A.
SPECIFIC AIMS Our efforts to determine how the Ssu72 CTD phosphatase component of the CPF complex interacts with the transcription initiation machinery led to our identification of 'gene loops' that juxtapose promoter and terminator regions (Ansari and Hampsey, 2005; Singh and Hampsey, 2007). We detected loops at genes ranging in length from 1 kb to 7 kb, suggesting that looping might be a general phenomenon of RNAP II transcription. We propose that gene loops are dynamic chromatin structures that form in a transcription- dependent manner. Our working model is that gene loops promote transcription activation, perhaps by facilitating translocation of RNAP II from the terminator back to the promoter of the same gene, although we do not exclude other possibilities for how loops affect transcription. Herein, we propose to continue our studies of gene loops in yeast, focusing on the following three Specific Aims:
Specific Aim #1 : Define the physical characteristics of gene loops. To date, we have analyzed six genes for juxtaposition of promoter-terminator regions; all were chosen for analysis because of physical characteristics that made them amenable to study by 3C. The objectives of this Aim are to refine our physical analysis of looping and to identify genes that will facilitate physiological analysis of looping. It is not our intention to perform a global analysis of looping, but instead to identify loops at genes whose regulatory properties are well defined. The successful outcome of this Aim will provide us with target genes for assessing the structural requirements for looping under repressing and activating conditions (Aim #2) and the physiological consequences of looping (Aim #3).
Specific Aim #2 : Define the molecular determinants of gene looping. We will expand our analysis of factors that affect promoter-terminator interactions, asking if known components of the initiation and 3'-end processing machineries mediate looping. Our initial studies revealed that the transcription initiation factor TFIIB and the Ssu72 and Pta1 components of the CPF 3'-end processing complex are required for looping. In this Aim we will focus on (i) Sub1, a protein that affects initiation, but has also been reported to function as a RNAP II anti-terminator; (ii) the Gal4 and Pho4 transcriptional activators; and (iii) Rat1, a 5' -> 3' exonuclease involved in transcription termination. We will also screen additional initiation, 3'-end processing and termination factors for affects on looping, and propose to use our expertise in classical yeast genetics to search for other factors that affect promoter- terminator interactions.
Specific Aim #3 : Define the physiological significance of gene loops. We recently discovered that gene loops at GAL10 persist following a cycle of galactose activation and glucose repression and that persistence of looping is required rapid reactivation of GAL10 transcription.
In Aim #3, we propose to confirm and extend these results. In addition to analysis of GAL10, we will determine whether looping affects activation/reactivation of the PHO5 gene. Our hypothesis is that looping is required for high levels of gene transcription and that looping underlies 'memory' of recent transcriptional activity. The refinement of 3C technology offers a unique opportunity to determine how DNA topology regulates gene expression. The successful outcome of the experiments described in this proposal will provide novel insight into transcriptional regulatory mechanisms. Moreover conclusion from our studies with yeast can be extrapolated to better understand mammalian gene expression as several recent studies have identified comparable gene loops in human cells, including gene loops at the BRCA1 tumor suppressor gene and between the 5'LTR promoter and 3'LTR poly(A) signal of HIV, the causative agent of AIDS.

