Progress 05/07/15 to 04/30/20
Outputs
Target Audience:That portion of the scientific community that is concerned with DNA damage and DNA repair systems, chromosome structure, gene regulation, and neural cell determination. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?These results were presented in seminars and in research publications What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the period of this project (5/7/2015 - 4/30/2020), we performed experiments designed to further our understanding of the role of the JIL-1 kinase. We did this by studying the effects of mutations of the JIL-1 kinase on genome stability, and we also conducted experiments investigating the new finding made during the 2016 - 2017 period about the role of the JIL-1 kinase in the regulation of the Achaete Scute complex (AS-C) of genes. These experiments resulted in several new discoveries that indicate that JIL-1 has an important role in maintaining the stability of the genome and also in the regulation of expression of essential developmental regulatory genes. 1. We determined that loss of function mutations of JIL-1 have an effect on genomic stability by measuring the frequency of meiotic recombination in specific regions of chromosomes 2 and 3 as well as in the X chromosome. These frequencies were measured in individuals heterozygous for the JIL-1 null allele z2 which have a reduced level of JIL-1 protein. Using a series of standard marking mutations, we determined that frequencies of meiotic recombination are altered in all three chromosomes. Curiously, some regions of the X, 2, and 3 show an increase in recombination frequency and others a decrease. Regions adjacent to the centromeres show a decrease and regions in more central portions of the chromosome arms show an increase. We have also completed a series of experiments in which standard marking mutations were used to demonstrate that the rate of somatic recombination in the X chromosome and the 3rd chromosome is increased in individuals heterozygous for the z2 allele. Our hypothesis is that these changes in meiotic and somatic recombination frequencies are the result of decreased levels of JIL-1 protein altering chromatin structure in a way that changes the repair of DNA double strand breaks, and, in particular, alters which repair pathway is used to repair such breaks, shifting repair of more DNA breaks into recombination and mutation inducing pathways. We tested this by examining the response of individuals with the z2 mutation to the chemical mutagen Methyl Methane Sulfonate (MMS), which is known to induce a high level of DNA double strand breaks. In these experiments individuals with the z2 mutation treated with MMS showed a significant increase in the frequency of repair-induced recombination in both the X and the 3rd chromosomes, suggesting that a significant shift to the recombination-inducing repair pathway has occurred. Further experiments to confirm this are planned. These results represent a novel discovery that the epigenetic chromatin modifications made by JIL-1 have an important role in maintaining genome stability. 2. In previous analyses we noted that chromosomes containing the z2 allele that had been maintained in a heterozygous condition in long-term stocks had accumulated numerous second-site recessive lethal mutations. However, these chromosomes had been maintained in our lab for more than 15 years and their origins were not clear (they had been produced in different experiments). We undertook two analyses of two specific chromosomes (z2A5 and z2430). These two chromosomes had been generated in our lab on specific dates (2014 for z2A5 and 2012 for z2430). These chromosomes had been generated by recombining the z2 allele onto wild-type chromosomes that were known to be free of other lethal mutations. Both of these chromosomes had been tested when they were created to demonstrate that they did not contain any other lethal mutations. We performed a series of experiments to determine whether new lethal mutations had arisen in these chromosomes in the relatively short time (4 - 6 years) since they were created. In a series of controlled crosses these z2 chromosome were allowed to undergo recombination with a wild type chromosome and 110 recombinant chromosome lines were established, each containing a different recombinant chromosome. Each recombinant chromosome was screened for lethality and for the presence of z2 (by PCR). Some of the recombinant chromosomes derived from z2A5 and from z2430 contained a recessive lethal mutation at a second site (z2A5 = 14/61 lines and z2430 = 12/49 lines), indicating that new lethal mutations had arisen in both z2 chromosomes. Control crosses using a wild type chromosome in similar crosses generated no new lethal mutations in more than 100 lines. These results indicate that in the short time of 4 years (z2A5) or 6 years (z2430), several new lethal mutations had been induced independently in both chromosomes. These results suggest that the z2 allele may act as a mutator mutation producing an increase in new mutations. Our hypothesis is that this increase is the result of the failure of individuals with reduced levels of JIL-1 protein to correctly repair DNA damage, and in particular DNA double strand breaks, and that this mis-repair results in new mutations. 3. During this grant period we discovered that the recessive lethality of the z2 allele of JIL-1 could be rescued by a duplication of a portion of the X chromosome. A series of experiments using defined duplications, deficiencies, cloned regions of the X chromosome, and gain-of-function and loss-of-function mutations of genes of the Achaete-Scute complex (AS-C) revealed that the z2 lethality is rescued by an increase in expression of one or more AS-C genes, indicating that normal levels of JIL-1 are required for normal action of the AS-C genes. The AS-C genes are well known neurogenic genes which control the formation of neuroblasts during development. This discovery represents the first identification of specific developmental regulatory genes whose normal expression requires JIL-1. In a series of further experiments we discovered that the lethal z2 individuals fail to produce the hormone ecdysone. Ecdysone production is normally triggered by the activity of a specific set of brain neurons, and experiments are currently in progress to determine whether these neurons are missing in z2 individuals. These results represent a truly novel discovery about the role of the epigenetic chromosomal modifications by JIL-1 in development and present a new opportunity for further study of the regulation of this important set of developmental regulatory genes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Christian Albig, Chao Wang, Geoffrey P. Dann, Felix Wojcik, Tama?s Schauer, Silke Krause, Sylvain Maenner, Weili Cai, Yeran Li, Jack Girton, Tom W. Muir, J�rgen Johansen, Kristen M. Johansen, Peter B. Becker, and Catherine Regnard. 2019 JASPer controls interphase H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila. Nat Commun. 2019 Nov 25;10(1):5343. doi: 10.1038/s41467-019-13174-6.
