Histochem Cell Biol (2008) 129:5–11 9 Fig. 3 Intranuclear retention and sequestration as a regulatory means. a Retention of proteins within intranuclear subcompartments regulates their nuclear concentration and their rate of nuclear export. b Seques- tration of proteins at the nuclear periphery, particularly via interaction with the lamina (blue) controls their nucleoplasmic concentration and their availability at target genes. Sequestration can act both as a nega- tive or a positive regulatory mechanism. Sequestration of an activator leads to repression, sequestration of a repression leads to activation. c Retention of partially or fully processed RNA within intranuclear com- partments modulates RNA export rate the nucleus. Several proteins are now known to accumulate, in particular nuclear locations in response to physiological cues and their sequestration is thought to contribute to their cellular function. Prototypical example of sequestration as a regulatory mechanism is the accumulation of the ubiquitin ligases MDM2 and the von Hippel-Lindau tumor suppressor pro- tein VHL in the nucleolus (Mekhail et al. 2005, 2007). These two proteins are responsible for the ubiquitination of the p53 tumor suppressor and the hypoxia-induced factor HIF, respectively. ModiWcation of these two targets leads to their nuclear export and degradation in the cytoplasm. Since ubiquitination is a major regulatory mechanism in their function, the concentration of the ubiquitin ligase in the nucleus signiWcantly determines their fate. Both MDM2 and VHL diVuse freely within the nucleus. However, in response to physiological cues, both MDM2 and VHL can become sequestered in the nucleolus, thus reducing their nucleoplasmic concentration and the extent of ubiquitina- tion of their targets (Mekhail et al. 2005, 2007) (Fig. 1a). Sequestration of these proteins appears to occur by increased retention of diVusing molecules within the nucleolus. How this retention is regulated is currently unclear (Mekhail et al. 2005, 2007). The possibility that retention is a highly controlled mechanism comes from observations on nucleostemin, a nucleolar protein impli- cated in stem cell maintenance and cancer proliferation (Meng et al. 2007; Tsai and McKay 2005). The retention of nucleostemin in the nucleolus is controlled by a GTP–GDP binding cycle which might be directly or indirectly linked to signaling pathways. In addition to nucleostemin the localization of several additional GTP-binding domain con- taining nucleolar proteins seems to be controlled in a simi- lar fashion (Meng et al. 2007; Tsai and McKay 2005). Intranuclear sequestration can also be a means of nega- tive regulation. The basic helix–loop–helix transcription factor Hand1 is expressed in trophoblast stem cells and is required for their diVerentiation along several lineages (Martindill et al. 2007). Hand1 is sequestered in the nucleo- lus and upon stimulation during diVerentiation is rapidly released and moves into the nucleoplasm where it presum- ably acts on target genes. Hand1 is retained in the nucleolus by its interaction with I-mfa, and this interaction is sensi- tive to phosphorylation of Hand1. Upon phosphorylation Hand1 dissociates from I-mfa and is released from the nucleolus. Interestingly, both the sequestration of Hand1 in the nucleolus and its release are functionally important. Premature release of Hand1 triggers diVerentiation, whereas the presence of Hand1 is also required to maintain the stem-cell potential of trophoblast stem cells. Thus nucleolar sequestration of this key cell fate regulator serves a dual regulatory role (Martindill et al. 2007). Most examples of intranuclear sequestration involve the nucleolus. However, the nuclear lamina has recently also emerged as a major site for transcription factor sequestra- tion (Heessen and Fornerod 2007) (Fig. 1b). Prominent transcription factors which associate with the nuclear periphery either via interaction with the lamins or with the inner nuclear membrane proteins include c-fos, Oct-1 and Rb (Imai et al. 1997; Ivorra et al. 2006; Johnson et al. 2004). It is tempting to speculate that the association of these factors with the periphery reduces the intranuclear concentration and thus their availability at target genes (Heessen and Fornerod 2007). A physiological role for such peripheral sequestration is most strongly suggested by observations on the pro-proliferation factor c-fos which associates with the lamina in quiescent cells but is released upon entry of cells into the cell cycle correlating with acti- vation of some of its target genes (Ivorra et al. 2006). Simi- larly, release of Oct-1 from the lamina correlates with activation of collagenase, one of its prime target genes (Imai et al. 1997). The sequestration of transcription factors at the nuclear periphery is a potentially powerful and sim- ple mechanism of transcriptional regulation. It will be important to determine how widespread this mechanism is and how precisely the association of transcription factors with the lamina is controlled. Dynamic traYcking of RNA is similarly used as a regu- latory mechanism (Prasanth and Spector 2007). In particu- lar, it appears that RNA retention can act as a quality 123