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Reconstitution of Pituitary Specific Transcription

垂体特异性转录的重建

基本信息

批准号：
7991550
负责人：
ARTHUR GUTIERREZ-HARTMANN
金额：
$ 3.51万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-03 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7991550
关键词：
Activins Address Alternative Splicing Amino Acids Binding Biochemical Biological Cell Line Cell Ontogeny Cell Proliferation Cells Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases DNA DNA Binding DNA Sequence Data Development Dwarfism Event Family Funding Gene Expression Gene Targeting Genes Genetic Transcription Goals Growth Hela Cells Histone Acetylation Histone Deacetylase Human Maps Mediating Modeling Molecular Molecular Biology Mus Nude Mice PRL gene Phenotype Pituitary Gland Protein Isoforms Proteins RNA Interference RNA Splicing Recruitment Activity Research Personnel Role Set protein Site Specificity Structure Testing Transcriptional Activation Transgenic Mice Transgenic Organisms cell type homeodomain in vivo insight lactotroph mutant novel programs promoter reconstitution response transcription factor transcription factor Pit-1 transgene expression tumor growth

项目摘要

The broad mandate of this proposal is to use transgenic, molecular and biochemical approaches to determine the in\ vivo biological effects specific to the Pit-IB isoform with regards to Pit-1-dependent pituitary cell ontogeny and gene expression, and to define the mechanism that mediates B-isoform-specific transcriptional responses in pituitary cells. The POU-homeodomain transcription factor, Pit-1, controls the development of somatotroph, lactotroph and thyrotroph pituitary cell-types, and regulates the cell-specific expression of GH, PRL and TSHB genes. The single Pit-1 gene is expressed in the Pit-1 lineage as two alternatively-spliced mRNAs, resulting in Pit-1 and Pit-IB proteins. Pit-IB contains a unique 26 amino-acid (AA) S-domain inserted at AA 48 of Pit-1, precisely in the middle of the transcription activation domain (TAD). However, Pit-1and Pit-IB share identical structures otherwise. Naturally occurring Pit-1mutations in mice and humans resulting in heritable dwarfism map to regions common to both Pit-1 and Pit-IB. However, the precise biological contributions of Pit-IB to pituitary cell-specific ontogeny and gene expression remain unknown. AlthoughPit- 1B has been overlooked after its initial description, during the previous funding period we made important, novel and unique contributions to our understanding of the precise molecular mechanisms by which the Pit-1 B-domain functions as a transcription switch motif resulting in B-isoform-specific transcriptional responses. We found that Pit-IB consistently inhibits PRL, GH and TSHB promoter activities in all pituitary cell lines tested, whereas Pit-IB consistently activates these same promoters in all nonpituitary cell lines tested. Moreover, Pit-IB inhibited basal, cAMP- and Ras- stimulated rPRL promoter activity in GH4 pituitary cells. We elucidated mechanism, demonstrating that the B-domain functions as a pituitary-specific represser motif acting via five hydrophobic amino acids that recruit HDAC activity to alter the histone acetylation state of the proximal rPRL promoter. Importantly, the B-domain can function as an autonomous, modular, active and HDAC-dependent represser motif when fused to the Gal4 DBD. Finally, adenoviral encoded HA-Pit-1B inhibits endogenous PRL and cyclinDI, but activates RB gene expression; and inhibits GH4 cell proliferation and tumor growth in nude mice, providing evidence of its biological effects. Thus, the Pit-1/Pit-IB pair provides a prototypical model to study transcription factor isoform-specific functions. We hypothesize that a pituitary- restricted represser complex that associates with the B-domain dictates Pit-IB isoform-specific transcriptional responses. A corollary hypothesis is that pituitary cells expressing increased Pit-IB- will have a distinct phenotype. To address this hypothesis, we propose four Specific Aims: (1) To determine whether HA-Pit-1B- transgene expression governs the ontogeny and/or expansion of the Pit-1 lineage in transgenic mice. (2) To determine the biological responses specifically induced by Pit-IB in GH4 cells. (3) To use RNAi to determine the biological role of Pit-IB and defined co-repressors in mediating B-specific responses. (4) To identify and functionally validate the represser complex associated with the B-domain. Insights gained from these studies will not only provide a better understanding of Pit-IB isoform-specific functions, but will also provide a conceptual and experimental framework to study other highly-related transcription factors that bind to overlapping DMA sites.

该提案的广泛授权是使用转基因，分子和生物化学方法来确定基因

项目成果

期刊论文数量（31）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type.

DOI：
10.1210/mend.6.12.1337142
发表时间：
1992-12
期刊：
Molecular endocrinology
影响因子：
0
作者：
C. Keech;S. M. Jackson;S. Siddiqui;K. Ocran;A. Gutierrez-Hartmann
通讯作者：
C. Keech;S. M. Jackson;S. Siddiqui;K. Ocran;A. Gutierrez-Hartmann

Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell-free extracts.

GH3 垂体无细胞提取物对大鼠催乳素基因的选择性转录和 DNase I 保护。

DOI：
10.1073/pnas.84.15.5211
发表时间：
1987
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Gutierrez-Hartmann,A;Siddiqui,S;Loukin,S
通讯作者：
Loukin,S

Lipoprotein lipase gene expression in rat adipocytes is regulated by isoproterenol and insulin through different mechanisms.

大鼠脂肪细胞中脂蛋白脂肪酶基因的表达受异丙肾上腺素和胰岛素通过不同机制的调节。

DOI：
10.1210/mend-4-9-1416
发表时间：
1990
期刊：
Molecular endocrinology (Baltimore, Md.)
影响因子：
0
作者：
Raynolds,MV;Awald,PD;Gordon,DF;Gutierrez-Hartmann,A;Rule,DC;Wood,WM;Eckel,RH
通讯作者：
Eckel,RH

Ras mediates Src but not epidermal growth factor-receptor tyrosine kinase signaling pathways in GH4 neuroendocrine cells.

Ras 介导 GH4 神经内分泌细胞中的 Src，但不介导表皮生长因子受体酪氨酸激酶信号通路。

DOI：
10.1073/pnas.91.18.8612
发表时间：
1994
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Pickett,CA;Gutierrez-Hartmann,A
通讯作者：
Gutierrez-Hartmann,A

Human IL-1 receptor antagonist promoter. Cell type-specific activity and identification of regulatory regions.

人 IL-1 受体拮抗剂启动子。