We have used the globin family of genes in chicken to study developmental regulation of gene expression, both at the level of individual interaction of trans-acting factors with local promoters and enhancers, and at the level of chromatin structure. Regulation of all members of the a- and [3-globin clusters is affected by the erythroid regulatory factor GATA-1. Separate mechanisms exist for regulation of individual members of the family. As an example, we describe the control mechanisms that play a role in the expression of the p-globin gene, which is expressed only in primitive lineage erythroid cells. In addressing the involvement of chromatin structure in gene activation, we have examined the role of locus control elements, and also considered the way in which RNA polymerase molecules might accommodate to the presence of nucleosomes on transcribed genes.

The globin gene family has served as a model for the study of developmentally regulated gene expression (Evans et al., 1990), allowing investigators to address the questions of how the entire family is activated in an erythroid-specific manner, and how individual family members are turned on and off at successive stages of erythroid development. We have focussed our attention on erythroid development in the chicken, in part because embryonic erythroid development in chickens is well described, and because cells at each developmental stage can be obtained in abundance. Furthermore, since avian erythrocytes retain their nuclei, many regulatory factors can be isolated readily from cells found in the adult circulation.

Two distinct erythroid lineages are produced in the developing chick embryo (Dieterlen-Lievre, 1988; Nikinmaa, 1990). The primitive lineage predominates in the circulation until about day 5 after fertilization; at that point the definitive lineage appears. Primitive lineage cells express the embryonic β-globin genes ρ and ε, and the α-globin genes, απ, αA and αD. Definitive cells on the other hand express βH and βA, as well as αA and αD (Fig. 1). The steps leading to the activation of these genes require the participation of typical trans-acting factors that bind to promoters and enhancers, as well as the modification of chromatin structure over the a and p domains. Understanding of the globin regulatory pathways thus requires an analysis of both local and long-range interactions.

Fig. 1.

Organization of the α-globin and β-globin clusters in chicken. The letters e and i mark the β- and α-cluster enhancers respectively.

Fig. 1.

Organization of the α-globin and β-globin clusters in chicken. The letters e and i mark the β- and α-cluster enhancers respectively.

Our earliest studies of globin gene expression were concentrated on the βA-globin gene, and were greatly aided by the development of a method for transfection of plasmids into primary erythroid cells (obtained from the embryonic circulation) with little or no damage to the cells (Hesse et al., 1986; Lieber et al., 1987). The method permits testing of the activity of globin promoters and enhancers in cells of both the primitive and definitive lineages. This led to the identification of a strong, erythroid-specific enhancer element just 3 ′ of the βA-globin gene (Hesse et al., 1986; Choi and Engel, 1986), which preferentially activates that gene in definitive lineage cells. As was subsequently shown (Nickol and Felsenfeld, 1988; Choi and Engel, 1988), the enhancer also functions in primitive lineage cells to activate the embryonic ε gene, located downstream of the enhancer.

The general erythroid factor GATA-1

DNase I footprinting in vitro and site mutagenesis allowed us to identify essential elements of the enhancer (Evans et al., 1988; Reitman and Felsenfeld, 1988). Of particular interest was a pair of DNA sequence motifs of the form (A/T)GATA(A/G), which we had already identified in the promoter regions of the p and αD genes (Kemper et al.,1987). Related binding sites for this factor (now called GATA-1, originally named Eryfl in chicken, and NF-E1 or GF-1 in human and mouse) were identified in the promoters and enhancers of all the globin genes (Evans et al.,1988). The cDNA from mouse (Tsai et al., 1989) and both the cDNA and gene from chicken, cGATA-1 (Evans and Felsenfeld, 1989; Hannon et al., 1991), have been cloned. The human GATA-1 cDNA and gene (Trainor et al., 1990; Zon et al., 1990; Zon and Orkin, 1992) have also been cloned. cGATA-1 is a 304 amino acid protein with two metal ion ‘finger’ motifs, which binds typically as a monomer to an asymmetric site (Evans and Felsenfeld,1989). Trans-activation studies in chick embryo fibroblasts (Evans and Felsenfeld, 1991) show that some promoters carrying a single copy of a GATA-1 binding site are strongly stimulated by expression of GATA-1. When plasmids carrying such a GATA-1 expression vector are cotransfected with an appropriate CAT reporter plasmid, a 360-fold stimulation of CAT activity is observed relative to antisense controls (Fig. 2). Remarkably, the presence of additional GATA-1 sites in the promoter does not increase this response in fibroblasts. A quite different effect is seen when the same group of reporters is introduced into primary embryonic erythroid cells: the reporter carrying multiple GATA-1 sites is much more active (Evans and Felsenfeld, 1991). This result has led us to suggest that cGATA-1 is interacting either with an activator present in fibroblasts, or an erythroid cell-specific repressor.

