Amanda Ames

Mentor: Ragunathan
Biological Chemistry

Areas of Interest

In eukaryotes, a major level of gene regulation comes in the form of epigenetics, a change in gene expression which occurs at the level of chromatin structure.  Histone tail post-translational modifications dictate whether a locus is condensed (heterochromatin) or accessible for transcription (euchromatin). Heterochromatin protects the cell from genome instability, contributes to proper chromosome segregation through centromere function, and dictates transcriptional patterns essential for cell differentiation. In fission yeast, heterochromatin formation is dependent on methylation at histone H3 lysine 9 (H3K9me) which is regulated by a variety of proteins through chromodomain binding. At the centromere, heterochromatin formation is an RNA interference (RNAi) dependent mechanism.  The centromere contains pericentromeric repeats which are transcribed and processed into small-interfering RNAs (siRNAs). These siRNAs are used by the RNA-induced initiation of transcriptional silencing (RITS) complex, which contains an argonaute protein that binds small RNAs.  Argonaute- siRNA binding works in tandem with Chp1, a chromodomain protein in RITS, to bind nascent RNA transcripts and H3K9me directly on chromatin.  Based on genetic studies, both RNAi and Chp1-mediated H3K9me binding are required for heterochromatin formation in vivo. This  highlights a paradoxical relationship between transcription of noncoding RNAs and the heterochromatin-mediated inhibition of transcription.  The mechanism of this process remains poorly understood and perplexing.  My work involves using a combination of genetics, in vitro biochemistry, and single-molecule microscopy to answer mechanistic questions about the RITS complex and other epigenetic factors in heterochromatin formation.