Beverly S. Chilton

Impaired DNA Damage Response, Genome Instability, and Tumorigenesis in SIRT1 Mutant Mice

In lower eukaryotes, Sir2 serves as a histone deacetylase and is implicated in chromatin silencing, longevity, and genome stability. Here we mutated the Sirt1 gene, a homolog of yeast Sir2, in mice to study its function. We show that a majority of SIRT1 null embryos die between E9.5 and E14.5, displaying altered histone modification, impaired DNA damage response, and reduced ability to repair DNA damage. We demonstrate that Sirt1(+/-);p53(+/-) mice develop tumors in multiple tissues, whereas activation of SIRT1 by resveratrol treatment reduces tumorigenesis. Finally, we show that many human cancers exhibit reduced levels of SIRT1 compared to normal controls. Thus, SIRT1 may act as a tumor suppressor through its role in DNA damage response and genome integrity.

Uteroglobin/Clara cell 10-kDa family of proteins: Nomenclature committee report

COMMITTEE CHAIR: J. KLUGa,b COMMITTEE MEMBERS: H. M. BEIER,c A. BERNARD,d B. S. CHILTON,e T. P. FLEMING, f R. I. LEHRER,g L. MIELE,h N. PATTABIRAMAN,i AND G. SINGHj bInstitut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität Marburg, D-35033 Marburg, Germany cDepartment of Anatomy and Reproductive Biology, RWTH University of Aachen, D-52057 Aachen, Germany dUnit of Industrial Toxicology and Occupational Medicine, Faculty of Medicine, Catholic University of Louvain, B-1200 Bruxelles, Belgium eDepartment of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA fDepartment of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA gDepartment of Medicine and Molecular Biology Institute, University of California (Los Angeles) School of Medicine, Los Angeles, California 90095-1690, USA hCardinal Bernardin Cancer Center and Department of Pathology, Loyola University Medical Center, Maywood, Illinois 60153, USA iAdvanced Biomedical Computing Center, SAIC-NCI/FCRDC, Frederick, Maryland 21702-1201, USA jDepartment of Pathology, University of Pittsburgh School of Medicine and VA Medical Center, Pittsburgh, Pennsylvania 15240, USA

Prolactin and growth hormone signaling.

Prolactin (PRL) and growth hormone (GH) act by way of their receptors as either hormones (systemically) or cytokines (locally). The Jak2/Stat5 pathway is the principal route by which PRL/GH activate target genes. The availability of knockout mice for each member of this signaling cascade has provided opportunities to understand their unique interactions. Jak2 is important in alternative signal transduction schema such as the MAP kinase and PI3K/Akt pathways. The putative Jak2/RUSH pathway is based on the fact that RUSH mediates the ability of PRL to augment progesterone-dependent gene transcription. New evidence shows that suppressors, regulators, and degraders control Jak2/Stat5. This review focuses on the most recent advances in the field of PRL/GH signal transduction.

Quantitative analysis of gene expression by ion-pair high-performance liquid chromatography

We have analyzed the utility of ion-pair reversed-phase HPLC for gene quantification by competitive reverse transcriptase polymerase chain reaction (RT-PCR). Competitive RT-PCR reactions employed various RNA competitors which shared high sequence similarity to the native transcripts for which they served as references. Competitive reactions resulted in the detection of two reaction products when reactions were analyzed by agarose gel electrophoresis, but three products when analyzed by HPLC. The third product was demonstrated to be a heteroduplex formed between mixed strands of native and competitor amplicons. Mathematical analysis of these competitive reactions indicated that identification and quantification of the heteroduplexes were essential to produce accurate gene quantification. PCR amplification efficiency was shown to be identical for native and competitor transcripts. However, RT efficiency differences were observed which may be sequence dependent. These differences were highly consistent across reactions for the same native and competitor inputs. Increasing the sequence similarity resulted in a competitor which had the same RT efficiency as the native transcript. Titration of various levels of competitor against native RNA resulted in the expected linear relationships which had slopes of unity. Quantitation could be performed with similar precision in single tube comparisons in which the initial abundance of the native transcript was calculated by knowledge of the final reaction product ratio and the initial competitor input level. The assay system is highly accurate, i.e. the measured level of gene expression reflected the actual copy number of the gene present in the sample. This was demonstrated by performing reactions in which known amounts of native transcript were quantified and the amount estimated by the assay was shown to be the same as the known amount added to the reaction. A similar approach has been devised for examining the relative levels of alternatively spliced isoforms. In this system, primers were selected to produce reaction products which served as their own internal competitors (by spanning the alternative splice site). Hormonal dependence of the ratio of abundance of two isoforms of the rabbit RUSH-1 gene was demonstrated.

An Sp1-NF-Y/progesterone receptor DNA binding-dependent mechanism regulates progesterone-induced transcriptional activation of the rabbit RUSH/SMARCA3 gene

