Методы изучения регуляторных районов генов презентация

I

Concepts, Strategies, and Techniques. 2000, Cold Spring Harbor Laboratory Press

either a single major transcription start site or several start sites within a narrow region of several nucleotides. Focused transcription is the predominant mode of transcription in simpler organisms. In dispersed transcription, there are several weak transcription start sites over a broad region of about 50 to 100 nucleotides. Dispersed transcription is the most common mode of transcription in vertebrates. For instance, dispersed transcription is observed in about twothirds of human genes. In vertebrates, focused transcription tends to be associated with regulated promoters, whereas dispersed transcription is typically observed in constitutive promoters in CpG islands.

варианты репортерных конструкций в векторе pGL4.10[luc2], слева обозначены нуклеотидные позиции относительно точки старта транскрипции гена Xist. На диаграмме отражена относительная люциферазная активность конструкций в перевиваемой культуре фибробластов линии Sd10. Стрелкой обозначена точка старта транскрипции Xist, черными точками – метилированные конструкции, О.Е. – относительные единицы активности люциферазы. Достоверность различий: *** - P≥0.999, ** - P≥0.99, * - P≥0.95. CNS1 – conservative noncoding sequence.

colocalization of orphan CGIs with sites of transcriptional initiation taken from Illingworth et al. (2010). CXXC (Cfp1) affinity purification identifies the locations of CGIs that overlap with H3K4me3, RNAPII, GRO-seq (Core et al. 2008), and CAGE tags (Faulkner et al. 2009). Genes (RefSeq) are annotated below the sequencing profiles, with those mapped to the positive and negative strand displayed above and below the chromosome line, respectively. Orphan CGIs are denoted by asterisks.

splice acceptor site in exon 2. Promoters 1B and 1C are GC rich and are present in previously published sequences. The hGR 1A promoter and exon are novel, and they are located approximately 27 kbp upstream of the start site for transcription for exon 1C.

Diagram of the hGR Gene Structure
and Organization

GR (C) genes.

Each gene contains a tandem array of multiple first exons in the variable region, each of which is separately spliced to a common set of downstream constant exons.The approximate length of each gene is shown below the corresponding panels.

Theresa Zhang, Peter Haws and Qiang Wu. Multiple Variable First Exons: A Mechanism for Cell- andTissue-Specific Gene Regulation. 2004 14: 79-89; Genome Res.

Множественные экзоны 1

one first exon.
The numbers are shown above each histogram.The inset shows an enlargement of the distribution of genes with more than three first exons.

Theresa Zhang, Peter Haws and Qiang Wu. Multiple Variable First Exons: A Mechanism for Cell- andTissue-Specific Gene Regulation. 2004 14: 79-89; Genome Res.

Множественные экзоны 1

A T C

Extended
primer

87 nt

Free excess
primer

Синтез меченых на 5’ конце праймеров
при помощи γ- 32P и Т4 полинуклиотидкиназы

Гибридизация меченого праймера со
специфической мРНК

5’

3’

Удлинение праймера до 5’ конца мРНК
c помощью обратной транскриптазы и dNTP

Анализ полученной меченой ДНК
На секвенирующем геле

Размер меченого продукта соответствует
расстоянию 5’ конца праймера
до старта транскрипции

Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques. 2000, Cold Spring Harbor Laboratory Press

Spring Harbor Laboratory Press

Hybridize primer 100–200 nucleotides from 5′ end of mRNA.

Extend primer to 5′ end of mRNA using reverse transcriptase
and dNTPs.

Ligate oligo of known sequence to 3′ end of cDNA using RNA
ligase. Alternatively, extend cDNA using terminal transferase
(TdT) and dGTP.

Perform PCR using primer complementary to ligated oligo and a downstream primer that is slightly internal to the primer used for
cDNA synthesis above.

(Optional)
Sequence PCR products

Sequence
Individual clones

Determine size on
sequencing gel (if
radiolabeled primer
used for PCR).

Insert PCR
product into
vector

Spring Harbor Laboratory Press

Plasmid includes region of gene containing
putativetranscription start site and 59 nucleotides
downstreamof this start site, fused to the SP6
promoter and flanked by convenient restriction sites.

Cut with HindIII to linearize plasmid.

Add transcription buffer, SP6 RNA polymerase, NTPs plus [α -32P]UTP (asterisks) to generate antisense probe.

Hybridize probe to isolated mRNA.

Digest with RNase T1 and RNase A
(cleaves single-stranded RNAs).

Creates a 59-nucleotide RNA-RNA hybrid.