Fig. 2.

Effect of expressing cGATA-1 in primary chicken embryo fibroblasts on transcription from various promoters containing GATA-1-binding sites. GATA-1 expression vectors were transiently cotransfected with CAT reporter plasmids carrying various promoters. Results were normalized for transfection efficiency by cotransfection of a β-galactosidase expression vector. The solid black boxes represent GATA-1-binding sites. (From Evans and Felsenfeld, 1991).

Fig. 2.

Effect of expressing cGATA-1 in primary chicken embryo fibroblasts on transcription from various promoters containing GATA-1-binding sites. GATA-1 expression vectors were transiently cotransfected with CAT reporter plasmids carrying various promoters. Results were normalized for transfection efficiency by cotransfection of a β-galactosidase expression vector. The solid black boxes represent GATA-1-binding sites. (From Evans and Felsenfeld, 1991).

It seems clear that cGATA-1 must play an important role in the activation of the erythroid program. We do not know how cGATA-1 itself is first activated in the early stages of development. Examination of the cGATA-1 promoter (Hannon et al., 1991; Fig. 3) reveals the presence of a cluster of three strong GATA-1-binding sites upstream of the site of transcription initiation. Trans-activation studies in primary chick embryo fibroblasts confirm that these sites contribute to the expression of GATA-1. Although this autoregulatory function may well serve to maintain expression of the gene, it clearly cannot initiate it. The first steps in activation of GATA-1 expression may involve either the binding to these sites of other members of the GATA family, or interaction of other factors with putative sites that we have identified in or near the GATA-1 promoter. Notable among these is a c-myb site and a site for an unidentified member of the steroid hormone receptor superfamily in the first intron (Fig. 3). We are presently investigating the possible role of these sites in control of GATA-1 expression in early erythroid progenitors.

Fig. 3.

Protein-binding sites on the promoter of the cGATA-1 gene. Lines represent sequences protected in DNase I footprinting experiments with extracts from erythroid cells. The steroid hormone receptor superfamily binding site is the palindromic sequence GGTCA.. at the 5 ′ end of the first intron. (From Hannon et al., 1991).

Fig. 3.

Protein-binding sites on the promoter of the cGATA-1 gene. Lines represent sequences protected in DNase I footprinting experiments with extracts from erythroid cells. The steroid hormone receptor superfamily binding site is the palindromic sequence GGTCA.. at the 5 ′ end of the first intron. (From Hannon et al., 1991).

Control of individual globin genes

GATA-1 clearly can serve as part of a general erythroid ‘switch’ mechanism, but how can it contribute to lineage-specific expression of individual globin family members? We choose as an example the ρ-globin gene, a member of the β-globin family that is expressed only in cells of the primitive lineage. When plasmid constructions containing the ρ-globin promoter are transiently transfected into primary erythroid cells, the same lineage specificity is observed (Fig. 4). The construction pCAT is active in primitive lineage cells, but not in the definitive lineage. As shown in Fig. 4, constructions carrying the βA promoter (PACATE) or the βA promoter (αACATI) also are expressed in a way consistent with their behavior in vivo. These results indicate that the information for lineage-specific (‘stage’-specific) control of ρ-globin gene expression is contained in the 456 bp-promoter segment of pCAT. To identify the controlling elements, we have made a series of deletion and site mutations in the pCAT promoter (Minie et al., 1992). The effect of the site mutations is summarized in Fig. 5. Two elements critical for expression from the p promoter in primitive cells are a GATA-1 motif at -221 bp from the cap site, and an Spl-binding site 50 bp 5’ of the transcriptional start site.

Fig. 4.

(A) Plasmid construction used in transient expression studies of the p-globin promoter. (B) Lineage specific expression of cat under the control of the ρ-, αA- and βA-globin promoters. (From Minie et al., 1992).

Fig. 4.

(A) Plasmid construction used in transient expression studies of the p-globin promoter. (B) Lineage specific expression of cat under the control of the ρ-, αA- and βA-globin promoters. (From Minie et al., 1992).

