The neuronal repressor REST (RE1-silencing transcription factor; also called NRSF) is expressed at high levels in mouse embryonic stem (ES) cells 1 , but its role in these cells is unclear. Here we show that REST maintains self-renewal and pluripotency in mouse ES cells through suppression of the microRNA miR-21. We found that, as with known self-renewal markers, the level of REST expression is much higher in self-renewing mouse ES cells than in differentiating mouse ES (embryoid body, EB) cells. Heterozygous deletion of Rest (Rest 1/2 ) and its short-interfering-RNA-mediated knockdown in mouse ES cells cause a loss of self-renewal-even when these cells are grown under self-renewal conditions-and lead to the expression of markers specific for multiple lineages. Conversely, exogenously added REST maintains self-renewal in mouse EB cells. Furthermore, Rest 1/2 mouse ES cells cultured under self-renewal conditions express substantially reduced levels of several self-renewal regulators, including Oct4 (also called Pou5f1), Nanog, Sox2 and c-Myc, and exogenously added REST in mouse EB cells maintains the self-renewal phenotypes and expression of these self-renewal regulators. We also show that in mouse ES cells, REST is bound to the gene chromatin of a set of miRNAs that potentially target self-renewal genes. Whereas mouse ES cells and mouse EB cells containing exogenously added REST express lower levels of these miRNAs, EB cells, Rest 1/2 ES cells and ES cells treated with short interfering RNA targeting Rest express higher levels of these miRNAs. At least one of these REST-regulated miRNAs, miR-21, specifically suppresses the self-renewal of mouse ES cells, corresponding to the decreased expression of Oct4, Nanog, Sox2 and c-Myc. Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self-renewal and pluripotency of mouse ES cells.REST is believed to be a major transcriptional repressor of neurogenesis 2-5 , and activation of REST target genes was found to be sufficient to convert neural stem/progenitor cells to neuronal phenotypes 6,7 . However, REST activity seems to depend on the cellular context; for example, REST can show both an oncogenic 8-10 and tumour-suppressor function 5 as well as involvement in haematopoietic and cardiac differentiation [3][4][5] . Embryonic stem (ES) cells are pluripotent cells that have the potential for both indefinite selfrenewal and differentiation into all three germ layers of the body 11 . Here we provide evidence that REST has a unique role as a protector of self-renewal and pluripotency in mouse ES cells, corresponding to the expression of critical regulators such as Oct4, Nanog, Sox2 and c-Myc.We began by assessing the levels of REST protein in mouse ES cells growing under self-renewal conditions and differentiation conditions ( Fig. 1a; ES and EB, respectively). As expected, western blotting showed that the ES cells had higher levels of REST expression and of the representative markers of self-renewal (proteins Oct4, Sox2 and c-M...

Linker DNA was found to be critical for the specific docking of ISW2 with nucleosomes as shown by mapping the physical contacts of ISW2 with nucleosomes at base-pair resolution. Hydroxyl radical footprinting revealed that ISW2 not only extensively interacts with the linker DNA, but also approaches the nucleosome from the side perpendicular to the axis of the DNA superhelix and contacts two disparate sites on the nucleosomal DNA from opposite sides of the superhelix. The topography of the ISW2-nucleosome was further delineated by finding which of the ISW2 subunits are proximal to specific sites within the linker and nucleosomal DNA regions by site-directed DNA photoaffinity labeling. Although ISW2 was shown to contact B63 bp of linker DNA, a minimum of 20 bp of linker DNA was required for stable binding of ISW2 to nucleosomes. The remaining B43 bp of flanking linker DNA promoted more efficient binding under competitive binding conditions and was functionally important for enhanced sliding of nucleosomes when ISW2 was significantly limiting.

In the present study, we demonstrated that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from the hippocampus and olfactory bulb (OB). Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampal and OB-derived neural stem cells. Our analysis indicated that the balance between Wnt3, which triggers the expression of insulin via NeuroD1, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on diabetic (DB) status. We also show that adult neural progenitors derived from DB animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of DB animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes.

