In the crowded and dynamic three-dimensional space of the nucleus, active genes often share
local neighborhoods with one another. Despite significant recent progress in analyses of 3D
positioning of genes, it is not yet fully understood how the co-localization of genes plays a role in
their transcriptional output. The long-term goal of this research is to understand how a gene’s
expression is influenced by its neighbors in space and time. The study of how co-localized
genes share local resources has the potential to shed significant light on fundamental
mechanisms of transcription, but progress has been severely limited by a reliance on methods
that examine transcription and 3D position separately in fixed or disrupted tissues. The objective
of this proposal is to uncover the impact of gene co-localization on transcriptional dynamics in
living cells to provide a dynamic spatiotemporal quantification of transcription for genes that
share a local 3D neighborhood. To address this objective, the proposal focuses on Drosophila
melanogaster and takes advantage of a phenomenon called transvection, where sequences at
allelic positions on homologous chromosomes are stably co-localized via a process called
somatic homolog pairing. Furthermore, the proposed experiments employ the MS2 and PP7
systems for live analysis of transcriptional dynamics, which permit simultaneous quantification of
transcriptional activity and assessment of the 3D positions of genes over time in living cells. The
central hypothesis guiding the proposal is that co-localization of genes via somatic homolog
pairing will result in the sharing of local stores of resources necessary for transcription, which
can be understood by analyzing how co-localization influences the parameters of transcriptional
dynamics, including the frequency, duration, and amplitudes of transcriptional bursts. This
hypothesis is tested in the context of two contrasting scenarios resulting from co-localization of
genes via transvection: in one scenario, co-localized promoters in cis and in trans to an
enhancer compete for activation, while in a second scenario, co-localized enhancers cooperate
to achieve augmented levels of transcriptional output. The proposed experiments have the
expected outcome of identifying precisely and quantitatively how interactions between co-
localized genes influence transcriptional dynamics, providing key insights into mechanisms by
which 3D genome organization controls gene expression.
