Progress 07/01/06 to 06/30/13
Outputs
Target Audience: Scientific community in the field of Immunology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Over the last year, our laboratory has trained 3 undergraduate researchers and two graduate students. Two of the undergraduates have graduated now, with one taking a job as a research technician at U. Colorado School of Medicine and one going to Optometry School. One of the graduate students is preparing to defend his thesis in the coming month. How have the results been disseminated to communities of interest? The undergraduates in the laboratory presented their work at the NCSU Undergraduate Research Symposium this Spring. The work on TEA priming is currently in preparation for submission as a peer reviewed journal article, with an anticipated publication date in 2014. A second manuscript on USF-1 and TCRbeta regulation will be submitted for publication in 2014. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have obtained a grant from NCSU to pursue our analyses of the role of USF-1 in regulating TCRbeta V(D)J recombination through its activation as a DNA damage response factor. Our data to date demonstrates that USF-1 binding to a critical promoter in TCRbeta is blocked when USF-1 is phosphorylated in response to recombination-induced dsDNA breaks. Loss of USF-1 binding leads to activation of the promoter and a redirection of recombinase to the gene segment most proximal to the promoter. In our study of TCRalpha gene priming, we have found that the protein CTCF binds the TEA promoter in the earliest stages of pre-implantation embryonic development, and thereby protects the promoter sequence against repressive hypermethylation that targets the flanking regions post-implantation. Loss of CTCF leads to rapid methylation of the promoter in embryonic stem cells. Work is underway to define the genomic breadth of this novel role for CTCF (ie, protection of regulatory sequences against hypermethylation), and also to define the functional role of hypermethylating the promoter's flanks.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Stone JL, McMillan RE, Skaar DA, Bradshaw JM, Jirtle RL, Sikes ML. DNA double-strand breaks relieve USF-mediated repression of D?2 germline transcription in developing thymocytes. J. Immunol. 188:2266-75.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: During 2010, we have completed our analyses of the mechanisms that govern transcription at the Dbeta2 gene segment of the T cell receptor (TCR) beta locus. These results have been submitted for publication and are currently under review. During this period, we have also made initial progress on mapping the epigenetic changes that occur at the TCRalpha promoter, T early alpha (TEA) in concert with early thymocyte development. These initial studies have suggested yet more experiments which are underway. We expect to complete our first manuscript on this topic in the coming months. A grant to fund research in this area was submitted to NIH in 2010 and received a fundable score. We are awaiting a federal budget. We have additionally published 2 other research manuscripts in collaboration with departmental colleagues using our chromatin immunoprecipitation technology to address epigenetic questions raised in their research programs, and have also disseminated our results in 2 review articles, one published in 2010, one to be published in the coming months. PARTICIPANTS: During 2010, the primary researchers on this project included myself (Michael Sikes, Ph.D.) and two graduate students: 1. Justin Bradshaw 2. Jennifer Stone Both have been directly mentored by my. Collaborators have included Dr. Brian Troxell and Dr. Hosni Hassan, Mr. Akinbolade Oyegunwa and Dr. Scott Laster and Dr. Frank Scholle. Additionally, the project has provided training opportunities for graduate students who have worked briefly in the laboratory to master our chromatin immunoprecipitation or electrophoretic mobility shift assays. These students have included Mr. Jason Wilson and Ms. Lipeng Ming. TARGET AUDIENCES: The primary target audiences for this project include other scientists working in the fields of epigenetics, V(D)J recombination, immunology, and developmental biology. Larger audiences include those interested in gene regulation, ontology, and cancer biology PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our