RelA and RelB transcription factors in distinct thymocyte populations control lymphotoxin-dependent interleukin-17 production in γδ T cells.

The NF-κB transcription factor regulates numerous immune responses but its contribution to interleukin-17 (IL-17) production by T cells is largely unknown. Here, we report that IL-17, but not interferon-γ (IFN-γ), production by γδ T cells required the NF-κB family members RelA and RelB as well as the lymphotoxin-ß-receptor (LTßR). In contrast, LTßR-NF-κB signaling was not involved in the differentiation of conventional αß Th17 cells. Impaired IL-17 production in RelA- or RelB-deficient T cells resulted in a diminished innate immune response to Escherichia coli infection. RelA controlled the expression of LT ligands in accessory thymocytes whereas RelB, acting downstream of LTßR, was required for the expression of RORγt and RORα4 transcription factors and the differentiation of thymic precursors into γδT17 cells. Thus, RelA and RelB within different thymocyte subpopulations cooperate in the regulation of IL-17 production by γδ T cells and contribute to the host's ability to fight bacterial infections.

Protein-tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling.

Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation, and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia. Little is known about the protein-tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells, or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable 32D myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. In particular, the sites pTyr-589, pTyr-591, and pTyr-842 involved in the FLT3 ligand (FL)-mediated activation of FLT3 were hyperphosphorylated the most. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Direct interaction of DEP-1 and FLT3 was demonstrated by "substrate trapping" experiments showing association of DEP-1 D1205A or C1239S mutant proteins with FLT3 by co-immunoprecipitation. Moreover, activated FLT3 could be dephosphorylated by recombinant DEP-1 in vitro. Enhanced FLT3 phosphorylation in DEP-1-depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Stable overexpression of DEP-1 in 32D cells and transient overexpression with FLT3 in HEK293 cells resulted in reduction of FL-mediated FLT3 signaling activity. Furthermore, FL-stimulated colony formation of 32D cells expressing FLT3 in methylcellulose was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL stimulation but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and that its loss may contribute to but is not sufficient for leukemogenic cell transformation.

Lack of nuclear factor-kappa B2/p100 causes a RelB-dependent block in early B lymphopoiesis.

Nuclear factor-kappaB (NF-kappaB) transcription factors regulate B-cell development and survival. However, whether they also have a role during early steps of B-cell differentiation is largely unclear. Here, we show that constitutive activation of the alternative NF-kappaB pathway in p100(-/-) knockin mice resulted in a block of early B-cell development at the transition from the pre-pro-B to the pro-B-cell stage due to enhanced RelB activity. Expression of the essential B-cell transcription factors EBF and in particular Pax5 was reduced in p100(-/-) B-cell precursors in a RelB-dependent manner, resulting in reduced mRNA levels of B lineage-specific genes. Moreover, enhanced RelB function in p100(-/-) B-cell precursors was accompanied by increased expression of B lineage-inappropriate genes, such as C/EBP alpha, correlating with a markedly increased myeloid differentiation potential of p100(-/-) progenitor B cells. Ectopic expression of Pax5 in hematopoietic progenitors restored early B-cell development in p100(-/-) bone marrow, suggesting that impaired early B lymphopoiesis in mice lacking the p100 inhibitor may be due to down-regulation of Pax5 expression. Thus, tightly controlled p100 processing and RelB activation is essential for normal B lymphopoiesis and lymphoid/myeloid lineage decision in bone marrow.

Molecular characterization of the pericentric inversion of chimpanzee chromosome 11 homologous to human chromosome 9.

In addition to the fusion of human chromosome 2, nine pericentric inversions are the most conspicuous karyotype differences between humans and chimpanzees. In this study we identified the breakpoint regions of the pericentric inversion of chimpanzee chromosome 11 (PTR 11) homologous to human chromosome 9 (HSA 9). The break in homology between PTR 11p and HSA 9p12 maps to pericentromeric segmental duplications, whereas the breakpoint region orthologous to 9q21.33 is located in intergenic single-copy sequences. Close to the inversion breakpoint in PTR 11q, large blocks of alpha satellites are located, which indicate the presence of the centromere. Since G-banding analysis and the comparative BAC analyses performed in this study imply that the inversion breaks occurred in the region homologous to HSA 9q21.33 and 9p12, but not within the centromere, the structure of PTR 11 cannot be explained by a single pericentric inversion. In addition to this pericentric inversion of PTR 11, further events like centromere repositioning or a second smaller inversion must be assumed to explain the structure of PTR 11 compared with HSA 9.

Breakpoint analysis of the pericentric inversion distinguishing human chromosome 4 from the homologous chromosome in the chimpanzee (Pan troglodytes).

The study of breakpoints that occurred during primate evolution promises to yield valuable insights into the mechanisms underlying chromosome rearrangements in both evolution and pathology. Karyotypic differences between humans and chimpanzees include nine pericentric inversions, which may have potentiated the parapatric speciation of hominids and chimpanzees 5-6 million years ago. Detailed analysis of the respective chromosomal breakpoints is a prerequisite for any assessment of the genetic consequences of these inversions. The breakpoints of the inversion that distinguishes human chromosome 4 (HSA4) from its chimpanzee counterpart were identified by fluorescence in situ hybridization (FISH) and comparative sequence analysis. These breakpoints, at HSA4p14 and 4q21.3, do not disrupt the protein coding region of a gene, although they occur in regions with an abundance of LINE and LTR-elements. At 30 kb proximal to the breakpoint in 4q21.3, we identified an as yet unannotated gene, C4orf12, that lacks an homologous counterpart in rodents and is expressed at a 33-fold higher level in human fibroblasts as compared to chimpanzee. Seven out of 11 genes that mapped to the breakpoint regions have been previously analyzed using oligonucleotide-microarrays. One of these genes, WDFY3, exhibits a three-fold difference in expression between human and chimpanzee. To investigate whether the genomic architecture might have facilitated the inversion, comparative sequence analysis was used to identify an approximately 5-kb inverted repeat in the breakpoint regions. This inverted repeat is inexact and comprises six subrepeats with 78 to 98% complementarity. (TA)-rich repeats were also noted at the breakpoints. These findings imply that genomic architecture, and specifically high-copy repetitive elements, may have made a significant contribution to hominoid karyotype evolution, predisposing specific genomic regions to rearrangements.

Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome.

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17.

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.