Research impact in the field



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Personal Statement, Benjamin A. Garcia

Research impact in the field

Histones are small proteins that complex with DNA to make our chromosomes and regulate gene expression through their diverse numbers of post-translational modifications (PTMs).1 My lab has been developing novel mass spectrometry (MS) based experimental and computational platforms to measure many facets of histone PTM biology that have been utilized by numerous research groups world-wide such as Neil Kelleher, Dick Smith, Ole Jensen, Yingming Zhao, Steve Carr and Donald Hunt to name a few.2-7 These platforms include methods for (i) high-throughput Bottom Up detection of novel histone PTMs and quantitative comparison of histone modifications from multiple cellular states, especially stem cell pluripotency, virus infection and cancer.8-14 (ii) quantitative tracking of combinatorial histone PTMs.15-17 (iii) monitoring in vivo histone PTM dynamics,18-24 and (iv) combining genomic approaches with MS to take any defined part of the genome and accurately quantify histone PTMs and non-histone proteins that reside on these distinct pieces of chromatin, and then map this proteomic data back to specific genomic locations.16, 24-27 These MS approaches have opened up new areas of chromatin biology and epigenetics research, and have proven to the histone field that MS is the premier tool for detection and quantification of histone PTMs.

Histone PTMs have been separately associated with either transcriptional activation or repression depending on the location of a single PTM site, but what type of effect distinct combinations of simultaneously occurring PTMs (Histone Codes) have upon transcriptional events remains unclear. My lab has specifically addressed this problem by developing novel Middle Down MS based methods for high-throughput quantitative tracking of hundreds of combinatorial Histone Codes in a single experiment.17 These methods are the only of their type for high-throughput proteomic sequencing of combinatorial Histone Codes, and to date we have performed experiments on human histones and have discovered hundreds of distinctly modified Histone Codes on histone H3. We have recently used this technology to determine the make-up of single nucleosomes and whether they occur in symmetric or asymmetric forms (i.e. both H3 molecules modified with the exact PTM patterns or not). This is an open question whether both copies of a histone within a nucleosome carry identical PTMs in vivo that has not been addressed. We find that mononucleosomes purified from embryonic cells exhibit both symmetrically and asymmetrically modified populations with respect to H3K27me3 and H4K20me1. Analysis of histone mark co-occurrence by the same approach revealed novel relations between histone marks and provides direct physical evidence for the existence of bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Further it was shown that the Polycomb Repressive Complex 2 (PRC2), while inhibited by symmetric trimethylation at H3K4 or H3K36, is able to catalyze H3K27 methylation on nucleosomes carrying asymmetric H3K4me3 or H3K36me3. These unpublished results uncover a mechanism for the establishment of nucleosomes with bivalent features. The existence of asymmetric modification of nucleosomes increases the number of functional nucleosome states and bears directly on the dynamics regulating the function and inheritance of chromatin states.

Second, although histone PTMs such as acetylation are known to be dynamically reversible processes, most studies only present a static snapshot of histone PTMs. To follow dynamic histone PTM flux/kinetics, the Garcia lab has have created novel approaches using quantitative MS in combination with in vivo metabolic labeling of specific PTMs for temporal analysis of the Histone Code. Using these approaches, we can monitor the progression and dynamics of specific histone PTMs during their cellular lifespan including histone methylation, acetylation and phosphorylation.18, 20, 22, 23 With these approaches, we have begun to define for the first time the steady-state turnover kinetics and half-lives of all known histone modification sites, finding interesting correlations with PTMs associated with active or silenced genes. We are also using this approach to identify the mechanisms for how histone methylation patterns are inherited from old to newly synthesized histones. Our metabolic labeling approach allows for distinction of old and new histones, and when combined with cell synchronizations can allow for tracking of histone modification pattern formation across the cell cycle. We find that two heterochromatic methyl marks H3K9 and H3K27 are initiated on new histones in S-phase, but do not become completely trimethylated until the following G1 phase. This suggests that the trimethylation does not need to be fully established before mitosis, but rather that monomethylation is sufficient to allow propagation of methylation to the higher degrees after mitotic division.

