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biochemistry and biophysics


Presidential Associate Professor of Biochemistry and Biophysics
Epigenetics Program
Department of Biochemistry and Biophysics

9-124 Smilow Center for Translational Research
3400 Civic Center Blvd.
Perelman School of Medicine
University of Pennsylvania
Philadelphia, PA 19104-6059
215-573-9423 (office); 215-573-9422 (lab)

B.S. University of California, Davis (2000) Chemistry
Ph.D.University of Virginia (2005) Chemistry
NIH NRSA Postdoctoral Fellow, Institute for Genomic Biology Postdoctoral Fellow, University of Illinois, Urbana-Champaign (2008)



Quantitative Proteomics for Analysis of Chromatin Structure and Function

The sequences of the human genome and genomes of many other organisms are now readily available and have revolutionized modern biological research. Nevertheless, the next challenge presently on the horizon (after the post-genome era) is the comprehensive characterization of proteins, the ‘active/expressed’ part of the genome. DNA sequence or mRNA levels alone cannot predict the dynamic aspects of cellular function. Proteins, their post-translational modifications (PTMs) and the multi-protein complexes they form are the driving forces of the cellular machinery. These observations have led to the emergence of a new sub-field of contemporary biology called Proteomics: the characterization of the protein complement expressed by a genome of a particular organism or tissue. At the heart of proteomic experiments is the use of nanoflow liquid chromatography-tandem mass spectrometry for the analysis of complex protein mixtures, which is arguably the most rapid, sensitive and accurate technique available for sequence characterization of proteins.

The Garcia laboratory is focused on developing novel mass spectrometry based proteomic methodologies for quantitatively characterizing changes in protein expression and post-translational modification state within a given proteome during significant biological events or in response to external perturbation. Our goal is to utilize large-scale proteomic data to improve our understanding of biological processes at the molecular level. Application of our proteomic technology spans several areas of cellular biology, but one main interest is described below.

A proteomic approach for systems-wide analysis of key molecular events during epigenetic processes

Epigenetic refers to stable heritable changes in gene expression that are not due to changes in DNA sequence, such as DNA methylation, RNA interference and histone modifications. These epigenetic changes are responsible for generating different cell types originating from the exact same genome. Additionally, even though all genes exist in every cell, only a small number of genes are expressed in any given cell type and these expression patterns can be “memorized”. Inheritance of these transcription patterns through DNA replication and chromatin remodeling that accompanies each cell division is crucial for cell survival, but the mechanisms by which this “memory” is achieved is not well understood. Emerging as one key regulator of cellular memory are histones. Histones are small basic proteins that function to package genomic DNA into repeating nucleosomal units (containing ~146 bp of DNA wrapped around two copies each of histones H3, H4, H2A and H2B) forming the chromatin fiber and hence our chromosomes. In general, the packaging of DNA into chromatin is recognized to be a major mechanism by which the access of genomic DNA is restricted. This physical barrier to the underlying DNA is precisely regulated, at least in part, by the PTMs of histones.

A wide number of studies show that several single covalent histone modifications such as methylation, acetylation, phosphorylation and ubiquitination located in the N-terminal tails correlate with both the regulation of chromatin structure during active gene expression, or heterochromatin formation during gene silencing. These histone PTMs occur on multiple but specific sites, suggesting that histones can act as signaling platforms for proteins that “read” these marks. In support, proteins that contain special domains that bind to single site-specific methylation and acetylation marks on histones have been discovered. Consequently, the “Histone Code” hypothesis and other theories have been put forward to explain how these histone marks can result in distinct cellular outcomes in terms of chromatin-regulated functions. Nevertheless, it is currently unknown what effects, if any, multiple combinations of histone modifications might exert, and translating the combinatorial modification patterns of histones into biological significance remains a significant challenge. Therefore, we feel that the utilization of advanced proteomic technology in chromatin biology will enhance investigations of histone modifications to a much higher scale.

For investigating such a complex mechanism, we are focusing on uncovering changes in “Histone Codes” as a result of exogenous stimuli, over-expression, or knockdown of proteins that affect chromatin remodeling networks. Additionally, we anticipate tracking the chromatin remodeling signaling cue to changes in overall global protein expression, alterations in signal transduction cascades (phosphoproteomics), and disruption of protein-protein interaction networks which will produce information concerning the dynamic mechanism of response in regard to the external perturbation. In combination with biochemical experimentation, bioinformatics analysis and other “omics” technologies, we feel that our large-scale proteomic data will help provide a systems biology outlook on epigenetic processes that will lay the foundation for development of drug treatments for human diseases and conditions that are believed to be of epigenetic origin.

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