7 B). transcribed into large and stable RNAs that remain associated with the 9q12 region, even throughout mitosis. This work not only reveals the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure, but also provides evidence for a transcriptional activity within a locus considered so far as heterochromatic and silent. polytene chromosomes (Nowak and Corces, 2000; Labrador and Corces, 2003), perhaps because human chromosome 9 sat III repeats are not structured as a canonical heat-shock promoter (unpublished data). Thus, nuclear stress granules contain acetylated core histones. Open in a separate window Figure 2. Stress-induced HSF1 granules contain acetylated core histones. HSF1 (green) and acetylated forms of each core histone (red) were codetected by immunofluorescence in HeLa cells submitted or not to a 1-h heat shock at 42C. At 37C, HSF1 is Bromocriptin mesylate diffusely distributed in the nucleus and cytoplasm, and acetylated histones exhibit a punctate distribution throughout the nucleus. After heat shock, acetylated forms of the four core histones are present to various extents within stress granules. Bar, 5 m. To confirm that the presence of acetylated histones within the granules is a stress-induced event, we investigated the possibility that a histone acetyltransferase (HAT) is also recruited to the granules upon stress. Therefore, we analyzed the distribution of transiently expressed fusion proteins encoding either GCN5, Tip60, or CREB binding protein (CBP) in HeLa cells (Col et al., 2001; Legube et al., 2002). GCN5 and Tip60 both displayed a punctate nuclear staining that was unaffected by stress (unpublished data). In contrast, a fraction of the overexpressed CBP protein was detected at 42C in a few granular structures that coincided with HSF1-containing granules, in addition to the persisting punctate nucleoplasmic staining (Fig. 3). CBP is thus able to relocate to the granules during stress, likely accounting for the stress-induced accumulation of acetylated histones within these structures. Open in a separate window Figure 3. CBP is recruited to nuclear stress granules during heat shock. Transiently expressed CBP-HA (red) was codetected with HSF1 (green) in HeLa cells. In nonCheat-shocked cells, CBP displays a fine punctate staining dispersed throughout the nucleoplasm. At 42C, a fraction of the protein colocalizes with HSF1 in the granules. Bar, 5 m. Stress induces the transcription of sat III repeats Based on these observations, we sought to determine if transcription occurs within the granules. Nuclear stress granules form primarily on the 9q12 juxtacentromeric region in humans, with putative secondary sites on chromosomes 12 and 15 and perhaps other chromosomes (Denegri et al., 2002; unpublished data). As the only characterized target sequence Bromocriptin mesylate for HSF1 within the granules is a sat III repeat of the 9q12 region characterized by the clone pHuR98 (Jolly et al., 2002), we investigated the possibility that these repeats could be inducibly transcribed during stress. We first performed FISH to detect transcripts with the clone pHuR98 as a probe (Grady et al., 1992). Results are presented in Fig. 4. Both at 37C and 42C, a diffuse nucleoplasmic and cytoplasmic staining was observed (Fig. 4 A). In addition, at Bromocriptin mesylate 42C, three to four bright nuclear foci were also visible in most cells. These granular foci corresponded to transcripts as confirmed by their absence in cells treated with RNase A before hybridization (Fig. 4 A). The intensity of the nuclear and cytoplasmic diffuse staining is comparable to the background level both at 37C and 42C, thus demonstrating that sat III transcripts are essentially concentrated in the three to four nuclear foci (Fig. 4 B). Similarly RNA FISH experiments performed with probes Bromocriptin mesylate corresponding to Bromocriptin mesylate Rabbit polyclonal to PAWR either chromosome 9 classical satellites (D9Z1), chromosome 9 centromeric repeats, or chromosome 16 sat II repeats (pHuR195) did not reveal any signal (not depicted). Codetection of sat III transcripts with HSF1 or RNA pol II showed a colocalization of these transcript foci with nuclear stress granules (Fig. 4 C). Finally, to confirm that the presence of three.