Twist-ing cell fate: mechanistic insights into the role of twist in lineage specification/differentiation and tumorigenesis.

Bone marrow-derived mesenchymal stem cells (MSC), are multipotent cells that give rise to multiple lineages including osteoblasts, adipocytes, muscle, and fibroblasts. MSCs are useful for clinical applications such as cell therapy because they can be isolated from an individual and expanded for use in tissue repair, as well as other therapeutic applications, without immune rejection. However, one of the key problems in the use of MSCs for these applications is the efficiency of these cells to engraft and fully regenerate damaged tissues. Therefore, to optimize this process, a comprehensive understanding of the key regulators of MSCs self-renewal and maintenance are critical to the success of future cell therapy as well as other clinical applications. The basic helix loop helix transcription factor, Twist, plays a master regulatory role in all of these processes and, therefore, a thorough understanding of the mechanistic insights in the role of Twist in lineage specification/differentiation and tumorigenesis is vital to the success of future clinical applications for the therapeutic use of MSCs. In this article, we highlight the basic mechanisms and signaling pathways that are important to MSC fate, maintenance, and differentiation, as well as the critical role that Twist plays in these processes. In addition, we review the known literature suggesting a critical role for Twist in the generation of cancer stem cells, as this information may contribute to a broader understanding of stem cell biology and stem-cell-based therapeutics.

Distinct promoter regions regulate spatial and temporal expression of the Drosophila caspase dronc.

DRONC is an apical Drosophila caspase essential for programmed cell death during fly development. During metamorphosis, dronc gene expression is regulated by the steroid hormone ecdysone, which also regulates the levels of a number of other critical cell death proteins. As DRONC protein levels are important in determining caspase activation and initiation of cell death, we have analyzed the regulation of the dronc promoter using transgenic flies expressing a LacZ reporter gene under the control of the dronc promoter. Our results indicate that dronc expression is highly dynamic during Drosophila development, and is controlled both spatially and temporally. We demonstrate that while a 2.3 kb dronc promoter region contains most of the information required for correct gene expression, a 1.1 kb promoter region is expressed in some tissues and not others. We further demonstrate that during larval-pupal metamorphosis, two ecdysone-induced transcription factors, Broad-Complex and E93, are required for correct dronc expression. Our data suggest that the dronc promoter is regulated in a highly complex manner, and provides an ideal system to explore the temporal and spatial regulation of gene expression driven by nuclear hormone receptors.

A NF-kappa B/Sp1 region is essential for chromatin remodeling and correct transcription of a human granulocyte-macrophage colony-stimulating factor transgene.

The GM-CSF gene is expressed following activation of T cells. The proximal promoter and an upstream enhancer have previously been characterized using transfection and reporter assays in T cell lines in culture. A 10.5-kb transgene containing the entire human GM-CSF gene has also been shown to display inducible, position-independent, copy number-dependent transcription in mouse splenocytes. To determine the role of individual promoter elements in transgene function, mutations were introduced into the proximal promoter and activity assessed following the generation of transgenic mice. Of four mutations introduced into the transgene promoter, only one, in an NF-kappaB/Sp1 region, led to decreased induction of the transgene in splenocytes or bone marrow-derived macrophages. This mutation also affected the activity of reporter gene constructs stably transfected into T cell lines in culture, but not when transiently transfected into the same cell lines. The mutation alters the NF-kappaB family members that bind to the NF-kappaB site as well as reducing the binding of Sp1 to an adjacent element. A DNase I hypersensitive site that is normally generated at the promoter following T cell activation on the wild-type transgene does not appear in the mutant transgene. These results suggest that the NF-kappaB/Sp1 region plays a critical role in chromatin remodeling and transcription on the GM-CSF promoter in primary T cells.

Nuclear factor of activated T cells contributes to the function of the CD28 response region of the granulocyte macrophage-colony stimulating factor promoter.

The granulocyte macrophage colony stimulating factor (GM-CSF) promoter contains a 10 bp element known as CK-1 or CD28RE that specifically responds to the co-stimulatory signal delivered to T cells via the CD28 surface receptor. This element is a variant NFkappaB site that does not function alone but requires an adjacent promoter region that includes a classical NFkappaB element, an Sp-1 site and a putative activator protein-1 (AP-1)-like binding site. The entire region is referred to as the CD28 response region (CD28RR). The GM-CSF CK-1 element has been shown to bind NFkappaB proteins, in particular c-Rel, whose binding and function is dependent on the architectural transcription factor HMGI(Y). It has been previously suggested that the nuclear factor of activated T cells (NFAT) family of proteins also plays a role in the activity of this region. We show here that recombinant NFATp but not AP-1 can bind to the GM-CSF CD28RR. NFATp present in activated Jurkat T cell extracts can also interact with the CD28RR. The binding of NFATp and Rel proteins requires the same core CK-1 sequences, and appears to be mutually exclusive. We investigated the functional significance of NFATp binding to CK-1 by overexpressing the protein in Jurkat T cells and found that NFATp cannot activate the CD28RR alone but can cooperate with signals generated by phorbol 12-myristate 13-acetate/calcium ionophore. The CD28RR is therefore a complex region that can bind and respond to a combination of transcription factors and signals.