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
Girton J., Johansen J., and Johansen K. The JIL-1 kinase is required for the normal function of the Achaete-scute complex in Drosophila melanogaster. in prepartion
Girton J., Johansen J., and Johansen K. The JIL-1 kinase is required for the normal production of ecdysone in Drosophila melanogaster. in preparation
Girton J., Johansen J., and Johansen K. The JIL-1 kinase is required for maintaining genomic stability in Drosophila melanogaster. in preparation
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The portion of the scientific community that is concerned with DNA damage and DNA repair systems, chromosome structure, gene regulation, and neural cell determination. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Material has been presented in seminars to interested faculty and graduate students. These results are currently being readied for publication in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?This project is scheduled to end in April 2020, so the next reporting period is less than a full year. During this reporting period we will prepare the results of our work for publication.
Impacts
What was accomplished under these goals?
IMPACT: The focus of our research is to discover new information about fundamental cellular processes of importance to animal agriculture. Our research findings this project period are of importance to our understanding of the role of chromatin structure and epigenetic changes in that structure in maintaining genome stability (the induction of high rates of mutations due to mis-repair of DNA DSB is a key process in the development of cancer by genes such as BRCA1). We also found new and important information about the fundamental mechanisms by which chromosomal structure regulates key gene activities during growth and development. This increase in our knowledge has implications for better understanding of how neural birth defects may arise through improper epigenetic chromosome modification. This project will end in April 2020, so in this reporting period (10/1/2018 - 9/30/2019) we performed experiments to complete our investigation of two effects of mutations of the JIL-1 kinase. The first experiments completed our analysis of the effect of JIL-1 mutations on genomic stability. Our results present a clear picture that the JIL-1 null allele (z2) increases rates of mutation, meiotic recombination, and somatic recombination in both the X chromosome and the autosomes, and that this effect occurs at several different places in these chromosomes. We conclude this is a general phenomenon and not due to special structures or DNA sequences at one location. These results indicate the normal kinase activity of JIL-1 is required to maintain genomic stability. We propose the reduction of JIL-1 kinase activity in z2 mutants results in small, localized euchromatic chromosome regions adopting a heterochromatic configuration, and that this change shifts the repair of any DNA DSB in those regions to a heterochromatin specific error-prone repair pathway. This finding is of importance to our understanding of the role of chromatin structure and epigenetic changes in that structure in maintaining genome stability (the induction of high rates of mutations due to mis-repair of DNA DSB is a key process in the development of cancer by genes such as BRCA1). The z2 allele of JIL-1 is a recessive lethal and homozygous z2 individuals (z2/z2) never survive to the adult stage. In previous periods we discovered that z2/z2 individuals whose genotype also contains an extra copy of one or more of the achaete - scute (ac - sc) genes survive. In this period we completed our experiments to demonstrate that the z2/z2 lethality can be rescued by an increase in the expression of ac - sc genes. We used three cloned copies of genomic DNA each containing an extra copy of different ac - sc genes. Each of these rescued z2/z2 lethality but this rescue was eliminated when a loss of function mutation of the cloned gene was present, reducing the number of functional copies of the ac - sc gene to the normal number. In a final test we used an allele (acHw-1) that overexpress the ac gene. This overexpressing allele rescued the z2 lethality of z2/z2 individuals. We also demonstrated the increased expression of acHw-1 in the mother, resulting in a higher level of maternal ac - sc gene product packaged into the egg, can rescue the z2 lethality in the larvae. We conclude the z2 lethality is due to the loss of JIL-1 function reducing the expression of ac - sc genes. The ac - sc genes are neurogenic, (initiate the developmental pathway leading to nerve cell formation), so these findings imply the chromatin structure changes controlled by JIL-1 have an important role in the creation of nerve cells and the establishment of the nervous system. This is new and important information about the fundamental mechanisms by which chromosomal structure regulates key gene activities during growth and development. This increase in our knowledge has implications for better understanding of how neural birth defects may arise through improper epigenetic chromosome modification.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Christian Albig, Chao Wang, Geoffrey P. Dann, Felix Wojcik, Tam�s Schauer, Silke Krause, Sylvain Maenner, Weili Cai, Yeran Li, Jack Girton, Tom W. Muir, J�rgen Johansen, Kristen M. Johansen, Peter B. Becker, and Catherine Regnard. 2019 JASPer controls interphase H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila (In Press, Nature Communications)
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:That portion of the scientific community that is concerned with DNA damage and DNA repair systems, chromosome structure, gene regulation, and neural cell determination. