The AML1/CBFbeta transcription factor complex, a frequent target of chromosomal translocations in leukemia, is essential for the generation of definitive hematopoietic stem cells. Paradoxically, expression of the acute myeloid leukemia-associated AML1-ETO fusion protein in mice results not in leukemia, but in embryonic lethality due to an absence of normal hematopoiesis. To bypass the embryonic lethality, we generated a mouse strain with a conditional AML1-ETO knockin allele that contains a loxP bracketed transcriptional stop cassette 5' to the AML1-ETO fusion site. Activation of this allele in vivo by Cre-mediated recombination resulted in an enhanced replating efficiency of myeloid progenitors, but it did not block their differentiation, nor was it sufficient to induce leukemia. However, induction of cooperating mutations resulted in the development of an acute myeloid disease that mimicked many of the features of human AML1-ETO-expressing leukemia.

The AML1/core binding factor ␤ (CBF␤) transcription factor is essential for definitive hematopoiesis; however, the downstream pathways through which it functions remain incompletely defined. Using a differential cloning approach to define components of this pathway, we have identified a novel gene designated HERF1 (for hematopoietic RING finger 1), whose expression during development is dependent on the presence of functional AML1/CBF␤. HERF1 contains a tripartite RING finger-B box-␣-helical coiled-coil domain and a C-terminal region homologous to the ret proto-oncogene-encoded finger protein. Expression of HERF1 during embryogenesis coincides with the appearance of definitive erythropoiesis and in adult mice is restricted to erythroid cells, increasing 30-fold during terminal differentiation. Importantly, inhibition of HERF1 expression blocked terminal erythroid differentiation of the murine erythroleukemia cell line MEL, whereas its overexpression induced erythroid maturation. These results suggest an important role for this protein in erythropoiesis.

To further elucidate the potential role of mitogens and cytokines in regulation of the kappa immunoglobulin light-chain locus, we have characterized the activation of transcription factor binding, kappa germ line transcription, DNase I hypersensitivity, and V -to-J recombination upon induction of model pre-B-cell lines. We find that both lipopolysaccharide (LPS) and gamma interferon (IFN-␥) are capable of activating germ line transcription, DNase I hypersensitivity, and recombination of the kappa locus. We also find that transforming growth factor ␤ is capable of completely inhibiting LPS activation of transcription and recombination but has no apparent effect on activation of transcription factor binding, It is becoming increasingly clear that the tissue-specific and developmentally regulated transcription and recombination of immunoglobulin (Ig) gene segments represent overlapping points of control during B-cell development. The concomitant activation of transcription and recombination of all Ig loci has led to the suggestion that transcription of unrearranged Ig genes plays a key role in regulating recombination (23, 31, 37, 44-46, 55, 60, 61). A number of observations have provided strong support for this hypothesis. First, while a common V(D)J recombinase is expressed in both B cells and T cells, functional recombination of Ig genes is restricted to B cells, where they are also transcriptionally active (62). While ectopic expression of the recombinase activating genes RAG-1 and RAG-2 has been shown to confer V(D)J recombinase activity to integrated and extrachromosomal substrates in nonlymphoid cells, the endogenous Ig loci, which are transcriptionally silent in these cells, do not undergo recombination (19,25,33). In addition, it has been demonstrated previously that induction of germ line transcription of the unrearranged kappa Ig lightchain and heavy-chain loci is accompanied by an increased frequency of gene rearrangement (44, 45). More direct evidence that cis-acting transcriptional regulatory elements are involved in recombination has been demonstrated by integration of recombination substrates by using transfection approaches (5, 6, 11, 24, 35), transgenic approaches (15-17, 20, 21), and targeted knockouts of endogenous sequences (18,48,54,59).While there is strong evidence that cis-acting transcriptional regulatory regions within the kappa Ig light-chain locus play a role in recombination, the mechanism by which they regulate recombination and the trans-acting factors involved have yet to be elucidated. The observation that cells are able to regulate the ability of the endogenous kappa locus to serve as a substrate for the V(D)J recombinase has led to the suggestion that transcription may target the locus by providing accessibility of the locus to the recombinase (see reviews in references 32, 39, and 47). The mechanism by which transcription may mediate accessibility is not clear; however, changes in local chromatin structure within the kappa locus have been shown to occur upon transcriptional ac...

Previous analyses of immunoglobulin V region promoters has led to the discovery of a common octamer motif which is functionally important in the tissue-specific and developmentally regulated transcriptional activation of immunoglobulin genes. The germline promoters (Ko) located upstream of the J region gene segments of the kappa locus also contain an octamer motif (containing a single base pair mutation and referred to as the variant octamer) which has been shown previously to bind Oct-1 and Oct-2 transcription factors in vitro. To further elucidate the role of this variant octamer motif in the regulation of germline transcription from the unrearranged kappa locus, we have quantitated the relative binding affinity of Oct-1 and Oct-2 for the variant octamer motif and determined the functional role of this octamer motif in transcriptional activation. We find that, although the variant octamer motif binds Oct-1 and Oct-2 in vitro with 5-fold lower affinity than the consensus octamer motif, mutation of the variant octamer motif to either a consensus octamer or non-octamer motif has no effect on transcriptional activation from the germline promoter. We also find significant differences in activation of germline and V region promoters by kappa enhancers. Our results suggest that the germline promoters and V region promoters differ in their dependence on octamer for activation and respond differently to enhancer activation. These findings have important implications in regulation of germline transcription as well as concomitant activation of the V-J recombination of the kappa light chain locus.

No abstract