Harinder Singh, Ph.D.

Gene Regulatory Networks Orchestrating Development of the Innate and Adaptive Immune System

Research Summary

My laboratory is assembling and analyzing gene regulatory networks that orchestrate the development of various cell types of the innate and adaptive immune system including B-and T lymphocytes, macrophages, neutrophils dendritic and mast cells. These networks comprise of inter-connected transcription factors, signaling molecules and miRNAs that regulate cell fate choice and developmental transitions. We are also interested in manipulating these regulatory networks to engineer stem cells to efficiently adopt particular immune cell fates. Currently, the laboratory has the following three research foci:


I. Gene regulatory networks that dictate cell fate choices in the immune system

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 super-family. We demonstrated that PU.1 was specifically required for the development of both the innate (macrophages and granulocytes) as well as the adaptive (B and T lymphocytes) lineages of the immune system. Importantly, PU.1 was suggested to function in a cell-intrinsic manner at the level of multipotential lymphoid-myeloid progenitors. Recently, hematopoietic intermediates that can generate macrophages, granulocytes as well as lymphocytes but lack erythrocytic and megakaryocytic developmental potentials have been characterized by other laboratories leading to a revised developmental framework for hematopoiesis.

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 comprised 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. Importantly, the experimental results have been used 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 (collaboration with A. Dinner).  

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: ChIP-on-Chip 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.

II. Regulation of discrete developmental transitions within the B-cell developmental pathway

We have obtained novel insights into two major developmental transitions within the B cell developmental pathway, the pre-B to B and the B to plasma cell, by pursuing the genetic analysis of 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 by collaborating with U. Storb’s group. The characterization of Pip (IRF-4) led to the identification of a second immune-specific IRF, ICSBP (IRF-8) that specifically interacts with PU.1. From a biochemical and structural standpoint, these complexes were particularly intriguing because Pip is recruited to its binding site on DNA by phosphorylated PU.1. Using a variety of biochemical and structural approaches, we have been analyzing the assembly of PU.1/Pip/DNA ternary complexes.

Pursuing the implications of our molecular studies, we demonstrated 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 recombination. Using IRF-4,8-/- pre-B cells we have proceeded to demonstrate that two pathways converge to synergistically drive light-chain rearrangement. We have proposed that stage-specific activation of light-chain recombination during B cell development is ensured by a combination of acquired pre-BCR and attenuated IL-7 signaling.

Curiously, IRF-4 also regulates the B to plasma cell transition. 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 R. Dalla-Favera have independently demonstrated that IRF-4 regulates both isotype switching as well as plasma cell differentiation. Our molecular analysis has revealed that IRF-4 regulates these processes by controlling the expression of the AID and Blimp-1 genes, respectively. Importantly, we have proposed a gene regulatory network in which graded expression of IRF-4 regulates the transition from isotype switching to plasma cell differentiation.

III. 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 been particularly focused 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.

Given the unusual structural organization of Ig gene loci (Mb size 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 intra-chromosomal dynamics. Using 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 immunoglobulin (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 a 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.

Selected Papers

Scott EW, Simon MC, Anastasi J and Singh H. (1994). Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science, 265:1573-1577.

Scott EW, Fisher RC, Olson MC, Kehrli EW, Simon MC and Singh H. (1997). PU.1 functions in a cell-autonomous manner to control the differentiation of multipotential lymphoid-myeloid progenitors. Immunity, 6:437-447.

Brass AL, Zhu AQ and Singh H. (1999). Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers. EMBO J., 18:977-991.

Singh H, DeKoter RP and Walsh JD. (1999). PU.1, a shared transcriptional regulator of lymphoid and myeloid cell fates. Cold Spring Harbor Symposia on Quantitative Biology, Vol. 64 Cold Spring Harbor Laboratory Press, NY.

DeKoter, R.P. and Singh, H. (2000). Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 288:1439-1441.

DeKoter, R.P., Lee, H.-J. and Singh, H. (2002). PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity 16:297-309.

