MeCP2 Expression in Human Cerebral Cortex and Lymphoid Cells: Immunochemical Characterization of a Novel Higher-Molecular-Weight Form

Abstract

Most cases of Rett syndrome are associated with mutations in the coding region of MECP2. Here we characterized a novel MeCP2 immunoreactivity, initially detected in normal cerebral cortex, by using a panel of MeCP2 antibodies and a combination of immunochemical techniques. We found that a novel higher-molecular-weight form (~ 100 kDa) of MeCP2 is detected in human frontal cortex nuclear and synaptic fractions and in lymphoid cells. Although in the cortex the higher-molecular-weight form is relatively more abundant than the standard ~ 75 kDa immunoreactivity, in extranuclear locations, lymphocyte lysates show a predominance of the standard 75 kDa band. Lymphoblasts revealed a more complex pattern of MeCP2 expression, with prominent higher-molecular-weight form and both higher-molecular-weight form and 75 kDa MeCP2 immunoreactivities encompassing several closely migrating bands. We also successfully immunoprecipitated both the 75 kDa immunoreactivity and the higher-molecular-weight form MeCP2 from cerebral cortex with a C-terminal antibody and confirmed their identities by immunoblotting with C- and N-terminal antibodies. Our data provide compelling evidence for the existence of a novel MeCP2 molecular form, most likely the result of post-translational modification. Detection in both brain and lymphoid cells suggests an important role for higher-molecular-weight form in MeCP2-dependent processes. The presence of higher-molecular-weight form MeCP2 in postsynaptic fractions indicates a possible involvement in linking synaptic activity and transcriptional repression that, in turn, could play a role in the pathogenesis of Rett syndrome and other neurologic disorders. (J Child Neurol 2003;18:675—682).

References

Naidu S. : Rett syndrome: A disorder affecting early brain growth. Ann Neurol 1997;42:3—10.

van den Veyver IB , Zoghbi HY : Mutations in the gene encoding methyl-CpG-binding protein 2 cause Rett syndrome. Brain Dev 2001 ;23(Suppl 1):S147—S151.

Nan X. , Campoy FJ , Bird A. : MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 1997;88: 471—481.

Ballestar E. , Wolfe AP : Methyl-CpG binding proteins: Targeting specific gene repression. Eur J Biochem 2001 ;268:1—6.

Akbarian S. , Chen RZ , Gribnau J. , et al: Expression pattern of the Rett syndrome gene MeCP2 in primate prefrontal cortex. Neurobiol Dis 2001;8:784—791.

Johnston MV , Jeon OH , Pevsner J. , et al: Neurobiology of Rett syndrome: A genetic disorder of synapse development. Brain Dev 2001;23(Suppl 1):S206—S213.

Shahbazian MD , Antalffy B. , Armstrong DL , Zoghbi HY : Insight into Rett syndrome: MeCP2 levels display tissue- and cell-specific differences and correlate with neuronal maturation. Hum Mol Genet 2002;11:115—124.

LaSalle JM , Goldstine J. , Balmer D. , Greco CM : Quantitative localization of heterogeneous methyl-CpG-binding protein 2 (MeCP2) expression phenotypes in normal and Rett syndrome brain by laser scanning cytometry. Hum Mol Genet 2001;10:1729—1740.

Aber KM , Nori P. , MacDonald S. , et al: Methyl-CpG-binding protein 2 is localized in the postsynaptic compartment: An immunochemical study of subcellular fractions. Neuroscience 2003;116: 77—80.

10.

Paratcha G. , Stein ML , Szapiro G. , et al: Experience-dependent decrease in synaptically localized Fra-1. Mol Brain Res 2000;70: 120—130.

11.

Eberwine J. , Miyashiro K. , Kacharmina JK , Job C. : Local translation of classes of mRNAs that are targeted to neuronal dendrites. Proc Natl Acad Sci U S A 2001;98:7080—7085.

12.

Kaufmann WE , Jarrar MH , MacDonald SM : MeCP2 expression in human neocortex. Brain Dev 2002;24:392.

13.

Brown V. , Jin P. , Ceman S. , et al: Microarray identification of FMRPassociated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 2001;107:477—487.

14.

Mole L. , Margolis D. , Carroll R. , et al: Stabilities of quantitative plasma culture for human immunodeficiency virus, RNA, and p24 antigen from samples collected in Vacutainer CPT and standard Vacutainer tubes. J Clin Microbiol 1994;32:2212—2215.

15.

Lewis JD , Meehan RR , Henzel WJ , et al: Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell 1992;105:487—497.

16.

Huttner WB , Scheibler W. , Greengard P. , De Camilli P : Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III: Its association with synaptic vesicle preparation. J Cell Biol 1983;96: 1374—1388.

17.

Sun H-T. , Cohen S. , Kaufmann WE : Annexin 1 is abnormally expressed in FRAGILE X syndrome: A two-dimensional electrophoresis study in lymphocytes. Am J Med Genet 2001; 103:81-90.

18.

Kaufmann WE , Abrams MT , Chen W. , Reiss AL : Genotype, molecular phenotype, and cognitive phenotype: Correlations in fragile X syndrome. Am J Med Genet 1999;83:286—295.

19.

Wan M. , Zhao K. , Lee SS , Francke U. : MECP2 truncating mutations cause histone H4 hyperacetylation in Rett syndrome . Hum Mol Genet 2001;10:1085—1092.

20.

Kudo S. , Nomura Y. , Segawa M. , et al: Functional analyses of MeCP2 mutations associated with Rett syndrome using transient expression systems. Brain Dev 2001;23(Suppl 1):S165—S173.

21.

Kaufmann WE , MacDonald SM , Altamura C. : Dendritic cytoskeletal protein expression in mental retardation: An immunohistochemical study of the neocortex in Rett syndrome. Cereb Cortex 2000;10:992—1004.

22.

Hoffbuhr KC , Moses LM , Jerdonek Ma , et al: Associations between MeCP2 mutations, X-chromosome inactivation, and phenotype. Ment Retard Dev Disabil Res Rev 2002;8:99—105.

23.

Oda Y. : Choline acetyltransferase: The structure, distribution and pathologic changes in the central nervous system. Pathol Int 1999;49:921—937.

24.

Traynor J. , Agarwal P. , Lazzeroni L. , Francke U. : Gene expression patterns vary in clonal cell cultures from Rett syndrome females with eight different MECP2 mutations. BMC Med Genet 2002;3:12.

25.

Balmer D. , Arredondo J. , Samaco RC , LaSalle JM : MECP2 mutations in Rett syndrome adversely affect lymphocyte growth, but do not affect imprinted gene expression in blood or brain. Hum Genet 2002;110:545—552.

26.

Geiman TM , Robertson KD : Chromatin remodeling, histone modifications, and DNA methylation—How does it all fit together? J Cell Biochem 2002 ;87:117—125.

27.

Shahbazian MD , Zoghbi HY : Molecular genetics of Rett syndrome and clinical spectrum of MECP2 mutations. Curr Opin Neurol 2001;14:171—176.

28.

Weaving LS , Williamson SL , Bennetts B. , et al: Effects of MECP2 mutation type, location and X-inactivation in modulating Rett syndrome phenotype. Am J Med Genet 2003;118A:103—114.

29.

Kaufmann WE : Cortical development in Rett syndrome: Molecular, neurochemical, and anatomical aspects, in Kerr A , Engerström IW (eds): The Rett Disorder and the Developing Brain. Oxford, UK, Oxford University Press, 2001, 85—110.