The modulation of higher order structure of chromatin has profound implications in the regulation of nuclear processes, like transcription, replication, recombination or DNA repair. Those processes require accessible DNA to recruit large protein complexes to function.
In humans, the architectural proteins of the “high mobility group nucleosomal binding domain” (HMGN) family participate in the opening of chromatin structure and the regulation of gene expression. HMGN proteins bind the nucleosome particle through a conserved nucleosomal binding domain (NBD) and compete with H1 for the binding to chromatin.
Of all HMGN family members, HMGN5 has the biggest effect on transcriptional regulation in mouse and human cells. Moreover, HMGN5 is also able to induce large-scale chromatin decondensation in vivo.
In the present work we study the functional role of HMGN5 in the opening of higher-order structure of chromatin and gene expression by biochemical and genome-wide methods.
We identified a novel and specific RNA binding domain overlapping with the NBD of HMGN5. Moreover, by in vitro competition assays we demonstrated that HMGN5 exhibits exclusive binding to nucleosomes or to RNA. Furthermore, we showed that the RNA binding activity is a feature of other HMGN members as well, highlighting a novel function for those proteins.
The overexpression and knockdown of HMGN5 in human cell lines affect the expression of about 3000 genes respectively, with 1287 overlapping target genes.
ChIP-seq analysis of HMGN5 revealed that HMGN5 mainly associates with active regulatory genomic regions, like promoters and CpG islands, and it localizes to DNase I hypersensitive sites (DHSs). Moreover, we found that the actively regulated target genes belong to the group of genes involved in RNA metabolic processes. HMGN5 binding overlaps with RNA polymerase II binding sites. CLIP-seq analysis of HMGN5-bound RNAs revealed that HMGN5 is able to bind nascent transcripts. In the light of the biochemical results, we propose that HMGN5 participates in the regulation of RNA metabolism by a dual mechanism that enables HMGN5 binding either chromatin or RNA since the HMGN5-bound RNAs have no functional relationship with the chromatin function of HMGN5.
Interestingly, by using quantitative mass spectrometry we identified CTCF, BANF1 and seven proteins associated with the pre-ribosomal RNA processing.
Strikingly, our ChIP-seq data revealed that HMGN5 co-localizes with HMGN5. As CTCF constitutes the major organizer of chromatin architecture, the results presented here suggest a cooperative role of both proteins in the organization of higher-order structure of chromatin.
Further functional characterization of the potential HMGN5-CTCF complex, may shed light on the regulation of higher-order chromatin organization.