The Integrator Complex: A Multi-Tool For Metazoan Transcription
RNA Polymerase II
Transcriptional regulation is fundamental for the function and proliferation of eukaryotic cells. Since the discovery of RNA Polymerase II (RNAPII), our understanding of the mechanisms of transcription has transformed what we know about basic cell biology and its impact on disease. Precise timing and coordination of gene expression is vital for the proper development of eukaryotes, and dysregulation of transcription is a hallmark of tumorigenesis. To translate our knowledge of cellular processes into innovations for cancer and developmental disease, a comprehensive understanding of transcription is essential. The Integrator Complex is a transcriptional co-regulator complex found only in metazoa. Integrator has been shown to regulate multiple facets of transcription, from processing UsnRNAs to regulating distinct steps of RNAPII at protein-coding genes. However, the functions of most Integrator subunits remain unknown. For this thesis, my goal was to investigate the contribution of subunit modules towards the complex’s multifaceted roles in transcriptional regulation. In the first chapter, I identify a novel submodule of the Integrator complex with protein phosphatase 2A (PP2A). I demonstrate that the subunit INTS6 recruits PP2A to sites of active transcription where it dephosphorylates numerous substrates, including the C-terminal domain (CTD) of RNAPII, counteracting the pro-elongation activity of the kinase CDK9. I show that loss of INTS6 confers resistance to CDK9 inhibition in normal and tumor cell lines, leading to amplified transcriptional responses. These data identify a submodule of the Integrator complex responsible for recruiting PP2A to RNAPII, leading to fine-tuned gene expression through a kinase-phosphatase checkpoint. In the second chapter, I show that INTS13 functions as an independent submodule of Integrator and regulates enhancer activation in monocyte-macrophage differentiation. I reveal that INTS13 associates with NAB2 and the transcription factor EGR1 to activate poised enhancers, eliciting chromatin looping and gene activation. Independent depletion of INTS13, NAB2, or EGR1 impairs monocytic differentiation in cell lines and primary human progenitors. These data demonstrate an additional submodule of Integrator that regulates transcription through enhancers for proper differentiation. Altogether, this thesis demonstrates that the Integrator complex utilizes distinct submodules to carry out its regulatory functions, which are integral for fine-tuned transcriptional responses in metazoans.
Rahul M. Kohli