Lastly, the Garcia lab is also taking an active approach to characterize more specific regions of the genome with the goal of analyzing all the histone PTMs and non-histone proteins such as transcription factors that are found on individual genes.16, 25-27 This work termed “proteogenomic mapping” for the characterization of local chromatin regions has proven vital for determining how histone PTMs and their interaction networks contribute to distinct transcriptional states, and represents the missing link between genomics based sequencing and quantitative MS based proteomics research to provide a proteomic snapshot of the chromosome landscape.

Contributions and achievements in research, teaching, clinical activities, service and administration

I have been involved in several modes of teaching, mentoring and service to my department and to the Penn community. In a formal capacity, I have taught lectures on mass spectrometry in BMB 508 graduate course. Every other spring I also co-teach BMB 626 Mass Spectrometry and Proteomics with David Speicher (Wistar Institute). Most recently, I have been involved in teaching in the Metabolism course for first year medical students (Med Rounds discussion). I am currently the BMB Chair of Graduate Admission, and have served on the Graduate Admissions committee for the last three years. For the last three years I have also been the Director of Diversity Recruitment for the BMB graduate program, where I have participated in diversity recruitment at conferences (i.e. SACNAS). Additionally, I have served on the Executive BMB committee and BMB Retreat Planning committee as well. Campus-wide, I have served on search committees for the Microbiology Chair search, and also for two searches for two junior faculty (Chemistry and Medicine departments). I am also currently starting a new Quantitative Proteomics Core for the SOM and will act as the Faculty Director. I have also served on 18 graduate student research committees (14 current).


Projects in Progress

How do alterations in the Histone Code affect cellular function at the proteome-wide level? This is one of the questions that drives us to take a large-scale view of the consequences of resetting the Histone Code. For these experiments, we are inhibiting histone modifying enzyme activity either by small molecule or RNA inhibition to induce a change in the Histone modification patterns normally observed in cells. Changes in these histone modification patterns coincide with changes in gene expression patterns, chromatin structure and transduce a multitude of further downstream effects. We have created a novel proteome-wide approach for identifying dynamic signaling cascades that allows us to look at rapidly phosphorylated proteins in a temporal manner. Additionally, using large-scale proteomic technology, we can quantitatively monitor the expression and post-translational modification state of thousands of distinct proteins in related experiments. Combining this "bird’s eye" proteomic working view of the entire cell (including distinct signaling pathways) with gene expression experiments will allow us to gain a holistic perspective of the systems biology level of epigenetic control. Additionally, we can ask the question in reverse, how do signaling pathways affect chromatin structure and gene expression? It is well established that after some type of cellular activation, changes in gene expression patterns are typically encountered. Our hypothesis is that to some extent after cellular activation, phosphorylation signaling pathways are activated and this results in chromatin remodeling, transcription factor binding and histone modification changes that then drive gene expression patterns. We are currently applying this type of systematic analyses to viral infections and cellular differentiation events.


Overall Goals

The goal of my research group is to characterize the role of the proteome and post-translational modifications (PTMs) on proteins under various cellular states. I have focused my entire career on analyzing protein PTMs (especially histone PTMs) from various biological states, developing approaches and informatics that are currently considered state-of-the-art in the field, and are used by many research groups world-wide for chromatin proteomics studies.



References
1. Strahl, B. D.; Allis, C. D., The language of covalent histone modifications. Nature 2000, 403, (6765), 41-5.

2. Tan, M.; Luo, H.; Lee, S.; Jin, F.; Yang, J. S.; Montellier, E.; Buchou, T.; Cheng, Z.; Rousseaux, S.; Rajagopal, N.; Lu, Z.; Ye, Z.; Zhu, Q.; Wysocka, J.; Ye, Y.; Khochbin, S.; Ren, B.; Zhao, Y., Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 2011, 146, (6), 1016-28.

3. Tian, Z.; Tolic, N.; Zhao, R.; Moore, R. J.; Hengel, S. M.; Robinson, E. W.; Stenoien, D. L.; Wu, S.; Smith, R. D.; Pasa-Tolic, L., Enhanced top-down characterization of histone post-translational modifications. Genome biology 2012, 13, (10), R86.