Denature and analyze by denaturing gel electrophoresis.

отдаленного
регуляторного района

отдаленный
регуляторный район

Трансфекция клеток
репортерной плазмидой

Инкубация в течении 24-72 часов
транскрипция для эписомных плазмид и
синтез белка

Измерение
активности
фермента
репортерного гена

Измерение уровня
репортерной мРНК

Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques. 2000, Cold Spring Harbor Laboratory Press

Методы исследования регуляторных районов

S. Collins, Lukas Wagner, Carolyn M. Shenmen, Gregory D. Schuler, Stephen F. Altschul, Barry Zeeberg, Kenneth H. Buetow, Carl F. Schaefer, Narayan K. Bhat, Ralph F. Hopkins, Heather Jordan, Troy Moore, Steve I. Max, Jun Wang, Florence Hsieh, Luda Diatchenko, Kate Marusina, Andrew A. Farmer, Gerald M. Rubin, Ling Hong, Mark Stapleton, M. Bento Soares, Maria F. Bonaldo, Tom L. Casavant, Todd E. Scheetz, Michael J. Brownstein, Ted B. Usdin, Shiraki Toshiyuki, Piero Carninci, Christa Prange, Sam S. Raha, Naomi A. Loquellano, Garrick J. Peters, Rick D. Abramson, Sara J. Mullahy, Stephanie A. Bosak, Paul J. McEwan, Kevin J. McKernan, Joel A. Malek, Preethi H. Gunaratne, Stephen Richards, Kim C. Worley, Sarah Hale, Angela M. Garcia, Laura J. Gay, Stephen W. Hulyk, Debbie K. Villalon, Donna M. Muzny, Erica J. Sodergren, Xiuhua Lu, Richard A. Gibbs, Jessica Fahey, Erin Helton, Mark Ketteman, Anuradha Madan, Stephanie Rodrigues, Amy Sanchez, Michelle Whiting, Anup Madan, Alice C. Young, Yuriy Shevchenko, Gerard G. Bouffard, Robert W. Blakesley, Jeffrey W. Touchman, Eric D. Green, Mark C. Dickson, Alex C. Rodriguez, Jane Grimwood, Jeremy Schmutz, Richard M. Myers, Yaron S. N. Butterfield, Martin I. Krzywinski, Ursula Skalska, Duane E. Smailus, Angelique Schnerch, Jacqueline E. Schein, Steven J. M. Jones, and Marco A. Marra
Contributed by Francis S. Collins, et al.

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences
Mammalian Gene Collection (MGC) Program Team*, 2000.

organisms.
Dreos R., Ambrosini G., Groux R., Cavin Périer R., Bucher P.
Nucleic Acids Res. 2017 Jan 4;45(D1):D51-D55. doi: 10.1093/nar/gkw1069. Epub 2016 Nov 28.

YSPTSPS

J. Good, M. S. Guyer, S. Kamholz, L. Liefer,
K. Wetterstrand, F. S. Collins).
Initial ENCODE Pilot Phase Participants:
Affymetrix, Inc. (T. R. Gingeras, D. Kampa, E. A. Sekinger,
J. Cheng, H. Hirsch, S. Ghosh, Z. Zhu, S. Patel, A. Piccolboni, A. Yang, H. Tammana, S. Bekiranov, P. Kapranov, R. Harrison, G. Church, K. Struhl); Ludwig Institute for Cancer Research (B. Ren, T. H. Kim, L. O. Barrera, C. Qu, S. Van Calcar, R. Luna, C. K. Glass, M. G. Rosenfeld); Municipal Institute of Medical Research (R. Guigo, S. E. Antonarakis, E. Birney, M. Brent, L. Pachter, A. Reymond, E. T. Dermitzakis, C. Dewey, D. Keefe, F. Denoeud, J. Lagarde, J. Ashurst, T. Hubbard, J. J. Wesselink, R. Castelo, E. Eyras); Stanford University (R. M. Myers, A. Sidow, S. Batzoglou, N. D. Trinklein, S. J. Hartman, S. F. Aldred, E. Anton, D. I. Schroeder, S. S. Marticke, L. Nguyen, J.Schmutz, J.Grimwood,M.Dickson, G. M. Cooper, E. A. Stone, G. Asimenos, M. Brudno); University of Virginia (A. Dutta, N. Karnani, C. M. Taylor, H. K. Kim, G. Robins); University of Washington (G. Stamatoyannopoulos, J. A. Stamatoyannopoulos, M. Dorschner, P. Sabo, M. Hawrylycz, R. Humbert, J. Wallace, M. Yu, P. A. Navas, M. McArthur, W. S. Noble); Wellcome Trust Sanger Institute (I. Dunham, C. M. Koch, R.M.Andrews,G. K.Clelland, S. Wilcox, J. C. Fowler, K.D.
James, P. Groth, O. M. Dovey, P. D. Ellis, V. L. Wraight, A. J. Mungall, P. Dhami, H. Fiegler, C. F. Langford, N. P. Carter,
D. Vetrie); Yale University (M. Snyder, G. Euskirchen, A. E.
Urban, U. Nagalakshmi, J. Rinn, G. Popescu, P. Bertone, S.

