Abstract: Transcription is one of the first steps in gene expression, and the many protein factors involved must
be tightly regulated to correctly carry out cellular processes. The Integrator complex, a 15-subunit metazoan-
specific complex, was initially discovered to be crucial for the proper transcription of certain non-coding RNAs.
Recently, it has been found that Integrator is a broad transcriptional regulator with roles at many protein-coding
and non-coding genes. Integrator acts as a negative regulator of RNA Polymerase II (RNAPII) transcription
through promoter-proximal termination and can alter its functionality in response to stress, such as DNA damage
or reactive oxygen species. Despite its critical role in fine-tuning transcriptional regulation, it is not yet understood
how Integrator is assembled and regulated, presenting a knowledge gap in the field.
Our preliminary experiments strikingly revealed that a key factor involved in DNA damage repair (DDR)
associates with a global regulator of RNAPII activity, thus providing a previously unknown connection between
these two processes. Specifically, the BRCA1-ATM activator 1 (BRAT1) associates with Integrator subunit 11
(INTS11), the endonuclease-containing subunit within the Integrator complex. BRAT1 is a cytoplasmic protein
that re-localizes to the nucleus upon DNA damage to act as a scaffold between the site of damage and repair
proteins. Our cryo-EM analysis of the INTS11-BRAT1 heterodimer reveals that BRAT1 forms a circular structure
around INTS11 and uses three conserved C-terminal residues to bind to the INTS11 active site, preventing its
cleavage of RNA. Additionally, the cryo-EM analysis showed that INTS9 can bind to form an INTS11-INTS9-
BRAT1 heterotrimer, suggesting interplay between BRAT1 as an inhibitor of INTS11 and other members of the
Integrator complex. The goal of this proposal is to examine the function and mechanism of action of these two
unique complexes. To examine the function, our lab generated BRAT1 null cell lines and a rapid depletion system
using the auxin-inducible degron (AID) tag to allow quick depletion of INTS11. Initial studies of these lines show
that BRAT1 loss results in an INTS11 depletion phenotype, presenting upregulation of genes negatively
regulated by Integrator. Overall, these data present a model where BRAT1 could regulate Integrator activity
through its association with INTS9/11. The hypothesis is that BRAT1 acts as a licensing factor, facilitating the
assembly of the Integrator complex and providing precise regulation, which is especially important during cellular
stress such as DNA damage. To test this hypothesis, three aims are proposed. First, the interaction between
BRAT1 and INTS9/11 will be biochemically defined to further probe the structure. Next, the impact of BRAT1 on
the canonical Integrator complex function will be determined. Finally, the impact of INTS11-INTS9-BRAT1 during
DNA damage repair will be elucidated. Successful completion of these aims will provide a more thorough
understanding of the regulation and assembly of the Integrator complex.