4. Sidoli, S.; Schwammle, V.; Ruminowicz, C.; Hansen, T. A.; Wu, X.; Helin, K.; Jensen, O. N., Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones. Proteomics 2014, 14, (19), 2200-11.

5. Zheng, Y.; Sweet, S. M.; Popovic, R.; Martinez-Garcia, E.; Tipton, J. D.; Thomas, P. M.; Licht, J. D.; Kelleher, N. L., Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, (34), 13549-54.

6. Papazyan, R.; Voronina, E.; Chapman, J. R.; Luperchio, T. R.; Gilbert, T. M.; Meier, E.; Mackintosh, S. G.; Shabanowitz, J.; Tackett, A. J.; Reddy, K. L.; Coyne, R. S.; Hunt, D. F.; Liu, Y.; Taverna, S. D., Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis. eLife 2014, 3, e02996.

7. Jaffe, J. D.; Wang, Y.; Chan, H. M.; Zhang, J.; Huether, R.; Kryukov, G. V.; Bhang, H. E.; Taylor, J. E.; Hu, M.; Englund, N. P.; Yan, F.; Wang, Z.; Robert McDonald, E., 3rd; Wei, L.; Ma, J.; Easton, J.; Yu, Z.; deBeaumount, R.; Gibaja, V.; Venkatesan, K.; Schlegel, R.; Sellers, W. R.; Keen, N.; Liu, J.; Caponigro, G.; Barretina, J.; Cooke, V. G.; Mullighan, C.; Carr, S. A.; Downing, J. R.; Garraway, L. A.; Stegmeier, F., Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia. Nature genetics 2013, 45, (11), 1386-91.

8. Leroy, G.; Dimaggio, P. A.; Chan, E. Y.; Zee, B. M.; Blanco, M. A.; Bryant, B.; Flaniken, I. Z.; Liu, S.; Kang, Y.; Trojer, P.; Garcia, B. A., A quantitative atlas of histone modification signatures from human cancer cells. Epigenetics & chromatin 2013, 6, (1), 20.

9. Lin, S.; Wein, S.; Gonzales-Cope, M.; Otte, G. L.; Yuan, Z. F.; Afjehi-Sadat, L.; Maile, T.; Berger, S. L.; Rush, J.; Lill, J. R.; Arnott, D.; Garcia, B. A., Stable Isotope labeled histone peptide library for histone post-translational modification and variant quantification by mass spectrometry. Molecular & cellular proteomics : MCP 2014.

10. O'Connor, C.; DiMaggio, P. A., Jr.; Shenk, T.; Garcia, B. A., Quantitative Proteomic Discovery of Dynamic Epigenome Changes that Control Human Cytomegalovirus Infection. Molecular & cellular proteomics : MCP 2014.

11. Plazas-Mayorca, M. D.; Bloom, J. S.; Zeissler, U.; Leroy, G.; Young, N. L.; DiMaggio, P. A.; Krugylak, L.; Schneider, R.; Garcia, B. A., Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown. Molecular bioSystems 2010, 6, (9), 1719-29.

12. Plazas-Mayorca, M. D.; Zee, B. M.; Young, N. L.; Fingerman, I. M.; LeRoy, G.; Briggs, S. D.; Garcia, B. A., One-pot shotgun quantitative mass spectrometry characterization of histones. Journal of proteome research 2009, 8, (11), 5367-74.

13. Sridharan, R.; Gonzales-Cope, M.; Chronis, C.; Bonora, G.; McKee, R.; Huang, C.; Patel, S.; Lopez, D.; Mishra, N.; Pellegrini, M.; Carey, M.; Garcia, B. A.; Plath, K., Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1gamma in reprogramming to pluripotency. Nature cell biology 2013, 15, (7), 872-82.

14. Britton, L. M.; Newhart, A.; Bhanu, N. V.; Sridharan, R.; Gonzales-Cope, M.; Plath, K.; Janicki, S. M.; Garcia, B. A., Initial characterization of histone H3 serine 10 O-acetylation. Epigenetics : official journal of the DNA Methylation Society 2013, 8, (10), 1101-13.

