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Biological Functions of Chromosomal Proteins

Michael Bustin

3 Collaborator(s)

Funding source

National Cancer Institute (NIH)
Chromatin regulates gene expression and therefore plays a key role in developmental processes and in the etiology of various diseases including Cancer. Nuclear protein such as histone H1 and HMGs have been shown to bind to, and alter the properties of the chromatin fiber. Changes in the expression of these architectural proteins are linked to various developmental defects and to various diseases including cancer. It is significant that in higher eukaryotes every cell contains H1 and HMGs. The ubiquitous presence of these proteins in numerous cells and their relative conserved structure is a strong argument that their biological function is important for the generation or maintenance of the cellular phenotype. However, in spite of numerous studies, the mechanism of action, and the exact cellular function of these proteins remains one of the most perplexing aspects of chromatin biology. Towards understanding the biological function and mechanism of action of HMGNs and other chromosomal proteins we have generated genetically modified mice that either lack, or overexpress, all the HMGN protein variants. An early, and very important general finding of these studies is the realization that previous observations with cell free systems or even with cells grown in tissue culture, do not fully explain the possible biological functions of the various proteins in the context of the entire organism. HMGN5/NSBPB1, a new member of the HMG protein family which we discovered, is highly expressed in preimplantation embryos but after implantation it is not expressed in embryonic tissues but it is highly expressed in the placenta and in trophoblast cells. At later developmental stages the protein is re-expressed in most cells, albeit at low levels. The migration of trophoblast cells during and after implantation has been used as a model for cell migration during metastasis. The high expression of HMGN5 in trophoblast cells, together with recent reports in the literature that the levels of HMGN5 are elevated in prostate and breast cancer, implicate the protein in tumorigenesis. Using transgenic mice in which we specifically targeted HMGN5 expression to embryonic tissues we found that aberrant expression of HMGN5 affects the viability and phenotype of the born mice. A significant number of newborn mice are either very small or die immediately after birth. Further analysis revealed abnormalities in the heart muscles. The muscle contained numerous abnormal nuclei in which the chromatin structure seems significantly perturbed. These studies demonstrate the biological consequences of structural alterations in the chromatin fiber. We have elicited conditional HMGN5-/- knock out mouse and are studying its phenotype. We found that HMGN3, a member of the HMGN protein family, is highly and specifically expressed in all the endocrine cells of Langernas pancreatic islets. Knock-out mice lacking HMGN3 protein are diabetic. In the blood of these mice the glucose levels are elevated and both glucagon and insulin levels are lower than those of normal mice. Detailed studies on alpha cells indicated that HMGN3 does not affect the synthesis or secretion of glucagon from alpha cells. On the other hand HMGN3 has significant effect of the transcription profile of the insulin-secreting beta cells. but both their We elucidated the molecular mechanism leading to this phenotype and demonstrated that in pancreatic beta cells, HMGN3 enhances the binding of the transcription factor PDX1 to the promoter of the Glut2 transporter. Loss of HMGN3 decreases the levels of Glut2 thereby impairing glucose import into beta cells, insulin secretion from these cells and glucose homeostasis of the mice. These studies demonstrate directly that the interaction of HMGN protein with chromatin has significant biological consequences. Our previous studies suggest that loss of HMGN1 protein may affect the tumorigenic potential of mice. HMGN1-/- mice cannot properly repair the damage induced in their DNA either by UV or by gamma irradiation. The impaired DNA repair of these mice can be linked directly to the interaction of HMGN1 protein with chromatin and to the effect of HMGN protein on the properties of the chromatin fiber. To address directly whether loss of HMGN could affect carcinogenesis we have initiated a study in which we treat wild type and HMGN1-/- mice with N-nitrosodiehylamine, a potent carcinogen leading to liver cancer. We are using standard protocols developed and extensively used by other laboratories at NCI, to test whether loss of HMGN1 affects the development of liver cancer. The gene coding for human HMGN1 is located in located on chromosome 21, in a region that contains only 33 genes, which are known to be critical for the development of Downss syndrome, one of the most prevalent genetic disease. We have generated a transgenic mice overexpressing HMGN1 at levels similar to those found in humans containing 3 copies of chromosome 21. Using these mice and also mice lacking HMGN1, we find that HMGN1 modulates the expression of genes known to lead to significant mental retardation and to other biological abnormalities. Potentially, these studies may provide new insights into molecular mechanisms underlying the generation of the Downs syndrome phenotype. Studies with a conditional HMGN2 knock-out moue which we have recently generated, reveal that proper expression of this protein during development is crucial for embryonic survival. Loss of the protein leads to embryonic lethality at E8.5 just before full mesoderm induction. The results suggest that HMGN2 may play a crucial role in the induction of this germ cell layer. These studies also serve as a classical example that studies with tissue culture cells may not always help elucidate the true biological function of a protein. Tissue culture cells lacking HMGN2 do survive, but mouse embryos do not. Studies with these mice will lead to an understanding of the mechanisms whereby HMGN regulate the orderly progression of gene expression that is needed for proper development and differentiation. Cell migration is essential for various physiological processes such as embryonic development, immunity and tissue repair. The metastasis of tumor cells involves gain of migration capabilities. Thus, elucidating the fundamental mechanisms regulating cell migration has important implication to the understanding of a wide range of biological processes and may have practical applications to the development of better cancer therapies. We have discovered that cell migration involves, and is contingent on, major reorganization in the structure of the chromatin fiber. This pioneering study which is the first to report that cell migration involves changes in the structure chromatin fiber points out to an additional cellular structure that can be targeted by drugs aimed at interfering with cell migration. In summary, we have developed new model systems for studies on the role of chromatin and chromosomal-binding proteins in various biological processes. These experimental systems provide necessary tools and experimental models for detailed studies on the role of chromatin and chromatin modifiers in a wide range of biological systems. We intend to share these mice with the entire scientific community thereby expanding the scope of our research efforts and contribute to the understanding of the molecular mechanisms whereby events occurring in chromatin ultimately impact the entire organism.An important resent finding is that HMGN1 plays a major role in the childhood leukemia seen in Downs syndrome children.

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