Rudolf Grosschedl

HMG domain proteins: architectural elements in the assembly of nucleoprotein structures.

The high-mobility group (HMG) domain is a DNA-binding motif that is shared abundant non-histone components of chromatin and by specific regulators of transcription and cell differentiation. The HMG family of proteins comprises members with multiple HMG domains that bind DNA with low sequence specificity, and members with single HMG domains that recognize specific nucleotide sequences. Common properties of HMG domain proteins include interaction with the minor groove of the DNA helix, binding to irregular DNA structures, and the capacity to modulate DNA structure by bending. DNA bending induced by the HMG domain can facilitate the formation of higher-order nucleoprotein complexes, suggesting that HMG domain proteins may have an architectural role in assembling such complexes.

Regulation of LEF-1/TCF transcription factors by Wnt and other signals.

LEF-1/TCF transcription factors mediate a nuclear response to Wnt signals by interacting with beta-catenin. Wnt signaling and other cellular events that increase the stability of beta-catenin result in transcriptional activation by LEF-1/TCF proteins in association with beta-catenin. In the absence of Wnt signaling, LEF-1/TCF proteins repress transcription in association with Groucho and CBP. The LEF-1/TCF transcription factors can also interact with other cofactors and play an architectural role in the assembly of multiprotein enhancer complexes, which may allow for the integration of multiple signaling pathways.

LEF-1, a Nuclear Factor Coordinating Signaling Inputs from wingless and decapentaplegic

wingless and decapentaplegic signal during endoderm induction in Drosophila to regulate expression of the homeotic gene Ultrabithorax. Here, we define a minimal wingless response sequence in the midgut enhancer of Ultrabithorax. We show that this sequence is recognized by the murine transcription factor LEF-1 (lymphocyte enhancer binding factor 1) in a ternary complex with armadillo protein, the cytoplasmic target of the wingless signaling pathway. In stable transformants, transcriptional stimulation of the Ultrabithorax enhancer by LEF-1 depends on armadillo. Furthermore, overexpression of LEF-1 bypasses the need for wingless signaling and causes phenotypes in the midgut, notum, and wing that mimic wingless hyperstimulation. Finally, efficient transcriptional stimulation by LEF-1 in the midgut depends also on the decapentaplegic response sequence and is limited spatially by decapentaplegic signaling. Thus, LEF-1 coordinates inputs from multiple positional signals, consistent with its architectural role in regulating the assembly of multiprotein enhancer complexes.

Wnt Signaling Regulates B Lymphocyte Proliferation through a LEF-1 Dependent Mechanism

Lymphocyte enhancer factor-1 (LEF-1) is a member of the LEF-1/TCF family of transcription factors, which have been implicated in Wnt signaling and tumorigenesis. LEF-1 was originally identified in pre-B and T cells, but its function in B lymphocyte development remains unknown. Here we report that LEF-1-deficient mice exhibit defects in pro-B cell proliferation and survival in vitro and in vivo. We further show that Lef1-/- pro-B cells display elevated levels of fas and c-myc transcription, providing a potential mechanism for their increased sensitivity to apoptosis. Finally, we establish a link between Wnt signaling and normal B cell development by demonstrating that Wnt proteins are mitogenic for pro-B cells and that this effect is mediated by LEF-1.

Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with beta-catenin.

ABSTRACT Wnt signaling is thought to be mediated via interactions between β-catenin and members of the LEF-1/TCF family of transcription factors. Here we study the mechanism of transcriptional regulation by LEF-1 in response to a Wnt-1 signal under conditions of endogenous β-catenin in NIH 3T3 cells, and we examine whether association with β-catenin is obligatory for the function of LEF-1. We find that Wnt-1 signaling confers transcriptional activation potential upon LEF-1 by association with β-catenin in the nucleus. By mutagenesis, we identified specific residues in LEF-1 important for interaction with β-catenin, and we delineated two transcriptional activation domains in β-catenin whose function is augmented in specific association with LEF-1. Finally, we show that a Wnt-1 signal and β-catenin association are not required for the architectural function of LEF-1 in the regulation of the T-cell receptor α enhancer, which involves association of LEF-1 with a different cofactor, ALY. Thus, LEF-1 can assume diverse regulatory functions by association with different proteins.