study of the epigenetic regulation of V(D)J recombination was particularly fruitful in 2010. In completing our analysis of Dbeta2 gene regulation, we discovered that differential regulation of the 2 Dbeta2 promoters was accomplished by USF-1-mediated repression of the more upstream promoter prior to V(D)J recombination. USF-1 binding at the recombinase targeting sequence serves to repress 5'PDbeta2 promoter activation before recombination, resulting in relatively inefficient remodeling of the Dbeta2 chromatin and the subsequent poor targeting of recombinase to Dbeta2. As recombination proceeds, we found that even inefficient generation of DNA breaks (the intermediate step in recombination) induces a P38 MAP kinase-dependent loss of USF-1 binding and the upregulation of 5'PDbeta2 activity, which in turn should allow for efficient targeting of the recombinase to Dbeta2 during the final steps of recombination. Our work is the first to offer a mechanistic interpretation for how closely related gene segments (Dbeta1 and Dbeta2) could target recombinase with different efficiencies and how this regulation might be tied to thymocyte development. Much work remains and is expected to have a significant impact on the more general study of recombinational accessibility. In our studies of TCRalpha epigenetics, we have found that the Jalpha gene segment chromatin is modified extremely early in stem cells, well before those cells have committed to the T lineage, that these epigenetic modifications are diverse and dynamic, switching formats at multiple stages of development. Work is underway to identify the protein factors responsible for depositing and removing the epigenetic marks, to identify the cis elements that direct their deposition, and to determine how these marks impact the competency of Jalpha segments for subsequent recombination. These studies should prove highly significant in the field and are expected to open a new area of investigation in our understanding of lymphocyte development and stem cell differentiation.
Publications
- Oyegunwa, E, Sikes, ML, Wilson, JR, Scholle, F, and Laster, SM. (2010) Tetra-O-methyl nordihydroguaiaretic acid (Terameprocol) inhibits NFκB-dependent transcription by preventing RelA from binding its cognate sites on DNA. J. Inflamm. 7, 59.
- Sikes, ML, McMillan, RE and Bradshaw, JM. (2010) The center of accessibility: Dβ control of V(D)J recombination. Archivum Immunologiae et Therapiae Experimentalis. 58, 427-433.
- Troxell, B, Sikes, ML, Fink, RC, Vazquez-Torres, A, Jones-Carson, J. and Hassan, HM (2010) Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Thyphimurium. J. Bacteriol. 193, 497-505.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: During the reporting period (10-1-08 to 9-30-09) the project "epigenetics of V(D)J recombination" has yielded a number a substantial outputs. As detailed elsewhere in this document, we have published 2 manuscripts and collaborated on a third, and have developed a powerful modification of the chromatin immunoprecipitation methodology. Our methodology was disseminated as a publication (see below) and has been met with interest in the field. Additionally, we have collaborated with GlaxoSmithKline to assess potential cross-reactivity of developmental HIV integrase inhibitors. Results were provided directly to GSK and are proprietary. PARTICIPANTS: Michael Sikes, PI Ruth McMillan, Post-doctoral fellow Justin Bradshaw, graduate student Jessica Lunsford, research technician Matt George, undergraduate student Clayton Morrison, graduate student Wendell Ivory, volunteer Partner Organizations: GlaxoSmithKline Collaborators: Jonathan Olson, Dept. of Microbiology, NCSU Hosni Hassan, Dept. of Microbiology, NCSU Scott Laster, Dept. of Microbiology, NCSU Michael Krangel, Dept. of Immunology, Duke University During the reporting period, this project provided direct and sustained training for 1 post-doctoral fellow, 2 graduate students, 1 technician, 1 undergraduate, and 1 volunteer. TARGET AUDIENCES: Our work is geared toward understanding the genetic and epigenetic programs that regulate gene usage. Our target audience includes all cellular and molecular biology researchers, and in particular focuses on researchers investigating the developmental programming the governs lymphocyte maturation. PROJECT MODIFICATIONS: No major changes during this period.
