Background on…. the Cell Cycle, Epigenetics, Aspergillus, snxA, etc.
In Dr. James’ lab we conduct cell cycle research in Aspergillus nidulans, a common fungus that also happens to be our model organism of choice. More specifically, we study the regulation of these cell cycle genes, something that we promise to discuss later in the blog.
The cell cycle simply (or rather complexly actually) refers to the normal cycle of growth and reproduction whereby a cell divides to form two identical daughter cells, a process called mitosis. During this cycle many well orchestrated and tightly controlled process occur, such as the dissolution of the protective membrane surrounding the nucleus, chromosome condensation (10-100 fold), and microtubule breakdown and reassembly. Following microtubule breakdown and reassembly into a spindle apparatus, the complex moves into the nucleus, attaches itself to the chromosomes, and pulls them apart to form two nuclei that contain identical genetic information, forming the basis for the two new daughter cells.
As you can imagine with a process this complex and essential to eukaryotic life, there are many key players, the majority of which are different genetically encoded proteins. In A. nidulans, regulation of the cell cycle depends upon a group of proteins that are highly conserved across all eukaryotes, making the fungus an excellent model organism for cell cycle research. What our group is especially focused on is understanding the regulation of a gene called snxA, which was originally identified as a suppressor of a second gene involved in mitotic induction (here is the cell cycle tie in). The way that this works is that when the second gene (named nimX) is mutated, mitotic entry is prevented and the cell dies. However, mutations in the snxA gene, named snxA1 and snxA2, are able to alleviate the mitotic defect conferred by nimX mutations, forming the basis of an interesting relationship (another way to think about this is that when snxA is mutated, the nimX gene is no longer needed for mitosis). After discovering this interesting genetic interaction, the James lab has embarked on a journey to understand the function and regulation of the snxA gene in Aspergillus.
What we have worked hard to reveal thus far is that the snxA mutations, snxA1 and snxA2, are not caused by changes to the snxA DNA sequence, and are actually the result of stable epigenetic modifications to the snxA DNA-protein complex. Simply put, the term epigenetics encompasses the changes above and beyond the DNA level, which determine whether a given gene is activated or repressed. In this way different chemical modifications added to the DNA and DNA associated proteins (termed “chromatin,” the DNA + protein architecture). In A. nidulans, however, the only source of epigenetic variance stems from the acetylation and methylation of the various histone proteins that comprise the majority of proteins found in chromatin. Through the strategic positioning of acetyl and methyl groups on these histones, genes can be more actively transcribed, as the addition of these groups negates the positive charge of amino acids in the histones, pushing the negatively charged DNA away from the chromatin. This process ‘opens’ up the DNA, allowing more active transcription of the genes by the cellular machinery.
Quick diagram showing the acetylation of histone proteins (on the left) marks active gene expression by allowing transcription factor (TF) access to DNA. Complex patterns of histone modifications occur in eukaryotes and can also act to compact the DNA and repress gene expression (right).
More Specifics on the Projects…
My project (Hi I’m Sarah) is very closely related to the epigenetic investigation of snxA, and involves investigating a suppressor of the snxA2 defect called set2. This gene in A. nidulans encodes protein SET2 that has been characterized as a histone H3K36 methyltransferase and normal function involves repressing transcription with the introduction of 3 methyl groups at lysine 36 of histone H3. Previous work using Western blotting, where proteins are identified using specific antibodies, has shown that a set2 gene with a point mutation (set2-sup59) in a snxA2 background has a high level of tri-methylated H3K36 protein. This is unusual if we assume that the set2 gene with a point mutation has eliminated the proper function of the protein. My project this summer will involve investigating this finding again but rather than using a point mutation, we will use the same Western blot technique to see if a ∆set2 (a complete deletion of the set2 gene) mutant will have the same unexpected high level of tri-methylated H3K36. This process hasn’t started yet, so in the meantime I will continue to assist other projects that Dr. James has in the works in the lab.
I (Morgan) am also largely focused on snxA this summer and my goal is to find what we consider the “regulator of snxA”—a gene that is genetically but not physically linked to snxA that controls snxA function from a location elsewhere in the genome. Previously, we detected this exact linkage (two genes are said to be “linked” if they are in close proximity to one another on the same chromosome) in an area on chromosome I. After whole genome sequencing of several snxA1 and snxA2 mutants, the only gene on chromosome I with a mutation (we believe the regulator of snxA gene must have DNA mutations in snxA1 and snxA2) in every mutant was AN6263. These gene codes for an AAA-ATPase, a protein involved in powering a wide range of processes throughout the cell by translating the energy released through ATP hydrolysis into conformational changes on various substrates. The AAA+ proteins make up one of the largest families of proteins found in living cells, with one of their functions including the regulation of gene expression—which is exactly what we expect is happening with snxA mutants. AN6263 in particular doesn’t share many domains with other ATPases known in A. nidulans and its close relatives, which means there’s a possibility we are working with a completely novel gene, which is exciting in-and-of itself.
My goal in these first few weeks has been to make copies of both the wild-type and mutant versions of the AN6263 gene in order to answer three questions: 1, will cloning the wild-type gene into a snxA mutant restore the wild-type phenotype; 2, will deleting the gene from a wild-type strain create a snxA mutant; and 3, will inserting the gene containing the point mutation that we discovered during whole-genome sequencing turn a wild-type strain into a snxA mutant? We’ve already used PCR to create the necessary genes, isolated and purified them, and transformed them into A. nidulans. Now all that is left is to wait and see which transformants display the snxA mutant phenotypes, and from there we can determine whether or not AN6263 is indeed the gene we’re looking for!
Now you should be mostly caught up on everything going on in the lab! Thanks for reading!