Jagan M. R. Pongubala

Crystal structure of PU.1/IRF-4/DNA ternary complex

The Ets and IRF transcription factor families contain structurally divergent members, PU.1, Spi-B and IRF-4 (Pip), IRF-8 (ICSBP), respectively, which have evolved to cooperatively assemble on composite DNA elements and regulate gene expression in the immune system. Whereas PU.1 recruits IRF-4 or IRF-8 to DNA, it exhibits an anticooperative interaction with IRF-1 and IRF-2. We report here the structure of the ternary complex formed with the DNA binding domains of PU.1 and IRF-4 on a composite DNA element. The DNA in the complex contorts into an unusual S shape that juxtaposes PU.1 and IRF-4 for selective electrostatic and hydrophobic interactions across the central minor groove. Together, the protein-protein and protein-DNA interactions provide insights into the stereochemical basis of cooperativity and anti-cooperativity between Ets and IRF factors.

Gene regulatory networks directing myeloid and lymphoid cell fates within the immune system

Considerable progress is being achieved in the analysis of gene regulatory networks that direct cell fate decisions within the hematopoietic system. In addition to transcription factors that are pivotal for cell fate specification and commitment, recent evidence suggests the involvement of microRNAs. In this review we attempt to integrate these two types of regulatory components into circuits that dictate cell fate choices leading to the generation of innate as well as adaptive immune cells. The developmental circuits are placed in the context of a revised scheme for hematopoiesis that suggests that both the innate (myeloid) and adaptive (lymphoid) lineages of the immune system arise from a common progenitor.

Normal Myeloid Development Requires Both the Glutamine-Rich Transactivation Domain and the PEST Region of Transcription Factor PU.1 but Not the Potent Acidic Transactivation Domain

ABSTRACT Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore,PU.1 −/− embryonic stem (ES) cells fail to differentiate into Mac-1+ and F4/80+macrophages in vitro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability ofPU.1 −/− ES cells to differentiate into macrophages. In this study, we take advantage of ourPU.1 −/− ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. ThePU.1 −/− ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages.

AKT Induces Transcriptional Activity of PU.1 through Phosphorylation-mediated Modifications within Its Transactivation Domain

Signal transduction by the antigen receptor complexes is critical for developmental progression of B-lymphocytes, which are defined by assembly and sequential expression of immunoglobulin genes, which in turn are regulated by the enhancer elements. Although proximal antigen-receptor signal transduction pathways are well defined, the precise nuclear factors targeted by these signals remained unknown. Previous studies have demonstrated that tissue-restricted transcription factors including PU.1 and PU. 1 interaction partner (PIP) function synergistically with c-Fos plus c-Jun to stimulate the κE3′-enhancer in 3T3 cells. In this study, we demonstrate that the functional synergy between these factors is enhanced in response to mitogen-activated protein kinase kinase kinase, in 3T3 cells, where the enhancer is inactive. However in S194 plasmacytoma cells, mitogen-activated protein kinase kinase kinase was able to stimulate the activity of PU.1 but unable to induce the κE3′-enhancer activity. We have found that Ras-phosphoinositide 3-kinase-dependent externally regulated kinase, AKT, induces κE3′-enhancer activity in both pre-B and plasmacytoma cells. AKT stimulation of the κE3′-enhancer is primarily due to PU.1 induction and is independent of PU.1 interaction with PIP. Activation of AKT had no effect on the expression levels of PU.1 or its protein-protein interaction with PIP. Using a series of deletion constructs, we have determined that the PU.1 acid-rich (amino acids 33–74) transactivation domain is necessary for AKT-mediated induction. Substitution analyses within this region indicate that phosphorylation of Ser41 is necessary to respond to AKT. Consistent with these studies, ligation of antigen receptors in A20 B cells mimics AKT activation of PU.1. Taken together, these results provide evidence that PU.1 is induced by AKT signal in a phosphoinositide 3-kinase-dependent manner, leading to inducible or constitutive activation of its target genes.

Gene Regulatory Networks that Orchestrate the Development of B Lymphocyte Precursors

The B cell developmental pathway represents a leading model within the hematopoietc system for the analysis of gene regulatory networks, which orchestrate cell fate specification and commitment. Considerable progress is being made in the characterization of regulatory components that comprise such networks and examining their connectivity. These components include the cytokine receptors Flk2 and IL-7R as well as the transcription factors PU.1, Ikaros, E2A, EBF and Pax-5. We review recent experimental evidence concerning the molecular functions of these regulatory components and attempt to connect them in sequentially acting and inter-dependent regulatory modules.

Developmentally regulated higher-order chromatin interactions orchestrate B cell fate commitment

Abstract Genome organization in 3D nuclear-space is important for regulation of gene expression. However, the alterations of chromatin architecture that impinge on the B cell-fate choice of multi-potent progenitors are still unclear. By integrating in situ Hi-C analyses with epigenetic landscapes and genome-wide expression profiles, we tracked the changes in genome architecture as the cells transit from a progenitor to a committed state. We identified the genomic loci that undergo developmental switch between A and B compartments during B-cell fate determination. Furthermore, although, topologically associating domains (TADs) are stable, a significant number of TADs display structural alterations that are associated with changes in cis-regulatory interaction landscape. Finally, we demonstrate the potential roles for Ebf1 and its downstream factor, Pax5, in chromatin reorganization and transcription regulation. Collectively, our studies provide a general paradigm of the dynamic relationship between chromatin reorganization and lineage-specific gene expression pattern that dictates cell-fate determination.