Causes and consequences of gene silencing
Causes and consequences of gene silencing
Gene silencing mechanisms are critical for genome stability and development. They go awry in a variety of human diseases, especially cancer. Key mechanisms underlying gene silencing include chromatin regulation and control by small RNAs. We study these questions in yeast systems (S. pombe and C. neoformans) because they are have mammalian-like silencing systems (H3K9me, H3K27me, DNA methylation and RNAi), yet are highly experimentally tractable (short generation times, haploid genetics, and numerous additional tools). This enables us to tackle questions that would be difficult or impossible to address in more complex systems.
How Long Can Cells Remember Their Past?
Cytosine methylated on the five position in DNA (5mC) is a repressive modification that can be epigenetically inherited in eukaryotes. Most cytosine residues in the human genome are methylated and defects in a methyl-cytosine “reader” protein causes Rett Syndrome, a devastating inherited neurological disorder with similarities to autistm. De novo DNA methyltransferases (Dnmts) catalyze the modification of 5mC on unmethylated DNA. Their action can be subsequently “remembered” by maintenance-type Dnmts that recognize a hemimethylated substrate.
We recently discovered that the sole Dnmt in the yeast Cryptococcus neoformans is a maintenance-type enzyme. This organism has no de novo enzyme, raising the question of how 5mC is established. Phylogenetic and experimental analysis revealed that the ancestral species harbored a de novo Dnmt, DnmtX. Remarkably, the gene for DnmtX was lost between 50-100 million years ago, raising the possibility of extremely long-term epigenetic memory. We are investigating how 5mC is maintained in the C. neoformans lineage without the de novo enzyme.
A Polycomb Memory System in A Budding Yeast
Polycomb is a conserved repression system critical for animal and plant development. It mediates epigenetic memory via a poorly understood chromatin-based mechanism that involves the writing and reading of H3-K27 methylation. We have characterized the Polycomb system that assembles repressive subtelomeric domains of H3K27 methylation (H3K27me) in the yeast Cryptococcus neoformans. Purification of this PRC2-like protein complex reveals orthologs of animal PRC2 components as well as a chromodomain-containing subunit, Ccc1, which recognizes H3K27me. Whereas removal of either the EZH or EED ortholog eliminates H3K27me, disruption of mark recognition by Ccc1 causes H3K27me to redistribute. Strikingly, the resulting pattern of H3K27me coincides with domains of heterochromatin marked by H3K9me. Indeed, additional removal of the C. neoformans H3K9 methyltransferase Clr4 results in loss of both H3K9me and the redistributed H3K27me marks. These findings indicate that the anchoring of a chromatin-modifying complex to its product suppresses its attraction to a different chromatin type, explaining how enzymes that act on histones, which often harbor product recognition modules, deposit distinct chromatin domains despite sharing a highly abundant and largely identical substrate-the nucleosome.
The yeast system does not appear to involve a PRC1-like complex, suggesting that this PRC2 might have both mark depositing and silencing effector functions. Using the power of yeast, we are investigating how this system is targeted to the correct chromosomal sites, how it represses transcription and testing whether it mediates epigenetic memory.
How is HP1 Heterochromatin Assembled at the Correct Sites?
Heterochromatin marked by heterochromatin protein 1 (HP1) is a conserved feature of eukaryotic chromosomes and plays myriad critical roles in genome stability, cell type programming, and human disease.
Landmark studies in the yeast S. pombe revealed that the RNAi machinery is required for constitutive heterochromatin formation marked by H3K9me. However, in S. pombe and in numerous other organisms, it is clear that RNAi-independent mechanisms are critical for heterochromatin assembly, yet these mechanisms remain mysterious.
The crux of the issue is how cells recognize and silence repeats and transposable elements when these tend to be among the most rapidly evolving sequences in genomes? In published work, we described a key component of the RNAi-independent pathway, a PolII associated RNA-binding protein called Seb1. Using Seb1 as tool, we are currently elucidating the molecular function of Seb1 and relating this function to its role in triggering heterochromatin. These studies are elucidating the role of noncoding transcription in triggering heterochromatin formation independently of the RNAi machinery. Such findings will likely apply to other organisms as heterochromatin transcription is a widely conserved phenomenon.
How Are Transposons Targeted for RNAi?
Transposons are a mortal threat to genomes as they are selfish elements whose only goal in life is to self-propagate. RNAi systems evolved to combat this threat, but it is not well understood how tranposons are distinguished from normal cellular genes. We discovered that in Cryptococcus, stalled spliceosomes are a signal for the production of endo-siRNAs. In other words, assembly onto the spliceosome but subsequent stalling due to poor introns is necessary for siRNA production. These studies have led us investigate the determinants of spliceosome assembly and catalysis using new genome-wide methods that have been developed in the laboratory (the “spliceosome profiling suite”).