My laboratory assembles and analyzes gene regulatory networks that orchestrate the development of various cell types of the hematopoietic and immune systems, including B and T lymphocytes, macrophages, dendritic cells, neutrophils, and mast cells. These networks comprise interconnected transcription factors that regulate cell fate choice and developmental transitions. We are also interested in manipulating these regulatory networks to engineer stem cells to adopt particular immune cell fates.
Gene Regulatory Networks That Dictate Cell Fate Choices in the Immune and Hematopoietic Systems
Our long-standing interest in the regulation of cell fate choices in the immune and hematopoietic systems was initiated by our genetic analysis of the transcription factor PU.1, a member of the Ets superfamily. We demonstrated that PU.1 is specifically required for the development of the innate (macrophages and granulocytes) and the adaptive (B and T lymphocytes) lineages of the immune system. PU.1 was suggested to function in a cell-intrinsic manner at the level of multipotential progenitors that give rise to both the innate and adaptive cells of the immune system. Our proposal has been supported by work from other laboratories involving the characterization of hematopoietic intermediates that can generate macrophages, granulocytes, and lymphocytes but lack the potential to develop into erythrocytes and megakaryocytes. This has resulted in a revised developmental framework for hematopoiesis.
To explore molecular functions of PU.1 in the development of the lymphoid-myeloid system, we were among the first to establish powerful cell culture complementation systems. These systems enabled us to complement the PU.1 mutation in distinct lineages, as well as to bypass selectively the various molecular functions of PU.1. These analyses revealed that a major function of PU.1 in hematopoiesis is to regulate the transcription of genes encoding key cytokine receptors. Furthermore, we showed that graded levels of PU.1, as well as its cooperative or antagonistic interplay with the GATA family of transcription factors, regulate the specification of distinct cell fates within the hematopoietic system.
We are analyzing the molecular mechanisms by which PU.1 regulates cell fate choice in the context of macrophages and neutrophils. We have elucidated a novel regulatory circuit composed of counter antagonistic repressors Egr/Nab and Gfi-1, which function to resolve an initial mixed-lineage pattern of gene expression into one that is specific for macrophages or neutrophils. In collaboration with Aaron Dinner (University of Chicago), we have used these results to assemble and mathematically model a gene regulatory network that exhibits both graded and bistable behaviors and more generally accounts for the onset and resolution of mixed-lineage patterns during cell fate determination.
Our continuing analysis of the role of PU.1 in early B cell development led to the discovery that PU.1 is required for the developmental induction of the transcription factor EBF that in turn functions as the key B cell fate determinant. This analysis led us to propose a comprehensive hierarchical regulatory network for specification and commitment to the B cell fate (see figure).
We are currently utilizing a combination of genetic, molecular, and mathematical modeling approaches to assemble and analyze gene regulatory networks orchestrating cell fate choices in the hematopoietic system. These include array-based chromatin-crosslinking analyses, to connect each transcription factor in a given network with its large set of target genes, and high-throughput functional screens of lineage- and stage-specific cis-regulatory elements.
Regulation of Discrete Developmental Transitions Within the B Cell Developmental Pathway
We have obtained novel insights into two major transitions within the B cell developmental pathway, the pre–B to B cell and the B cell to plasma cell, by genetically analyzing the transcription factor Pip (IRF-4). Pip (PU.1 interaction partner) is an immune system–specific member of the interferon regulatory factor (IRF) family that my laboratory cloned in collaboration with Ursula Storb's group (University of Chicago). The characterization of Pip (IRF-4) led to the identification of a second immune-specific IRF, ICSBP (IRF-8), which specifically interacts with PU.1. These complexes were biochemically and structurally intriguing because Pip is recruited to its binding site on DNA by phosphorylated PU.1. We have used a variety of biochemical and structural approaches to analyze the assembly of PU.1/Pip/DNA ternary complexes.
Given that IRF-4 and IRF-8 interact with PU.1 to regulate the activity of immunoglobulin (Ig) light-chain gene enhancers, we reasoned that the two factors may function interchangeably to control light-chain gene recombination and the pre–B to B cell transition. We confirmed our prediction by demonstrating that B-lineage cells lacking IRF-4 and IRF-8 undergo a precise developmental arrest at the cycling pre–B cell stage and are blocked for light-chain gene recombination. Using IRF-4,8–/– pre–B cells, we have recently shown that two molecular pathways converge to drive light-chain rearrangement synergistically. We propose that stage-specific activation of light-chain recombination during B cell development is ensured by a combination of acquired pre-BCR (pre–B cell receptor) and attenuated IL-7 (interleukin-7) signaling.
Curiously, IRF-4 also regulates the transition from B cell to plasma cell. This terminal differentiation program involves a transient developmental state (germinal center B cell) that enables Ig gene class switching and somatic hypermutation. Our laboratory and that of Riccardo Dalla-Favera (Columbia University) have independently demonstrated that IRF-4 regulates both isotype switching and plasma cell differentiation. Our analysis has revealed that IRF-4 regulates these processes by controlling the expression of the AID and Blimp-1 genes, respectively. We have proposed and generated a mathematical model for a gene regulatory network in which graded expression of IRF-4 regulates the transition from isotype switching to plasma cell differentiation.
Nuclear Compartmentalization, Transcription, and Recombination Dynamics of Immunoglobulin Loci
My laboratory has had a long-standing interest in the regulation of Ig gene transcription and recombination. We have focused particularly on cell biological and molecular mechanisms that could facilitate long-range interactions between transcriptional elements such as promoters and enhancers or between DNA recombination signals flanking widely separated Ig gene segments. Using altered DNA-specificity mutants, we uncovered a fundamental mechanism that enables Ig gene enhancers to activate transcription from a distal promoter. The mechanism involves localized recruitment of TBP (TATA-box–binding protein) via the POU DNA-binding domain of the transcription factor Oct-1. Our results suggest that in order for enhancers to function from long-range, TBP complexes need to be preassembled at promoters via localized recruitment by a proximal activator.
Given the unusual structural organization of Ig gene loci (megabase domains containing large numbers of iterated variable gene segments), we hypothesized that the transcription and recombination of these loci may also be regulated by nuclear compartmentalization and exceptional intrachromosomal dynamics. Using three-dimensional immuno-FISH, we were the first to demonstrate that Ig loci undergo developmentally regulated nuclear compartmentalization. The germline loci are associated with the nuclear lamina in multipotential hematopoietic progenitors and move away from the nuclear periphery in developing B- but not T-lineage cells as they prepare to undergo recombination. Furthermore, widely separated Ig gene segments appear to be more closely positioned in B-lineage nuclei, suggesting the involvement of a structure that could facilitate long-range DNA recombination via DNA looping.
While studies on Ig and other loci have correlated positioning at the nuclear lamina with gene repression, the functional consequence of this compartmentalization has remained untested. We have devised a new approach for inducible tethering of genes to the inner nuclear membrane (INM) and have demonstrated repositioning of chromosomal regions to the nuclear lamina. Such repositioning results in gene repression, and a similar mechanism likely contributes to the lineage-restricted activity of Ig loci. Our future studies in this area are directed at identifying and analyzing cis-elements and trans-factors that regulate the positioning of the IgH locus at the INM–nuclear lamina compartment.
