The main focus of my laboratory is to determine the role of ETS transcription factors in epithelial cell
development and tumorigenesis, with a focus on pituitary, mammary and, most recently, GI epithelial cell model systems.
With regards to the pituitary project, we study how the combinatorial action of ETS factors, Ets-1 and GABP,
acting in concert with the POU-homeodomain transcription factor, Pit-1, serve to regulate the lactotroph-specific
basal and hormone-regulated expression of the prolactin gene. Utilizing biochemical, structural, molecular and
transgenic approaches, we have discovered that ETS factors play a critical role in specifying lactotroph cell
identity in pituitary ontogeny. We have expanded both the Pit-1 and Ets factor projects into transgenic mice,
showing that targeting a dominant-negative ETS transgene to the pituitary lactotroph lineage abrogates pituitary
lactotroph development. We are also studying the biological role of Pit-1ß, an alternative-splice form of Pit-1,
which appears to act as a negative regulator of Pit-1 functions.
With regards to our breast cancer project, we study the role of the epithelial-specific ETS transcription factor,
Ese-1, in human mammary epithelial cell tumorigenesis. The Ese-1 gene is an ETS member that is particularly
relevant to breast cancer, since the Ese-1 chromosomal locus is amplified in 50% of early breast cancers and
Ese-1 mRNA is over-expressed in human breast ductal carcinoma in situ (DCIS). We have shown that Ese-1 confers a
transformed phenotype to immortalized, but nontransformed MCF-12A epithelial mammary cells. These Ese-1-transformed
cells display increased motility, invasiveness, growth factor-independent growth and MAPK activation, colonies in
soft agar, and that they form tumor-like colonies in a 3D organoid assay. The most exciting and significant recent
contribution is our discovery that Ese-1 transforms human mammary epithelial cells via a novel cytoplasmic
mechanism. We have mapped the transforming domain of Ese-1 to a unique, 40-AA serine- and aspartic rich (SAR)
domain and we have shown that exclusive cytoplasmic targeting of this subdomain is both necessary and sufficient
for the transformation response. These studies have not only defined a new ETS factor transformation mechanism,
but they have also established a novel paradigm for cell transformation in general. Finally, in our GI project,
we have targeted a dominant-negative ETS to the proximal intestine, and these transgenic mice show abnormal
radial branching morphogenesis. We have crossed these dnETS mice with APCmin mice to test whether the dnETS
transgene blocks APC-induced GI tumorigenesis.
We are making a significant commitment to transgenic approaches to decipher the role of specific
POU-homeodomain and ETS transcription factors in mediating the ontogeny, maintenance and tumorigenesis of
epithelial cells in the pituitary, mammary and GI systems.
Figure 4. Mapping of chemical shift perturbations to the Pithd structure.
Surface density representations (inlays: ribbon representation in the same orientation) of the crystal structure of the
Pithd bound to DNA. These fragments were taken from the crystal structure as resolved by Jacobson et al (1997).
- The results from figure 2C have been indicated by color coding. Chemical shift changes > 25 Hz are colored red, changes
between 25 and 15 Hz dark red and between 15 and 10 pink. Unaffected residues are colored yellow. In green are the solvent
exposed residues of the first helix, which showed a periodicity of three residues in chemical shift perturbation. The amide
proton of W261, which is deeply buried, is colored in blue.
- The hydrophobic pocket around W261 formed by V257, V258, I222 and W261 itself is colored in beige. Overlaying the
pocket is K226, colored in blue. The DNA has been deleted for clarity.
- Model for association of Ets-1 to the Pithd. K226 is shown in blue, the hydrophobic pocket in beige and T220 in green.
Since the crystal structure does not extend beyond residue K273, an extended tail was added to represent the C-terminus of
the Pithd. Ets-1 could use L288 for initial binding and subsequently, depending on the post-translational state of the Pithd,
dock in the hydrophobic pocket next to W261. Additional stabilizing contacts may be made at the C-terminus of the DNA
recognition helix (helix 3). These pictures were generated using MSI's Weblab viewer Lite version
(www.msi.com).