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Material has been presented in seminars at ISU to interested faculty and graduate students. These results are currently being readied for publication in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?In this period we will conclude our analysis of the effect of ac -sc alleles of the lethality of z2/z2 individuals by studying the effect of the acHw-1 allele. Unlike the ac - sc alleles studied previously this allele is the result of a transposon insertion into the ac locus and it has a well-documented gain of function (overexpression) phenotype. We will test this allele with our three rescue duplications to see what effect it has on the rescue of the viability of z2/z2 individuals. We will also begin our molecular analysis of the effects of JIL-1 mutations on DNA double strand break (DSB) repair. Specifically, we will use a reporter construct to analyze a population of repaired DSB to determine whether the lower level of JIL-1 functioning product in z2/+ genotypes alters the choice of repair pathway. This type of analysis makes use of a reporter construct, one in which DSB can be induced and whose sequence can be readily determined after repair. We will make use of the DR-white reporter construct (DRw). DRw is a genetically engineered construct that was designed to assay the frequency with which different pathways repair DSBs. This construct will allow us to determine whether the DSB has been repaired by the Nonhomologous End Joining pathway (NHEJ), the Single Strand Annealing pathway (SSA), or the Homologous Repair pathway (HR). The DRw construct contains a rare I-SceI, restriction site. This site is not found elsewhere in the Drosophila genome and it can be cut by a restriction enzyme with essentially 100% efficiency, generating a DSB at the restriction site in every cell. The restriction site is inserted into a cloned copy of the coding region of the white gene, which contains a mutation making it nonfunctional. A heat-shock inducible restriction enzyme construct will be used to generate the restriction enzyme at a defined developmental phase. This will induce the formation of a DSB at the restriction site in male germ line cells during the premeiotic development stage. The progeny of the treated males will be assayed for characteristics of the different DSB repair pathways. For example, repair by the SSA pathway results in the deletion of an insert adjacent to the SceI site. Repair by NHEJ pathways gives mutations (insertions or deletions) at the restriction site. Repair by HR pathways results in the conversion of the base sequence of the modified white insert. The different base sequences resulting from each of these different repair pathways can be detected by PCR amplification of the DNA surrounding the restriction site from the progeny followed by sequencing. This system has been used by others to measure the effects of mutations on the distribution of use of different DSB repair pathways. We will use a similar approach, comparing repair pathway frequencies in individuals that have a z2/+ or other JIL-1 mutant genotype that has reduced JIL-1 protein to those in individuals who do not (+/+). If our hypothesis about the role of JIL-1 is correct we should observe significant differences in the frequency with which different repair pathways are used in these individuals, and the magnitude of the difference should be correlated with the severity of the JIL-1 loss-of-function genotype. Once the reporter strains have been developed, batches of individuals will be collected as embryos, heat treated to induce the production of the restriction enzyme, and collected as adults. Male will be mated and single fly DNA isolation from progeny will be followed by PCR using primers that flank the reporter site, and the PCR products will be sequenced. A significant number of cases must be done to establish the frequency of use of each pathway in the control and in the JIL-1 mutant strains. Once the genetic strains have been created the work does not require any highly sophisticated analytical procedures and is well suited for undergraduate research projects.
Impacts
What was accomplished under these goals?
In this reporting period (10/1/2017 - 9/30/2018) we performed experiments designed to further our understanding of the effects of mutations of the JIL-1 kinase affect DNA stability. We also conducted experiments investigating the new finding made during the 2016 - 2017 period about the basis for the recessive lethality of JIL-1 mutations. In our last report we presented evidence that loss of function mutations of JIL-1 induce increased rates of mutation, meiotic recombination, and somatic recombination. Our hypothesis is that these increases result from changes in chromatin structure that make euchromatic chromosome structure prone to mis-repair of DNA double strand breaks. In this report period we performed two experiments demonstrating that z2, the null allele of JIL-1, does induce new mutations at a rapid rate. We also continued to study our finding that the recessive lethality of JIL-1 mutations is related to the function of the achaete - scute complex (ac - sc). Duplications containing an extra copy of one or more of the ac-sc genes will rescue the lethality of z2/z2 individuals. In genetics such an interaction often indicates a functional interaction, and may lead to an understanding of the biochemical/molecular basis of the action of the genes involved. In the current period we have performed a series of experiments designed to determine if the observed rescue of z2/z2 lethality is indeed a result of the over expression of ac - sc genes. These experiments have generated important new knowledge of the role of JIL-1 and epigenetic changes in chromatin structure in gene regulation during development. Our findings represent a significant increase in our knowledge of how genome stability and gene expression are regulated by epigenetic changes in chromatin structure. Given that the ac - sc genes establish neural cell fate (activation of ac - sc genes directs an embryonic or stem cell to become a nerve cell), this discovery may have implications for our basic understanding of nervous system development and potentially important medical implications. Induction of new mutations by the z2 allele In our previous analysis of the null allele of JIL-1 (z2) we noted that chromosomes containing z2 that had been maintained in a heterozygous condition in long-term stocks had numerous lethal mutations. However, these chromosomes had been maintained in our lab for more than 15 years and their origins were not clear (they had been produced in different experiments). We undertook two analyses of two specific chromosomes (z2A5 and z2430). These two chromosomes had been generated in our lab on specific dates (2014 for z2A5 and 2012 for z3430). These chromosomes had been generated by recombining the z2 allele onto wild-type chromosomes that were known to be free of other lethal mutations. As well, both chromosomes had been carefully tested after they were created using the JIL-1 rescue construct (GF29.1) that contains a functional copy of the wild-type JIL-1 coding region. Individuals homozygous for either z2A5 or z2430 could be rescued by GF29.1 at a level of essentially 100%, indicating these two chromosomes did not contain any other lethal mutations at the time they were created. We performed a series of experiments to determine whether z2 had induced new lethal mutations in these chromosomes in the relatively short time (4 - 6 years) since they were created. In these experiments flies with z2A5 or z2430 chromosomes were crossed to a wild-type strain to generate heterozygous females, within which recombination occurred between the z2 chromosome and the wild type chromosome. These females were mated to males carrying the marked balancer chromosome TM6, and heterozygous progeny, each with one recombinant chromosome, were recovered. 