A directly repeated sequence in the beta-globin promoter regulates transcription in murine erythroleukemia cells.

Abstract

We have identified a previously undetected cis-acting element in the mouse p-major globin promoter region that is necessary for maximal transcription levels of the gene in the inducible preerythroid murine erythroleukemia (MEL) cell line. This element, termed the j-globin direct-repeat element (jDRE), consists of a directly repeated 10-base-pair sequence, 5'-AGGGCAG(G)AGC-3', that lies just upstream from the TATA box of the promoter. The 13DRE motif is highly conserved in all adult mammalian P-globin promoter sequences known. Mutation of either single repeat alone caused less than a twofold decrease in transcript levels. However, simultaneous mutation of both repeated regions resulted in a ninefold decrease in accumulated transcripts when the gene was transiently transfected into MEL cells. Attachment of the ,IDRE to a heterologous promoter had little effect on levels of accumulated transcripts initiated from the promoter in undifferentiated MEL cells but resulted in a threefold increase in transcript levels in induced (differentiated) MEL cells. Similarly, a comparison of the relative effects of mutations in the 13DRE in uninduced and induced MEL cells indicated that the element was more active in induced cells. The increase in OiDRE activity upon MEL cell differentiation and the more pronounced effects of mutations in both repeats of the iDRE have implications for the mechanism of action of the element in regulating 13-globin transcription and for mutational studies of other repetitive or redundant transcription elements.The developmental-stage and tissue-specific regulation of the mammalian ,B-globin locus has provided a challenging system for understanding the molecular mechanisms that mediate gene activation during terminal differentiation. An important step in understanding this regulation is identification of the cis-acting regulatory sequences that are required for expression of globin genes in erythroid cells. The murine erythroleukemia (MEL) cell model system for adult erythrocyte development (13) has been useful in this characterization for the adult ,B-globin genes. MEL cells are arrested at the proerythroblast stage of erythroid development and can be induced to terminally differentiate in vitro in a process that closely mimicks the events of normal erythropoiesis (for a review, see reference 31). MEL cell differentiation is characterized by a large increase (10-to 50-fold) in the steady-state level of ,B-globin mRNA. This increase is due in part to an increase in the rate of transcriptional initiation from the ,B-globin promoter (3,19,47) and in part to an increase in globin mRNA stability (44). Because transfected 1-globin genes are regulated similarly to the endogenous genes when transferred into MEL cells (3, 46), it has been possible to identify cis-acting elements that play a role in 1-globin transcriptional regulation by mutagenesis experiments (1,4,6,47).Introduction of cloned hybrid genes into transgenic mice (24, 43) and MEL cells (1, 5, 6, 47) has implicated sequences both 5' and 3' to th...

Discrimination among potential activators of the beta-globin CACCC element by correlation of binding and transcriptional properties.

“…A large body of work has demonstrated that this element is required for wild-type levels of transcription of transfected ,3-globin-like genes in HeLa, L, and 3T6 cells (12,39,54). Similarly, this element is also required for transcription of transfected ,B-globin genes in erythroid cells (7,49). More striking, several different naturally occurring single base substitution (point) mutations in the CACCC element of the human 3-globin promoter that are known to decrease transcription cause 1-thalassemia (Table 1) (18,32,40,41,43,51).…”

“…Three (39,49). These mutations, -95A, -94T, -92C, -92T, -90T and -92C/ -93T, were all constructed by site-directed mutagenesis (33).…”

“…The 1-globin-metallothionein and H4-metallothionein fusion RNAs were quantitated by an S1 nuclease protection assay as described previously (7). Single-stranded, end-labeled probe DNAs complementary to the expected products of the transfected genes were made as described previously (7,49) (Fig. LA).…”

Regulation of Globin Gene Expression in Erythroid Cells

“…Common sets of transcription factors may serve to coordinate globin synthesis with other genes important for erythroid differentiation. Within the promoters of globin genes additional motifs have been recognized, including an extended CACC motif, a binding site for a putative stage-specific factor possibly analogous to chicken NF-E4, a direct repeat sequence (DRE) [39], and a conventional CCAAT box.…”

“…Two candidates have been identified. Within the P-globin gene promoter a conserved direct repeat element (DRE) may bind a transcription factor enriched in adult cells [39,1011. Within the embryonic E-globin promoter a positive regulatory element (E-PRE 11) may be the target of a distinct, embryonic binding activity [102].…”