A new approach for studying gene regulation by distant DNA elements in transgenic mice

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Download Full-text

Short interspersed DNA elements and miRNAs: a novel hidden gene regulation layer in zebrafish?

Chromosome Research ◽

10.1007/s10577-015-9484-6 ◽

2015 ◽

Vol 23 (3) ◽

pp. 533-544 ◽

Cited By ~ 4

Author(s):

Margherita Scarpato ◽

Claudia Angelini ◽

Ennio Cocca ◽

Maria M. Pallotta ◽

Maria A Morescalchi ◽

...

Keyword(s):

Gene Regulation ◽

Dna Elements

Download Full-text

Cell-Specific Expression of the Mouse Glycoprotein Hormone α-Subunit Gene Requires Multiple Interacting DNA Elements in Transgenic Mice and Cultured Cells

Molecular Endocrinology ◽

10.1210/mend.12.5.0103 ◽

1998 ◽

Vol 12 (5) ◽

pp. 622-633 ◽

Cited By ~ 2

Author(s):

Michelle L. Brinkmeier ◽

David F. Gordon ◽

Janet M. Dowding ◽

Thomas L. Saunders ◽

Susan K. Kendall ◽

...

Keyword(s):

Transgenic Mice ◽

Pituitary Cells ◽

Subunit Gene ◽

High Level Expression ◽

Glycoprotein Hormone ◽

Α Subunit ◽

Peripheral Tissues ◽

Cell Specificity ◽

Dna Elements ◽

High Level

Abstract The glycoprotein hormone α-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between −4.6 and −3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to −341/+43 mouseα -subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact −4.6 kb/+43 construct. Deletion of sequences between− 341 and −297 resulted in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a− 341/+43 α-promoter in both αTSH and αT3 cells, but it did not enhance α-promoter activity significantly in CV-1 cells. Removal of sequences between −341 and −297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the α-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from− 341 to −297 are critical for restricting expression to the anterior pituitary.

Download Full-text

Inducible ablation of adipocytes in adult transgenic mice expressing the E. coli nitroreductase gene

Journal of Endocrinology ◽

10.1677/joe.0.1750487 ◽

2002 ◽

Vol 175 (2) ◽

pp. 487-498 ◽

Cited By ~ 9

Author(s):

R Felmer ◽

W Cui ◽

AJ Clark

Keyword(s):

Adipose Tissue ◽

Transgenic Mice ◽

Present Model ◽

Complete Disappearance ◽

Mouse Development ◽

New Approach ◽

E Coli ◽

Fat Depots ◽

Extensive Cell ◽

Higher Doses

We describe the use of an enzyme prodrug system based on E. coli nitroreductase (NTR) to achieve the specific ablation of adipose tissue. Transgenic mice expressing the NTR gene specifically in the adipose tissue were generated using the adipocyte specific promoter aP2. After treatment with the prodrug CB1954 these mice showed extensive cell depletion in all fat depots; this was directly correlated to both the dose of prodrug and the levels of NTR expression. Higher doses of CB1954 resulted in complete disappearance of visible adipose stores in some transgenic mice. These mice exhibited an impaired ability to thermoregulate body temperature. Lower doses of CB1954 resulted in a partial reduction of the adipose tissue leaving non-expressing cells that escape ablation. These animals show normal levels of blood glucose and triglycerides but have reduced leptin levels. After 30 days they were able to regenerate the fat depots and leptin levels returned to normal but, interestingly, no NTR-expressing cells were detectable. The present model provides a new approach to manipulate the number of adipocytes at different stages of mouse development and provides a new system for the study of fat metabolism especially in abnormal conditions such as obesity and its modulation through manipulation of the target cell population.

Download Full-text

Ligand Selectivity and Gene Regulation by the Human Aryl Hydrocarbon Receptor in Transgenic Mice

Molecular Pharmacology ◽

10.1124/mol.109.054825 ◽

2009 ◽

Vol 75 (6) ◽

pp. 1412-1420 ◽

Cited By ~ 82

Author(s):

Colin A. Flaveny ◽

Iain A. Murray ◽

Chris R. Chiaro ◽

Gary H. Perdew

Keyword(s):

Gene Regulation ◽

Transgenic Mice ◽

Aryl Hydrocarbon Receptor ◽

Ligand Selectivity ◽

Aryl Hydrocarbon

Download Full-text

Gene regulation through chromatin remodelling by members of the nuclear receptor superfamily

Biochemical Society Transactions ◽

10.1042/bst0280369 ◽

2000 ◽

Vol 28 (4) ◽

pp. 369-373 ◽

Cited By ~ 15

Author(s):

I. J. McEwan

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Transcription Factors ◽

Thyroid Hormones ◽

Target Gene ◽

Protein Complexes ◽

Nuclear Receptor Superfamily ◽

Target Gene Expression ◽

Dna Elements ◽

Introductory Review

The intracellular receptors for steroid hormones, thyroid hormones, retinoids and vitamin D3 are known to bind to specific DNA elements and thus regulate target gene expression. This introductory review and the following papers address some of the mechanisms underlying this action. In particular, the ability of this family of transcription factors to recruit multi-protein complexes that have the capacity to remodel chromatin structure in order to silence or activate target gene expression is discussed.

Download Full-text

Encode and a new landscape for psychiatric genetics

The British Journal of Psychiatry ◽

10.1192/bjp.bp.113.127191 ◽

2013 ◽

Vol 203 (2) ◽

pp. 84-85 ◽

Cited By ~ 3

Author(s):

Akshay Nair ◽

Robert Howard

Keyword(s):

Gene Regulation ◽

Environment Interaction ◽

Psychiatric Genetics ◽

Encode Project ◽

Polygenic Risk ◽

Gene Environment Interaction ◽

Gene Environment ◽

Rigorous Research ◽

Dna Elements ◽

Insight Into

SummaryDespite rigorous research into the genetics of neuropsychiatric disorders, the mechanism by which polygenic risk leads to complex clinical phenotypes remains unclear. The Encyclopedia of DNA Elements (ENCODE) project gives us new insight into gene regulation, and gene–gene and gene–environment interaction.Better understanding of these key genomic mechanisms may provide the answers we have been searching for.

Download Full-text