HMGN1 modulates estrogen-mediated transcriptional activation through interactions with specific DNA-binding transcription factors.

The HMGN family of proteins modulates the chromatin structure in vertebrates. These small, highly charged, abundant proteins bind with higher affinity to nucleosomes than to double-stranded DNA. Nucleosomal binding is due to both direct interactions with nucleosomal DNA and interactions of HMGN proteins with histones (10, 18, 24, 46, 66, 71, 75). The binding of HMGN to chromatin relaxes the higher order chromatin structure. In particular, HMGN1 counteracts the ability of histone H1 to compact chromatin through competition for H1 binding sites on the chromatin. Such structural mechanisms result in the general transcription stimulation by HMGN proteins on chromatin templates in vitro (20, 74). The binding of HMGN proteins to nucleosomes also alters the posttranslational modification of nucleosomal histones at several distinct sites, which can result in either transcriptional stimulation or inhibition (42, 43, 77). Despite the seemingly indiscriminate effects of HMGN on chromatin compactness and histone modifications, the overexpression or knockout of HMGN proteins modulates the expression of only a small subset of genes, some of which are activated and others are inhibited by HMGNs (42, 63, 79). These results raised the question as to why HMGN proteins would affect transcription only at specific promoters. HMGN proteins exhibit no nucleosome binding preference based on the underlying DNA sequences in the nucleosomes (72). Thus, it is unlikely that the targeting of HMGNs to gene regulatory regions can occur through the recognition of specific DNA sequences. Alternatively, the targeting of HMGN proteins could be mediated by specific protein-protein interactions (34). Consistent with such a hypothesis, HMGN1/2 exist in multiple complexes in HeLa cells (41); however, the prominently associated proteins in this case were hnRNPs and annexin II, which sheds little light on how the specificity of HMGN1/2 could be obtained at chromatin targets. We have investigated the hypothesis that HMGN proteins are recruited to gene regulatory regions through direct interactions with specific DNA-binding transcription factors. A link between HMGN1 in particular and the estrogen receptor was suggested by early experiments in which HMGN1 was selectively modified in response to estrogen in the uteri of guinea pigs. This modification was connected to transcription activity, as an estrogen receptor antagonist prevented this acetylation of HMGN1 (5, 54). Estrogen-regulated gene expression in breast cancer cells provides an extensively studied model system for examining multiple modes of gene induction (51, 65). First, in the canonical genomic estrogen response pathway, the association of estrogen with nuclear estrogen receptor α (ERα) induces its binding to estrogen response elements (EREs) within the regulatory regions of target genes. One well-characterized gene that is regulated in this manner is TFF1 (11, 47, 67, 68). Upon the stimulation of MCF-7 cells with estrogen, the binding of ERα to the ERE within the TFF1 promoter induces a cyclic recruitment to the promoter of an array of both positive transcription cofactors (including histone acetyltransferases [HATs], histone methyltransferases [HMTs], p68 RNA helicase, p160 coactivators, Mediator, and the SWI/SNF ATP-dependent nucleosome-remodeling complex) and negative transcription cofactors, including histone deacetylases (HDACs) and the AAA proteins independent of O0S (APIS) 19S proteasome subunit (47, 69). The concomitant cyclic changes in chromatin modification and organization of the nucleosomes on the promoter promotes transcription activation and at the same time allows the transcription rate of the gene to respond rapidly to different stimuli by restricting the duration of activation. Second, estrogen activates the transcription from gene regulatory regions through the protein-protein interaction between ERα and other promoter-bound DNA-binding transcription factors, such as Sp1 (64). In such cases, ERα need not interact directly with the DNA. This type of regulation has been suggested to occur on the IGFBP4 gene promoter (59). Indeed, Sp1 is critical for the induction of this gene by estrogen, although Sp1 binding to this promoter is at a very low constitutive level (44). Finally, gene expression is activated downstream of estrogen interaction with plasma membrane-associated ERα via “nongenomic” signal transduction pathways (40). The extracellular signal-regulated kinase pathway, one of the major targets of estrogen stimulation (9, 40, 61), impacts gene expression by multiple mechanisms, including rapid activation of the serum response factor (SRF)/Elk-1 complex. Consequences include stimulation of the transcription of FOS (22, 23), a highly characterized SRF target gene. We have focused our study of the effects of HMGN1 on the induction of two estrogen-responsive genes, TFF1 and FOS, which are regulated through an ERE and a serum response element, respectively. Using these two particular genes, we tested the hypothesis that HMGN1 targets genes for regulation through interaction with DNA-binding transcription factors. We demonstrate, first, that HMGN1 inhibits the degree of estrogen induction at both genes. In addition, HMGN1 interacts specifically with both ERα and SRF, which regulate the responses of TFF1 and FOS to estrogen, respectively. Upon estrogen treatment, HMGN1 is recruited to the TFF1 gene regulatory region, but not to genomic regions lacking EREs, in parallel with the binding of ERα. Unexpectedly, although the regulation of the TFF1 gene expression by HMGN1 requires binding to specific transcription factors, it does not require high-affinity nucleosomal DNA-binding activity of HMGN1. Taken together, these results indicate that HMGN1 is targeted to specific gene regulatory regions through protein-protein interactions with transcription factors and that such interactions are required for HMGN1 to modulate transcriptional regulation. Regarding the mechanism of gene regulation, HMGN1 reduces the level of acetylation of Lys9 on histone H3 (AcLys9H3) at ERE-containing genes, such as TFF1, in which HMGN1 inhibits induction in response to estrogen. Thus, HMGN1 displays another means for negative feedback regulation to limit the extent of induction by estrogen, which is functionally analogous to, although mechanistically distinct from, the negative coregulators recruited to the promoter in an alternating manner with coactivators.