101 lines were established, each starting with one individual and the recombinant chromosomes in each were screened for lethality and for the presence of z2 (by PCR). We discovered that some of the recombinant chromosomes derived from z2A5 and from z2430 contained a recessive lethal mutation that was not z2 (z2A5 lines = 14/61 lines and z2430 = 12/49 lines), indicating that new lethal mutations had been induced in both z2 chromosomes. We then tested a random selection of the z2 containing chromosomes for the ability to be rescued by the GF29.1 rescue construct. 1/10 of the z2A5 lines and 5/12 of the z2430 lines could not be rescued, indicating that they still contained a recessive lethal mutation that had not been removed by recombination. These results indicate that in the short time of 4 years (z2A5) or 6 years (z2430), new lethal mutations had been induced independently in both chromosomes. This confirms that z2 is an active mutator gene. Interaction of JIL-1 with achaete - scute. The z2 allele of JIL-1 is a strong recessive lethal, and z2/z2 individuals never survive to the adult stage. However, a significant number of z2/z2 individuals whose genotype contains a duplication of a small portion of the X chromosome do survive. We discovered that such rescue duplications contained one or more members of the achaete - scute (ac - sc) complex of genes. The ac - sc complex is a tightly clustered group of four duplicated genes whose products are basic helix-loop-helix, DNA binding regulatory proteins with major roles in regulating gene expression and cell fate during development. They have overlapping functions and the loss of one gene can be compensated by an increase in expression of another. In this period, we have performed a number of experiments to demonstrate that the z2/z2 rescue effect is due to an increase in the expression of the ac - sc genes. Each of the three rescue duplications (Dc007, Dc430, Dc009) gives about a 30% rate of rescue of z2/z2 adults. In the current experiments we generated z2/z2 individuals that contained one of these duplications, and also one of a series of point mutations of ac or sc. These point mutations (ac1, sc1, sc8) have been well studied and each is known to be a loss of function allele. If our hypothesis about z2/z2 lethality being rescued because the Dc duplication provides an increase in ac - sc expression is correct, then these loss of function ac - sc alleles should decrease the level of ac - sc expression, counteracting the effect of a Dc duplication, and z2/z2 should no longer be rescued. This is, in fact, what we observed. In a series of experiments, all 9 combinations of ac1, sc1, sc8 and the duplications Dc007, Dc009, Dc430 were tested for z2/z2 rescue. In all crosses internal controls (male z2/z2 siblings with one of the rescue duplications but with a wild-type ac - sc allele on the X chromosome) had a rescue rate of about 30%. In all 9 crosses no experimental males (z2/z2 males with one of the rescue duplications and one of the three ac - sc mutations) survived, a rescue rate of 0%. Each cross was done in triplicate and the failure to observe any experimental flies is not a statistical fluke due to low numbers of flies. These results support for our hypothesis that the z2/z2 rescue is caused by an increase in expression of genes of the ac - sc complex. They suggest that the loss of JIL-1 function is responsible for a decrease in the level of expression of ac - sc genes, and that is responsible for the z2/z2 lethality. Given the ac - sc genes are essential, it is reasonable that a decrease in expression could lead to lethality, and that restoring the levels of expression by adding an additional copy of ac - sc genes could restore expression. The observation that z2 lethality appears to be related to the expression of one specific gene complex, and that this complex is a key regulatory complex that among other things controls the direction of uncommitted stem/embryonic cells into a neural cell identity is a significant finding.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Yao, Chao Wang, Yeran Li, Michael Zavortink, Vincent Archambault, Jack Girton, Kristen M Johansen, Jorgen Johansen
Evidence for a role of spindle matrix formation in cell cycle progression by antibody perturbation PLOS1 -D-18-23551R1
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:That portion of the scientific community that is concerned with DNA damage and DNA repair systems Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Results have been presentd to interested faculty and students in seminars at Iowa State. What do you plan to do during the next reporting period to accomplish the goals? In this period we will begin our molecular analysis of the effects of JIL-1 mutations on DSB repair. Specifically, we will use a reporter construct to analyze a population of repaired DSB to determine whether the lower level of JIL-1 functioning product in z2/+ genotypes alters the choice of repair pathway. This type of analysis makes use of a reporter construct in which DSB can be induced and whose sequence can be readily determined after repair to determine which repair pathway was used for the repair. We will make use of the DR-white reporter construct (DRw). DRw is a genetically engineered construct that was designed to assay the frequency with which different pathways repair DSBs. This construct will allow us to determine whether the DSB has been repaired by the Nonhomologous End Joining pathway (NHEJ), the Single Strand Annealing pathway (SSA), or the Homologous Repair pathway (HR). The DRw construct contains a rare I-SceI, restriction site. This site is not found elsewhere in the Drosophila genome and it can be cut by a restriction enzyme with essentially 100% efficiency, generating a DSB at the restriction site in every cell. The restriction site is inserted into a cloned copy of the coding region of the white gene, which contains a mutation making it nonfunctional. A heat-shock inducible restriction enzyme construct will be used to generate the restriction enzyme at a defined developmental phase. This will induce the formation of a DSB at the restriction site in male germ line cells during the premeiotic development stage. The progeny of the treated males will be assayed for characteristics of the different DSB repair pathways. For example, repair by the SSA pathway