Selected Publications
Wasylyk B,
Hagman, J, Gutierrez-Hartmann, A. ETS transcription factors:
Nuclear effectors of the Ras/MAPK signaling pathway. Trends in
Bioch. Sci. 23: 213-216, 1998.
Diamond SE,
Chiono M, Gutierrez-Hartmann A. Reconstitution of the protein
kinase A response of the rat prolactin promoter: Differential effects
of distinct Pit-1 isoforms and functional interaction with Oct-1.
Molecular Endo 13:228-238, 1998.
Farrow KN,
Gutierrez-Hartmann A. Transforming Growth Factor-ß1 inhibits rat
prolactin promoter activity in GH4 neuroendocrine cells. DNA & Cell
Biol 18:863-873, 1999.
Bradford AP,
Brodsky KS, Diamond SE, Kuhn LC, Liu Y, Gutierrez-Hartmann A.
The Pit-1 homeodomain and ß-domain interact with Ets-1 and modulate
synergistic activation of the rat prolactin promoter. J. Biol.
Chem. 275: 3100-3106, 2000.
Diamond SE,
Gutierrez-Hartmann A. The Pit-1ß-domain dictates active
repression and alteration of histone acetylation of the proximal rat
prolactin promoter. J. Biol. Chem. 275: 30977-30986, 2000.
Schweppe RE,
Gutierrez-Hartmann A. Pituitary Ets-1 and GABP bind to the
growth factor regulatory sites of the rat prolactin promoter. Nucl.
Acids Res. 29:1251-1260, 2001.
Dunkelburg
JA, Gutierrez-Hartmann A.LZ-FYVE: A novel developmental
stage-specific leucine zipper, FYVE-finger protein. DNA & Cell
Biology 20:403-412, 2001.
Augustijn KD*,
Duvall DL*, Wechselberger R, Kaptein R, Gutierrez-Hartmann A,
van der Vliet, PC. Structural characterization of the Pit-1/Ets-1
interaction: Pit-1 phosphorylation regulates Pit-1/Ets-1 binding.
Proc. Natl. Acad. Sci. USA 99:12657-12662, 2002. (* dual first
authors).
Pickett CA,
Manning N, Akita Y, Gutierrez-Hartmann A. Role of specific
protein kinase C isozymes in mediating epidermal growth factor,
thyrotropin-releasing hormone, and phorbol ester regulation of the rat
prolactin promoter in GH4/GH4C1 pituitary cells. Mol Endocrinol.
16:2840-2852, 2002.
Eckel KL,
Diamond SE, Tentler JJ, Cappetta GJ, Gutierrez-Hartmann A. The
epithelial-specific ETS transcription factor ESX/ESE-1/Elf-3 modulates
breast cancer-associated gene expression. DNA & Cell Biol.
22:79-94, 2003.
Tentler JJ,
AP Bradford, RE Schweppe, Gutierrez-Hartmann A.
Selective repression of prolactin gene transcription by stable
expression of dominant-negative Ets in GH4 rat pituitary cells.
Endocrine, in press, 2003.
Schweppe RE,
Melton AA, Eads M, Aveline L, Resing, K, Ahn NG, Gutierrez-Hartmann
A. Purification and mass spectrometric identification of GABP as
the functional pituitary Ets factor binding to the basal transcription
element of the prolactin promoter. J. Biol. Chem., in press,
2003.
Schedin PJ,
Eckel KL, McDaniel SM, Prescott JD, Brodsky KS, Tentler JJ,
Gutierrez-Hartmann A. ESX induces transformation and functional
epithelial to mesenchymal transition in MCF-12A mammary epithelial
cells. Submitted.
Duval DL, Jean
A, Gutierrez-Hartmann A. Ras signaling and transcriptional
synergy at a flexible Ets-1/Pit-1 composite element is defined by the
assembly of selective activation domains. Submitted.
CU Faculty PubMed Publications |
|
All PubMed Publications |
Back to Top
