Chromatin structure and histone modifications in gene regulation

Abstract: In the living cell, DNA is densely packed into a chromatin structure constituting nucleosomal arrays. One nucleosome core particle includes a disc shaped protein octamer consisting of pairs of histones H2A, H2B, H3 and H4. 146 bp of DNA is wrapped in almost two turns around this protein complex. The N-terminal tails of the histone proteins protrude out from the nucleosome core. These tails are highly flexible and are targets for multiple covalent modifications coupled to gene regulatory events. A linker histone H1 binds at the DNA entry/exit site of the nucleosome and organises the linker DNA and influence higher order chromatin structure. In order to induce gene expression the transcription factors, coactivators and also the transcriptional machinery has to get access to the DNA. This is obtained by modulation of chromatin structure by specific energy-depen dent chromatin remodelling complexes that has the ability to locally unfold the chromatin. In addition, nucleosomes may be positioned to expose specific regulatory DNA segments. The hormone-activated glucocorticoid receptor (GR) driven mouse mammary tumor virus (MMTV) promoter, fused to a thymidine kinase reporter gene was injected into Xenopus laevis oocytes. This was used for studies on chromatin structure coupled to gene regulation. Specific histone acetylations at H3K9, H3K14 and H4K16 were found to correlate with hormone-induced chromatin remodelling but to precede the transcription step. These histone modifications were reversible upon sequential treatment of a hormone antagonist, RU486, which correlated with the disruption of chromatin opening event. Hormone- in ducti on was also found to mediate transient trimethylation of H3K4 during the initiation phase of transcription. Hyperacetylation of histories by Trichostatin A (TSA) mediated complex effects on the chromatin structure. TSA induced a general relaxation of chromatin and a specific rearrangement of nucleosomes in the distal part of the MMTV promoter. This correlated with an enhanced basal transcription. Hormone-activation was impaired by TSA treatment and this correlated with topological changes and an overall increased sensitivity to MNase digestion. The nuclear factor 1 (NF1) was shown to bind to the non-induced MMTV promoter. Upon hormone induction, (NF1) and hormone-activated GR formed an enlarged MNase- resistant structure, that may function as a binding platform for general transcription factors. (NF1) and octamer transcription factor 1 (Oct1) expressed together were found to cooperatively bind and to induce partial nucleosome positioning. This resulted in a faster and stronger hormone response and also correlated with a faster induction of trimethylation of H3K4. Together this serves as a functionally MMTV chromatin presetting. Linker histone H1 was expressed and incorporated into the MMTV chromatin where it dissociated specifically from the active locus upon hormone-induced remodelling. A subsaturated level of specific nucleosome binding of H1 correlated with enhanced binding of hormone-activated GR and also with enhanced transcription. At this specific H1 concentration, a reduction of the general DNA access was seen thus indicating an H1concentration dependent rearrangement of the chromatin fibre. Together this support a mechanism where H1-concentration dependent stimulation of GR binding is achieved through GR-induced disruption of H1 at active locus resulting in preferential HI masking of the nonspecific DNA.