15. Molden, R. C.; Garcia, B. A., Middle-Down and Top-Down Mass Spectrometric Analysis of Co-occurring Histone Modifications. Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 2014, 77, 23 7 1-23 7 28.

16. Voigt, P.; LeRoy, G.; Drury, W. J., 3rd; Zee, B. M.; Son, J.; Beck, D. B.; Young, N. L.; Garcia, B. A.; Reinberg, D., Asymmetrically modified nucleosomes. Cell 2012, 151, (1), 181-93.

17. Young, N. L.; DiMaggio, P. A.; Plazas-Mayorca, M. D.; Baliban, R. C.; Floudas, C. A.; Garcia, B. A., High throughput characterization of combinatorial histone codes. Molecular & cellular proteomics : MCP 2009, 8, (10), 2266-84.

18. Evertts, A. G.; Zee, B. M.; Dimaggio, P. A.; Gonzales-Cope, M.; Coller, H. A.; Garcia, B. A., Quantitative dynamics of the link between cellular metabolism and histone acetylation. The Journal of biological chemistry 2013, 288, (17), 12142-51.

19. Mews, P.; Zee, B. M.; Liu, S.; Donahue, G.; Garcia, B. A.; Berger, S. L., Histone methylation has distinct dynamics from histone acetylation in cell cycle re-entry from quiescence. Molecular and cellular biology 2014.

20. Molden, R. C.; Goya, J.; Khan, Z.; Garcia, B. A., Stable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination. Molecular & cellular proteomics : MCP 2014, 13, (4), 1106-18.

21. Zee, B. M.; Britton, L. M.; Wolle, D.; Haberman, D. M.; Garcia, B. A., Origins and formation of histone methylation across the human cell cycle. Molecular and cellular biology 2012, 32, (13), 2503-14.

22. Zee, B. M.; Levin, R. S.; Dimaggio, P. A.; Garcia, B. A., Global turnover of histone post-translational modifications and variants in human cells. Epigenetics & chromatin 2010, 3, (1), 22.

23. Zee, B. M.; Levin, R. S.; Xu, B.; LeRoy, G.; Wingreen, N. S.; Garcia, B. A., In vivo residue-specific histone methylation dynamics. The Journal of biological chemistry 2010, 285, (5), 3341-50.

24. Evertts, A. G.; Manning, A. L.; Wang, X.; Dyson, N. J.; Garcia, B. A.; Coller, H. A., H4K20 methylation regulates quiescence and chromatin compaction. Molecular biology of the cell 2013, 24, (19), 3025-37.

25. Leroy, G.; Chepelev, I.; Dimaggio, P. A.; Blanco, M. A.; Zee, B. M.; Zhao, K.; Garcia, B. A., Proteogenomic characterization and mapping of nucleosomes decoded by Brd and HP1 proteins. Genome biology 2012, 13, (8), R68.

26. Ratnakumar, K.; Duarte, L. F.; LeRoy, G.; Hasson, D.; Smeets, D.; Vardabasso, C.; Bonisch, C.; Zeng, T.; Xiang, B.; Zhang, D. Y.; Li, H.; Wang, X.; Hake, S. B.; Schermelleh, L.; Garcia, B. A.; Bernstein, E., ATRX-mediated chromatin association of histone variant macroH2A1 regulates alpha-globin expression. Genes & development 2012, 26, (5), 433-8.

27. Wang, C. I.; Alekseyenko, A. A.; LeRoy, G.; Elia, A. E.; Gorchakov, A. A.; Britton, L. M.; Elledge, S. J.; Kharchenko, P. V.; Garcia, B. A.; Kuroda, M. I., Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila. Nature structural & molecular biology 2013, 20, (2), 202-9.

28. Garcia, B. A.; Pesavento, J. J.; Mizzen, C. A.; Kelleher, N. L., Pervasive combinatorial modification of histone H3 in human cells. Nature methods 2007, 4, (6), 487-9.






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