Dissecting the regulatory roles and cellular functions of mammalian ZSCAN5B and primate-specific paralogs

Certain subfamilies of zinc finger transcription factors (ZNF-TFs), especially those including KRAB or SCAN protein-interaction domains, have evolved rapidly in mammals due to repeated rounds of gene duplication, deletion, and divergence. The functions of the encoded proteins, and the evolutionary role of their duplication and divergence, are not well understood. I have focused on mammalian ZNF subfamilies that are rooted by relatively unique, conserved members but that have duplicated to generate groups of divergent paralogous transcription factors in primate species. One such family is the ZSCAN5 subfamily, represented by a single, conserved founding member, ZSCAN5B, and clustered paralogs that arose early in primate history, and this clustered subfamily has been the subject of my thesis work. I have shown that three of the primate paralogs, ZSCAN5A, B, and D, are expressed in different tissues and cell types while ZSCAN5C is not detectably expressed in most tissues. Using chromatin immunoprecipitation (ChIP-seq) in human cell lines and mouse placenta tissue, I have identified mammalian ZSCAN5B as a protein that binds with high specificity to tRNA genes (tDNA) and other polymerase III (Pol III) transcripts including several types of transposable elements (TEs). Primate-specific paralogs ZSCAN5A and ZSCAN5D also bind tDNAs, although ZSCAN5D preferentially recognizes a subset of TE-derived "extra-TFIIIC" or ETC sites. I also used siRNA knockdown followed by transcriptome sequencing (RNA-seq) to identify biological functions of the dominantly expressed paralog ZSCAN5A and the ancestral ZSCAN5B gene. In addition to tDNAs and other Pol III transcripts, I show that ZSCAN5A and ZSCAN5B gene knockdown also dysregulates many Pol II genes including some that are located near the DNA binding regions. Significant overlap between the differentially expressed gene (DEG) sets detected after ZSCAN5A or ZSCAN5B knockdown suggested that the two proteins may act cooperatively through their similar SCAN domains. The DEG sets thus suggested similar biological functions for ZSCAN5A and ZSCAN5B, but also highlighted some potentially distinct activities. In particular, based on these data I hypothesize that ZSCAN5B primarily regulates ribosome biogenesis and tRNA processing, whereas ZSCAN5A has an especially important role in mitotic progression. Consistent with this latter function, I show that ZSCAN5A as well as its human paralogs are cell-cycle regulated with peak expression around the time of mitosis. To test these hypotheses about gene function, I generated a series of resources including both human cell lines and transgenic mice engineered to overexpress or knock down ZSCAN5 genes specifically. Focusing on the predicted role of ZSCAN5A in cell cycle progression, I showed that ZSCAN5A knockdown dysregulates cell cycle progression in cultured human cells, leading to an accumulation of cells in mitotic phase and the appearance of aneuploid cells. These data suggest that, either through a cyto-architectural function, regulation of Pol III transcription, position effects on Pol II genes, or a combination of these roles, ZSCAN5A has an essential role in maintenance of chromosome integrity in human cells. These data open up many new avenues for future exploration. Together these data define unexpected functions for a conserved mammalian gene and two paralogs found only in primate genomes. The human paralogs have evolved from the basic tDNA-binding function of the conserved founder gene, ZSCAN5B, to bind more widely to Pol III transcripts and other TFIIIC binding sites and affect the transcription of a wider selection of nearby genes. Data presented here indicate that despite being duplicated only in primates, ZSCAN5A has been integrated into the control of a very ancient process, namely chromosome segregation during mitosis, and plays an important role in chromosome integrity. The new tools and resources I have developed provide essential tools needed to explore those functions in further depth