Kosak, S.T., Skok, J.A., Medina, K.L, Riblet, R., Le Beau, M.M., Fisher, A.G. and Singh, H. (2002). Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296:158-162.

Bertolino, E. and Singh, H. (2002). POU/TBP cooperativity: a mechanism for enhancer action from a distance. Molecular Cell 10:397-407.

Walsh, J.C., DeKoter, R.P., Lee, H.-J., Smith, E.D., Lancki, D.W., Gurish, M.F., Friend, D.S., Stevens, R.L., Anastasi, J. and Singh, H. (2002). Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates. Immunity 17:665-676.

Escalante, C.R., Brass, A.L., Pongubala, J.M.R., Shatova, E., Singh, H. and Aggarwal, A.K. (2002). Crystal structure of PU.1/IRF-4/DNA ternary complex. Molecular Cell 10:1097-1105.

Lu, R., Medina, K.L., Lancki, D.W. and Singh, H. (2003). IRF-4,8 orchestrate the pre-B-to-B transition in lymphocyte development. Genes Dev. 17:1703-1708.

Dahl, R., Walsh, J.C., Lancki, D., Laslo, P., Iye, S.R., Singh, H. and Simon, M.C. (2003). Regulation of macrophage and neutrophil cell fates by the PU.1 to C/EBPa ratio and G-CSF. Nature Imm. 4:1029-1036.

Medina, K.L., Pongubala, J.M., Reddy, K.L., Lancki, D.W., DeKoter, R., Kieslinger, M., Grosschedl, R., and Singh, H. (2004). Assembling a gene regulatory network for specification of the B cell fate. Developmental Cell 7:607-617.

Singh, H., Medina, K.L., and Pongubala, J. M.R.  (2005) Contingent gene regulatory networks and B cell fate specification.  Proc. Natl. Acad. Sci. USA. 102:4949-4953.

Bertolino, E., Reddy, K., Medina, K.L., Parganas, E., Ihle, J. and Singh, H. (2005)  Regulation of IL-7 dependent immunoglobulin heavy-chain gene rearrangements by transcription factor Stat5.  Nature Imm. 6:836-843.

Laslo, P., Spooner, C.J., Warmflash, A., Lancki, D., Lee, H-J., Sciammas, R., Gantner, B., Dinner, A. and Singh, H. (2006) Multilineage Transcriptional Priming and Determination of Alternate Hematopoietic Cell Fates. Cell 126:755-766.

Sciammas R., Shaffer A.L., Schatz J.H., Zhao H., Staudt L.M. and Singh H. (2006) Graded expression of interferon regulatory factor-4 coordinates isotype switching with plasma cell differentiation. Immunity 25:225-236.

Singh H. and Sciammas, R. (2006) Shedding B Cell Identity. Immunity 24:239–247.

Gantner, B and Singh, H. (2007) Immunology.  Short-term memory.  Nature 447:916-917.

Littman, DR and Singh H. (2007) Immunology. Asymmetry and immune memory. Science 315(5819):1673-4.

Singh, H. (2007) Shaping a helper T cell identity. Nature Immunology 8(2):119-20.

Pongubala, J.M., Northrup, D.L., Lancki, D.W., Medina, K.L., Treiber, T., Bertolino, E., Thomas, M., Grosschedl, R., Allman, D. and Singh, H. (2008) Transcription Factor EBF Restricts Alternate Lineage Options and Promotes B Cell Fate Commitment Independently of Pax5. Nat Immunol 9:203-215.

Johnson, K., Hashimshony, T., Sawai, C.M., Pongubala J.M., Skok, J.A., Aifantis, I. and Singh, H. (2008) Regulation of Immunoglobulin Light-Chain Recombination by the Transcription Factor IRF-4 and the Attenuation of Interleukin-7 Signaling.  Immunity 28:335-345.

Reddy, K., Zullo, J., Bertolino, E., Singh, H. (2008) Transcriptional Repression Mediated by Repositioning of Genes to the Nuclear Lamina. Nature 452:243-247.