The stable contact of ISW2 with nucleosomal DNA ϳ20 bp from the dyad was shown by DNA footprinting and photoaffinity labeling using recombinant histone octamers to require the histone H4 N-terminal tail. Efficient ISW2 remodeling also required the H4 N-terminal tail, although the lack of the H4 tail can be mostly compensated for by increasing the incubation time or concentration of ISW2. Similarly, the length of extranucleosomal DNA affected the stable contact of ISW2 with this same internal nucleosomal site, with the optimal length being 70 to 85 bp. These data indicate the histone H4 tail, in concert with a favorable length of extranucleosomal DNA, recruits and properly orients ISW2 onto the nucleosome for efficient nucleosome remodeling. One consequence of this property of ISW2 is likely its previously observed nucleosome spacing activity.ATP-dependent chromatin remodeling subfamilies ISWI, SNF2, CHD1, and INO80 have all been shown to alter the structure of chromatin to make DNA accessible for DNAbinding proteins during various regulatory processes inside the cell (10). These multiprotein complexes function in different ways to move or displace nucleosomes in order to increase the accessibility of DNA (4,12,14,26,27). Regulation of the activity of these enzymes in the cell is crucial, as perturbations can lead to neoplasias and other diseases (5).Most ISWI complexes consist of two to four subunits (25) and are involved in chromatin assembly (17, 37), spacing of nucleosomal arrays (17, 34), and moving mononucleosomes in ϳ10-bp steps (20,40). ISWI is fully stimulated only by nucleosomes with intact histone amino-terminal tails (1). Further studies have revealed that the basic patch R 17 H 18 R 19 of the histone H4 tail was required for remodeling by ISWI in vitro (6,7,15) and in vivo (13). Acetylation of K16 on H4 was also found to impede chromatin remodeling by ISWI (8). The H4 tail has been shown to interact with nucleosomal DNA near SHL2 (superhelical location 2), two helical turns away from the dyad axis, by chemical cross-linking (11). Consistent with this finding are the recent reports showing that yeast ISW2 makes strong contacts with the SHL2 site (18) and that this contact is critical for chromatin remodeling by ISW2 (41). It has not been shown directly if the H4 tail is required for ISW2 to contact the SHL2 site or for ISW2 remodeling.Given the recent finding that the histone H4 tail is involved in the formation of higher-order chromatin structure (31), it is important to determine how this structural role of the H4 tail may relate to its functional role in ISWI remodeling. Histone tails generally interact with linker DNA within ϳ25 bp from the edge of nucleosomes (38). The major binding sites for histone tails have been shown by UV cross-linking to be with linker DNA and not with the core nucleosome particle (2, 32). Specifically, extranucleosomal DNA causes a change in the contacts of the histone H2A C-terminal tail from the dyad axis to close to the entry/exit sites of nucleosomes (21, 36). The his...

Summary Distinct stages in ATP-dependent chromatin remodeling are found as ISW2, an ISWI type complex, forms a stable and processive complex with nucleosomes upon hydrolysis of ATP. There are two conformational changes of the ISW2-nucleosome complex associated with binding and hydrolysis of ATP. The initial binding of ISW2 to extranucleosomal DNA, the entry site and near the dyad axis of the nucleosome is enhanced by ATP binding; while subsequent ATP hydrolysis is required for template-commitment and causes ISW2 to expand its interactions with nucleosomal DNA to an entire gyre of the nucleosome and a short ~3–4 bp site on the other gyre. The histone-fold like subunit Dpb4 associates with nucleosomal DNA ~15 bp from the ATPase domain as part of this change and may help disrupt histone-DNA interactions. These additional contacts are independent of the ATPase domain tracking along nucleosomal DNA and are maintained as ISW2 moves nucleosomes on DNA.

Histone fold proteins Dpb4 and Dls1 are components of the yeast ISW2 chromatin remodeling complex that resemble the smaller subunits of the CHRAC (Chromatin Accessibility Complex) complex found in Drosophila and humans. DNA photoaffinity labeling found that the Dpb4 subunit contacts extranucleosomal DNA 37-53 bp away from the entry/exit site of the nucleosome. Binding of Dpb4 to Isw2 and Itc2, the two largest subunits of ISW2, was found to require Dls1. Even after remodeling and nucleosome movement, Dpb4 tends to remain bound to its original binding site and likely serves as an anchor point for ISW2 on DNA. In vitro, only minor differences can be detected in the nucleosome binding and mobilization properties of ISW2 with or without Dpb4 and Dls1. Changes in the contacts of the largest subunit Itc1 with extranucleosomal DNA have, however, been found upon deletion of the Dpb4 and Dls1 dimer that may affect the nucleosome spacing properties of ISW2.

RE-1 silencing transcription factor (REST), a master negative regulator of neuronal differentiation, controls neurogenesis by preventing the differentiation of neural stem cells. Here we focused on the role of REST in the early steps of differentiation and maturation of adult hippocampal progenitors (AHPs). REST knockdown promoted differentiation and affected the maturation of rat AHPs. Surprisingly, REST knockdown cells enhanced the differentiation of neighboring wild-type AHPs, suggesting that REST may play a non-cellautonomous role. Gene expression analysis identified Secretogranin II (Scg2) as the major secreted REST target responsible for the non-cell-autonomous phenotype. Loss-of-function of Scg2 inhibited differentiation in vitro, and exogenous SCG2 partially rescued this phenotype. Knockdown of REST in neural progenitors in mice led to precocious maturation into neurons at the expense of mushroom spines in vivo. In summary, we found that, in addition to its cell-autonomous function, REST regulates differentiation and maturation of AHPs non-cell-autonomously via SCG2.

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