Impacts
As noted above, we have recently developed a modified and highly streamlined chromatin immunoprecipitation protocol. This technique is critical to the study of epigenetics, allowing the investigator to directly assess interactions between DNA and nuclear proteins as it relates to gene usage. Although chromatin immunoprecipitation is the central method for epigenetic studies, the standard protocol is exceedingly long, complicated, and requires enormous amounts of cellular input. Our streamlined method reduces the protocol from 3 days to 5 hours, greatly simplifies individual steps, and requires as little as 1/1000 of the starting material of the standard protocol. We have already used our improved method to characterize the transcriptional control of the mouse T cell receptor beta gene, to characterize the role of molybdenum in regulation of Campylobacter jejuni physiology, and are currently collaborating with 3 separate labs to study inflammatory responses in macrophages, transcriptional control of Salmonella pathogenicity islands, and regulatory T cell development. We expect our technique to greatly impact the field of epigenetics in the coming years.
Publications
- Sikes, ML, Bradshaw, JM, Ivory, WT, Lunsford, JL, McMillan, RE, and Morrison, CR. (2009) A streamlined method for rapid and sensitive chromatin immunoprecipitation. J. Immunol. Meth. 15, 58-63.
- McMillan, RE and Sikes ML. (2009) Promoter activity 5 prime of Dbeta2 is coordinated by E47, Runx-1, and GATA-3. Mol. Immunol. 46, 3009-17.
- Taveirne, M, Sikes, ML, and Olson, JW. (2009) Molybdenum and Tungsten in Campylobacter jejuni: characterization of their role in physiology and the identification of separte transporters regulated by a single ModE-like protein. Molecular Microbiology. 74, 758-71.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The work covered by this project was presented in 2008 at two separate national immunology meetings: The 2008 Experimental Biology symposium (San Diego, CA, April 9, 2008), where the work was presented at separate poster and talk presentations; and the 2008 Gene expression in the Immune System symposium (Cold Spring Harbor, NY, April 22) where the work was presented as a poster. In addition, our research has been presented this year at the monthly informal Triangle Immunology Interest Group sessions, and at separate graduate student and postdoctoral research meetings at NCSU. PARTICIPANTS: Ruth McMillan, postdoctoral fellow has continued her training in the laboratory under this project Justin Bradshaw, graduate student, has continued his training in the laboratory under this project Wendell Ivory, graduate student, has training in the laboratory as an informal volunteer during this year. Jessica Lundsford, technician, has worked in the laboratory during this year Our collaborations continue with Dr. Michael Krangel (Duke University) and Eugene Oltz (Vanderbilt University). TARGET AUDIENCES: Research on this project has been primarily focused toward fellow scientists in the developmental immunology field. However, our published findings are of broader impact among developmental biologists and molecular biologists in general, and we hope our continued research will filter into instructional texts also. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The inability to complete recombination impairs lymphocyte development leading to immunodeficiencies of varying severity. Conversely, inappropriate targeting of recombination can result in chromosomal translocations that lead to lymphoid malignancies. Our research over the past year has provided provided a molecular framework for understanding how rearrangement of one gene locus, the T cell receptor beta locus, is sequentially ordered. Additionally, it provides a possible mechanism for expanding both T cell receptor diversity and thymocyte survival at early stages of development. Given the involvement of multiple transcription control elements in V(D)J recombination, our studies additionally promise more global insights into the general developmental mechanisms that underlie tissue-specific gene activation and oncogenesis.
