hir protein discovery- lacZ fusion to HTA1-HTB1 promoter. Screen for mutants that o/e lacZ. found that 5 complementation groups lost S phase specific expression in all but HTA2-HTB2 which remained s phase specific.

Shows FACT destabilizes nucleosomes so that H2A-H2B dimers are removed. This was shown in vitro with recomb hFACT. -they show SSRP1 (a fusion of Pob3-Nhp6) interacts with H3/4 but not H2A-H2B. -showed hFACT does have chaperone activity (deposites core histones onto DNA in vitro.

-paper solved structure of N term domain of Spt16 in FACT. This domain of spt16 and Pob3-M have overlapping rokes in binding the C-term extension of H2A. Therefore FACT has additive contacts with nucleosomes. -H2A binding site is in POb3-Q308 region previously described as binding RPA. -trypsin treated nucleosomes that have lost their tails no longer bind yFACT in vitro. -Spt16-Pob3 binds H3 and H4 tails in vitro by SPR. The Spt16NTD is not required for this interaction. The complex does not bind H2A tail. Didn't test H2A globular domain. This paper never tests H2A C-term extension mutants for FACT binding. Only tested genetically by synthetic interactions between FACT mutants and H2A mutants. Not very convincing that a physical interaction exists b/w H2A C term and FACT.

-this paper solves the crystal structure for Pob3, a member of FACT. -pob3 has an unusual double PH domain. A mutant w/in this domain that causes defects in replication is rescued by a mutation in RPA a ssDNA binding factor. This indicates that FACT coordinated with RPA during DNA replication. -The binding surface is on the N terminal PH domain. This domain is much less conserved between Pob3 and Rtt106. The C term is v conserved (likely the site of histone binding). -FACT and RPA display a weak physical interaction. -FACT alters nulceosomes independent of ATP therefore it's referred to as a reorganizer rather than a remodeler. -human FACT was purified by its ability to promote RNA pol II transcription on chromatin templates in vitro. -there are 6 PH domain subgroups. Pob3 PH domains are distinct and therefore make up a 7th group. -PH domain suggests it binds to a ligand but it doesn't suggest what the ligand it (lipid, peptid, etc). -PH domain: 7 stranded anti-parallel beta barrel that is capped at one end by a helix. -to identify pob3 regions involved in DNA replication they performed random mutagenesis on all of pob3, and screened for mutants that were sensitive to HU and were able to grow at 37 C. This indicates replication defects and that weren't a result of protein instability. -screen revealed Q308R and Q308K. This is in an external loop of the N term PH domain. -found supressor mutation in RPA. H2B: previously reported that replication defects caused by pob3 alleles can be rescued by increasing the ratio of H2A/H2B to H3/H4. This rescue is thought to be associated with transcription rather than replication. -pob3Q308K strains displayed growth defects when HTA2-HTB2 was o/e compared to HTA1/HTB1. This growth defect was exacerbated 75mM HU (a condition permisable to pob3-Q308K. -this trend was also observed in the RPA mutant background. Overexpressing H2B copy to was more detrimental that o/e copy 1. This may be because copy 1 promoter self regulates and therefore not as much protein was expressed. -They think maybe because copy 2 isn't transcriptionally repressed by the Hir proteins. The mutant pob3 has diminished tolerence for increased expression of H2A/H2B to H3/H4.

-rtt106KOcac1KO cells show mis-localized Sirs, reduced binding at TEL. -Sir4 initial recruitment to TEL was unaffected in rtt106cac1KO but spreading was affected in both. Cac1 simgle mutants are known not to affect sir localization at TEL. Rtt106 single wasn't tested. -At HMR Caf1 and Rtt106 are required for the initial recruitment of Sir2 and 3 but not 4 to the HMR-e silencer. -Spreading of all sirs from e to a1 is defecting in caf1KOrtt106KO -increased HMR silencing defect in caf1KOrtt106KOsir1ko, indicated chaperone importance in spreading. Single mutants also showed defects at HMR. Double was synergistic. Singles were identical. Spreading is delayed but the amount of Sir4 spread catches up w. wt after 4 hrs. Sir2 and 3 never catch up to wt. -single mutants showed little effect on sir3 and 4 nulcear periphery localization, but double mutant showed defects by GPF supporting lack of Sir-telomere assoc. -Rtt106 interacts with sir4 in vitro and in vivo. Sir4-Ta pulled down Rtt106-FLAG. Rtt106 does not interact with Sir2 or Sir3 in vitro.

