|Abstract:||KRAB-associated C2H2 zinc-finger (KRAB-ZNF) proteins are the products of a rapidly evolving gene family that traces back to early tetrapods, but which has expanded dramatically to generate an unprecedented level of species-specific diversity. While most attention has been focused on the more recently evolved primate KRAB-ZNF genes, the vertebrate roots of the KRAB-ZNF families have remained mysterious. We recently mined ZNF loci from seven sequenced genomes (opossum, chicken, zebra finch, lizard, frog, mouse, and human genome) and found hundreds of KRAB-ZNF proteins in every species we examined, but only three human genes were found with clear orthologs in non-mammalian vertebrates. These three genes, ZNF777, ZNF282, and ZNF783, are members of an ancient familial cluster and encode proteins with similar domain structures. These three genes, members of an ancient familial cluster, encode a noncanonical KRAB domain that is similar to an ancient domain which is prevalent in non-mammalian species. In contrast to the mammalian KRAB, which is thought to function as a potent repressor, this ancient domain serves as a transcriptional activator. Our evolutionary analysis confirmed the ancient provenance of this activating KRAB and revealed the independent expansion of KRAB-ZNFs in every vertebrate lineage. This finding led us to ask the question: what are the functions of these ancient family members and why, of such a large and diverse family group, were these three genes conserved so fastidiously over hundreds of millions of years?
In chapter 2, I report the regulatory function of ZNF777, combining chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) with siRNA knockdown experiments to determine genome-wide binding sites, a distinct binding motif, and predicted targets for the protein in human BeWo choriocarcinoma cells. Genes neighboring ZNF777 binding sites can be either up- or down- regulated, suggesting a complex regulatory role. Our studies revealed that some of this complexity is due to the generation of HUB-containing and HUB-minus isoforms, which are predicted to have different regulatory activities. Based on these experiments, we hypothesize that ZNF777 regulates pathways best known for their roles in neurogenesis and axon pathfinding, but also recently shown to play critical roles in placental development.
Since ZNF777 is also expressed in embryonic brain, we sought to further investigate the functional role of this ancient gene in neuron development. In chapter 3, I show that mouse Zfp777 is expressed in neuronal stem cells (NSC) cultured from early mouse embryos, with a pattern that changes over the course of neuron differentiation in vitro. Using the NSC platform, I characterized the binding landscape of Zfp777 in undifferentiated NSC. To circumvent the roadblock posed by the lack of a ChIP-grade antibody for the mouse protein, I exploited the CRISPR-Cas9 technique to tag the endogenous Zfp777 protein with FLAG epitopes. Our results revealed a novel Zfp777 binding motif that bears significant similarity to a motif predicted in in vitro studies, and found that Zfp777 binds to promoters of genes encoding transcription factors, Wnt and TGF-beta pathways components, and proteins related to neuron development and axon guidance. Since these same functions were also found to be regulated by ZNF777 in BeWo cells, these results suggested that the mouse and human Zfp777 and ZNF777 proteins regulating similar genes and pathways, most classically associated with axon guidance, in diverse tissues.