The production of IL-10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4(+)CD25(+) naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.     

Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses.

The sequence of 363 nucleotides near the 3' end of the pol gene and 564 nucleotides from the 5' terminus of the env gene in an endogenous murine leukemia viral (MuLV) DNA segment, cloned from AKR/J mouse DNA and designated as A-12, was obtained. For comparison, the nucleotide sequence in an analogous portion of AKR mink cell focus-forming (MCF) 247 MuLV provirus was also determined. Sequence features unique to MCF247 MuLV DNA in the 3' pol and 5' env regions were identified by comparison with nucleotide sequences in analogous regions of NFS -Th-1 xenotropic and AKR ecotropic MuLV proviruses. These included (i) an insertion of 12 base pairs encoding four amino acids located 60 base pairs from the 3' terminus of the pol gene and immediately preceding the env gene, (ii) the deletion of 12 base pairs (encoding four amino acids) and the insertion of 3 base pairs (encoding one amino acid) in the 5' portion of the env gene, and (iii) single base substitutions resulting in 2 MCF247 -specific amino acids in the 3' pol and 23 in the 5' env regions. Nucleotide sequence comparison involving the 3' pol and 5' env regions of AKR MCF247 , NFS xenotropic, and AKR ecotropic MuLV proviruses with the cloned endogenous MuLV DNA indicated that MCF247 proviral DNA sequences were conserved in the cloned endogenous MuLV proviral segment. In fact, total nucleotide sequence identity existed between the endogenous MuLV DNA and the MCF247 MuLV provirus in the 3' portion of the pol gene. In the 5' env region, only 4 of 564 nucleotides were different, resulting in three amino acid changes between AKR MCF247 MuLV DNA and the endogenous MuLV DNA present in clone A-12. In addition, nucleotide sequence comparison indicated that Moloney-and Friend-MCF MuLVs were also highly related in the 3' pol and 5' env regions to the cloned endogenous MuLV DNA. These results establish the role of endogenous MuLV DNA segments in generation of recombinant MCF viruses.     

Upregulation of human angiotensinogen (AGT) gene transcription by interferon-gamma: involvement of the STAT1-binding motif in the AGT promoter

Mechanisms to maintain blood pressure in the face of infection are critical to survival. The angiotensinogen (AGT) gene locus is an important component of this response. Thus the AGT gene, expressed predominantly by liver cells, is known to be a positive acute phase reactant. We have previously demonstrated activation of the AGT promoter in hepatocytes through the IL6/STAT3 signaling mechanism. We have now investigated whether IFN-gamma, a cytokine also induced in response to diverse infections, can regulate AGT gene expression, and have elucidated the molecular mechanism involved. IFN gamma treatment up-regulated AGT mRNA level and promoter activity in Hep3B hepatocytes. Sequential deletion of the promoter from the 5' side suggested the major IFN gamma responsive DNA element to be between -303 and -103. This region contained a candidate STAT1-binding site between -271 and -279. EMSA and chromatin immuno-precipitation (ChIP) assays confirmed that IFN-gamma treatment induced the binding of STAT1 to this element. Reporter constructs containing this AGT promoter derived element in a multimerized context but not a mutant version were responsive to IFN gamma. Moreover mutating this STAT1 element in the context of the wild-type AGT holo promoter reduced responsiveness to IFN gamma. In contrast to the clear synergism between dexamethasone and IL 6 in the upregulation of the AGT promoter (through interaction between GR and STAT3), the combination of IFN gamma with IL 6 or with dexamethasone did not further increase AGT promoter activity suggesting that the IFN gamma/STAT1 pathway represents a separate signaling mechanism. These data highlight the redundancy in cytokine-mediated host response pathways aimed at the maintenance of blood pressure during infection.     

Autocrine IL-6/STAT3 signaling aids development of acquired drug resistance in Group 3 medulloblastoma

Medulloblastoma (MB) is a high-grade pediatric brain malignancy that originates from neuronal precursors located in the posterior cranial fossa. In this study, we evaluated the role of STAT3 and IL-6 in a tumor microenvironment mediated drug resistance in human MBs. We established that the Group 3 MB cell line, Med8A, is chemosensitive (hence Med8A-S), and this is correlated with a basal low phosphorylated state of STAT3, while treatment with IL-6 induced robust increases in pY705-STAT3. Via incremental selection with vincristine, we derived the stably chemoresistant variant, Med8A-R, that exhibited multi-drug resistance, enhanced IL-6 induced pY705-STAT3 levels, and increased IL6R expression. Consequently, abrogation of STAT3 or IL6R expression in Med8A-R led to restored chemosensitivity to vincristine, highlighting a prominent role for canonical IL-6/STAT3 signaling in acquired drug resistance. Furthermore, Med8A-S subjected to conditioning exposure with IL-6, termed Med8A-IL6+ cells, exhibited enhanced vincristine resistance, increased expression of pY705-STAT3 and IL6R, and increased secretion of IL-6. When cocultured with Med8A-IL6+ cells, Med8A-S cells exhibited increased pY705-STAT3 and increased IL-6 secretion, suggesting a cytokine feedback loop responsible for amplifying STAT3 activity. Similar IL-6 induced phenomena were also observed in the Group 3 MB cell lines, D283 and D341, including increased pY705-STAT3, drug resistance, IL-6 secretion and IL6R expression. Our study unveiled autocrine IL-6 as a promoter of STAT3 signaling in development of drug resistance, and suggests therapeutic benefits for targeting the IL-6/STAT3 signaling axis in Group 3 MBs.Subject terms: Cancer microenvironment, CNS cancer, Paediatric cancer     

The Human Cystathionine β-Synthase (CBS) Gene: Complete Sequence, Alternative Splicing, and Polymorphisms

Cystathionine β-synthase [CBS; -serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains 5 kb of the 5′ flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5′ UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3′ UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number ofAlurepeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians.     

The nucleotide sequence of the chick cytoplasmic beta-actin gene

The nucleotide sequence of the chick beta-actin gene was determined. The gene contains 5 introns; 4 interrupt the translated region at codons 41/42, 120/122, 267, 327/328 and a large intron occurs in the 5' untranslated region. The gene has a 97 nucleotide 5'-untranslated region and a 594 nucleotide 3'-untranslated region. A slight heterogeneity in the position of the poly A addition site exists; polyadenylation can occur at either of two positions two nucleotides apart. The gene codes for an mRNA of 1814 or 1816 nucleotides, excluding the poly(A) tail. In contrast to the chick skeletal muscle actin gene the beta-actin gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. In the 5' flanking DNA, 15 nucleotides downstream from the CCAAT sequence, is a tract of 25 nucleotides that is highly homologous to the sequence found in the same region of the rat beta-actin gene.     

Excision in vitro of the DNA bound carcinogen, 4-nitroquinoline 1-oxide.

It has been shown that 4-hydroxyaminoquinoline 1-oxide, the proximate form of the carcinogen 4-nitroquinoline 1-oxide, binds covalently to the purine bases of DNA. Here we report that carcinogen-bound nucleotides can be excised from DNA by a 5' leads to 3' exonuclease associated with DNA polymerase I of E. coli in the forms of either mononucleotides or oligonucleotides. Beef spleen phosphodiesterase II (5' leads to 3') also split carcinogen-bound nucleotides, while a 3' leads to 5' exonuclease of DNA polymerase I and E. coli exonuclease III (3' leads to 5') could not excise the modified nucleotide.