Regulation of the interleukin-7 receptor alpha promoter by the Ets transcription factors PU.1 and GA-binding protein in developing B cells.

Signaling through the IL-7 receptor (IL-7R) is required for development and maintenance of the immune system. The receptor for IL-7 is heterodimeric, consisting of a common gamma chain (gammac, encoded by Il2rg) and an alpha subunit (IL-7Ralpha, encoded by Il7r). The Il7r gene is expressed specifically in the immune system in a developmental stage-specific manner. It is not known how the Il7r gene is transcriptionally regulated during B cell development. The goal of this study is to elucidate the function of the Il7r promoter region in developing B cells. Using a combination of 5' rapid amplification of cDNA ends analysis, transient transfection assays, and DNase I hypersensitivity mapping, we identified the location of the Il7r promoter. Using a combination of electrophoretic mobility shift analysis, chromatin immunoprecipitation experiments, and RNA interference experiments, we found that the Ets transcription factors PU.1 and GA-binding protein (GABP) activate the Il7r promoter by interacting with a highly conserved Ets binding site. In committed B lineage cells, GABP can promote Il7r transcription in the absence of PU.1. However, the results of retroviral gene transfer experiments suggest that PU.1 is uniquely required to initiate transcription of the Il7r locus at the earliest stages of progenitor B cell generation. In summary, these results suggest that Il7r transcription is regulated by both PU.1 and GABP in developing B cells.

PU.1 immortalizes hematopoietic progenitors in a GM-CSF-dependent manner.

OBJECTIVE: The Ets family transcription factor PU.1 is essential for both myeloid and lymphoid development. PU.1 was discovered because of its involvement in murine erythroleukemia. We previously described that infection with a retroviral vector encoding PU.1 immortalizes fetal liver progenitor cells in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. In this study, we sought to characterize PU.1-immortalized progenitor (PIP) cells. METHODS: PIP cells were characterized using microscopy, reverse-transcriptase polymerase chain reaction analysis, and flow cytometric analysis. In addition, progenitors were immortalized with a retrovirus containing a PU.1 cDNA flanked by loxP sites. The differentiation potential of immortalized progenitors was tested by Cre-mediated excision of the proviral PU.1 cDNA. RESULTS: PIP cells are blastlike in morphology and express cell surface markers indicative of myeloid development. Immortalization of progenitor cells requires both an acidic activation domain and an intact DNA-binding domain of PU.1. Gene expression analysis of PIP cells demonstrated the expression of genes of both myeloid and erythroid lineages. Proliferation of PIP cells was GM-CSF dependent and restricted. Upon Cre-mediated excision of proviral PU.1 cDNA, increased expression of myeloid and erythroid-specific genes was observed; as well as the appearance of both macrophages and erythrocytes in culture. CONCLUSION: We demonstrate that ectopic expression of PU.1 is sufficient to immortalize a hematopoietic progenitor with myeloid and erythroid differentiation potential in response to GM-CSF. These data highlight the importance of the level of PU.1 expression at critical stages of hematopoiesis.

Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells.

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1(-/-) fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcgammaRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcgammaRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcgammaRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3' regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

The reverse transcriptase sequence of human immunodeficiency virus type 1 is under positive evolutionary selection within the central nervous system.

The human immunodeficiency virus type 1 (HIV-1) enters the central nervous system (CNS) during the acute phase of infection and causes AIDS-related encephalitis and dementia in 30% of individuals. Previous studies show that HIV-1 sequences derived from the CNS of infected patients, including the sequence encoding reverse transcriptase (RT), are genetically distinct from sequences in other tissues. The hypothesis of the current study is that the RT sequence of HIV-1 is under positive selection within the CNS. Multiple alignments of non-CNS-derived and CNS-derived HIV-1 RT sequences were constructed using the ClustalW 1.8 program. The multiple alignments were analyzed with the Synonymous/Nonsynonymous Analysis Program. Codon positions 122-125, 135-149, and 166-212 of the CNS-derived RT sequences underwent a greater accumulation of nonsynonymous than synonymous substitutions, which was markedly different from the analysis results of the non-CNS-derived RT sequences. These residues are located in the finger and palm subdomains of the RT protein structure, which encodes the polymerase active site. The analysis of CNS-derived partial-length RT sequences that encompass these regions yielded similar results. A comparison of CNS-derived RT sequences to a non-CNS-derived RT consensus sequence revealed that a majority of the nonsynonymous substitutions resulted in a specific amino acid replacement. These results indicate that reverse transcriptase is under positive selection within the CNS. The amino acid replacements were visualized on a three-dimensional structure of HIV-1 RT using the Sybyl software suite. The protein structure analysis revealed that the amino acid replacements observed among the CNS-derived sequences occurred in areas of known structural and functional significance.