Kruppel-associated box zinc-finger proteins (KRAB-ZFPs) make up the largest family of transcription factors in humans. These proteins emerged in the last common ancestor of coelacanth and tetrapods, and have expanded and diversified in the mammalian lineage. During evolution, KRAB-ZFPs and retrotransposons are co-expanding and co-diversifying in the vertebrate genomes, and growing evidence from loss of function studies in the mouse and genome-wide binding studies strongly supports that the “arm race” between KRAB-ZFPs and retrotransposons provides an engine that facilitates mammalian and human adaptations. Previous studies have shown that transcriptome fluctuations of retro-transposons are remarkable features for early embryonic development. However, which KRAB-ZFPs are responsible for their transcriptional regulation is still need to be identified. My long-standing interest has been to characterize KRAB-ZFPs in embryonic development and uncover their molecular mechanisms. To this end, I have developed CRISPR/Cas9 gene editing system to generate KRAB-ZFPs floxed knockin mice and cell lines. With the combination of conditional Cre mice and cell lines, we have been able to efficiently and accurately knockout KRAB-ZFP genes temporally and spatially. Our lab focus on the following aspects:

1.     Establish the networks between KRAB-ZFPs and retrotransposons

Thousands of retrotransposons occupy a large proportion of the genome ~10%, and the number of KRAB-ZFPs also reaches up to hundreds. Previous studies suggested that the relationship between the two families is not a one-to-one correspondence. For example, IAPEz (intracisternal A particles) of mouse retrotransposon ERVK family has 26,000 copies in the genome, with large amounts of H3K9me3 signals enriched in its region. However, knockout of a single bound KRAB-ZFP gene could not reactivate the retrotransposon, indicating that IAPEz may be co-regulated by multiple KRAB-ZFPs simultaneously. On the other hand, the KRAB-ZFP-regulated retrotransposon elements are widely present in a variety of retrotransposons. For example, the PBS-pro (proline tRNA primer-binding site) element specifically recognized by ZFP809 is present in both MMVL30 and RLTR6 retrotransposons. Therefore, there is strong evidence suggesting complex regulatory networks exists between KRAB-ZFPs and retrotransposons. With combinatory approaches integrating biochemistry, high-throughput sequencing, unbiased screening, and CRISPR/Cas9 gene-editing system, we attempt to identify the regulatory networks between the key KRAB-ZFPs and their corresponding retrotransposons during embryonic development.

2.     Identify the epigenetic interactions between KRAB-ZFPs and imprinted genes

The epigenetic features of the imprinted genes are retained during early embryonic development. Recent studies have reported that multiple ZFPs are involved in the process. For example, ZFP57, ZFP568, and CTCF play essential roles in the transcriptional regulation of imprinted gene IGF2 during early embryonic development. By genome-wide mapping of the overlapping regions between the imprinting genes and the KRAB-ZFPs binding sites, we have been able to predict some KRAB-ZFPs that may regulate the imprinted genes. We are going to knock out these  KRAB-ZFPs in CAST/Eij; C57BL/6 hybird lines followed by high throughput sequencing analysis.

3.     Explore the antagonistic mechanisms of KRAB-ZFPs and CTCF on chromatin structure

CTCF acts as a chromatin insulator to prevent the spread of heterochromatin genome-wide and to maintain proper chromatin higher-order structure. Our study has found that CTCF binding sites in the genome may overlap with the target sites of specific KRAB-ZFPs  (especially the retrotransposon element sequences and imprinted regions),  indicating that KRAB-ZFPs may compete CTCF for DNA binding, thus affect the high-order structure of chromatin. With chromatin capture technique, we attempt to study the interaction among KRAB-ZFPs, CTCF, and retrotransposons/imprinted genes, thus further illustrating the epigenetic mechanisms of chromatin structure dynamics, especially heterochromatin structure dynamics, during embryonic development.