Developmental Changes in the Expression of High Mobility Group Chromosomal Proteins*

The high mobility group (HMG) chromosomal pro- teins may modulate the structure of distinct regions in chromatin, thereby affecting processes such as devel- opment and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14-day embryos, whereas circulating cells from adult an-imals are devoid of HMG and most other mRNAs. Nu- clear run-off experiments and Northern analysis of RNA from various developmental stages and from Per- coll-fractionated cells indicate that the genes are transcribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-l4a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied by a change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a conse- quence or

The high mobility group (HMG) chromosomal proteins may modulate the structure of distinct regions in chromatin, thereby affecting processes such as development and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14day embryos, whereas circulating cells from adult animals are devoid of HMG and most other mRNAs. Nuclear run-off experiments and Northern analysis of RNA from various developmental stages and from Percoll-fractionated cells indicate that the genes are transcribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-l4a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied by a change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a consequence or a prerequisite for proper differentiation.
The HMG protein class is among the most abundant and ubiquitous nonhistones found in the nuclei of higher eucaryotes (1, 2). HMG-1 and -2 are single strand DNA-binding proteins (3) which recognize specific DNA structures rather than sequences (4, 5). HMG-14 and -17 are nucleosomal binding proteins (6), which may modulate the chromatin structure of transcriptionally active genes (7, 8). Chicken erythrocytes contain two forms of HMG-14. The main HMG-14 component, HMG-l4a, has a higher molecular weight than the minor component, named HMG-l4b, which is the true homologue of the mammalian HMG-14 (9).
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ll To whom correspondence should be addressed: Bldg. 37, Rm.
During chicken embryogenesis the circulating erythrocytes have a defined developmental program, involving two distinct cell lineages. The primitive lineage, the cells of which synthesize the embryonic @' and @", predominates from the start of erythropoiesis to day 5. After this time, the definitive erythroid lineage, the cells of which synthesize the later globins (pH and PA), gradually becomes the major population throughout the rest of embryogenesis. The red cells are readily available at every stage of maturity and remain nucleated throughout life, thereby allowing the analysis of the structure, as well as the expression of genes of interest. The putative involvement of HMG proteins in chromatin structure and gene expression lead us to investigate the expression of the proteins during erythropoiesis. In this study we report that the relative levels of the HMG proteins and their mRNAs change significantly during erythropoiesis.

MATERIAL AND METHODS
Celk-Fertilized eggs and normal adult chicken erythroids were purchased from Truslow Farm (Chestertown, MD). Erythrocytes from embryos were obtained by nicking an amniotic blood vessel. Reticulocytes were obtained by wing bleeding of adult chickens made anemic by injections of I-acetyl-3-phenylhydrazine.
R N A Analysis-Total RNA was extracted from erythrocytes of different age embryos and from reticulocytes of adult anemic chicken by the guanidinium/cesium chloride method, as described (IO). For Northern analysis, the RNA was fractionated on 1% (w/v) agaroseformaldehyde gel, transferred to Genescreen Plus membranes and hybridized with the probes described in the legend to Figs. 1 and 2, according to the method of Church and Gilbert (11). Primer extension and nuclear run-offs were done as described (10). The probe used for primer extension experiments is described in the text.
Protein Extraction and Gel Electrophoresis-HMG proteins were extracted with 5% (w/v) perchloric acid (1) and fractionated on 15% polyacrylamide-sodium dodecyl sulfate gels, as described (19). Labeled proteins were obtained by suspending cells in lysine-depleted minimal essential medium containing dialyzed fetal bovine serum for 20 h with [14C]lysine (0.1 pCi/ml). The proteins were extracted, fractionated, and stained in polyacrylamide gels. The gels were dried and exposed to Amersham Hyperfilm 0-max for 30 days a t -70 "C.

