or the SH2 domain, with an affinity (K[subscript D]) of 2.74 [micrometer], and disrupts the binding of Stat3 to the cognate pTyr-peptide, GpYLPQTV-NH2, with an IC₅₀ of 23 [micrometer]. Moreover, S3I-201.1066 selectively blocks the association of Stat3 with the epidermal growth factor receptor (EGFR), and inhibits Stat3 tyrosine phosphorylation and nuclear translocation in EGF-stimulated mouse fibroblasts. In cancer cells that harbor aberrant Stat3 activity, S3I-201.1066 inhibits constitutive Stat3 DNA-binding and transcriptional activities. By contrast, S3I-201.1066 has no effect on Src activation or the EGFR-mediated activation of the Erk1/2MAPK pathway. S3I-201.1066 selectively suppresses the viability, survival, and malignant transformation of the human breast and pancreatic cancer lines and the v-Src-transformed mouse fibroblasts harboring persistently active Stat3. Treatment with S3I-201.1066 on malignant cells harboring aberrantly active Stat3 down regulated the expression of c-Myc, Bcl-xL, Survivin, matrix metalloproteinase 9, and VEGF, which are known Stat3-regulated genes important in diverse tumor processes. The in vivo administration of S3I-201.1066 induced significant anti-tumor response in mouse models of human breast cancer, which correlates with the inhibition of constitutively active Stat3 and the suppression of known Stat3-regulated genes. Further computer-aided lead optimization derives higher-affinity (K[subscript D], 504 nM), orally bioavailable Stat3 SH2 domain-binding ligand, BP-1-102 as a structural analog of S3I-201.1066. The most significant modification is the pentafluorobenzene sulfonamide component of BP-1-102, which permits accessibility of a third sub-pocket of the Stat3 SH2 domain surface. BP-1-102-mediated inhibition of aberrantly-active Stat3 in human pancreatic cancer, Panc-1, breast cancer, MDA-MB-231, and prostate (DU145) cancer cells and in the mouse transformed fibroblasts harboring aberrantly-active Stat3. It also disrupts Stat3-NF[kappa]B cross-talk and suppresses the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule-1, macrophage-migration-inhibitory factor/glycosylation-inhibiting factor, interleukin-1 receptor antagonist and the serine protease inhibitor (serpin) protein 1, and the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, and vascular endothelial growth factor expression in vitro and in vivo. Inhibition of tumor cell-associated constitutively-active Stat3 further suppresses focal adhesion kinase and paxillin induction, enhances E-cadherin expression, and down-regulates Kruüppel-like factor 8 expression. Consequently, BP-1-102 selectively suppresses anchorage-dependent and independent growth, survival, migration and invasion of Stat3-dependent tumor cells in vitro. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of mouse xenografts of human breast and lung tumors. Computer-aided lead optimization has therefore derived a more suitable small-molecule inhibitor as a drug candidate. Our studies of the Stat3 SH2 protein surface and of the interactions between lead agents and the SH2 domain provided significant data to facilitate the structural optimization. From S2I-201 to S3I-201.1066 and to BP-1-102, we note the substantial gain in potency and efficacy, and the pharmacokinetic improvements. The oral bioavailability of BP-1-102 represents a substantial advancement in the discovery of small-molecule Stat3 inhibitors as novel anticancer agents.