Frank J. Rauscher, III, Ph.D.
Establishing, maintaining, and controlling differentiation of the stem cell niche in many diverse tissues is undoubtedly critical for mammalian development and homeostasis. These cells must retain and exploit the ability to reverse “memorized” states of gene activation and repression and be able to essentially “re-set the switches” of complete transcriptomes in order to spawn new cell phenotypes. It is also clear that a new breed of therapeutic agents may have the ability to target these mechanisms. In order to both develop new transcriptional therapies and exploit the stem cells therapeutically, scientists must continue to define the molecular mechanisms that allow for coordinated activation or repression of specific sets of target genes.
Targeting the enzymatic machinery, which modifies chromatin structure, is a promising tactic as this has emerged as a primary mechanism of gene regulation. Post-transcriptional modification of core histone tails via phosphorylation, acetylation/de-acetylation, methylation and ubiquination and sumoylation are key as are the proteins that recognize these modifications.
Research in the lab has been focusing on proteins such as HP1 that directly recognize modified histone tails and lead to gene silencing. HP1 recognizes histone H3 that is modified by methylation at Lysine 9 and this is required for gene silencing. HP1 expression is lost during the progression of breast cancers to metastatic potential. Reversing HP1 mediated gene silencing must be overcome in order to re-program gene expression in both cancer and normal cells. The lab has been interested in how this can be accomplished in mammalian stem cells and in cancer stem cells. Heterochromatin protein 1 (HP1) in Drosophila is required for stable epigenetic gene silencing classically observed as position effect variegation of a transgene integrated adjacent to constitutive heterochromatin. However, mammalian HP1 proteins may be euchromatic, can be deposited on active genes by specific corepressors and anchored there by histone H3 containing the lysine 9-methylation mark. Little is known about the physical properties of chromatin that contains euchromatic genes that are silenced via HP1 recruitment.
The lab discovered that the KRAB-zinc finger superfamily of silencers, via association with their obligate co-repressor KAP1 can coordinate histone deacetylation, histone methylation and HP1 deposition to silence euchromatic genes. In recent work, a mammalian cell culture based system for hormone-regulated recruitment of this machinery to an expressed transgene has resulted in the following discoveries. In the presence of hormone, the transgene is rapidly repressed, the gene is spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with HP1/KAP1 over a highly localized region centered around the promoter.
Remarkably, once repression is established (via a 48 hour pulse of hormone) the silenced state is stably maintained for >50 population doublings in the absence of hormone at high frequency in clonal cell populations. This stable silencing is maintained in the absence of the DNA binding component and is highly reminiscent of HP1 dependent gene variegation in flies. The frequency of silent clone generation is increased by HP1alpha dose but not by HP1gamma. However, unlike variegation in flies, the silent state does not spread to adjacent euchromatic transcriptional units. Detailed analysis of clonal silent cell lines has shown a region comprising only 3-4 nucleosomes at the promoter is highly enriched in trimethylated H3-MeK9, the SETDB1 methyltransferase and KAP1, thus showing that once the machinery is nucleated via a DNA binding protein, it can be maintained at the locus in its absence. Thus, in mammalian cells, highly localized recruitment of HP1 to a euchromatic promoter establishes a mitotically heritable silenced chromatin state. In current studies the lab discovered that silencing by this system requires post-translational modification be the Ubiquitin-like protein SUMO. They mapped the sites of SUMOylation and discovered the E3 ligase required for SUMO addition.
Another project on cancer-related gene silencing concerns the SNAIL zinc-finger proteins that repress E-Cadherin (and other genes) during the Epithelial-Mesenchymal Transition (EMT) de-differentiation pathway, which occurs during metastatic progression. The lab cloned and characterized a novel co-repressor for the SNAIL and showed that it is required for E-Cadherin repression. The protein is a novel member of the LIM domain family and exists both in the cytoplasm and the nucleus. Current studies are underway to define the genomic binding sites and associated proteins. To this end the lab recently discovered that the LIM protein associates directly with an enzyme, which displays arginine, methyltransferase activity and that this enzyme is required for silencing.