Redundant Regulation of T Cell Differentiation and TCRα Gene Expression by the Transcription Factors LEF-1 and TCF-1

Lymphoid enhancer factor 1 (LEF-1) and T cell factor 1 (TCF-1) are closely related transcription factors that are both expressed during murine T cell differentiation and that regulate the T cell receptor alpha (TCRalpha) enhancer in transfection assays. Targeted gene disruption of either the Tcf1 or Lef1 gene in mice did not affect TCRalpha gene expression and resulted in an incomplete defect or no defect in thymocyte differentiation. Here, we examine a potential redundancy of these transcription factors by analyzing double-mutant mice. In fetal thymic organ cultures from Lef1-/- Tcf1-/- mice, alpha/beta T cell differentiation is completely arrested at the immature CD8+ single-positive (CD8+ ISP) stage and is markedly impaired at an earlier stage. In addition, we find that sorted CD8+ ISP cells from Lef1-/- Tcf1-/- mice express TCRbeta but show a severely reduced level of TCRalpha gene transcription. Together, these data show that LEF-1 and TCF-1 are redundant in the regulation of T cell differentiation and gene expression.

Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A.

The transcription factors EBF and E2A are required at a similar step in early B cell differentiation. EBF and E2A synergistically upregulate transcription of endogenous B cell-specific genes in a non-B cell line. Here, we examine a genetic collaboration between these factors in regulating B lymphopoiesis. We find that Ebf+/- E2a+/- mice display a marked defect in pro-B cell differentiation at a stage later than observed in the single homozygous mutant mice. Pro-B cells from Ebf+/- E2a+/- mice show reduced expression of lymphoid-specific transcripts, including Pax5, Rag1, Rag2, and mb-1. We also show that EBF directly binds and activates the Pax5 promoter. Together, these data show collaboration between EBF and E2A and provide insight into the hierarchy of transcription factors that regulate B lymphocyte differentiation.

EBF and E47 Collaborate to Induce Expression of the Endogenous Immunoglobulin Surrogate Light Chain Genes

Early B cell factor (EBF) and E47 participate in the transcriptional control of early B lymphocyte differentiation. With the aim of identifying genetic targets for these transcription factors, we stably transfected cDNAs encoding EBF or a covalent homodimer of E47, individually or together, into immature hematopoietic Ba/F3 cells, which lack both factors. In combination, EBF and E47 induce efficient expression of the endogenous immunoglobulin surrogate light chain genes, lambda5 and VpreB, whereas other pre-B cell-specific genes remain silent. Multiple functionally important EBF and E47 binding sites were identified in the lambda5 promoter/enhancer region, indicating that lambda5 is a direct genetic target for these transcription factors. Taken together, these data suggest that EBF and E47 synergize to activate expression of a subset of genes that define an early stage of the B cell lineage.

Matrix attachment region-dependent function of the immunoglobulin μ enhancer involves histone acetylation at a distance without changes in enhancer occupancy

ABSTRACT Nuclear matrix attachment regions (MARs), which flank the immunoglobulin μ heavy-chain enhancer on either side, are required for the activation of the distal variable-region (VH) promoter in transgenic mice. Previously, we have shown that the MARs extend a local domain of chromatin accessibility at the μ enhancer to more distal sites. In this report, we examine the influence of MARs on the formation of a nucleoprotein complex at the enhancer and on the acetylation of histones, which have both been implicated in contributing to chromatin accessibility. By in vivo footprint analysis of transgenic μ gene constructs, we show that the occupancy of factor-binding sites at the μ enhancer is similar in transcriptionally active wild-type and transcriptionally inactive ΔMAR genes. Chromatin immunoprecipitation experiments indicate, however, that the acetylation of histones at enhancer-distal nucleosomes is enhanced 10-fold in the presence of MARs, whereas the levels of histone acetylation at enhancer-proximal nucleosomes are similar for wild-type and ΔMAR genes. Taken together, these data indicate that the function of MARs in mediating long-range chromatin accessibility and transcriptional activation of the VHpromoter involves the generation of an extended domain of histone acetylation, independent of changes in the occupancy of the μ enhancer.

Transcriptional control of lymphoid development: lessons from gene targeting

Abstract Cis-acting control regions have been defined for many hematopoietic genes and used to identify DNA-binding factors. The orderly expression of these factors is hypothesized to control entry into and progression along the various differentiation pathways. Gene-knockout technology is now unveiling the essential functions of these candidate differentiation control factors. Here, Hans Clevers and Rudolf Grosschedl discuss the results and implications of a number of such gene-disruption experiments.