Fig. 5.

Effect of mutation in GATA-1-, Spl- and TFIID-binding sites of the ρ-globin promoter on expressed CAT activity. (Minie et al., 1992).

Fig. 5.

Effect of mutation in GATA-1-, Spl- and TFIID-binding sites of the ρ-globin promoter on expressed CAT activity. (Minie et al., 1992).

Since GATA-1 and Spl are present in both primitive and definitive lineages, it is perhaps difficult to see how they could be responsible for lineage-specific expression. However, measurement of the nuclear concentrations of both factors in primitive and definitive lineages reveals that there are major differences between the two lineages. As shown in Fig. 6, the concentrations of GATA-1 and Spl (as measured by their ability to bind to their specific DNA sites in a gel shift assay) decrease by almost an order of magnitude as the primitive cells are replaced by the definitive lineage. We have also measured GATA-1 mRNA levels: these decrease by 3-to 4-fold.

Fig. 6.

(A) Abundance of cell lineages in the embryonic circulation during chicken development. (B) Levels of globin and GATA-1 mRNAs during development. (C) Developmental variation in the abundance of Spl, GATA-1 and the NF-1 family member PAL, measured by gel shift assay. The inset shows a calibration curve for the assay. (From Minie et al., 1992).

Fig. 6.

(A) Abundance of cell lineages in the embryonic circulation during chicken development. (B) Levels of globin and GATA-1 mRNAs during development. (C) Developmental variation in the abundance of Spl, GATA-1 and the NF-1 family member PAL, measured by gel shift assay. The inset shows a calibration curve for the assay. (From Minie et al., 1992).

Since we know the approximate nuclear concentrations of the factors, and their specific binding constants, we can estimate the effect of the decrease in nuclear abundance of factors on the occupancy of sites on the p promoter within the nucleus. We calculate that the observed concentration decrease might result in a reduction in the number of doubly occupied promoter sites by almost two orders of magnitude (Minie et al., 1992). The binding affinities of Spl and GATA-1 sites in the control elements of individual globin genes vary considerably. We have suggested that this combination of varying affinity with varying concentration is important in differential regulation of individual globin family members. Although we cannot exclude the possibility of contributions to lineage-specific regulation by other factors and elements not included in the pCAT construction, sufficient information for such regulation appears to be contained within the proximal promoter region of the ρ-globin gene. Thus, regulation of p-globin expression can be controlled by factors that are not confined to a particular lineage. A similar concentration-dependent mechanism (involving additional factors) appears to account for lineage-specific expression of the απ-globin gene (J. Knezetic and G. Felsenfeld, unpublished). On the other hand, control of the βA-globin gene appears to depend upon the presence of a lineage-specific factor, NF-E4 (Gallarda et al., 1989).

Role of chromatin structure

Distant regulatory elements also exert control over globin gene expression. The locus control region (LCR) of the human P-globin domain is 10 to 20 kb away from the globin gene cluster (Grosveld et al., 1987; reviewed by Evans et al., 1990). Our search for equivalent elements in the chicken p locus has led us to a further examination of the properties of the p/e enhancer described above. We find that transgenic mice carrying the enhancer, coupled to the βA-globin gene and its promoter, express the chicken gene in a copy-number-dependent fashion, i.e. the amount of chicken ρ-globin mRNA made is directly proportional to the number of integrated copies of the gene (Fig. 7; Reitman et al., 1990). This result shows that LCR elements need not be located at the ends of a putative loop domain: the β/ε enhancer is situated in the middle of the P cluster.

Fig. 7.

Levels of chicken βA-globin mRNA in mice made transgenic with a DNA segment containing the βA-globin promoter, gene and 3 ′ ρ/ε enhancer. The RNA:DNA ratio is the amount of transgene RNA, per transgene copy, expressed as a percentage of the mouse βmaJ RNA, per gene copy. (From Reitman et al., 1990).

Fig. 7.

Levels of chicken βA-globin mRNA in mice made transgenic with a DNA segment containing the βA-globin promoter, gene and 3 ′ ρ/ε enhancer. The RNA:DNA ratio is the amount of transgene RNA, per transgene copy, expressed as a percentage of the mouse βmaJ RNA, per gene copy. (From Reitman et al., 1990).