results in the deletion of an insert adjacent to the SceI site. Repair by NHEJ pathways gives mutations (insertions or deletions) at the restriction site. Repair by HR pathways results in the conversion of the base sequence of the modified white insert. The different base sequences resulting from each of these different repair pathways can be detected by PCR amplification of the DNA surrounding the restriction site from the progeny. This system has been used by others to measure the effects of mutations on the distribution of use of different DSB repair pathways. We will use a similar approach, comparing repair pathway frequencies in individuals that have a z2/+ or other JIL-1 mutant genotype that has reduced JIL-1 protein to those in individuals who do not (+/+). If our hypothesis about the role of JIL-1 is correct we should observe significant differences in the frequency with which different repair pathways are used in these individuals, and the magnitude of the difference should be correlated with the severity of the JIL-1 loss-of-function genotype. Once reporter strains have been developed containing the reporter construct, the inducible restriction enzyme construct, and also z2, batches of individuals will be collected as embryos, heat treated to induce the production of the restriction enzyme, and collected as adults. Male will be mated and single fly DNA isolation from progeny will be followed by PCR using primers that flank the reporter site, and the PCR products will be sequenced. A significant number of cases must be done to establish the frequency of use of each pathway in the control and in the JIL-1 mutant strains. Once the genetic strains have been created the work does not require any highly sophisticated analytical procedures and is well suited for undergraduate research projects.
Impacts
What was accomplished under these goals?
In this reporting period (10/1/2016 - 9/30/2017) we completed experiments in objectives 1 and 2 aimed at discovering new information about fundamental cellular processes of importance to agriculture. This has generated new knowledge of the molecular mechanisms organisms used to repair double strand breaks in DNA (DSB). In the course of these experiments we made an important new discovery about the interaction of JIL-1 with a group of key regulatory genes. This is a completely new observation and represents a significant increase in our knowledge of how gene expression is regulated by chromatin structure. Objective 1... We will expand on our discovery that a loss-of-function mutation of the JIL-1 kinase gene increases the frequency of meiotic recombination in the X chromosome. Meiotic recombination is a carefully regulated process involving recombination complexes that generate and resolve DNA double strand breaks. We previously discovered the JIL-1 loss of function allele z2 alters the rate of meiotic recombination in one region of the X chromosome. To determine whether this effect was a general one, we completed additional experiments measuring the rate of meiotic recombination in a separate region of the X chromosome (between genes f and su(f)) and also in two regions of the second chromosome (between b and cn, and between cn and vg). The recombination frequency was significantly increased in the y - w and f - su(f) regions, was significantly decreased in the b - cn region, and was unchanged in the cn - vg region. This indicates the JIL-1 effect on meiotic recombination is a general one, not confined to one specific region of the genome. Objective 2... We will expand our analysis to determine whether the effect on recombination of loss-of-function-mutations of JIL-1 occurs in somatic as well as germ-line cells. We wished to determine if the z2 effect on recombination also occurred in somatic cells. Somatic cells undergo recombination that results from the repair of random DSB. The frequency of somatic recombination is measured in experiments in which somatic recombination in individuals heterozygous for a mutation gives rise to patches of homozygous cells whose phenotype can be scored. The frequency of these patches of cells is used as a measure of the frequency of somatic recombination. We used standard mutations, such as multiple wing hairs (mwh) that changes the shape of the hairs normally found on the surface of the wing, and yellow (y) and forked (f) that change the shape and color of bristles.We determined that somatic recombination frequency is significantly increased in the third chromosome and the X chromosome in z2/+ individuals compared to +/+ individuals. To determine if these changes result from the reduction in JIL-1 activity we repeated these experiments using strains that contained cloned DNA constructs containing all or portions of the JIl-1 gene. A full length construct eliminated the increase in recombination as did a partial construct that contained the JIL-1 chromosome binding region.A construct missing this region did not block the increase. We conclude the increase in recombination is caused by a reduction in JIL-1 protein levels, and that reduced binding of JIL-1 kinase to the chromosome is a key factor. To test whether this increase in somatic recombination is the result of changing the repair of DSB we performed additional experiments in which we induced treated larvae with the chemical mutagen Methyl Methane Sulfonate (MMS) and measured the frequency of recombination in the X chromosome. MMS is a widely used mutagen that is known to cause DNA DSB. As expected, this treatment increased the frequency of somatic recombination in control (+/+) strains.However, it caused a significantly greater increase in the experimental (z2/+) strains, indicating the z2 effect is the result of an alteration in the repair of DSB. Analysis of the chromosomal location of the recombination events indicated the frequency of recombination in z2/+ individuals was not increased in the euchromatic portion of the chromosome but was greatly increased in the heterochromatic region near the centromere. Our hypothesis explains these results in terms of the effect of JIL-1 kinase on chromatin structure.Normal JIL-1 action phosphorylates H3S10, generating an open, gene-active chromatin structure.This phosphorylation blocks histone methylation that produces the heterochromatic, closed chromatin structure.A reduction in the level of JIL-1 results in heterochromatic proteins migrating from the centric heterochromatin to form ectopic heterochromatic islands in euchromatic regions.We believe meiotic recombination complexes (which bind only to euchromatic chromatin) to avoid those ectopic regions, and cluster in the remaining euchromatic regions, producing lower rates of meiotic recombination