The intracellular localization, and thereby the function, of a number of key regulator proteins tagged with a short leucine-rich motif (the nuclear export signal or NES) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [1]. A common characteristic of these regulators is their transient action in the nucleus during either a specific phase of the cell cycle or in response to specific signals [1]. Here, we show that a particular member of the class II histone-deacetylases mHDA2/mHDAC6 [2] belongs to this family of cellular regulators that are present predominantly in the cytoplasm, but are also capable of shuttling between the nucleus and the cytoplasm. A very potent NES present at the amino terminus of mHDAC6 was found to play an essential role in this shuttling process. The sub-cellular localization of mHDAC6 appeared to be controlled by specific signals, since the arrest of cell proliferation was found to be associated with the translocation of a fraction of the protein into the nucleus. Data presented here suggest that mHDAC6 might be the first member of a functionally distinct class of deacetylases, responsible for activities not shared by other known histone deacetylases.

Recently we identified a new family of histone deacetylases in higher eukaryotes related to yeast HDA1 and showed their differentiation-dependent expression. Data presented here indicate that HDAC5 (previously named mHDA1), one member of this family, might be a potent regulator of cell differentiation by interacting specifically with determinant transcription factors. We found that HDAC5 was able to interact in vivo and in vitro with MEF2A, a MADS box transcription factor, and to strongly inhibit its transcriptional activity. Surprisingly, this repression was independent of HDAC5 deacetylase domain. The N-terminal non-deacetylase domain of HDAC5 was able to ensure an efficient repression of MEF2A-dependent transcription. We then mapped protein domains involved in the HDAC5-MEF2A interaction and showed that MADS box/MEF2-domain region of MEF2A interacts specifically with a limited region in the N-terminal part of HDAC5 which also possesses a distinct repressor domain. These data show that two independent class II histone deacetylases HDAC4 and HDAC5 are able to interact with members of the MEF2 transcription factor family and regulate their transcriptional activity, thus suggesting a critical role for these deacetylases in the control of cell proliferation/differentiation. Acetylation of chromatin proteins and transcription factors is part of a complex signaling system that is largely involved in the control of gene expression (1). Thus far, the specific involvement of histone acetyltransferases and deacetylases in the control of individual gene expression has been clearly established (2, 3). One major role of these enzymes is the control of cell differentiation in response to specific signals. Evidence exists for the participation of the RPD3-related members in this process (4). Recently, however, a new family of higher eukaryotic histone deacetylases, distinct from the already characterized RPD3-related members, has been identified (5-7). These enzymes are related to yeast HDA1 histone deacetylase and within the cloned members, two show sequence homology and the same domain organization and are called HDAC4 and HDAC5 (5, 6). Despite this homology, HDAC4 and HDAC5 are probably capable of exerting distinct functions, since immunoprecipitation experiments showed that in cells they can be associated with different partners (6). A member, named mHDA2/HDAC6, shows unique features within deacetylases, in that it possesses two HDA1 homology domains (5, 6). In contrast to the RPD3-related members, the expression of these genes is not ubiquitous. HDAC5 and mHDA2/HDAC6 expression is activated upon cell differentiation (5). These observations suggest that members of the class II histone deacetylases may play a specific role in the regulation of cell differentiation. Looking for potential partners of HDAC5, we obtained evidence of interaction between HDAC5 and MEF2 transcription factors. We therefore focused our efforts on investigating this issue. The MEF2 family of transcription factors belongs to the large family of MADS...

Histone H1° is a differentiation-specific member of the histone H1 family. The accumulation of the protein is associated with the terminal stage of cell differentiation and is regulated at various levels. In mouse, the analysis of the expression of the single copy gene encoding H1° has shown that another HlO-related mRNA species (0.9 kb) is present in addition to the usual 2.1-kb mRNA. In this study, we have cloned and sequenced the smaller HlO-related mRNA. This mRNA seems to be produced by the use of an additional polyadenylation signal present in the 3' untranslated region (UTR) of the initial transcript. This smaller H1"-encoding mRNA is expressed only in mouse and is transferred into polysomes as efficiently as the larger version upon the induction of cell differentiation. The use of the described polyadenylation site removes over 1 kb of the 3' UTR of H1" mRNA and seems to be involved in the regulation of H1° mRNA stability.The expression of histone H1 protein is developmentally regulated [l -51. Indeed, three main classes of somatic histone H1 are expressed among vertebrates. Embryonic H1 is present only during the early stages of the embryonic development of amphibians [3]. The regular H1 group is composed of several closely related proteins. These proteins, although absent during early stages of development, accumulate in cells at the same time as zygotic expression begins [2, 4, 51. Finally, the arrest of cell proliferation and the appearance of the differentiated phenotype in the embryo is associated with the expression of the third class of histone H1, a differentiation-specific subtype (adult type H1) [6, 71. Hl", a member of this adult-type histone H1 family, was first described in mammals [8] and it is now clear that it is widely expressed in vertebrates. Indeed, genomic and cDNA sequence have been published for human [9, lo], mouse [ l l , 121, rat [13] and xenopus H1" [14, 71 and there is evidence for its expression in birds [15], reptiles [16] and fishes [17]. Since this protein is expressed in relation to the commitment of cells to many differentiation programs, the regulation of its expression may be a key process in the establishment of a particular genetic program. In mouse, a 0.9-kb mRNA is expressed in addition to the cloned 2.1-kb H1° mRNA [ll]. In order to understand the different levels of the regulation of H1" gene expression better, we have characterized the smaller HlO-related mRNA. This mRNA is a truncated version of the 2.1-kb H1° mRNA containing only about 130 bases of 3' UTR. An additional polyadenylation signal present in the 3' UTR of the 2.1-kb mRNA seems to be used to convert a fraction of the initial transcript into the 0.9-kb species. It is efficiently transferred into polysomes upon induction of cell differentiation. The shortening of the 3' UTR by this alternative polyadenylation seems to be involved in the stabilization of H1"-encoding message under certain conditions, i.e. during the process of dedifferentiation. MATERIAL AND METHODS Cell lines and cultureMurine ery...