CSL Behring.

Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in ''knockout'' mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the ␤-like globin gene cluster. The insertion of PGK-Neo into the granzyme B gene, the most 5 gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the ␤-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3 to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK-Neo can be ''captured'' by the regulatory domain into which it is inserted. These results suggest that the PGK-Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

To examine the function of murine ␤-globin locus control region (LCR) 5 hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5 HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human ␥-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5 HS3 reduces expression of the linked embryonic y-and ␤H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult ␤ (␤major plus ␤minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5 HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult ␤-globin gene expression in adult erythrocytes.The human ␤-globin gene cluster consists of a set of five functional genes and one pseudogene. The cluster is active exclusively in erythroid cells, and the individual ␤-like globin genes are expressed during specific developmental stages (33). Sequences local to the individual globin genes are sufficient to direct developmental stage-specific and tissue-specific expression (3,4,6,24,37,38), but high-level expression of the globin genes depends on distant regulatory sequences collectively called the locus control region (LCR) (33). The LCR is composed of a series of erythroid cell-specific, developmentally stable DNase I-hypersensitive sites (HSs) (5, 12, 39) that contain the sequences essential for LCR activity (7,11,13,16,24,29,36).Recent efforts have been directed toward determining the essential sequences within the LCR. Homology comparisons between the LCRs of humans, mice, goats, and rabbits have revealed conservation of the HS cores but not the intervening sequences (17-19, 22, 23, 25, 31). This finding suggests that the conserved sequences define the HS; functional assays have confirmed this hypothesis (12-14, 17, 18, 29, 30, 36). The HSs have been tested individually in transient and stable assays in tissue culture cells and in transgenic mice; 5Ј HS2, -3, and -4 do indeed contain the information that directs high-level expression of linked genes.Recent studies have focused on the activities of the individual HSs within the context of the entire ␤-globin cluster. These approaches have been undertaken to avoid the complications of studying small transgenes in which distant or undefined regulatory influences may be missing. Transgenic animals have now been made by using large DNA fragments derived from the ␤-globin cluster (1,8,15,27,28,35); although these tra...

Nearly 40% of cases of acute myelogenous leukemia (AML) of the M2 subtype are due to a chromosomal translocation that combines a sequence-specific DNA binding protein, AML1, with a potent transcriptional repressor, ETO. ETO interacts with nuclear receptor corepressors SMRT and N-CoR, which recruit histone deacetylase to the AML1-ETO oncoprotein. SMRT-N-CoR interaction requires each of two zinc fingers contained in C-terminal Nervy homology region 4 (NHR4) of ETO. However, here we show that polypeptides containing NHR4 are insufficient for interaction with SMRT. NHR2 is also required for SMRT interaction and repression by ETO, as well as for inhibition of hematopoietic differentiation by AML1-ETO. NHR2 mediates oligomerization of ETO as well as AML1-ETO. Fusion of NHR4 polypeptide to a heterologous dimerization domain allows strong interaction with SMRT in vitro. These data support a model in which NHR2 and NHR4 have complementary functions in repression by ETO. NHR2 functions as an oligomerization domain bringing together NHR4 polypeptides that together form the surface required for high-affinity interaction with corepressors. As nuclear receptors also interact with corepressors as dimers, oligomerization may be a common mechanism regulating corepressor interactions.Nearly 40% of cases of acute myeloid leukemia (AML) M2 are associated with the t(8;21)(q22;q22) chromosome translocation (39). This translocation creates a fusion between the AML1 gene on chromosome 21 and the ETO gene (also known as MTG8/CDR) on chromosome 8. The resulting chimeric protein AML1-ETO contains the DNA-binding domain (DBD) of AML1 and nearly all of ETO (5,24,38,42). The underlying mechanism of AML1-ETO leukemogenic activity is not fully understood.AML1 is a hematopoietic cell-specific transcription factor and is essential for definitive hematopoietic development (43,44,51). At least two potentially related mechanisms have been proposed for the AML1 function. The first is that AML1 synergistically interacts with other adjacent transcription factors, including C/EBP (55) and myb (2, 53). The second is that AML1 is able to recruit the p300/CBP coactivator complex to activate its target gene expression (23). In t(8;21), the activation domain of AML1 is replaced by the ETO protein. Transient-transfection assays indicate that AML1-ETO interferes with AML1 transactivation from certain potential AML1 target genes (9, 36).ETO is expressed at high levels in brain (38) and has also been detected in hematopoietic cells (6). Recently, we and others discovered that ETO as well as AML1-ETO physically associates with nuclear receptor corepressors called N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid receptors) (11,33,49). This discovery provides a mechanism for the repression activity of AML1-ETO. N-CoR and SMRT recruit class I and class II histone deacetylases (HDACs) (19)(20)(21), and this enzyme activity leads to a repressive chromatin state. Consistent with this mechanism, ETO and AML1-ETO are both associ...

The 8;21 translocation produces a fusion between the ETO gene and that encoding the myeloid transcription factor AML1. The AML1-ETO fusion substitutes the majority of the ETO protein for the coregulator recruitment domains of AML1. Biochemical analyses of ETO have led to the identification of numerous interacting proteins including many corepressors. Importantly, the proteins interacting with ETO are different from those of wild-type AML1, suggesting that altered coregulator recruitment underlies the oncogenic properties of AML1-ETO. The list of corepressors capable of binding ETO includes histone deacetylases (HDACs) and components of distinct HDAC core complexes. These investigations have provided mechanistic insight into corepressor recruitment by ETO and clues to the leukemogenic activity of AML1-ETO.

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Bosutinib 200-600 mg with food was safe and well tolerated. Under fed conditions, bosutinib exposures were linear and dose proportional, and C (max) increased by ~1.5-fold. The t (1/2) supported a once-daily dosing regimen.

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