Locus control regions are distinguishable from other kinds of regulatory elements that may affect chromatin structure. The above results suggest that LCRs are not likely to mark domain boundaries. Although matrix attachment regions (MARs) may occur nearby, there is no evidence that LCR activity depends on the presence of an MAR. The LCR is also different in properties from DNA sequences, such as the ses elements in Drosophila (Kellum and Schedl, 1991, 1992) and the A elements in the chicken lysozyme locus (Stief et al., 1989), that appear to isolate genes from the effects of m-acting sequences or chromatin structures which lie outside the region they flank. To a first approximation, when a pair of ses sequences surrounds a gene and its local regulatory elements, the gene is expressed at levels governed only by those elements, regardless of where it is inserted in the genome. The ses functions passively, perhaps by forming closed loop domains that sterically restrict access from the outside. Locus control regions appear to be more closely related to conventional enhancers, though many enhancers do not function as LCRs, and there is some evidence that LCR elements may not always be enhancers.

It is possible that LCRs serve to establish locally ‘open’ chromatin domains at the promoters of their target genes (see Felsenfeld, 1992). In any case, the LCR is probably an ‘active’ control region that exerts its effect by perturbing chromatin structure and stabilizing interactions of trans-acting factors with sites on local promoters and enhancers.

Transcription on chromatin templates

It is well established that (with the exception of promoters and enhancers) transcriptionally active eukaryotic genes to a large extent retain their bound histones (Ericsson et al., 1990; Postnikov et al., 1991; Tazi and Bird, 1990; Felsenfeld, 1992). Except at very high rates of transcription, nucleosome cores and even some higher order chromatin structure are likely to be present (Kimura et al., 1983; Caplan et al., 1987; Fisher and Felsenfeld, 1986). How does RNA polymerase transcribe through such structures? We have investigated the possibility that histone octamer displacement accompanies a transient wave of positive supercoiling generated by the advance of the polymerase. The generation of positive supercoils ahead of the polymerase, and negative supercoils behind, has been observed in vivo and in vitro (Liu and Wang, 1987; Tsao et al., 1989; Brill and Sternglanz, 1988). Since formation of nucleosome cores requires the introduction of negative supercoils, it might be expected that positive supercoiling would disfavor nucleosome core formation. We found, however, that in reconstitution experiments in vitro, histone octamers form normal nucleosome structures on positively supercoiled DNA, as judged by their circular dichroic properties, and the chemical reactivity and cross-linking behavior of the histones (Clark and Felsenfeld, 1991). Thus the presence of positive supercoiling does not automatically result in the displacement of a histone octamer. On the other hand, if positive and negative supercoiled plasmids are present together in solution, added histone octamers, under reversible binding conditions, will always prefer the negatively supercoiled DNA. This has led us to propose a model (Fig. 8) in which histone octamers are transferred, one at a time, from a position directly in front of the advancing polymerase to a position directly behind. We have not yet been able to test this hypothesis, but we have undertaken experiments to determine what happens to individual histone octamers during transcription. Our results (Clark and Felsenfeld, 1992) show that nucleosome cores are displaced from their original binding site to other sites on the same plasmid. Although the latter experiments were carried out with a prokaryotic RNA polymerase (SP6), it seems likely that transcription by eukaryotic polymerases will take advantage of the same mechanism, which appears to make use of an intrinsic property of the histones.

Fig. 8.

A hypothetical model for the passage of RNA polymerase through DNA covered with histone octamers, in which one octamer at a time is transferred from the path of the advancing polymerase, driven in part by local supercoiling differences. (From Clark and Felsenfeld, 1991).

Fig. 8.

A hypothetical model for the passage of RNA polymerase through DNA covered with histone octamers, in which one octamer at a time is transferred from the path of the advancing polymerase, driven in part by local supercoiling differences. (From Clark and Felsenfeld, 1991).

The globin gene system provides a model for the study of developmental regulation of gene expression. Within this system it is possible to explore mechanisms of regulation at every level of organization, from the relatively simple interactions of tran.v-acting factors with their sites in local promoters and enhancers, to the still poorly understood effects of distant elements that appear to mediate chromatin structure. The role of chromatin structure is now being addressed in many laboratories. We do not yet know how promoters and enhancers are cleared of histone octamers when genes are activated; activation is likely to involve different mechanisms for different kinds of genes (Felsenfeld, 1992). We also have only a vague idea of how chromatin structure of transcriptionally active domains differs from that of compact chromatin, or how the structure is generated. The globin system of genes can be used to address all of these questions.

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