A human Hox 1 homeobox gene exhibits myeloid-specific expression of alternative transcripts in human hematopoietic cells

Abstract

As part of a survey of the expression of homeobox-containing genes in human hematopoietic cells, we identified a novel gene (PL1) expressed only in cells of the myelomonocytic lineage (Shen et al., Proc. Natl. Acad. Sci, USA 86, 8536, 1989). On Northern gel analysis, major transcripts of 3.0 and 2.2 kb length are observed. Alternatively spliced homeobox-containing cDNAs, corresponding to the major transcripts, have been cloned from two myeloid leukemia cell libraries. The two cDNAs share the homeodomain and 3' flanking region but have unique 5' flanking regions. The longer transcript, would encode a 496 amino acid homeobox-containing protein, while the shorter message would encode a 94 amino acid homeobox-containing protein lacking the extended amino-terminal region. These two transcripts are differentially expressed in human leukemia cell lines. The larger transcript is exclusively expressed in cells with myelomonocytic features, while the smaller transcript is expressed in a variety of hematopoietic cell types. PL mRNA is also detectable in normal human bone marrow by RNAse protection. Neither transcript is expressed in uninduced teratocarcinoma cells or in the adult human tissues surveyed. The homeodomain is identical to the genomic sequence for Hox 1H, a newly identified member of the Hox 1 locus (Acampora et al. Nucl. Acids Res. 17, 10385, 1989). The PL1 gene was localized to chromosome 7 using chromosome specific blots and sublocalized to region pI4-21 by in situ hybridization of chromosomal spreads, confirming its location within the Hox 1 complex.

Activation of SHP2 Protein-tyrosine Phosphatase Increases HoxA10-induced Repression of the Genes Encoding gp91PHOX and p67PHOX

“…Protein Expression Vectors-The human HoxA10 cDNA was obtained from Dr. C. Largman (University of California, San Francisco) (35). This cDNA sequence represents the major transcript in mammalian hematopoietic cells, encoding a 393-amino acid 55-kDa protein.…”

“…The latter reflects increased transcription of the CYBB and NCF2 genes (45,46). This cell line has relative overexpression of HoxA10 as one of the genetic lesions involved in the transformed phenotype (35). Therefore, these stable U937 transfectant pools were analyzed for gp91 PHOX and p67 PHOX expression during IFN-␥-induced differentiation.…”

“…For these experiments, SHP1-GST was used as a positive control, and GST protein was used as a negative control for HoxA10 coprecipitation. SHP2-GST, SHP1-GST, or control GST was incubated with in vitro translated 35 S-labeled HoxA10, and incubation mixtures were affinity-precipitated on glutathione-agarose. Precipitates were separated by SDS-PAGE, and proteins were identified by Coomassie Blue staining and autoradiography.…”

“…For these experiments, HoxA10 and E76K SHP2 were expressed as in vitro translated proteins. 35 S-Labeled HoxA10 was incubated with unlabeled E76K SHP2 or control lysate. Proteins were immunoprecipitated under denaturing conditions with anti-phosphotyrosine antibody or irrelevant control antibody.…”

“…B, in vitro translated HoxA10 copurifies with either SHP1 or SHP2 expressed as a GST fusion protein. 35 S-Labeled HoxA10 was in vitro translated in reticulocyte lysate and used in affinity purification assays with SHP1 or SHP2 expressed as a GST fusion protein in E. coli. GST protein was used as a negative control in these experiments.…”