Progress 12/01/10 to 11/30/15
Outputs
Target Audience:The audience is molecular biologists working in academic research labs (graduate students, post-doctoral fellows and faculty). Changes/Problems:There weren't really any major changes but as mention previously, Objective 3 became the main focus due to time and resource limitations. This was my first grant proposal as a newly appointed assistant professor and I made the all too common mistake of writing an over zealous set of experiments. But the good news is, significant progress was made on understanding Objective 3; much more than anticipated. What opportunities for training and professional development has the project provided?Provided molecular biology training to undergrautes, graduate and post-doctoral students. How have the results been disseminated to communities of interest?Published journal articles What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The major accomplishments surrounded Objective 3 becasue work on that aim has some degree of overlap with an NIH-funded project providing significant resources toward understanding the molecular mechanisms surrounding facultative heterochromatin in circadian- and light-regulated gene expression. In short, the molecular mechanism of facultative heterochromatin at the central clock gene consists of a sense protein coding gene and a natural antisense transcript. When these 2 transcripts are co-transcribed, they generate siRNA molecules that direct heterochromatin formation via a model analgous to RNAi medated heterochromatin.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Raduwan, H., Isola, A.L., and Belden, W.J. (2013) Methylation of histone H3 on lysine 4 by the lysine methyltransferase SET1 protein is needed for normal clock gene expression. Journal of Biological Chemistry 288(12): 8380-8390.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Joska T.M., Mashruwala A, Boyd J.M, and Belden W.J. (2014) A universal cloning method based on yeast homologous recombination that is simple, efficient and versatile. J Microbiol Methods 100: 46-51.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Li, N., Joska, T.M., Ruesch, C.E., Coster, S.J., and Belden, W.J. (2015) The frequency Natural Antisense Transcript First Promotes, Then Represses, frequency Gene Expression via Facultative Heterochromatin. Proc Natl Acad Sci USA 112: 4357-4362.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Joska, T.M., Zaman, R., and Belden, W.J. (2014) Regulated DNA methylation and the circadian clock: implications in cancer. Biology 3: 560-577.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Ruesch, C.E., Ramakrishnan, M., Park, J., Li, N., Chong, H.S., Zaman, R., Joska, T.M., and Belden, W.J. (2014) The Histone H3 Lysine 9 Methyltransferase DIM-5 Modifies Chromatin at frequency and Represses Light-Activated Gene Expression. G3: Genes, Genomes, Genetics 5: 93-101.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Our target audiences are academic scientists ranging from graduate students to Professors. The results of our reasearch are published in scientific journals with broad readership. I also present the findings at scientific conferences with large audiences. Changes/Problems: The only major problem we have encountered are lingering effects of Hurricane Sandy. My laboratory lost power and all of our freezers thawed which has caused some problems with strains in our freezer collection. Some have been contaminated and have needed to be cleaned up. This has posed minor set backs in lost time. We now have a better system to identify problems and solve them instead of having the problems creep through to our data generation and analysis. What opportunities for training and professional development has the project provided? The project has provided training and development for both students (graduate and undergraduate) and post-docs. These burgeoning researchers have learned a variety of techniques that encompass molecular biology and molecular genetics. Some of these include real-time reverse transcription PCR (RT-PCR) for RNA quantification, Western blotting for protein detection, DNA methylation analysis and chromatin immunoprecipitation. In addition to these technical activites, the students and post-docs have learned critical thinking, hypothesis development, and some basics in manuscript preparation. How have the results been disseminated to communities of interest? Some of the work has been presented at scientific conferences. One conference was the Neurospora Fungal Genetics Conference in Asilomar, California and the second was at the Society for Research in Biological Rhythms (SRBR). There have been no manuscripts for this specific reporting period. However, we do have a manuscript accepted for publication that will be reported in the next progress report. We also have another manuscript that has been resubmitted for re-review. What do you plan to do during the next reporting period to accomplish the goals? Heterochromatin is established in response to diurnal light dark cycles. Therefore, we are going to focus on the role of CSW-1 in the light response and try to pinpoint the defects in light-adaptive represson. To accomplish this, we will try to determine the kinetic relationship of CSW-1 with other chromatin marks in heterochromatin formation. We are going to perform a comprehensive analysis on what marks are missing in the CSW-1 mutant and then try to determine how it is bound and recruited to chromatin.
Impacts
What was accomplished under these goals?
