EXPERIMENTAL CELL RESEARCH 198,268-275 (1992) A Novel Nonhistone Protein (MENT) Promotes Nuclear Collapse at the Terminal Stage of Avian Erythropoiesis S. A. GRIGORYEV,’ V. 0. SOLOVIEVA, K. S. SPIRIN, AND I. A. KRASHENINNIKOV Department of Molecular Biology, Moscow State University, Moscow, U.S.S.R. The terminal stage of differentiation of nucleated chicken erythrocytes is associated with an overall gene repression and a condensation of the repressed chroma- tin portion. Two-dimensional DNP electrophoresis has been used to separate transcriptionally active and re- pressed chromatin of mature chicken erythrocytes. The repressed chromatin fraction is shown to be enriched with histone HS as well as with a 42-kDa nonhistone chromosomal protein. The 42-kDa protein designated here as MENT (mature erythrocyte nuclear termina- tion stage-specific protein) is hyperexpressed at the ter- minal stage of chicken erythropoiesis and is accumu- lated in adult chicken erythrocyte nuclei. This protein was purified by ion-exchange chromatography from 0.4 M NaCl extracts of the erythrocyte nuclei. It ap- peared to be a basic polypeptide (~19.2) which, how- ever, precipitated at low PH. When reconstituted in ui- tro with immature erythrocyte nuclei, MENT promoted condensation of intact nuclear chromatin and enhanced the solubilization of nuclease-digested polynucleo- somes, thus mimicking the processes occuring in uiuo at the final stage of erythrocyte maturation. The extent of dissociation of specific gene sequences from the nuclear matrix in MENT-treated nuclei is in striking correla- tion with their transcriptional activity. No other basic proteins (HS, cytochrome c, RNase A) added to the nu- clear preparation at the same level as MENT (protein/ DNA = 0.005) caused any effect on nuclear organiza- tion. No alterations were observed when MENT was mixed with erythroblasts and nonerythroid nuclei hav- ing little or no histone H5. We propose that MENT coop- erates with histone HS to complete the nuclear collapse in mature nucleated erythrocytes. Q 1992 Academic PZ~SS. I~C. INTRODUCTION In addition to trans-acting factors eukaryotes have an alternative mechanism for gene repression that is the heterochromatization (chromatin condensation) of par- ticular genome domains. Certain heterochromatic re- ‘To whom correspondence and reprint requests should be ad- dressed at present address: Zoology Department, University of Mas- sachusetts at Amherst, Amherst, MA 01993. gions (constitutive) are established early in embryogen- esis and are inherited in cell lines in a position-depen- dent manner. Other chromatin regions become heterochromatic (facultative) in the course of cell dif- ferentiation [l, 21. One can distinguish three levels of heterochromatin propagation: the lower level is asso- ciated with repression of limited genomic regions ex- tended for several dozen kbp (e.g., locus “white” of Dro- sophila [3] ); at the second level, heterochromatin is ex- tended over a whole X-chromosome of female mammals [4]; and, finally, at the third level, the bulk of interphase chromatin in a given cell type, e.g., in the nucleated erythrocytes of lower vertebrates, becomes hypercon- densed and transcriptionally inert [5-71. The mecha- nism and the driving force of progressive heterochro- matization remain unknown. It is also unclear whether there are any differences in chromatin structure be- tween euchromatin and heterochromatin fractions or whether these two chromatin portions differ only in the amount of free space between the chromatin fibers. We have isolated a nonhistone chromosomal protein (MENT, mature erythrocyte nuclear termination stage-specific protein) which is selectively bound to the repressed chromatin fraction. Hyperexpression of MENT coincides with complete metabolic inactivation and heterochromatization of mature chicken erythro- cytes. When reconstituted with immature erythroeyte nuclei in vitro this protein induced the structural transi- tions (condensation) observed for the chromatin depos- ited in the nuclei but not for the soluble chromatin. Thus MENT is a probable candidate for a factor induc- ing the heterochromatization of chromatin at the termi- nal stage of erythroid differentiation. Its ability to disso- ciate polynucleosomes from the nuclear framework sug- gests that MENT may act by releasing the euchromatin domain boundaries, thus allowing chromatin condensa- tion to spread from neighboring heterochromatic re- gions. MATERIALS AND METHODS Cells, nucki, and microscopy. Erythrocytes were obtained from the blood of lo-day-old (EE-10) and 15-day-old (EE-15) embryos and from the blood of adult chickens (AE). An erythroblast cell line (HD3) transformed by a temperature-sensitive virus of avian erythro- blastosie was obtained from Dr. T. Graf (Heidelberg, Germany) and 0014~4827192 $3.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form resewed 268