in some regions and higher rates in others.DSB in heterochromatin in somatic cells are mostly repaired by nonhomologous end joining (NHEJ), which results in no recombination. DSB in euchromatin are repaired by homologous repair (HR), which can result in recombination. We believe in z2/+ individuals the migration of heterochromatic proteins away from the centric heterochromatin generates a more "open" centric chromatin structure, which allows more of the MMS-induced DSB to be repaired by HR pathways, causing the observed increase in recombination. New observation: Interaction of JIL-1 and the achaete - scute complex. The z2 allele of JIL-1 is a strong recessive lethal, and z2/z2 individuals never survive to the adult stage.However, in the course of our experiments we observed that a significant number of z2/z2 individuals whose genotype contained a duplication of a small portion of the X chromosome did survive.We proposed this was the result of a specific gene, whose presence in three copies was able to counteract the lethal effect caused by the loss of JIL-1 function (a "triplo-rescue" gene).Such interactions have been reported for other genes and the rescue genes invariably have related functions or physically interact with the target gene, in such a way that increasing the dose of the product of one gene can compensate or overcome reduced function of the other. This was an exciting finding, as it suggested a way to discover important information about the action of JIL-1. We therefore performed a series of experiments to precisely locate the "triplo-rescue" factor.We tested the rescue ability of a series of duplications that subdivided the original rescue region and have localized the effect to one small region. We had originally hoped this region would contain one gene but instead discovered three small duplications that all rescue but do not overlap, so there is no one gene present in all of them. What we did discover was that each of these duplications contained one or more members of the achaete - scute complex (ac-sc) of genes. The ac - sc complex has been extensively studied in Drosophila and is a tightly clustered group of four duplicated genes whose products are basic helix-loop-helix, DNA binding regulatory proteins with major roles in regulating gene expression.They have genetically complex interactions and have been shown to have overlapping functions such that the loss of one gene can sometimes be compensated by an increase in expression of another.Our findings suggest JIL-1 may play a previously undetected role in ac-sc function.This finding will be followed up in a new, separate project in the future. Objective 3... We will test the hypothesis that the effects of loss-of-function mutations of JIL-1 on recombination and mutation are the result of changes in the frequency with which different DNA repair pathways are used to repair DNA double strand breaks. These experiments are still being designed and will be pursued at a later stage of the project.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Sengupta, S., Rath, U., Yao, C., , Zavortink, M., , Wang, C., Girton, J., Johansen, K.M., and Johansen, J. 2016 Digitor/dASCIZ has multiple roles in Drosophila development. PLOS1 11:e0166829 doi 10.1371.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Li, Y., Wang, C., Cai, W., Sengupta, S., Zavortink M., Deng, H., Girton, J., Johansen, J., and Johansen, K.M. 2017 H2av facilitates euchromatic h3s10 phosphorylation but is not required for heat shock-induced chromatin decondensation or transcriptional elongation. Development 144, 3232-3240
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:That portion of the scientific community that is concerned with DNA damage and DNA repair systems. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We are currently undertaking two additional series of SMART experiments to follow up on those described in objective 2. The first is designed to demonstrate that the effects we have observed on somatic recombination frequencies are in fact caused by the z2 mutation. We have developed a series of Drosophila strains that carry cloned DNA fragments containing all or parts of the coding region of JIL-1. These fragments have been inserted into constructs that contain appropriate promoters that allow the sequence to be expressed and these constructs have been transformed into flies to establish these strains. One fragment (labeled 29.1) is a full length JIL-1 cDNA. Expression of one copy of 29.1 produces sufficient functional JIL-1 product to rescue the lethality of homozygous JIL-1 mutants (z2/z2). Since z2 is a null mutation that produces no functional JIL-1 product, this shows that 29.1 can supply all of the normal JIL-1 function. We made use of this to do a SMART test in which we are measuring the frequency of somatic recombination in flies that were heterozygous for mwh and were heterozygous for z2, as we did before, but were also heterozygous for 29.1. If the increase in 3rd chromosome somatic recombination frequency we observed in our previous experiments is due to the lack of JIL-1 product caused by the z2 allele, then the expression of 29.1 in these individuals should replace that missing product and eliminate the increase in somatic recombination frequency. Our preliminary results suggest that this is the case. We will complete this experiment in the next reporting period. This is an important experiment as it indicates that the effect we have been observing is caused by the z2 allele itself and not by some other, undetected, mutation that happens to be on the same chromosome as z2. We are also doing two additional SMART tests in which we will measure the frequency of 3rd chromosome somatic recombination in flies heterozygous for mwh and z2, and which also contain DNA fragments containing only portions of the JIL-1 coding region. This will allow us to determine which region of the gene is responsible for the observed effect on recombination. The final SMART test we are currently undertaking involves the treatment of flies with the chemical mutagen methyl methane sulfonate (MMS). This mutagen is known to cause DNA double strand breaks. We plan to measure the frequency of somatic recombination in flies that are heterozygous for mwh and for z2 which have been treated with MMS at a concentration that is standardly used to generate significant numbers of DNA double strand breaks. We will compare the frequency of recombination in these flies with that of flies that are heterozygous for mwh but which have normal (+/+) alleles of JIL-1. This is an important experiment as it will show that the effect we have observed in z2/+ flies is indeed related to DNA double strand breaks.
