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Pedro
Rodriguez-Collazo, Ph.D.
B.S. Biology, Dept. of Biochemistry, State Univ. of Odessa, Odessa, Ukraine M.S. Biology, Dept. of Biochemistry, State Univ. of Odessa, Odessa, Ukraine Ph.D. Biochemistry & Molecular Biology, Dept. of Biochemistry, McGill Univ., Montreal |
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I
finished my Ph.D. with Maria Zannis-Hadjopoulos in McGill University
where I co-discovered nucleosome assembly protein 2 (NAP-2). Sanford
attracted me to come to Pittsburgh as a Research Assistant Professor
in the Department of Cell Biology and Physiology. My research interests
are in chromatin and the epigenetic control of chromatin associated
processes. |
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Research Interest
BackgroundThe nucleosome fold is the basic structural and functional unit of the chromatin, the complex between DNA, proteins and RNA, and consists of approximately 180 base pairs of DNA wrapped around an octameric complex of core histones 2(H2A, H2B, H3, H4) and linker histone H1. The DNA array codifies the primary structure of the proteins, and at the same time a variety of proteins, for instance the histones, regulate the DNA-template processes through a variety of mechanisms, which do not change the genetic code and in some cases can be inherited. Such inheritable changes in gene function and expression that do not involve changes in the DNA nucleotide sequence are commonly known as "epigenetic". It is thought that the combination of covalent histone modifications might generate a sort of "histone code" through which the cell could sensor its external and internal environmental status ('environmental sensor'). Scientific Interests We focus on histone epigenesis and their role in DNA-template processes. My current research interests are on chromatin epigenesis and the role of histones modifications in such processes. We study how changes in histone modifications in response to a variety of stimulus direct the DNA-template processes by affecting chromatin structure. We are developing for these studies an easy and sensitive assay for extracting histones from intact cells, in one single step without affecting the original posttranslational pattern of modifications. In addition, we are using variety of techniques and approaches, ranging from cell biology to biochemical analysis to get insights on the relationship between chromatin structure and function. The past three years in Dr. Leuba’s lab as a Research Assistant Professor have been very productive for me. Focusing on changes in histone modifications linked to cell transformation and cancer progression, I was awarded an NCI KO1 grant for five years to study “Regulation of Histone Phosphorylation in Breast Cancer Cells”. Using cell culture, we were able to gain deeper mechanistic insights on cAMP-induced histone dephosphorylation and its role in cell-cycle progression. This work is in continuous development and is part of my actual and future work. To understand the role of histone phosphorylation in chromatin structure and function, we developed unique methods for histone isolation, preserving their native covalent modifications. The USA-Patent Office has issued a non-provisional patent. The patent has raised a great interest among biotech companies, and Active Motif has currently commercialized the use of it. We have been systematically characterizing the role of hyper- and hypo-phosphorylated histones isolated from cultured cells on chromatin-template processes. We are using a plasmid supercoiling technique, as a means to measure changes in the kinetics of nucleosome assembly by histone chaperones that can be affected by histone phosphorylation and other modifications. We are using nuclease digestion, salt extraction, and recently we started using cryo-electron microscopy to elucidate the role of histone phosphorylation in higher order chromatin structure. Based on these studies, we recently submitted a manuscript and another is under development. In a friendly environment here at the University of Pittsburgh, we are collaborating with several labs involved in chromatin epigenesis studies. With Dr. Paul Sammak, we are developing biochemical techniques to characterize chromatin from pluripotent and differentiated neuronal cells. We made available our unique protocol for histone extraction, from whole cells, to Dr. Vesna Rapic-Otrin that is focused on DNA repair. In addition, we collaborate with Proteomic and Genomic Department at University of Pittsburgh (Dr. Billy Day and Paul Wood) to further characterize changes in histone post-translational modification in response to changes in cellular environment. A cryo-electron microscopy characterization of nucleosomal arrays with different degree of modifications is underway in collaboration with Dr. Peijun Zhang of the Department of Structural Biology. Issued patent 1 Pedro Rodriguez-Collazo, Sanford Leuba and Jordanka Zlatanova. Method of Extraction Chromatin Fractions from Intact Cells. 2008. Publication No. US-2008-0241845-A1. 10/02/2008. Access through http://www.uspto.gov/patft/. The present invention describes a simple and reliable method for H1, H3, H4, H3-H4, and H2A-H2B isolation, from intact cells, in which their covalent post-translational modifications are preserved. We conclusively demonstrated that highly purified H1, H3, H4, H3/H4 and H2A-H2B can be obtained from a variety of cell lines and tissues, in one or two steps, with high yield, bypassing the need for nuclei isolation, chromatin DNA digestion and gradient-ultra centrifugation of the samples that would artificially affect such modifications. The extracted histones can be efficiently assembled, by histone chaperones, into nucleosomal structure to be used in DNA-template process studies. Moreover, the purity of the isolated histones allows direct mass spectrometry analysis of histone’s covalent biomarkers from normal and pathological tissues. |
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