RESULTS AND DISCUSSION
Northern analyses of total RNA extracted from circulating erythroids of embryos, and anemic and normal chickens indicate that the steady-state levels of HMG mRNAs decrease during embryogenesis (Fig. 1). The HMG messages are absent from reticulocytes, with the exception of HMG-14b mRNA, the level of which is greatly reduced. The mRNA extracted from 5-day erythroids contains significantly more HMG message than that extracted from 7-day or older embryos. Compared with 5-day red cells, 14-day red cells contain 10% HMG-14a mRNA and 40% mRNA for either HMG-l4b, -17, or -1, indicating that the HMG-14a message decreases more rapidly than that of the other HMGs. As an internal control to the experimental system, we have used the well characterized expression of the @*-globin gene. The data in Fig. 1 indicate that, during the same period, the mRNA level of @*-globin increases significantly.
The decrease of HMG mRNA levels suggests that the messages are synthesized a t early stages of differentiation. To define better the developmental stage a t which these HMG mRNAs are actually transcribed, nuclear run-off experiments were done. In both 5-and 9-day embryos the levels of HMG- /3"-Globin represents embryonic transcripts; BA-globin represents the adult transcript. Erythrocytes from 6-day embryos were fractionated on Percoll gradients (12). The RNA was prepared from the unfractionatedcells (Total) and from the top (T) or hottom ( R ) hand. Note t.hat the bottom band is devoid of BA-glohin. R, Northern analysis of HMG mRNA. Note that the top hand of day 6 (67') is highly enriched in HMG mRNAs as compared with the bottom band ( 6 R ) . (v/v) perchloric acid (1 ), fractionated on 15% polyacrylamide-sodium dodecyl sulfate gels (19) and stained with Coomassie Blue. The numbers a t the top of the lanes indicate the age of the embryos (days post-fertilization) from which the samples were taken. E, erythrocytes; R, reticulocytes.
14b and HMG-17 mRNA synthesis are barely detectable and significantly lower than those of globin mRNA which were used as controls (not shown). The results suggest that most  of the HMG mRNA detected in circulating erythrocytes is actually transcribed at an earlier stage of maturation. T o examine further this possibility, we analyzed the earlier blast cell stages of the definitive lineage, separate from both the primitive and definitive mature erythrocytes. This is accomplished by fractionating erythrocytes from 6-day embryos on Percoll density gradients (12) to yield two defined bands of cells, each of which is collected for RNA extraction. The cell lineage comprising each band is determined by primer extension analysis of the type of globin mRNA the cells synthesize, c, p, or A. The sequence spanning nucleotides +98 to +118 of PA-globin, which is common to all chicken @-globin mRNAs, is used as the primer. Since this common primer sequence is located a t different distances from the respective CAP sites (+1) of each @-globin transcript, each transcript's extension product of different length is unmistakably identified and quantified. This analysis and microscopic examination of the cells in the bands indicate that the top band ( Fig. 2 A , lune  67') of the Percoll gradients is a composite containing a small proportion of definitive 6-day erythroblasts and primitive erythrocytes, whereas the bottom band ( Fig. 2 A , lune 6B) contains pure erythrocytes of the primitive lineage. Northern analysis of the same fractions (Fig. 2R) indicates that the top band is highly enriched in HMG mRNAs as compared with either the total or the bottom band. The results support our conclusion that HMG gene transcription is an early event in the differentiation of both the primitive and definitive cell lineages. The messages detected in circulating cells reflect the relative stability of the HMG mRNAs and the percentage of various erythroid cells in the circulation. However, the data do not exclude the possibility that the decrease in the mRNA levels reflects degradation of maternal messages, which were stored in precursor cells. Analysis of the steady-state levels of HMG proteins at various stages of erythropoiesis reveals that the protein profile also changes during development (Fig. 3). In 3-and 5-day cells the major protein is HMG-14a and HMG-17 is barely visible, whereas HMG-14b cannot be detected. The ratio of HMG-14b or -17 to HMG-14a increases up to 9 days and remains constant throughout the following developmental stages. Densitometric analyses indicate that the ratio of HMG-17 to HMG-14a in erythroid cells from 7-day embryos is 4-fold lower than in erythrocytes from adult chickens. The changes are due mainly to a decrease in the amounts of HMG-14a.
The synthesis of HMG proteins during erythropoiesis is analyzed to see if it corresponds with the steady-state HMG protein levels and can be correlated to the switch from the primitive to the definitive erythroid lineage of circulating erythrocytes. Erythroid cells are incubated with ['4C]lysine, and the HMG proteins are fractionated by electrophoresis and visualized by Coomassie Blue staining and autoradiography (Fig. 4). In 5-day embryos the most prominent band corresponds to HMG-14a. The HMG-17 signal is weak, whereas HMG-14b is not visible on the autoradiograms even when the gel is overloaded. In 7-day embryos, the relative synthesis of HMG-14a decreases significantly and the incorporation of ['4C]lysine into HMG-14b is detectable. At 9 days the synthesis of HMG-14a is lower than that of HMG-14b. The changes in HMG synthesis, are most obvious when the relative ratios between the proteins are calculated (Table I). Between day 5 and 9 the relative ratio of HMG-17 to HMG-14a increases 10-fold. Between day 7 and 9 the relative ratio of HMG-14b to HMG-14a increases %fold, and the ratio of HMG-17 to HMG-14b decreases approximately 2-fold. Although the synthesis of HMG-14a is significantly reduced, this protein quantitatively remains the major member of the HMG-14/-17 family, most probably due to the low turnover rate of HMG proteins. The change in the synthesis of the two HMG-14 proteins coincides with the population change from primitive to definitive cell lineage in the embryonic circulation.
The results indicate that the steady-state levels of HMG mRNAs decrease during chicken erythrocyte development in a differentially timed way. As the circulating red cells mature from erythroblasts to erythrocytes and as the definitive lineage replaces the earlier primitive lineage there is a major shift in HMG-14 synthesis, from -14a to -14b, and also more subtle changes in the synthesis of the other HMG proteins. The shift in HMG synthesis is similar to that observed for histones H1 and H5 (20). Reduction in HMG mRNA levels also occurs during myogenesis in rat (21) suggesting that down-regulation of HMG mRNA may be associated with tissue differentiation. The proteins of the HMG-14/-17 family have been associated with transcriptionally active chromatin (7, 8). Our data suggest that the synthesis of HMG proteins during the early stages of erythrocyte maturation is correlated with a high activity of gene transcription. Furthermore, the shift in HMG-14 synthesis, which corresponds to the replacement of the primitive erythroid lineage by the definitive lineage in the population of circulating cells, indicates the possibility that the two different forms of HMG-14 may play a role in the selection of the cell-specific genes transcribed. It is not presently clear whether the switch in HMG gene expression is a consequence or a prerequisite for proper differentiation.