Hartman, J. Rozowsky, O. Emanuelsson, T. Royce, S. Chung, M. Gerstein, Z. Lian, J. Lian, Y. Nakayama, S. Weissman, V. Stolc, W. Tongprasit, H. Sethi).
Additional ENCODE Pilot Phase Participants:
British Columbia Cancer Agency Genome Sciences Centre (S. Jones, M. Marra, H. Shin, J. Schein); Broad Institute (M. Clamp, K. Lindblad-Toh, J. Chang, D. B. Jaffe, M. Kamal, E. S. Lander, T. S. Mikkelsen, J. Vinson, M. C. Zody); Children’s Hospital Oakland Research Institute
(P. J. de Jong, K. Osoegawa,M.Nefedov, B. Zhu); National Human Genome Research Institute/Computational Genomics Unit (A. D. Baxevanis, T. G. Wolfsberg); National Human Genome Research Institute/Molecular Genetics Section (F. S. Collins, G. E. Crawford, J. Whittle, I. E. Holt, T. J. Vasicek, D. Zhou, S. Luo); NIH Intramural
Sequencing Center/National Human Genome Research Institute (E. D. Green, G. G. Bouffard, E. H. Margulies, M. E. Portnoy, N. F. Hansen, P. J. Thomas, J. C. McDowell, B. Maskeri, A. C. Young, J. R. Idol, R. W. Blakesley); National Library of Medicine (G. Schuler); Pennsylvania State University (W. Miller, R. Hardison, L. Elnitski, P. Shah); The Institute for Genomic Research (S. L. Salzberg, M. Pertea, W. H. Majoros); University of California, Santa Cruz (D. Haussler, D. Thomas, K. R. Rosenbloom, H. Clawson, A. Siepel, W. J. Kent).
ENCODE Technology Development Phase Participants:
Boston University (Z. Weng, S. Jin, A. Halees, H. Burden, U. Karaoz, Y. Fu, Y. Yu, C. Ding, C. R. Cantor); Massachusetts General Hospital (R. E. Kingston, J. Dennis); NimbleGen Systems, Inc. (R. D. Green,M. A. Singer, T. A. Richmond, J. E. Norton, P. J Farnham, M. J. Oberley, D. R. Inman); NimbleGen Systems, Inc. (M. R. McCormick, H. Kim, C. L. Middle, M. C. Pirrung); University of California, et al!

The ENCODE Project Consortium. 2004. The ENCODE (ENCyclopedia Of DNA Elements) Project. Science 306: 636–640.

The ENCODE Project Consortium

Project. Science 306: 636–640.

Functional genomic elements being identified by the ENCODE pilot phase.
The indicated methods are being used to identify different types of
functional elements in the human genome

отдаленного
регуляторного района

отдаленный
регуляторный район

Трансфекция клеток
репортерной плазмидой

Инкубация в течении 24-72 часов
транскрипция для эписомных плазмид и
синтез белка

Измерение
активности
фермента
репортерного гена

Измерение уровня
репортерной мРНК

Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques. 2000, Cold Spring Harbor Laboratory Press

Методы исследования регуляторных районов

clustering of promoter activity among 16 diverse cell lines.
Each row indicates the promoter activity of a fragment in each of the cell lines, with red indicating the degree of activity and black indicating no activity.
Promoter activity has been normalized and log transformed to reflect comparable values between cell lines.
Area A represents a cluster of promoter fragments with strong, ubiquitous activity in all cell lines and area B represents a cluster of promoter fragments that exhibit variable function across the 16 cell types.

Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome


Sara J. Cooper, Nathan D. Trinklein, Elizabeth D. Anton, Loan Nguyen and Richard M. Myers Genome Res., 2006 16: 1-10;

Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome/ Sara J. Cooper, Nathan D. Trinklein, et al. Genome Res., 2006 16: 1-10