The major goal of this study is to examine whether the various members of the HMGN protein family can affect the cellular transcription profile in an HMGN-variant-specific manner. Although previous studies indicated that the binding of HMGN protein to chromatin alters the cellular transcription profile, the degree to which these changes are HMGN-variant specific has not yet been investigated.
The dynamic nature of HMGN binding to chromatin and the lack of any DNA sequence specificity in their chromatin interactions, taken together with the conservation of their nuclear-binding domain and similarities in their overall organization and physical properties, raised the possibility that the individual HMGN variants would be functionally redundant and have similar effects on the cellular transcription profile. Conversely, the widespread expression of HMGN1 and HMGN2, but not HMGN3 and HMGN5, in most tissues and the sequence specificity of their C-terminal domains suggest that potentially the proteins may have variant-specific effects on the transcription profile. Indeed, in vitro experiments revealed variant-specific effects on histone modifications, and experiments with genetically altered mice also suggest that the HMGN variants are not fully functionally redundant.
Our experiments suggest that each HMGN variant can affect the expression of numerous genes, especially when overexpressed, and by enlarge in an HMGN-specific manner. The amplitude of transcriptional changes was moderate; for most of the affected genes being in the limits of 2-fold difference. Importantly, nearly equal amount of genes were either up- or downregulated by each HMGN, suggesting that HMGNs are neither transcriptional activators nor repressors. The GO analyses indicated that multiple cellular processes were affected by individual HMGNs or by combinations of several HMGNs, suggesting that the HMGNs are general modulators of the cellular transcriptional fidelity.
Two molecular mechanisms whereby HMGN affect the transcription profile could be envisioned. One possibility is that by binding to nucleosomes, HMGN induce structural changes that alter the ability of transcriptional regulators, either positive or negative, to interact with their chromatin targets. A second possibility is that the HMGN interact with specific regulators and affect their chromatin interactions. Both possibilities suggest that the ability of HNGN variants to bind to chromatin is a major effect on the transcriptional output. Indeed, our previous experiments (50
), and our present analyses of the HMGN5-S19,23E mutant, indicate that HMGNs affect transcription by binding to nucleosomes.
While nucleosome binding seems to be an absolute requirement for any noticeable effects on transcription, the variant-specific effects on the transcription profile suggest that additional properties of these proteins play a role in determining their biological specificity. Because the C-terminal domain of HMGNs is highly variable in sequence between individual HMGN proteins, we tested the possibility that the specific transcriptional effects of HMGNs reside in this domain and expressed several HMGN swap mutants in MEF cells. Surprisingly, the transcriptional outcome following the expression of these swap mutants with a common NBD from HMGN1, and a C-terminal domain from HMGN1, HMGN2 or HMGN3 was different from either one of their ‘source’ proteins. Thus, the variant-specific effects of HMGNs on transcription are the consequence of coordinate effects of the various structural domains of each variant. In other words, neither the NBD nor the C-terminal domain alone defines the transcriptional effect of each HMGN protein, but rather the entire structure of the protein defines its specific role in transcription (E).
In considering the molecular mechanisms leading to HMGN-variant-specific effects on transcription, we note that early structural studies indicated that HMGNs have little ordered structure (52
), and our computational analysis () reveal that HMGNs are among the most intrinsically disordered proteins known. Intrinsically disordered proteins can interact with multiple protein partners with relatively low affinity and acquire more ordered structures (45–47
). It has been recently reported that the harmful effect of elevated cellular levels of many proteins is correlated with the degree of their disorderness (25
). At the same time, cells with decreased amount of these proteins function robustly and do not demonstrate significant changes in cellular functions. Our observation that knock out of HMGNs has significantly smaller effect on transcription supports this theory and strongly argues that disordered structure of HMGNs is one of the major functional properties of these proteins.
Variations in structure of HMGN proteins due to interaction with different protein partners can modulate the effects of HMGN variants on local nucleosome structure, global chromatin architecture and transcription (E). Indeed, both HMGN1 and HMGN2 have been shown to form multiple metastable macromolecular complexes (15
), and specific protein partners have been identified for several HMGN variants. Thus, HMGN3 interacts specifically with the thyroid hormone receptor (59
) and with the transcription factor PDX1 (19
), HMGN1 forms a complex with ERalpha and SRF (15
), and HMGN2 was shown to interact with PITX2 (60
). Our observation of cell-specific effects of HMGN3a protein on transcription in the pancreatic derived MIN6 cell line, but not in MEFs (19
), supports the idea of existence of specific protein partners for individual HMGN proteins.
In conclusion, our results reveal both specific and redundant roles of HMGN variants in the global regulation of gene expression. Each HMGN preferentially affects a unique set of genes with little or no specificity for defined cellular processes. Thus, changes in the expression of an HMGN may disrupt the fidelity of the cellular transcription and render the organism more susceptible to further damage. Indeed, experiments with genetically altered mice and with cells derived from these mice indicate that loss of HMGN1 leads to an impaired DNA damage repair response and increased tumorigenicity (27
). Likewise, loss of HMGN3, which is highly expressed in beta cells of the pancreatic islets, affects insulin secretion leading to a mild diabetic phenotype (19
). The transcriptional specificity of the HMGN variants is similar to that of the H1 variants. It seems that the dynamic interaction of HMGN, H1 and other structural proteins with chromatin is part of the mechanism that ultimately fine tunes the transcription profile to optimize cellular function.