Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction

The previously identified LRS (Loss of rDNA Silencing) domain of the nucleosome is critically important for silencing at both ribosomal DNA and telomeres. To understand the function of the LRS surface in silencing, we performed an EMS mutagenesis screen to identify suppressors of the H3 A75V LRS allele. We identified dominant and recessive mutations in histones H3, H4, and dominant mutations in the BAH (Bromo Adjacent Homology) domain of SIR3. We further characterized a surface of Sir3p critical for silencing via the LRS surface. We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles. Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4. Mutations in H3 and H4 genes that lose silencing tend to make the LRS surface more electronegative, whereas mutations that increase silencing make it less electronegative. Conversely, suppressors of LRS alleles in either SIR3, histone H3, or H4 also tend to make their respective surfaces less electronegative. Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain. Based on these findings, we propose an electrostatic model for how an extensive surface on the Sir3p BAH domain may regulate docking onto the LRS surface

Effects of DNA supercoiling on chromatin architecture

Disruptions in chromatin structure are necessary for the regulation of eukaryotic genomes, from remodelling of nucleosomes at the base pair level through to large-scale chromatin domains that are hundreds of kilobases in size. RNA polymerase is a powerful motor which, prevented from turning with the tight helical pitch of the DNA, generates over-wound DNA ahead of itself and under-wound DNA behind. Mounting evidence supports a central role for transcription-dependent DNA supercoiling in disrupting chromatin structure at all scales. This supercoiling changes the properties of the DNA helix in a manner that substantially alters the binding specificity of DNA binding proteins and complexes, including nucleosomes, polymerases, topoisomerases and transcription factors. For example, transient over-wound DNA destabilises nucleosome core particles ahead of a transcribing polymerase, whereas under-wound DNA facilitates pre-initiation complex formation, transcription factor binding and nucleosome core particle association behind the transcribing polymerase. Importantly, DNA supercoiling can also dissipate through DNA, even in a chromatinised context, to influence both local elements and large chromatin domains. We propose a model in which changes in unconstrained DNA supercoiling influences higher levels of chromatin organisation through the additive effects of DNA supercoiling on both DNA-protein and DNA-nucleosome interactions. This model links small-scale changes in DNA and chromatin to the higher-order fibre and large-scale chromatin structures, providing a mechanism relating gene regulation to chromatin architecture in vivo

Analysis of clinical studies with natural and synthetic luteinizing hormone releasing hormone in man

It was first determined that luteinizing hormone-releasing hormone (LH-RH) obtained from porcine bovine and ovine sources was biologically active in man. When the structure of porcine LH-RH was determined synthetic material was also found active in man. Studies indicated that FSH as well as LH were released from the pituitary into the peripheral circulation by both the natural and synthetic LH-RH. Although blood basal levels of LH and FSH are increased after the menopause a further increase was produced by LH-RH either naturally or synthetically. In other conditions (Klinefelters and Turners syndromes) with naturally occurring elevations of blood levels of LH and FSH further release was also obtained by both natural and synthetic LH-RH. In subjects pretreated with 200 mg/day clomiphene additional rapid increase was obtained. In patients with pituitary tumors and acromegaly the response was variable. However 1 patient who did not release gonadotropins after LH-RH did release thyrotropin (TSH) after receiving TSH-releasing hormone. The effects of LH-RH administration on other steroids in the blood are currently being investigated. Routes other than the intravenous administration are being tried e.g. subcutaneous and intramuscular. It has been found that a childs pituitary can release LH and FSH after administration of porcine LH-RH. In Kallmanns syndrome of hypogonadotropic hypogonadism with anosmia a small increase of gonadotropin release has been shown after rapid intravenous injection of LH-RH. Long duration of intravenous infusion of LH-RH with 2 supplementary intramuscular injections has produced ovulation in a patient primed with an FSH-containing material (Pergonal). In other experiments patients pretreated with clomiphene had an increased incidence of ovulation. In men with oligospermia only slight improvement has been obtained. Most of the data presented were obtained using highly purified material of porcine origin