We have made some progress in understanding the role of the chromatin remodeling enzymes Clockswitch (CSW-1) and Chromodomain Helicase DNA-binding 1 (CHD1) in circadian clock-regulated gene expression. It appears that CSW-1 is needed to establish facultative heterochromatin where as CHD1 is required to remove the heterochromatic marks via an unknown mechanism. Over the past year we have determined that our CSW-1 mutant lacks heterochromatin and appears to be devoid of all DNA methylation, whereas the CHD1 mutant has a hyper DNA methylation phenotype. It is still not entirely clear how CSW-1 is involved in heterochromatin formation but we are testing ongoing hypotheses. Heterochromtin and DNA methylation in Neurospora is established by a conserved process where a protein complex called DCDC is used to establish histone H3 lysine 9 methylation (H3K9me3). Heterochromatin protein 1 (HP1) associated with the DNA methyltransferase DIM-2 binds to H3K9me3 and establishes DNA methylation. We are testing whether or not CSW-1 is needed for DCDC recruitment and H3K9me3. In contrast, CHD1 appears to be needed to remove H3K9me3, but we still don't know how this is happening. Further studies should unveil how this occurs.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Belden, W. The frequency natural antisense transcript first promotes, then represses frq gene expression via facultative heterochromatin. 14th Biennial Meeting of the Society for Research on Biological Rhythms, Big Sky Resort, Montana. June 2014
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Belden, W. DIM-5 is required for DNA methylation at the Neurospora clock gene frequency. 27th Fungal Genetics Conference, Asilomar, CA, March, 2014
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The target audiences are scientists in academic and industry laboratories, along with graduate students and post doctoral fellows. Specifically, scientists, graduate students, and post-doctoral fellowswhose research disciplines span the gamut of research involving circadian rhythms, transcriptional regulation, chromatin remodeling and general aspects of fungal molecular biology research.. Changes/Problems: There have been no significant changes at this time. However, our hypotheses are subject to change based on experimental data acquired from our continuing efforts to understand chromatin remodeling and modification and the central clock genefrq in Neurospora crassa. What opportunities for training and professional development has the project provided? Students have learned how to perform some molecular biology experiments,including RT-PCR, Methylation sensitive Southern Blotting, phenotypic analysis and Chromatin immunoprecipitations. Moveover, there have been extensive discussions of the current literature and molecular methodologies of experimental techniques. How have the results been disseminated to communities of interest? Tthe work has been published in Primary Journal Publications. The characterization of set1 was published in the Journal of Biological Chemistry What do you plan to do during the next reporting period to accomplish the goals? We will continue with the experiments outlined in the proposal. Specifically, we are hoping to do some kinetic analysis of modifications and binding and do some invitro pulldowns.
Impacts
What was accomplished under these goals?
The circadian oscillator controls time-of-day gene expression by a network of interconnected feedback loops and is reset by light. The requisite for chromatin regulation in eukaryotic transcription necessitates temporal regulation of histone-modifying and chromatin-remodeling enzymes for proper clock function.CHD1 is known to bind H3K4me3 in mammalian cells, and Neurospora CHD1 is required for proper regulation of the frequency (frq) gene.Based on this, we examined a strain lacking histone H3 lysine 4 methyltransferaseSET1 to determine the role of H3K4 methylation in clock and light-mediated frq regulation. The expectations were that it may be similar to the chd1 deletion. We found that expression of frq was altered in strains lacking set1 under both circadian and light-regulated gene expression. There is a delay in the phasing of H3K4me3 relative to the peak in frq expression. White Collar 2 (WC-2) association with the frq promoter persists longer in strains lackingset1, suggesting a more permissible chromatin state. Surprisingly, SET1 is required for DNA methylation in the frq promoter, indicating a dependency on H3K4me for DNA methylation. These observations collective indicated that SET1 had a completely opposite phenotype from CHD1 and more resembled the phenotype in CSW-1. We then tested CSW-1 for DNA methylation and found that it was similar to SET1. This has changed our orignal hypothesis and now believe that CSW-1 is binding to H3K4me3;ongoing experiments will test this hypothesis.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Raduwan, H., Isola, A. L., and Belden, W. J. (2013) Methylation of histone H3 on lysine 4 by the lysine methyltransferase SET1 protein is needed for normal clock gene expression, J Biol Chem 288, 8380-8390.