Public Health Relevance

Several recent studies have revealed that DNA forms loops that juxtapose the ends of the gene. As examples; the human BRCA1 tumor suppressor gene and the HIV virus that causes AIDS were reported to form gene loops during their expression. We recently discovered that genes in yeast form similar DNA loops. We propose to use a powerful combination of molecular; genetic and biochemical methods to study gene loops in yeast to provide insight into gene expression mechanisms that go awry in cancers and other human diseases. Project NarrativeGM39484M. HampseySeveral recent studies have revealed that DNA forms loops that juxtapose the endsof the gene. As examples; the human BRCA1 tumor suppressor gene and the HIVvirus that causes AIDS were reported to form gene loops during their expression.We recently discovered that genes in yeast form similar DNA loops. We propose touse a powerful combination of molecular; genetic and biochemical methods to studygene loops in yeast to provide insight into gene expression mechanisms that goawry in cancers and other human diseases. __SpecificAimsTextDelimiter__ A. SPECIFIC AIMSOur efforts to determine how the Ssu72 CTD phosphatase component of the CPF complexinteracts with the transcription initiation machinery led to our identification of ''gene loops''that juxtapose promoter and terminator regions (Ansari and Hampsey; 2005; Singh andHampsey; 2007). We detected loops at genes ranging in length from 1 kb to 7 kb;suggesting that looping might be a general phenomenon of RNAP II transcription. Wepropose that gene loops are dynamic chromatin structures that form in a transcription-dependent manner. Our working model is that gene loops promote transcription activation;perhaps by facilitating translocation of RNAP II from the terminator back to the promoter ofthe same gene; although we do not exclude other possibilities for how loops affecttranscription. Herein; we propose to continue our studies of gene loops in yeast; focusingon the following three Specific Aims:Specific Aim #1: Define the physical characteristics of gene loops. To date; we haveanalyzed six genes for juxtaposition of promoter-terminator regions; all were chosen foranalysis because of physical characteristics that made them amenable to study by 3C. Theobjectives of this Aim are to refine our physical analysis of looping and to identify genes thatwill facilitate physiological analysis of looping. It is not our intention to perform a globalanalysis of looping; but instead to identify loops at genes whose regulatory properties arewell defined. The successful outcome of this Aim will provide us with target genes forassessing the structural requirements for looping under repressing and activating conditions(Aim #2) and the physiological consequences of looping (Aim #3).Specific Aim #2: Define the molecular determinants of gene looping. We will expandour analysis of factors that affect promoter-terminator interactions; asking if knowncomponents of the initiation and 3'-end processing machineries mediate looping. Our initialstudies revealed that the transcription initiation factor TFIIB and the Ssu72 and Pta1components of the CPF 3'-end processing complex are required for looping. In this Aim wewill focus on (i) Sub1; a protein that affects initiation; but has also been reported to functionas a RNAP II anti-terminator; (ii) the Gal4 and Pho4 transcriptional activators; and (iii) Rat1;a 5' -> 3' exonuclease involved in transcription termination. We will also screen additionalinitiation; 3'-end processing and termination factors for affects on looping; and propose touse our expertise in classical yeast genetics to search for other factors that affect promoter-terminator interactions.Specific Aim #3: Define the physiological significance of gene loops. We recentlydiscovered that gene loops at GAL10 persist following a cycle of galactose activation andglucose repression and that persistence of looping is required rapid reactivation of GAL10transcription. In Aim #3; we propose to confirm and extend these results. In addition toanalysis of GAL10; we will determine whether looping affects activation/reactivation of thePHO5 gene. Our hypothesis is that looping is required for high levels of gene transcriptionand that looping underlies ''memory'' of recent transcriptional activity.The refinement of 3C technology offers a unique opportunity to determine how DNAtopology regulates gene expression. The successful outcome of the experiments describedin this proposal will provide novel insight into transcriptional regulatory mechanisms.Moreover conclusion from our studies with yeast can be extrapolated to better understandmammalian gene expression as several recent studies have identified comparable gene loopsin human cells; including gene loops at the BRCA1 tumor suppressor gene and between the5'LTR promoter and 3'LTR poly(A) signal of HIV; the causative agent of AIDS.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM039484-19
Application #
8710992
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Sledjeski, Darren D
Project Start
1988-08-01
Project End
2014-03-31
Budget Start
2013-07-01
Budget End
2014-03-31
Support Year
19
Fiscal Year
2012
Total Cost
$324,903
Indirect Cost
$116,632
Name
Rbhs-Robert Wood Johnson Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
078795875
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Lamas-Maceiras, Mónica; Singh, Badri Nath; Hampsey, Michael et al. (2016) Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast. J Biol Chem 291:8960-8
Olayanju, Bola; Hampsey, James Jensen; Hampsey, Michael (2015) Genetic analysis of the Warburg effect in yeast. Adv Biol Regul 57:185-92
Singh, Badri Nath; Hampsey, Michael (2014) Detection of short-range chromatin interactions by chromosome conformation capture (3C) in yeast. Methods Mol Biol 1205:209-18
Rosado-Lugo, Jesús D; Hampsey, Michael (2014) The Ssu72 phosphatase mediates the RNA polymerase II initiation-elongation transition. J Biol Chem 289:33916-26
Yadon, Adam N; Singh, Badri Nath; Hampsey, Michael et al. (2013) DNA looping facilitates targeting of a chromatin remodeling enzyme. Mol Cell 50:93-103
Hampsey, Michael (2012) Molecular biology. A new direction for gene loops. Science 338:624-5
Goel, Shivani; Krishnamurthy, Shankarling; Hampsey, Michael (2012) Mechanism of start site selection by RNA polymerase II: interplay between TFIIB and Ssl2/XPB helicase subunit of TFIIH. J Biol Chem 287:557-67
Hampsey, Michael; Singh, Badri Nath; Ansari, Athar et al. (2011) Control of eukaryotic gene expression: gene loops and transcriptional memory. Adv Enzyme Regul 51:118-25
Seibold, Steve A; Singh, Badri Nath; Zhang, Chunfen et al. (2010) Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase. Biochim Biophys Acta 1799:575-87
Singh, Badri Nath; Ansari, Athar; Hampsey, Michael (2009) Detection of gene loops by 3C in yeast. Methods 48:361-7

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