Impacts
What was accomplished under these goals?
During the current reporting period (10/1/2015 - 9/30/2016) we have carried out a number of experiments designed to achieve the goals outlined in objectives 1 and 2 of this project. This research will result in an increase in our knowledge of the molecular mechanisms that organisms use to repair double strand breaks in DNA. These breaks are constantly being generated by a variety of agents / events and organisms have evolved elaborate mechanisms to detect and repair them. Many important aspects of these systems remain unknown. DNA damage is a major cause of new mutations and is a factor in cancer and birth defects, so this is an important area of study. In our preliminary experiments we obtained results suggesting that the tandem kinase JIL-1 in Drosophila is involved in double strand break repair, and that loss of function mutations of JIL-1 have an effect, even in the heterozygous condition, on this repair. This is a completely new observation and if it is in fact correct, and if we can determine the parameters of JIL-1 action, it would be a significant increase in our knowledge of the DNA repair process and of the genes / proteins involved. The finding that JIL-1 is involved in DNA repair would open important new areas of investigation of DNA repair. Our current objectives are to demonstrate conclusively that this JIL-1 effect exists, and to define its parameters. Objective 1... We will expand on our discovery that a loss-of-function mutation of the JIL-1 kinase gene increases the frequency of meiotic recombination in the X chromosome. In our preliminary work we demonstrated that the rate of meiotic recombination in one region of the X chromosome is significantly increased in individuals heterozygous for the loss of function JIL-1 allele z2. Meiotic recombination is a result of one process (homologous repair) used to repair DNA double strand breaks. Meiotic recombination has been widely studied as a part of genetic mapping studies and as a process involving controlled DNA double strand breaks. This JIL-1 effect on meiotic recombination was discovered by measuring the rate of recombination between two well-known genes (y and w) in individuals heterozygous for z2 (z2/+) and comparing that with the rate of recombination between these same two genes in individuals that had normal alleles of JIL-1 (+/+). We observed a statistically significant increase in the recombination frequency in z2/+ flies and wished to determine if this effect was a general one, that could be found elsewhere in the genome, or if it was restricted to this particular chromosomal region. In the current reporting period we performed two different mapping experiments, measuring the rate of meiotic recombination in a second, separate region of the X chromosome (between the genes f and su(f)) and also in a region on the second chromosome (between the genes b and cn). To do these experiments we had to generate a number of strains with different combinations of genetic markers and mutant alleles by a process of selective breeding that required several generations. The resulting strains were then expanded to generate the large number of flies needed to determine whether any observed effects were statistically significant. The results indicate that for both of these regions there was a statistically significant change in the frequency of meiotic recombination in z2/+ individuals compared with +/+ individuals. These results indicate that the JIL-1 effect on meiotic recombination is a general one, not confined to one specific region of the genome. Objective 2... We will expand our analysis to determine whether the effect on recombination of loss-of-function-mutations of JIL-1 occurs in somatic as well as germ-line cells. The experiments in objective 1 were designed to measure the effect of the z2 allele on meiotic recombination, which occurs in germ-line cells. In objective 2 we wished to determine if the z2 effect also occurred in somatic cells. These cells do not undergo meiotic recombination, but do undergo somatic (also called mitotic) recombination. Somatic recombination also results from DNA double strand breaks, although it uses different systems of DNA repair than meiotic recombination. The frequency of somatic recombination is commonly measured in SMART (somatic mutation and recombination tests) experiments. These experiments make use of the fact that somatic recombination in individuals heterozygous for a mutation can give rise to patches of cells that are homozygous for the mutation, and which then display the mutant phenotype. The frequency of appearance of these patches of homozygous mutant tissue can be used as a measure of the frequency of somatic recombination, and hence of the frequency of DNA double strand breaks. Drosophila, with its wealth of known mutations and ease of genetic manipulation is an ideal system for such studies. We made use of standard Drosophila marker mutations, such as multiple wing hairs (mwh) that changes the shape of the hairs normally found on the surface of the wing. This gene is located on the 3rd chromosome so individuals heterozygous for mwh (mwh/+) and also heterozygous for z2 (z2/+) were generated by a selective breeding scheme and the frequency of recombinant patches of mwh cells in the wings was measured to determine the frequency of somatic recombination in the 3rd chromosome. We observed that there was a highly significant increase in somatic recombination frequency in z2/+ individuals. The frequency was four times greater than the rate in individuals who were homozygous for a normal allele of JIL-1 (+/+). We then generated a second series of genetically marked strains and performed a second SMART experiment in which we measured the frequency of somatic recombination using a marker mutation on the X chromosome to measure the frequency of somatic recombination in the X. This also showed a highly significant increase in the frequency of somatic recombination in z2/+ individuals. From these experiments we conclude that the loss of JIL-1 function in individuals heterozygous for the z2 allele does have an effect on somatic recombination, and that this is a general effect that is not confined to a single chromosome. These findings strongly support our hypothesis that this z2 effect involves the generation and/or repair of DNA double strand breaks. We are currently in the process of undertaking two additional SMART experiments to follow up on the success of these first experiments (described below in our plans for the next reporting period). Objective 3... We will test the hypothesis that the effects of loss-of-function mutations of JIL-1 on recombination and mutation that we have discovered are the result of these mutations altering the frequency with which different DNA repair pathways are used to repair DNA double strand breaks. We have not yet begun these experiments. These are planned for a later period in the project, once we have completed our experiments to demonstrate that the effects of JIL-1 on recombination are real and significant, and once we have generated the necessary genetic strains.