Discovery: Screen for enhancers of silencing defect of a PCNA (pol30) mutant -there are 2 PCNA alleles, one that prevents Caf1 from contributing to silencing and one that prevents Asf1 from contributing. Silencing is affects at TEL (URA reporter) and HML (mating assay). In the screen they used a PCNA mutant that prevented both Asf1 and Caf1 from silencing. Used the KO collection and a strain with GFP at HMR. The Pol30 mutant silencing defects at HMR were weak compared to asf1KOcac1KO. Defects were the same at TEL. Therefore they screened for mutants that enhanced the silencing phenotype of the pol30 mutant. -silencing assays show synergistic defects in cac1ko background but not in asf1KO. Therefore Rtt106 likely functions in the Asf1 pathway for silencing. -rtt106 binds H3/H4 in vitro and in vivo and shows nucleosome assembly activity in vitro. -rtt106 does not bind H2A/H2B in vitro. -rtt106 binds the Caf-1 complex through cac1. -only tests for asf1 binding in vitro. -Humans have 2 Asf1 sequence homologues Asfla and Asf1b. Both stimulate Caf1 to deposit H3/H4. -Yeast show no functional asf1 homologue. Rtt106 could be it. Alternatively Rtt106 may be unique to yeast. -because rtt106 assoc with the caf1 complex it likely also acts as an S phase chaperone.

-Asf1 participates in recplication coupled and replication independent histone deposition reactions. -Asf1 binds to and stimulates histone deposition by Caf-1 and Hir complexes. -2.2A structure of the N term immunoglobulin fold bound to H3 residues 121-134. -the Asf1 binding surface on H3 suggests that Asf1 sterically blocks the H3H3 interactions in H3/H4 tetramers. -point mutants within the H3 binding site of asf1 have silencing defects at TEL. -Propose the Asf1 chaperone forms a terinary complex in which Asf1 acts like a bridge to stabilize contacts between H3/H4 and the assisting chaperone.

Background: Dot1 is one of the only modifying enzymes with a chromatin requirement. Can act on free histones. -A basic patch on the H4 tail (K16,R17,R19) interacts with Dot1 and it essential for H3K79 methylation. -Sir3 inhibits H3K79 methylaiton. This inhibition is H4K16. -Sir3 and Dot1 bind the same basic H4 N term basic patch. Sir3 also bind the H3K79 region. Therefore Sir3 and Dot1 have overlapping chromatin targets. Model for heterochromatin boundary establishment at TEL: H4K16Ac displaces Sir3 which allows dot1 to bind and methylate H3K79 which further blocks Sir3 spreading.

Background: Sir3 purified form yeast Show preference for unAc H4K16 peptide. But sir3 purified from insect cells and E coli don't care about H4 Ac state. -Sir3 BAH domain binds H4 tail and H3 globular domain. -cells lacking dot1, sas2, and set1 show increased Sir3-histone interactions. -H4K16Q abolishes sir3BAH-histone interaction. H3K79A also lost interaction. K79R preserved interaction. K16R increased interaction. -Used SPR to show binding -orc BAH can replace Sir3 and complement silencing defects. Showed OrcBAH does bond histones but binding was affected by a distinct set of point mutants. -sir3deltaBAH shows silencing defects in reporter assays and decreased Sir3 ChIP to TEL. -mixed nucleosomes, Sir2,3,4 and NAD and got 20nm Sir-Nucleosome filaments shown by EM. -See methods for Sir2/4 purification