Thomas Fazzio received his B.S. degree from the University of Utah in 1997, where he studied the genetics of Vitamin B12 metabolism in Salmonella typhimurium, in the laboratory of John Roth. Tom received his Ph.D. from the University of Washington and Fred Hutchinson Cancer Research Center in 2004 for work on yeast chromatin regulation in the lab of Toshio Tsukiyama. Tom did postdoctoral work on chromatin regulation in stem cells at the University of California, San Francisco in the labs of Barbara Panning and J. Michael Bishop. This work was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research and a Pathway to Independence Award from the NIH. Tom joined the Program in Gene Function and Expression at the University of Massachusetts Medical School in spring 2010.
Chromatin Regulation in Stem Cells
In eukaryotes, relatively large amounts of DNA must be packed into microscopic nuclei within each cell. This is achieved via the formation of highly-organized, yet dynamic chromatin structure in cells. Chromatin affects nuclear processes like gene expression, DNA replication and recombination by several mechanisms, including inhibition of transcription factor binding and localization of genes to transcriptionally active or inactive regions of the nucleus.
Stem cells – cells that are capable of generating any cell type of a tissue or organism – need to remain developmentally flexible in order to be able to self-renew (generate more stem cells) or differentiate into various somatic cell types. To maintain this flexibility, embryonic stem cells maintain a unique chromatin structure that is unusually dynamic and exhibits an atypical pattern of histone modifications at the regulatory sequences of some developmentally regulated genes. In spite of this, the functions of chromatin structure in regulation of stem cell fate remain largely unknown.
We are interested in the mechanisms by which chromatin structure and chromatin regulatory proteins impact gene expression, self-renewal and differentiation in stem cells. To study these processes, we utilize an array of molecular, cellular, genetic, biochemical and systems level approaches.
Chromatin Regulation and Embryonic Stem Cell Self-Renewal
Chromatin structure in embryonic stem (ES) cells is distinct from differentiated cells. ES cells lack large domains of heterochromatin and have unusual patterns of histone modifications at the regulatory regions of some genes. In addition, the chromatin of ES cells is extremely dynamic, exhibiting rapid exchange of chromatin proteins on and off of DNA. However, the functions of this unusual chromatin structure in ES cell self-renewal and differentiation are mysterious.
In an RNA-interference (RNAi) screen of most chromatin proteins in mouse, we recently identified a number of chromatin regulatory proteins necessary for ES cell proliferation or self-renewal. However, the targets of these proteins, along with their cellular functions in ES cell self-renewal, remain largely unknown. Currently, we are examining the functions of some of these chromatin regulators, their regulation, and how they fit into the known transcriptional network governing ES cell pluripotency.
Functions and Regulation of the Tip60-p400 Chromatin Remodeling Complex
One important chromatin regulator in ES cells is the Tip60-p400 complex. Tip60-p400 has both histone exchange and histone acetyltransferase (HAT) activities, and functions in gene regulation and DNA damage repair.
We found that RNAi-mediated knockdown (KD) of any of the 17 subunits of the Tip60-p400 complex in ES cells inhibited self-renewal. However, the molecular functions of Tip60-p400 necessary for self-renewal remain unclear. Although the Tip60 HAT activity has been implicated in gene activation in somatic cells, the primary effect of Tip60 KD in ES cells was activation of genes induced during differentiation. The promoters of many of these genes are directly bound and acetylated by Tip60-p400, raising the question of how this complex might be repressing transcription (Figure 1). We are currently working to dissect the mechanisms by which Tip60-p400 regulates genes in ES cells, and how Tip60-p400 activity is regulated upon ES cell differentiation.
Figure 1. Possible mechanisms for Tip60-p400-mediated repression. (A) Acetylation-mediated activation of a repressor. Tip60, which also acetylates non-histone proteins, may activate a transcriptional repressor (TR) by acetylation, causing it to bind its target sequence and repress transcription. (B) Dual-modification repressor binding. Tip60-mediated histone acetylation, plus a second chromatin modification (or alternatively, a specific DNA sequence), might recruit a repressor specific for chromatin harboring both modifications. HMC: histone modifying complex. Adapted from Fazzio et al., Cell Cycle 7:21, 3302-3306.
Chromatin Regulation and Cancer Stem Cells
While embryonic stem cells, adult stem cells and cancer stem cells (cells within some tumors that can self-renew indefinitely and reconstitute the entire tumor on their own) have very different cellular properties, their chromatin structures share some common features. One example is the polycomb repressive complex 1 (PRC1) protein Bmi-1, which is required for self-renewal in a number of stem cell types. It is unclear whether there are many other chromatin regulators are similarly required for maintenance of developmental potency. We are addressing this question by testing whether chromatin regulators necessary for proliferation or self-renewal of ES cells are also essential in cancer stem cells. Our goals for these experiments are to learn more about the common features of chromatin structure in developmentally plastic cells, as well as identify potential new targets for drugs that target stem cells within tumors.