The interaction of regulatory factors with specific genomic regions constitutes a critical step in gene regulation. Most regulatory factors bind to specific sites as multiprotein complexes that affect the local structure of chromatin and recruit modifiers that alter the structure of chromatin to either enhance or repress transcriptional activity. This is well exemplified by modifications of core histone proteins. For example, acetylation of histones catalyzed by histone acetyltransferases (HATs) favors the generation of open chromatin regions, which in most instances enhances transcription. In contrast, deacetylation by histone deacetylases (HDACs) generates closed regions that are inhibitory to transcription. A balance between the two activities is essential for proper transcriptional regulation (52
). Such opposing forces also control the positioning of nucleosomes, which is also an important component of transcriptional regulation. Nucleosome remodeling is an important process in the dynamic alteration of chromatin structure. Studies done in Saccharomyces cerevisiae
have shown that while the ATP-dependent SWI/SNF complexes activate transcription by remodeling nucleosomes, ISWI family enzymes, especially ISW2, repress transcription by positioning nucleosomes such that regulatory regions are inaccessible (25
Our results indicate that the architectural protein HMGN1 is enriched at nucleosomes that are well positioned in regulatory regions. CTCF binding to linker DNA and the presence of well-positioned nucleosomes surrounding the binding sites have been previously demonstrated (20
). Our results indicate that the nucleosomes surrounding YY1 binding sites exhibit a similar profile. HMGN1 binds to the edges of nucleosomes surrounding CTCF and YY1 binding sites, a finding that is in full agreement with the known position of nucleosomes at the entry-exit points of the nucleosomal DNA (1
). Furthermore, recent work by Rattner et al. demonstrated a role for HMGN1 in stabilizing nucleosomes by inhibiting the chromatin remodeling activity (43
). Thus, our results suggest that HMGN1 plays a role in maintaining a nucleosome occupancy profile that enhances the accessibility of regulatory factors to their target. Consistent with this, at the HeLa-specific CTCF binding sites in CD4+
cells, nucleosomes occlude the binding site (20
). The absence of HMGN1 in these sites further reinforces the idea that the protein plays a specific role in the maintenance of nucleosome positions at regulatory sites. One of the possible mechanisms through which HMGN1 could influence nucleosome positioning is through the inhibition of the SWI/SNF chromatin remodeling complexes, as Rattner and colleagues suggest (43
However, it is also conceivable that HMGN1 associates with regulatory factors involved in the generation of open chromatin and may act to stabilize these structures. Chromatin remodeling activity of the INO80 complex has been suggested as a prerequisite for YY1 binding to its target sites (11
). Further investigation is necessary to test whether HMGN1 is associated with any such complexes.
Partial loss of the canonical −1 nucleosome upstream of the TSS and precise positioning of other surrounding nucleosomes are characteristic features of promoters of genes which are being actively transcribed (45
). It has been suggested that the periodically positioned nucleosomes found at the CTCF binding sites and promoters of active genes could be an outcome of one or two nucleosomes being precisely positioned and the neighboring nucleosomes being positioned relative to these nucleosomes (36
). Thus, it is possible that HMGN1 stabilizes the position of the nucleosomes, perhaps anchoring them at the boundaries of the CTCF and YY1 binding sites. Our identification of a significant enrichment of HMGN1 in the nucleosome-depleted region in active promoters is consistent with previous data indicating that HMGN1 preferentially binds to “anchor” nucleosomes. Due to variation in the lengths of nucleosome-depleted regions of different promoters, the distribution of HMNG1 binding at promoters is broader than at CTCF and YY1 binding sites. Therefore, it is possible that the HMGN1 peaks that we observe at the nucleosome-depleted region of the promoter could be due to an averaging of the binding that occurs at the edges of the flanking nucleosomes. Increased rate of digestion by micrococcal nuclease at the HSP70
promoter locus is higher in Hmgn1+/+
cells than in Hmgn1−/−
cells, suggesting a role for HMGN1 at the promoters of active genes (4
). Consistent with this, we detected enrichment of HMGN1 at active promoters and only very low levels of HMGN1 binding at silent gene promoters.
HMGN1 may play a similar role at the DNase I HS sites, for which we found extensive colocalization. The hypersensitivity of the DNase I HS sites was also higher in regions with stronger HMGN1 signals. Conventionally, DNase I HS sites have been used in the identification of regulatory elements (6
). Our findings indicate that HMGN1 binding data can be used in conjunction with DNase I HS site data to identify functional regulatory elements with higher confidence.
Taken together with previous analyses (12
), our results suggest that although HMGN1 moves rapidly through the nucleus, its residence time at regulatory sites is longer than at nonregulatory sites. Thus, from a functional view, HMGN1 preferentially turns over at chromatin regulatory sites, a finding that is in agreement with the observation that Hmgn1−/−
mice have an altered phenotype (31
). It remains to be seen whether HMGN1 is involved in the generation or maintenance of the regulatory sites, or whether it preferentially recognizes these preformed sites, perhaps targeted to specific chromatin regions as part of a metastable regulatory complex (35