Publications
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Progress 05/07/15 to 09/30/15
Outputs
Target Audience:These results are of interest to scientists interested in epigenetics, genome stability, DNA double stranded breaks, and/or DNA repair. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The experiments being prepared (as discussed above) will be carried out and the results will be reported in publications submitted to peer-reviewed journals, and presented at national / international conferences.
Impacts
What was accomplished under these goals?
We began this project in May 2015. Since then we have made progress towards all three objectives. Much of this progress involves preparation of strains of Drosophila containing the specific genetic mutations and/or DNA constructs needed for our proposed experiments. These strains must be generated in our lab and this involves multiple generations of selective breeding followed by careful testing (often by DNA sequence analysis) of the resulting strains. Once the correct strains have been generated they must be expanded to produce the populations needed for our experiments. This process of preparation has begun for all three objectives, and the greatest amount of progress has been made towards objective 1. Objective (1) We will expand on our discovery that a loss-of-function mutation of the JIL-1 kinase gene increases the frequency of meiotic recombination in the X chromosome. The results of our previous experiments indicated that flies heterozygous for the JIL-1 null allele z2 (z2/+) have an increased frequency of meiotic recombination between genes on the X chromosome. We have begun a similar experiment involving genes on the second chromosome. We have generated strains that allow us to generate flies heterozygous for the marker mutations cinnabar (cn), black (b) and also for z2 (z2/+), and also flies heterozygous for cn and b but not for z2 (+/+). In our experiment these heterozygous females are mated and their progeny screened to determine the frequency of meiotic recombination between cn and b. In our first preliminary analysis of 5402 progeny the recombination frequency between cn and b was higher in the z2/+ flies (9.73%) than in the +/+ flies (5.35%). This difference is statistically significant (P<.001). This strongly suggests that the effect of z2 on meiotic recombination is a general effect, not confined to the X chromosome. Objective (2) We will expand our analysis to determine whether the effect on recombination of loss-of-function-mutations of JIL-1 occurs in somatic as well as germ-line cells. The frequency of somatic recombination is typically measured in Drosophila by generating flies heterozygous for cell-autonomous recessive visible "marker" mutations (m/+). Mitotic recombination in somatic cells during development in such heterozygotes generates homozygous cells (m/m) that can be detected by their expression of the marker mutation phenotype. Such cells once generated by recombination grow during development producing patches or somatic clones of marked cells. The frequency with which such clones of marked cells are detected in a tissue is a measure of the frequency of somatic recombination. We are using the marker mutation multiple wing hairs (mwh) to measure the frequency of somatic recombination in the third chromosome in wing cells. We are currently generating strains containing mwh and z2 that will allow us to generate flies heterozygous for mwh (mwh/+) and also for z2 (z2/+), and also flies heterozygous for mwh but not for z2 (+/+). We will collect these individuals as adults and then dissect and examine the wings for clones of mwh/mwh cells. Should z2 have an effect in somatic cells similar to that seen in meiotic cells we should see a higher frequency of such clones in z2/+ individuals compared to +/+ individuals. We are also currently constructing marked strains that will allow us to make a similar measurement of the frequency of somatic recombination in the X chromosome to determine whether this z2 effect also occurs in the X chromosome. Objective (3) We will test the hypothesis that the effects of loss-of-function mutations of JIL-1 on recombination and mutation that we have discovered are the result of these mutations altering the frequency with which different DNA repair pathways are used to repair DNA double strand breaks. We plan on using the repair reporter system (Rr3) in Drosophila, which has been used by others to measure the frequency of double strained DNA breaks and the frequency with which such breaks are repaired by three different double strand break (DSB) repair systems. This system uses a DNA insert (the repair reporter construct) containing a rare restriction site at which DSB can be induced. The DNA sequences in the insert surrounding the restriction site have been engineered to allow the detection of the characteristics of these three different DSB repair systems. We have obtained strains containing this insert and also strains with an insert that contains the gene coding for the restriction enzyme. We are currently adapting these strains by a program of selective breeding that will insert z2 and allow us to compare the frequency of DSB and of the three types of DSB repair in z2/+ individuals with that in +/+ individuals. This will allow us to determine the effects of z2 on the frequency of DSB and also on the frequency with which each of the three different DSB repair systems are used.
Publications
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