Project Summary: Heterochromatin refers to the compacted and transcriptionally suppressed chromatin state
that typically includes repeat-rich regions near chromosomal arm ends, transposable elements (TEs), as well
as some genes. Heterochromatin plays important architectural and regulatory roles, and its misregulation leads
to aberrant gene expression and chromosome instability associated with cancers, aging and in germ cells,
embryonic lethality and sterility. Large fraction of heterochromatin from yeast to humans is marked by histone
H3 lysine 9 trimethylation (H3K9me3). H3K9me3 is deposited by histone mark “writer” complexes which can be
recruited to genomic targets by DNA binding proteins or small RNA guides. Many aspects of heterochromatin
establishment and maintenance in the cell and in development remain poorly understood. Heterochromatin
regulation is the central focus of this proposal. In germ cells, Piwi proteins and associated Piwi-interacting
small RNAs (piRNAs) guide a writer complex to install the H3K9me3 mark and induce transcriptional silencing
at TE targets. TE repression by piRNAs is essential for animal fertility, yet its mechanism is not known.
Candidate's previous work showed that localization to chromatin of the conserved SUMO E3 ligase Su(var)2-
10 induces heterochromatin formation in germ cells of the Drosophila ovary. Data led to a model that Su(var)2-
10 forms a complex with piRNA-Piwi at genomic targets, and deposits SUMO at yet-to-be-established factor(s),
which in turn recruits the H3K9me3 writer dSetDB1. Su(var)2-10 also controls H3K9me3 deposition at piRNA-
independent loci, including genes of several silencing factors, indicating a novel negative feedback mechanism
between heterochromatin levels and silencing factors that can explain how germ cells maintain
heterochromatin levels to ensure proper genome function. This proposal presents a strategy to elucidate the
role of Su(var)2-10/SUMO in piRNA-guided silencing, and to investigate the auto-regulation and developmental
inheritance of Su(var)2-10 dependent heterochromatin. The candidate will characterize the substrates of
SUMO modification by Su(var)2-10 using state-of-the-art proteomics coupled with RNAi (Aim 1), and use
biochemical and genetic approaches to investigate the mechanisms that lead to Su(var)2-10 localization and
SUMO-dependent dSetDB1 recruitment to genomic targets (Aim 2). In the long term, the candidate will
investigate the proposed model of heterochromatin regulation by negative feedback, and study the stability of
repressed chromatin states induced by Piwi and Su(var)2-10 across development (Aim 3). Together, this
project will provide deep mechanistic insight into heterochromatin formation in germ cells, and address
fundamental principles of epigenetic regulation relevant to normal cell function and disease states. Aim 1 and 2
will be initiated during the K99 phase in Dr. Alexei Aravin's lab at Caltech. This environment will provide all
necessary research facilities and training to achieve the proposed goals, and to generate reagents and data for
future studies, allowing a smooth transition to an independent researcher phase (Aim 3/R00).
