THE ROLE OF CHROMODOMAIN PROTEIN 1 (CDP-1) AT NEUROSPORA CRASSA HETEROCHROMATIN
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Proper heterochromatin I formation is a significant factor in chromosome organization and gene regulation. Therefore, it is vital to study the role of all proteins that contribute to proper heterochromatin formation in order to form a comprehensive understanding of the importance of heterochromatin. In Neuro~pora crassa2, heterochromatin formation depends on a number of well-studied proteins. However, we still have an incomplete understanding of this process. In order to improve our understanding of heterochromatin formation in N. crassa, the Selker lab has identified many novel proteins involved in this process. One novel protein is chromodomain protein I (CDP-I) that specifically localizes to heterochromatin. We hypothesized that CDP-I is involved in proper heterochromatin formation. To begin understanding the role of CDP-I at heterochromatin, we asked: 1 Heterochromatin- generally, it is a tightly packed fonn of chromatin (organizes DNA) that can (though not always) prevent certain proteins from accessing certain regions of DNA 2 Neurospora crassa (N. crassa)-fungal species used in the Selker Jab, as well as other labs, as a genetic model ii iii 1) Is CDP-1 required for proper heterochromatin formation at specific regions, such as the centromeres, telomeres, and interspersed heterochromatic regions? 2) Which domains3 of CDP-1 are required for proper protein function? 3) Do changes in gene expression occur when the cdp-1 gene is deleted? 4) Do proteins or protein complexes associate with CDP-1? If so, do they play a role in CDP-1 function? To test if CDP-1 is required for proper silencing at heterochromatic regions, my mentor knocked out4 the cdp-1 gene and inserted antibiotic resistance genes at various heterochromatic regions (centromere on linkage group5 I and VI, a telomere on linkage group VII, and in interspersed heterochromatin). I screened strains for the antibiotic resistance genes and the knockouts of certain genes of interest (e.g. cdp-1, hpo6) using Southern blots7. I tested the growth of the strains on antibiotic media to determine if heterochromatin was still forming properly at these regions with cdp-1 deleted. Once I determined where CDP-1 is required for proper heterochromatin formation, I tested which domains of CDP-1 are required for CDP-1 function. I transformed8 a cdp-1 knockout strain with plasmids9 that contained the cdp-1 gene with 3 Domains—proteins contain amino acid sequences which form a three-dimensional structure that carries out specific functions for the protein (catalytic, binding). CDP-1 has 2 types of domains: a chromodomain that allows CDP-1 to recognize and bind to chromatin and AT hooks that help CDP-1 bind to AT-rich DNA regions. 4 Knocked out/knockout—deletion (or replacement) of gene from genome 5 Linkage group—a chromosome in N. crassa 6 hpo—a gene encoding the protein, HP1, in N. crassa that is essential for heterochromatin formation. It is a highly conserved protein in higher organisms. 7 Southern blot—assay to detect the placement of restriction sites around a specific DNA sequence. See methods. 8 Transformed/Transformation—genetic technique to insert a specific gene into the genome of an organism. See methods. iv specific point mutations (chromodomain (CD), AT hook 1, AT hook 2, and AT hook 1/2 double mutant)10. These strains also had antibiotic resistance genes inserted into the genome (CenVIR::bar or CenIL::nat-1). I will test the CDP-1 mutant strains for growth on antibiotic media and antibiotic resistance gene expression using qRT-PCR. RNA sequencing11 (RNA-seq) showed that there were some genes whose expression levels changed when cdp-1 was deleted. Based on the RNA-seq results, I focused on the expression of six different genes to confirm the data from the RNA-seq. We confirmed that one gene, NCU1671812, had significantly higher expression levels when cdp-1 was deleted compared with wild type. This gene is located in the subtelomeric region of linkage group III, indicating that CDP-1 is required for silencing of this gene at telomere IIIR. Finally, to determine if CDP-1 interacts with other proteins or protein complexes in vivo, I examined four different proteins that immunoprecipitated with CDP-1 in a large scale pull-down assay performed by a former member of the Selker lab. The four proteins (referred to as interacting partners from here on) that I am interested in are CRF8-1, SPT-16 and POB-3 (FACT complex), and HIR-1 (HIRA complex). Each interacting partner has a role at chromatin and potentially associates with CDP-1 in N. crassa. I tagged13 all four interacting partners with 3xHA::hph14 and crossed15 them to 9 Plasmid—circular bacterial DNA that can be manipulated for genetic experiments, such as transformations. 10 Point mutations—mutations in the DNA sequence that alters one nucleotide into another nucleotide changing the amino acid sequence of the protein encoded by the DNA. 11 RNA sequencing—technique to measure RNA levels 12 NCU16718—a hypothetical gene in N. crassa genome whose function has yet to be defined. 13 Epitope tag—a nucleotide sequence genetically inserted at the end of a gene that is translated with the gene and can be detected with antibodies 14 3xHA::hph—3 sequence repeats of HA tag that can be detected with HA antibodies. The hph antibiotic resistance gene is used for the selection of the tag  v another strain with 3xflag::hph16 tagged CDP-1. I screened for strains with both CDP-1 and interacting partners tagged as well as strains with only interacting partners tagged. I then performed a Co-immunoprecipitation (Co-IP)17 to determine if each interacting partner is pulled down with CDP-1 when CDP-1 is immunoprecipitated. My results demonstrate that CDP-1 is required for silencing at centromere I and VI, as well as telomere IIIR. However, CDP-1 is not required for silencing at telomere VIIL or at interspersed heterochromatic regions. In addition, my results demonstrate that CDP-1 may interact with other proteins in order to carry out its function at heterochromatin. Further testing of the association between CDP-1 and the interacting partners, as well as, further efforts to produce stable mutant CDP-1 proteins will expand our understanding of CDP-1 function at heterochromatin. However, the preliminary findings that CDP-1 is required for proper heterochromatin formation at only certain heterochromatic regions in N. crassa contribute to our understanding of heterochromatin. 15 Crossed—2 N. crassa strains were mated, see methods 16 3xflag::hph—similar to 3xHA::hph, repeated sequence for flag tag that can be detected with flag antibodies 17 Co-immunoprecipitation—target protein co-precipitates a protein partner, see methods.