Steroids regulate alternative splicing of rabbit RUSH/SMARCA3, an SWI/SNF-related transcription factor. Transactivation was evaluated in 2057 bp of genomic sequence. Truncation analysis identified a minimal 252-bp region with strong basal promoter activity in transient transfection assays. The size of the 5′-untranslated region (233 bp) and the transcription start site were determined by primer extension analysis. The transcription start site mapped to a consensus initiator (Inr) element in a TATA-less region with a downstream promoter element (+29). These elements were authenticated by mutation/deletion analysis. The Inr/downstream promoter element combination is conserved in the putative core promoter (-35/+35) of the human ortholog, suggesting that transcription initiation is similarly conserved. Two Sp1 sites that are also conserved in the putative promoter of human SMARCA3 and a RUSH binding site (-616/-611) that is unique to the rabbit promoter repress basal transcription. These sites were variously authenticated by gel shift and chromatin immunoprecipitation assays. Analysis of the proximal promoter showed the -162/+90 region was required for progesterone responsiveness in transient transfection assays. Subsequent mutation/deletion analysis revealed a progesterone receptor half-site mediated induction by progesterone. An overlapping Y-box (in the reverse ATTGG orientation) repressed basal transcription and progesterone-induced transcriptional activation in the presence of the Sp1 sites. The specificity of progesterone receptor and transcription factor NF-Y binding were authenticated by gel shift assays. Chromatin immunoprecipitation assays confirmed the Y-box effects were mediated in a DNA binding-dependent fashion. This represents a unique regulatory scenario in which ligand-dependent transactivation by the progesterone receptor is subject to Sp1/NF-Y repression.

Quantification of alternatively spliced RUSH mRNA isoforms by QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency.

Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and the ion-pair reverse-phase (IP-RP)-HPLC product purification and detection system were developed to facilitate the isolation and proportional quantification of alternatively spliced RUSH mRNAs. RUSH isoforms result from alternative splicing of a 57-bp exon and encode SNF/SWI-related proteins that bind to the uteroglobin promoter. QRT-PCR was performed using total RNA, and a pair of primers designed to flank the 57-bp exon. When more than one splice variant was expressed, IP-RP-HPLC identified the specific homoduplex products, as well as the heteroduplexes formed as a consequence of partial sequence complementarity between the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes in the relative levels of RUSH message isoforms. The preferential expression of the RUSH-1alpha isoform by all the tissues except estrous uterine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold difference in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endometrium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and versatile tools for the detection and quantitation of mRNA splice variants.

Quantification of Specific Nucleic Acids, Regulated RNA Processing, and Genomic Polymorphisms Using Reversed-Phase HPLC

The ability to make accurate quantitative measurements of specific nucleic acid molecules is becoming increasingly significant in biomedical science. One important clinical application is the quantification of viral nucleic acids in patients’ serum and tissues to assess viral burden for determination of disease course and efficacy of treatment. In this case, accuracy and precision of measurement are of primary importance, as decisions concerning selection of medication and dose may depend entirely on perceived change in viral load in an otherwise asymptomatic patient. A second utility in basic biomedical research is the quantification of specific mRNA molecules in functionally characterized cell types. These measurements are useful because they provide a prediction of protein abundance in cell samples in which proteins cannot be quantified directly. These studies often require microdissection to separate the tissues of interest from neighboring tissues. Again, accurate quantification is important for the effective comparison of the expression of multiple genes in multiple tissues and at different laboratories, often under conditions of physiological or pharmacological manipulation.

Helicase-like transcription factor (Hltf) regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac transcription networks, and collagen biogenesis.

HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum) brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05) - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf’s regulation of the G2/M transition (p=9.726E-15) of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2) and lysyl (Plod2) collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3) for collagen trimerization, and lysyl oxidase (Loxl2) for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps) was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and fragmented. Thus, silencing Hltf during heart organogenesis compromised DNA double-strand break repair, and caused aberrant collagen biogenesis altering the structural network that transmits cardiomyocyte force into muscle contraction.

The reproductive importance of P-type ATPases

P-type ATPases are integral membrane proteins that use the free energy of ATP hydrolysis to generate transmembrane electrochemical ion gradients to support a variety of cellular processes. They have eight signature motifs, eight or ten transmembrane domains, highly conserved phosphorylation and ATP-binding sites, and similar hydropathic profiles. This review summarizes recent insights in the relationship of P-type ATPases to successful reproduction, and the hormone dependence of some family members. Because protein topology is central to understanding the pump action of this family of enzymes, this review also describes the dramatic change in the primary structure of one family member that may mediate transcription in the uterus.

Prolactin Signals Through RUSH/SMARCA3 in the Absence of a Physical Association with Stat5a

Abstract Jak/Stat-mediated prolactin signal transduction culminates in the sequence-selective binding of Stat5a. However, in the absence of Stat-binding sites, a RUSH-binding element mediates the prolactin signal in the rabbit uteroglobin promoter. Speculation about the existence of a Jak/RUSH pathway prompted this series of experiments to examine potential interactions between RUSH and Stat5a. Profiles of Jak/Stat pathway-specific genes by RT-PCR showed that mRNA for Jak2 and Stat5a is expressed in the endometrium of estrous, progesterone-treated, and 5-day pseudopregnant rabbits. Interspecies microarrays showed that transcripts for Stat5a were present at equal concentrations in the endometrium regardless of hormone treatment. The absence of a physical interaction between RUSH and individual Stat proteins bound to enhancer sites was demonstrated with transcription factor interaction arrays. These studies confirm that transmission of the prolactin signal through RUSH occurs in the absence of a physical association with Stat5a. Although a strong physical interaction between RUSH and Egr-1 was identified with the same arrays, no Egr-1 consensus sites were found in the region of the uteroglobin promoter (−175/−80) that contains the authentic RUSH site. Because the major transducer molecules (Jak2, Stat5a) are activated by tyrosine phosphorylation, Western analysis of immunoprecipitated samples, and gel shift assays were used to show that tyrosine phosphorylation is required for RUSH-DNA binding. The precise role for Jak2 in this process remains undefined. By comparison, serine-threonine-specific protein phosphorylation had no effect on RUSH-DNA binding.