Role of an Integrator-EGR axis in the regulation of myeloid enhancers Objective:
My research aims to identify how enhancer-based regulation determines monocytic cell fate. Specifically, we seek to understand the function of a newly identified regulatory axis, comprising the Integrator complex, the EGR-1/EGR-2 transcription factors and their co-factor NAB2.
We aim at determining how these chromatin regulators: a) prime myeloid enhancers for activation; b) remodel nucleosome accessibility and reshape 3D genome conformation to impose a monocytic-specific transcriptome. We seek to characterize the first lineage-specific function of the Integrator protein complex, which was previously believed to serve a general role in transcription. Proposed research: The process of myelopoiesis is governed by an elaborate gene expression program that originates in the bone marrow, and is brought to maturity in the peripheral blood and tissues. The transcriptional process that generates mature myeloid cells from hematopoietic stem cells (HSCs) is initiated by sequence-specific transcription factors (TFs) that instruct enhancers to elicit gene activation. However, how these myeloid TFs engage the basal transcriptional machinery and activate lineage-specific enhancers is poorly understood. We uncovered that the INTS13 subunit of the Integrator complex plays an essential role in myelopoiesis and we propose that Integrator carries the previously unidentified ability to target myeloid-specific TFs and modulate cell fate determination via enhancer regulation. We will molecularly dissect the function of Integrator, and its functional partners EGR-1/2 and NAB2, in monocytic/macrophagic differentiation by leveraging our expertise in biochemistry and functional genomics through the following aims. 1) We will define the enhancer network that determines myelopoiesis. We hypothesize that INTS13 modulates Integrator?s activity at EGR-targeted enhancers during monocytic commitment. Therefore, we will profile INTS13 and EGR-1/2 recruitment in human cells, and we will determine the status of targeted enhancers by monitoring their histone methylation and acetylation levels (H3K27Ac and H3K4Me1). Further, we will evaluate the transcriptional effect of INTS13, EGR-1/2 and NAB2 depletion on myeloid genes by RNA-seq. 2) We will determine the changes in chromatin architecture mediated by INTS13, EGR-1/2, and NAB2. We hypothesize that these chromatin regulators prime enhancers for activation and execute cell fate commitment. Our data suggest that INTS13, EGR-1/2, and NAB2 control the active/poised status of enhancers and oversee genome topology during differentiation. We will functionally test our hypothesis by analyzing nucleosome remodeling at enhancers (pioneer activity) and by determining how Integrator and EGR-1/2 modulate chromosomal looping and 3D genome conformation using Hi-C and 3C assays. Lastly, we will characterize the biochemical properties of Integrator to identify the myeloid-specific module that regulates EGR-activity in monocytes.
This proposal seeks to understand the molecular mechanism that regulates the commitment of multipotent progenitor cells in the bone marrow to become fully differentiated monocytes and macrophages. These findings will be important to appreciate the physiology of myeloid differentiation and may reveal new druggable nodes for Acute Myeloid Leukemia and other myeloproliferative diseases.