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: My laboratory focuses on understanding how chromatin remodeling assists circadian clock-regulated gene expression while simultaneously exploring how the clock controls chromatin/genome structure to ensure that the appropriate timing and amplitude of gene expression is achieved. Circadian rhythms are controlled by mechanistically conserved positive and negative transcriptional/translational feedback loops (1, 2). The core feedback loop consists of a heteromeric transcriptional activator complex that drives expression of the negative elements that inhibit their own transcription. Because chromatin regulation is a requisite for proper transcription, we have been examining a number of factors needed for the transcriptional negative feedback loop. Thus far we have been examining the trans tail pathway where histone H2B ubiquitination is needed for histone H3 lysine 9 di and tri methylation (H3K4me2/3). We have determined that loss of SET1, BRE1, and RAD6 all cause unique clock phenotypes. SET1 is a histone H3K4 lysine methyltransferase (KMT2), which catalyzes the mono-, di- and tri-methylation of lysine 4. RAD6, and its partner BRE1, are involved in the monoubiquitination of H2B (H2Bub) on lysine 130 and H2Bub is a requisite for H3K4 di- and tri-methylation. Further experiments are underway to elucidate how H2Bub and H3K4 methylation are involved in establishing and maintaining circadian rhythms. PARTICIPANTS: William J. Belden (PI) Hamidah Raduwan (Graduate Student) TARGET AUDIENCES: The Target Audience are scientists whose research involves circadian rhythms, chromatin remodeling or fungal biology. These include PIs, Post-docs, and graduate students. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The outcomes thus far indicate that expression of the clock gene frq was altered in strains lacking set1 under both circadian and light-regulated gene expression. There is a delay in the phasing of H3K4me3 relative to the peak in frq expression. White Collar 2 (WC-2) association with the frq promoter persists longer in Dset1, suggesting a more permissible chromatin state. SET1 is required for DNA methylation in the frq promoter, providing clues to an uncharacterized mechanism for H3K4me-dependent DNA methylation. The data support a model where SET1 is needed for proper regulation and may modulate the down regulation of frq as well as a subset of WCC target genes. The requirement for SET1, combined with the fact that H3K4me2 and H3K4me3 exclusively require H2B monoubiquitination, led us to examine the role of RAD6 and BRE1 in circadian gene expression. Normally, RAD6 and BRE1 are involved in the monoubiquitination of H2B (H2Bub), and H2Bub is a requisite for SET1-dependent H3K4 di- and tri-methylation. Strains lacking mus-8 (gene encoding RAD6 in Neurospora) and bre1 have different but overlapping phenotypes. The mus-8 deletions strain, but not the bre1 deletion, has a defect in the molecular oscillations of frq, indicating a complex molecular function for trans-histone crosstalk that may have compensatory mechanisms. RAD6 and BRE1 are also needed for efficient light-activated gene expression and Dbre1 possess a vvd-like phenotype. White Collar (WC-2) binding at the frq promoter is also compromised in the mus-8 null strain and there appears to be a low amplitude rhythm. The data support a model where RAD6 is required for frq activation and normal expression and BRE1 appears to be dispensable for oscillations but is involved in assisting light-activated expression and determining period length.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The Belden Laboratory is continually performing experiments to understand molecular interactions between chromatin remodeling and the biological clock in the model fungus, Neurospora crassa with the ultimate goal of publishing our empirically determined results. We currently have three manuscripts in progress that we plan to submit in the coming months. As an active lab we are routinely involved in mentoring, training and teaching both undergraduate and graduate students. Additionally, we actively participate in national and international scientific conferences where our data is presented in symposia and/or as poster presentations. Over the past year, we have presented our work at the American Society for Cell Biology annual meeting, the Keystone Symposium "Histone Code" Fact or Fiction, and the International Society of Chronobiology. PARTICIPANTS: Two Graduate students who work in my lab have participated in this project. Hamidah Raduwan Ph.D Candidate, Program in Microbial Biology, SEBS, Rutgers, New Brunswick, NJ 08901. Allison Isola Ph.D. Candidate, Joint Graduate Program in Toxicology, Rutgers, New Brunswick, NJ 08901 TARGET AUDIENCES: Our target audience is Academic Scientists across a broad range of disciplines. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The Belden Laboratory at Rutgers University is working on understanding the coordinate regulation of the biological clock and chromatin in a model fungal system using molecular biology, biochemistry and modern genetics. Fungal pathogens are a significant cause of phytopathology and represent one of the most costly elements in agricultural food production. Fungi reproduce sexually and asexually and the resulting spores can be easily spread via the air. Although fungicides are effective and used routinely, fungi can develop resistance creating a systematic burden in agriculture. Understanding the molecular mechanisms underlying the biological clock and spore production may facilitate the development of novel fungicides that are safer and more cost effective. Additionally, the research we perform is of great importance to health related issues because defects or insults to the biological clock are implicated in numerous diseases. Over the past year, we have been working on understanding how the biological clock controls chromatin and how defects in chromatin structure affect the clock.
Publications
- Froehlich, AC, Chen CH, Belden WJ, Madeti C, Roenneberg T, Merrow M, Loros J, Dunlap, (2010) Genetic and molecular characterization of a cryptochrome from the filamentous fungus Neurospora crassa. Eukaryotic Cell, 9:738-750.
- Belden, W.J., Lewis Z., Selker E.U., Loros, J.J. and Dunlap, J.C. (2011). CHD1 Remodels Chromatin and Influences Transient DNA Methylation at the Clock Gene frequency. PLoS Genetics.7.
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