Exploring Transposable Elements Regulated by ZNF649
Engineering
Senior Thesis 195
KRAB Zinc Fingers Proteins (KZNFs) are the largest family of human transcription factors. Many have evolved to battle against transposable elements (TEs) and protect the genome. When TEs are not repressed, they can disrupt genes and cause diseases. Throughout evolution, different KZNFs have emerged to battle invading TEs, leading to primate specific KZNF-TE interactions. In my thesis project, I investigated different families of transposable elements that were regulated by a specific KZNF, ZNF649, which is expressed in heart, skeletal muscle, and brain tissues of human adults. By analyzing ChIP-seq data of KZNFs and transposable elements, nucleotide KZNF recognition motifs were calculated and validated experimentally. With a binding reporter assay, I discovered that ZNF649 represses the TE L1PA4. Additionally, I showed how specific nucleotide mutations in the calculated ZNF649 DNA recognition motif are vital for ZNF649 to repress L1PA4. The TATA box was also found to be essential for repression of L1PA by ZNF649. To study other TEs that ZNF649 may have regulated, I tested to see if ZNF649 repressed the TE SVA, since it bound the VNTR region of SVA in the ChIP-seq data. Data from my binding assay in mouse embryonic stem cells (mESCs) revealed that ZNF649 does not repress certain SVA elements. However, computational analysis has shown that the tested SVA elements do not contain the consensus sequence defined by SVA elements that bind ZNF649. Furthermore, I used CRISPRi to knockdown ZNFs and study the change in expression levels of the ZNF and the TE it regulates. I determined the molecular requirements for ZNF649 to repress TEs, which includes the required nucleotides of the recognition motif and finger protein domains that recognize the motif, leading to the repression of the TE by ZNF649. Finally, I created a large KZNF library, which established a powerful platform to study how KZNF-TE interactions have influenced human-specific biology. My study will help the Haussler lab identify new KZNF-TE interactions and elucidate the relationships between ZNF649 and the TEs it regulates.
