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Full Developmental Silencing of theEmbryonic ζ-Globin Gene ReflectsInstability of its mRNA

J. ERIC RUSSELL,a–c ALICE E. LEE,b AND

STEPHEN A. LIEBHABERb,d,e

Departments of bMedicine, cPediatrics, and dGenetics, and the eHowardHughes Medical Institute, University of Pennsylvania School ofMedicine, Philadelphia, Pennsylvania 19104, USA

Three highly homologous α-like globin genes are expressed in humans. The α1- and α2-globin genes, which encode identical protein products, are co-expressed at low levels in

embryonic erythrocytes, and at high levels in fetal and adult erythrocytes. By comparison,ζ-globin is expressed at high levels in embryonic erythrocytes but is not detected in normaladult erythrocytes. The silencing of ζ-globin expression and reciprocal induction of α-glo-bin expression at the embryonic-fetal transition (post-conception weeks 6–8) is due in partto changes in the transcriptional activity of their cognate genes. Recent evidence, however,suggests that post-transcriptional mechanisms that determine globin mRNA stability mayalso be crucial effectors of normal patterns of globin gene expression. This paper (1)reviews evidence supporting the hypothesis that post-transcriptional controls affectingmRNA stability contribute to human (h) ζ-globin gene silencing; (2) describes a mousemodel system that has been instrumental in physiologically relevant evaluations of α- andζ-globin mRNA stability; and (3) presents data demonstrating that hζ-globin mRNA is sig-nificantly destabilized in adult erythroid cells relative to hα-globin mRNA.

EVIDENCE SUPPORTING A ROLE FOR POST-TRANSCRIPTIONALREGULATION OF ζ-GLOBIN GENE EXPRESSION

Three independent lines of evidence suggest that post-transcriptional controls play amajor regulatory role in hζ-globin gene silencing in fetal and adult erythrocytes.Significant quantities of hζ-globin mRNA can be detected in nuclear run-on experimentsutilizing bone marrow erythroid progenitor cells from normal human volunteers.1 In con-trast, hζ-globin mRNA is nearly undetectable in normal adult reticulocytes.2 Consideredtogether, these data indicate a post-transcriptional defect in the accumulation of hζ-globinmRNA in adult erythroid cells. The post-transcriptional nature of this effect was substan-tiated in our lab using mice transgenic for either the hζ-globin or hα-globin transcribedregions, each flanked by identical 5′ and 3′ hζ-globin transcriptional control elements.3

During fetal development, levels of hζ-globin mRNA fell dramatically, while levels of hα-globin mRNA were less severely affected. These data suggest that the difference in theaccumulation of the two mRNAs is effected by elements within their transcribed regions

aAddress for correspondence: J. Eric Russell, M.D., Abramson Research Building, Room 316F,Children’s Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA 19104. Tel: 215-590-3880; Fax: 215-590-4834; E-mail: jeruss@mail.med.upenn.edu

which dictate differences in their transcriptional or post-transcriptional regulation. Closeinvestigation of hα-globin gene expression in cultured cells and transgenic mice suggeststhat these elements regulate expression post-transcriptionally. It is clear that normalexpression of human α-globin is crucially dependent on the high stability of its mRNA, acharacteristic mediated through assembly of a multicomponent RNP complex (α-com-plex) on a specific 20 nt sequence within its 3′UTR.4,5 The importance of mRNA stabilityto hα-globin gene expression suggests that this property, or its absence, might also be uti-lized for the control of hζ-globin expression.

EVALUATION OF HUMAN GLOBIN mRNA STABILITY IN TRANSGENIC MICE

For the purpose of assessing globin mRNA stability, transgenic mice offer several dis-tinct advantages over other commonly employed methods.6,7 Transcriptional chase exper-iments in cultured cells require actinomycin D, which globally arrests gene transcriptionand may artifactually affect mRNA stability.8 The reliability of mRNA stabilities deter-mined from genes linked to serum-inducible promoters is poor for highly stable mRNAs,such as globin mRNAs, as the transfected cells require a prohibitively long preparatoryperiod of serum starvation to clear previously transcribed globin mRNAs. The transgenicmouse system is not subject to either of these limitations and offers the additional advan-tage that globin mRNA metabolism occurs in vivo in erythroid progenitor cells undergo-ing physiologic terminal differentiation. As in humans, murine erythroid cells undergotranscriptional silencing in the bone marrow and subsequently migrate into the peripheralcirculation as mRNA-rich reticulocytes (FIG. 1A). mRNAs recovered from the marrow andreticulocytes of individual mice represent sequential time points in a physiologic tran-scriptional chase experiment. Transgenic mRNA levels in each tissue are determined rela-tive to levels of endogenous mouse (m) α-globin mRNA using a quantitative RNaseprotection assay (FIG. 1B).6,7 A fall in the relative level of transgenic mRNA in the tran-scriptionally silent interval between the marrow and reticulocyte stages of terminal ery-throid differentiation indicates instability of the transgenic mRNA relative to themα-globin mRNA control. Results from these experiments are highly reproducible amongdifferent mice from the same transgenic line.6,7