Publications
- McMillan, RE and Sikes ML. (2008) Differential activation of dual promoters alters Dbeta2 germline transcription during thymocyte development. J. Immunol. Mar 1;180(5):3218-28.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Over the past year, we have worked to characterize the transcriptional control of the T cell receptor Dbeta2 element. As stated above, transcriptional regulatory elements have been implicated in all recombination events studied to date. We began these studies expecting to find a D-associated promoter similar to the one I identified upstream of the Dbeta1 element. Unexpectedly, we have identified not one, but two promoters associated with Dbeta2; one positioned downstream of the D and activated during the earliest stages of T cell development, and one upstream of the D and held inactive until the downstream promoter and a linked repressor element have been deleted by D-to-J recombination. This highly complex regulatory scheme is expected to be fundamental to directing the changes in timing and efficiency of Dbeta2 rearrangement that have heretofore defied explanation. After many unexpected turns, work is now complete. In response to the strength of our findings, we have
received a $200,000 R56 grant from the NIH. A complementary study is now underway to correlate differential promoter usage with regulation of Dbeta2 recombination. We have also pursued a third line of study assessing the timing of transcriptional activation at each of the 35 different Vbeta elements in isolated thymocyte subsets, and measuring the role of transcriptional control elements in directing delayed Vbeta-DJbeta rearrangement. These three research projects have been supported by NCARS and a research grant from the Elsa U. Pardee Cancer Foundation. The work outlined here has been presented in a Master's thesis and doctoral dissertation as well as one manuscript, and will be presented at the American Association of Immunologists annual meeting in 2008.
PARTICIPANTS: The follow individuals have worked on the project: Michael Sikes (PI): supervised all aspects of research and training for the project, as well as worked directly on several experiments related to publication of our first manuscript. Ruth McMillan (student): worked in partnership with Dr. Sikes to finish experiments and writing related to our first manuscript and her doctoral dissertation. Collaborators: Michael Krangel (Duke University) Juan Carlos Zuniga Pfluker (U. Toronto) Pierre Ferrier (INSERMS, Marseilles, FR) Jianzhu Chen (MIT) Mark Schlissel (U. California at Berkeley) The project has provided training for the following students: Ruth McMillan (doctoral student) Tim Orcutt (master's student) Jennifer Moss (undergraduate summer intern) Jessica Lundsford (undergraduate student) Justin Bradshaw (graduate student)
TARGET AUDIENCES: The project has provided masters education in molecular immunology to Tim Orcutt, who graduated with a MS degree from the laboratory this year. The project has provided doctoral education in molecular immunology to Ruth McMillan who graduated with a Ph.D. degree from the laboratory this year.
Impacts
The inability to complete recombination impairs lymphocyte development leading to immunodeficiencies of varying severity. Conversely, inappropriate targeting of recombination can result in chromosomal translocations that lead to lymphoid malignancies. Our research will provide a molecular framework for understanding the strict ordering of antigen receptor gene assembly. Given the involvement of multiple transcription control elements in V(D)J recombination, our studies additionally promise more global insights into the general developmental mechanisms that underlie tissue-specific gene activation and oncogenesis.
Publications
- McMillan, RE and Sikes ML. 2007. Transcriptional Control of Dbeta2. Doctoral dissertation. North Carolina State University, North Carolina.
- Orcutt, TM and Sikes ML. 2008. Dissecting the Epigenetic Regulation of Vbeta Recombination. Master's thesis. North Carolina State University, North Carolina.
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Progress 10/01/05 to 09/30/06
Outputs
Over the previous year, work has on this project has led to the characterization of a complex network of transcriptional promoters and repressors that govern expression of the Dbeta2 gene segment within the mouse t cell receptor beta gene. This gene is critical to the development of a functional immune system, and the Dbeta2 segment in particular is a central player in establishing the full range of our antigen recognition repertoire. We have identified two promoters flanking Dbeta2, and characterized their use during T cell development. This work has been submitted for publication to the Journal of Immunology. Current work is focused on defining the roles of each promoter in Dbeta2 recombination, as well as defining the mechanisms by which one of the two promoters is repressed at the earliest stages of T cell development.
Impacts
Our findings provide key insights into the mechanisms that orchestrate the development of the full range of receptors our immune cells must generate in order to respond to a vast array of different pathogens. These findings will guide additional studies that will help to define and/or predict certain genetic immune deficiencies or cancers.
Publications
- McMillan, RE and Sikes ML. (2007) Differential activation of dual promoters alters Dbeta2 germline transcription during thymocyte development. J. Immunol. (manuscript in submission).
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