UNCORRECTED PROOF MOLBIO 9602 1–12 Molecular & Biochemical Parasitology xxx (2005) xxx–xxx Trypanosoma cruzi histone H1 is phosphorylated in a typical cyclin dependent kinase site accordingly to the cell cycle 3 4 Julia Pinheiro Chagas da Cunha a , Ernesto S. Nakayasu b , Maria Carolina Elias a,1 , Daniel C. Pimenta c , Maria Teresa Tellez-Inon d , Federico Rojas d , Mu˜ noz Manuel d , Igor C. Almeida b , Sergio Schenkman a,∗ 5 6 7 a Departamento de Microbiologia, Imunologia e Parasitologia, R. Botucatu 862-8 a , EPM-UNIFESP, S˜ ao Paulo, SP 04023-062, Brazil 8 b Departamento de Parasitologia, ICB, USP, S˜ ao Paulo, Brazil 9 c Centro de Toxinologia Aplicada, CAT/CEPID, Instituto Butantan, S˜ ao Paulo, SP, Brazil 10 d Instituto de Investigaciones en Ingenieria Gen´ etica y Biologia Molecular (INGEBI, CONICET), Vta de Obligado 2490, 1428 Buenos Aires, Argentina 11 Received 16 September 2004; received in revised form 20 December 2004; accepted 21 December 2004 12 Abstract 13 Histone H1 of most eukaryotes is phosphorylated during the cell cycle progression and seems to play a role in the regulation of chromatin structure, affecting replication and chromosome condensation. In trypanosomatids, histone H1 lacks the globular domain and is shorter when compared with the histone of other eukaryotes. We have previously shown that in Trypanosoma cruzi, the agent of Chagas’ disease, histone H1 is phosphorylated and this increases its dissociation from chromatin. Here, we demonstrate using mass spectrometry analysis that T. cruzi histone H1 is only phosphorylated at the serine 12 in the sequence SPKK, a typical cyclin-dependent kinase site. We also found a correlation between the phosphorylation state of histone H1 and the cell cycle. Hydroxyurea and lactacystin, which, respectively, arrest parasites at the G1/S and G2/M stages of the cell cycle, increased the level of histone H1 phosphorylation. Cyclin-dependent kinase-related enzymes TzCRK3, and less intensely the TzCRK1 were able to phosphorylate histone H1 in vitro. Histone H1 dephosphorylation was prevented by treating the parasites with okadaic acid but not with calyculin A. These findings suggest that T. cruzi histone H1 phosphorylation is promoted by cyclin dependent kinases, present during S through G2 phase of the cell cycle, and its dephosphorylation is promoted by specific phosphatases. 14 15 16 17 18 19 20 21 22 23 © 2004 Published by Elsevier B.V. 24 Keywords: Histone H1; Phosphorylation; Cell cycle; Trypanosoma cruzi; Phosphatase; CDK 25 26 Abbreviations: a.m.u., atomic mass unit; ESI-TOF-MS, electrospray ionization-time of flight-mass spectrometry; ESI-IT-MS, electrospray ionization-ion trap-mass spectrometry; m/z, mass to charge ratio; CDKs, cy- clin dependent kinases; HU, hydroxyurea; OA, okadaic acid; AUT-PAGE, polyacrylamyde gel electrophoresis containing acetic acid; urea and Triton- DF116; TzCRK1, T. cruzi cyclin related kinase 1; TzCRK3, T. cruzi cyclin related kinase 3. ∗ Corresponding author. Tel.: +55 115 751 996; fax: +55 115 571 5877. E-mail address: sergio@ecb.epm.br (S. Schenkman). 1 Present address: Laboratorio de Parasitologia, Instituto Butantan, S˜ ao Paulo, SP, 05503-900, Brazil. 1. Introduction 27 Histone H1, also known as linker histone, consists of a 28 conserved central globular domain flanked by a relatively 29 short amino- and a long carboxy-terminal tail. The globular 30 domain seems to interact with linker DNA outside the nucle- 31 osome core, and the tails with the linker DNA and with the 32 amino-terminal tails of core histones [1]. Histone H1 affects 33 many features of chromatin structure and function. It stabi- 34 lizes the high-order structure of chromatin [2,3] and affects 35 nucleosome position and spacing [4,5]. It is involved in chro- 36 matin assembly during replication [6], chromatin remodeling 37 [7] and condensation [8], gene transcription [9] and cell apop- 38 1 0166-6851/$ – see front matter © 2004 Published by Elsevier B.V. 2 doi:10.1016/j.molbiopara.2004.12.007