HUMAN ζ-GLOBIN mRNA IS DESTABILIZED IN ADULT HUMANERYTHROID CELLS

We assessed the stabilities of hα- and hζ-globin mRNAs in adult mice from 10 and 16independent transgenic lines, respectively. Modifications of ζ-globin gene promoter andsilencer elements insured transcription of adequate levels of hζ-globin mRNA withoutaffecting its structure.6,7 Relative to mα-globin mRNA, the level of hα-globin mRNAremained stable in the transcriptionally silent interval between bone marrow and periph-eral reticulocytes (FIG. 2A). During the same interval, the level of hζ-globin mRNA fellrapidly. The difference in the average stability of hα- and hζ-globin mRNAs from all 26transgenic lines was highly significant (FIG. 2B). The fourfold difference in the relativestabilities of hα- and hζ-globin mRNAs was confirmed in single mice generated by theappropriate genetic crosses, which expressed both transgenes (FIG. 2C). These results areconsistent with the hypothesis that post-transcriptional mechanisms that direct destabi-lization of ζ-globin mRNA are crucial to its full silencing in fetal and adult erythroid cells.We are currently engaged in determining the mechanism(s) that underlie this facet of ζ-globin gene expression.

RUSSELL et al.: ζ-GLOBIN SILENCING 387

388 ANNALS NEW YORK ACADEMY OF SCIENCES

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RUSSELL et al.: ζ-GLOBIN SILENCING 389

FIGURE 2. Relative instability of human ζ-globin mRNA in situ in adult erythroid cells. (A) Stabilityof human α- and ζ-globin mRNAs relative to mouse α-globin mRNAs in mouse erythroid cells. Levelsof transgenic hα-globin mRNA (left) and hζ-globin mRNA (right) were determined relative to mα-globin mRNA using the RPA described in FIGURE 1. Autoradiographs from two representative hα-glo-bin and two representative hζ-globin transgenic mice are shown. The positions of the protected hα-,hζ- and mα-globin mRNA fragments are indicated. Unique numbers (top) identify independent trans-genic lines. B=bone marrow; R=reticulocyte. (B) ζ-globin mRNA is significantly less stable than α-globin mRNA in terminally differentiating adult erythroid cells. The mean stabilities of hα- andhζ-globin mRNAs determined from 10 and 16 independent transgenic lines, respectively, is plotted.The stability of endogenous mα-globin mRNA (defined as 1.0) is indicated by a dashed horizontalline. Error bars indicate ± 2 S.E. The difference between the mean stabilities of human α- and ζ-glo-bin mRNA is significant at p < 0.005. (C) Direct comparison of human α- and ζ-globin mRNA sta-bilities in mice co-expressing both transgenes. Mice expressing the hα- and hζ-globin transgenes weremated and offspring expressing both transgenes identified by Southern analysis. Levels of hα- and hζ-globin mRNA in bone marrow (B) and peripheral blood reticulocytes (R) from these mice were deter-mined by RPA. An autoradiograph from a representative study is illustrated, with the positions of theprotected human α- and ζ-globin mRNA fragments indicated to the left.

REFERENCES

1. YAGI, M. et al. 1996. Chromatin structure and developmental expression of the human α-globincluster. Mol. Cell. Biol. 6: 1108–16.

2. ALBITAR, M. et al. 1989. Theta, zeta, and epsilon globin messenger RNAs are expressed inadults. Blood 74: 629–637.

3. LIEBHABER, S. A. et al. 1996. Developmental silencing of the embryonic ζ-globin gene: con-certed action of the promoter and the 3′ flanking region combined with stage-specific silenc-ing of the transcribed segment. Mol. Cell. Biol. 16: 2637–2646.

4. WANG, X. et al. 1995. Detection and characterization of a 3′ untranslated region ribonucleopro-tein complex associated with human α-globin mRNA stability. Mol. Cell. Biol. 15:1769–1777.

5. KILEDJIAN, M. et al. 1995. Identification of two KH domain proteins in the α-globin mRNP sta-bility complex. EMBO J. 14: 4357–4364.

6. MORALES, J. et al. 1997. Destabilization of human α-globin mRNA by translation anti-termina-tion is controlled during erythroid differentiation and paralleled by phased shortening of thepoly(A) tail. J. Biol. Chem. 272: 6607–6613.

7. RUSSELL, J. E. et al. 1998. Sequence divergence in the 3′ untranslated regions of human ζ- andα-globin mRNAs mediates a difference in their stabilities and contributes to efficient α-to-ζgene developmental switching. Mol. Cell Biol. 18: 2173–2183.

8. WISDOM, R. et al. 1991 The protein coding region of c-myc mRNA contains a sequence thatspecifies rapid mRNA turnover and induction by protein synthesis inhibitors. Genes Develop.5: 232–243.

390 ANNALS NEW YORK ACADEMY OF SCIENCES