Yanping Li


October 2007

Document Type


Degree Name



Dept. of Biochemistry and Molecular Biology


Oregon Health & Science University


The evolutionarily conserved facilitating chromatin transcription (FACT) complex is a heterodimer of structure-specific recognition protein-1 (SSRP1) and Spt16. FACT has been shown to regulate transcription elongation through a chromatin template and has histone chaperone activity in vitro. In vivo, it is recruited to several actively transcribed genes similar to RNA polymerase II (RNAPII) in yeast and Drosophila. However, its global role in transcription regulation in human cells remains largely elusive. In Chapter Two, I conducted spotted microarray analysis using arrays harboring 8308 human genes to assess the gene expression profile after knocking down SSRP1 or Spt16 levels in human non-small cell lung carcinoma (H1299) cells. I found SSRP1 and Spt16 shared common and individual targets. A subset of genes was regulated by SSRP1 independent of Spt16. Further analyses of some of these genes not only verified these observations but also identified the serum-responsive gene, early growth response gene 1 (egr1), as a novel target for both SSRP1 and Spt16. Using chromatin immunoprecipitation (ChIP), I showed that SSRP1 and Spt16 were recruited to the coding region of egr1 after serum stimulation and important for the progression of elongation RNAPII on the egr1 gene. In addition to the role in transcription, FACT has been purified in a UV-responsive p53 Ser 392 kinase complex in our laboratory. The kinase in the complex is casein kinase 2 (CK2). The association of FACT with CK2 changes the CK2 substrate specificity toward p53 over other tested substrates. CK2, SSRP1, and Spt16 bind to each other via non-overlapping regions in vitro and in cells. In addition, SSRP1 is an efficient substrate for CK2 in vitro. In Chapter Three, we investigated the regulation of SSRP1 function by CK2. Phosphorylation of SSRP1 by CK2 inhibited the nonspecific DNA-binding activity of SSRP1 and the FACT complex in gel mobility shift assays. Using in vitro kinase assay with synthetic peptides as substrates, we identified serines 510, 657, and 688 as phosphorylation targets of CK2 in vitro. Mutagenesis of the three serines revealed that serine 510 was more important for the regulation of SSRP1's DNA-binding activity. Much of our knowledge about the in vivo roles of the mammalian FACT subunits regards human SSRP1 and its mouse homolog T160. SSRP1 has fundamental roles in cell growth control. SSRP1 is abundant in rapidly dividing cells and down-regulated during cell differentiation. Mice homozygous for the ssrp1 deletion mutant were lethal at "embryonic day 3.5". However, it is unclear how SSRP1 is involved in cell growth control. In Chapter Four, we reported the identification of SSRP1 as a novel regulator of microtubule (MT) dynamics. SSRP1 co-localizes with the mitotic spindle and midbody in human cells and associates with MTs in vitro. Purified SSRP1 facilitates tubulin polymerization and MT bundling in vitro. Depletion of SSRP1 leads to disorganized spindles, and midbodies in cells. Hence, SSRP1 plays a crucial role in MT growth and regulation during mitosis and contributes to the cell growth control. In summary, my thesis identified the Spt16-dependent and -independent roles of SSRP1 in regulating gene transcription in human cells, characterized the regulation of SSRP1 by CK2, and uncovered the novel role of SSRP1 during mitosis. These findings will benefit our understanding of SSRP1 function.




School of Medicine



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