Structure and biological importance of the Spn1-Spt6 interaction, and its regulatory role in nucleosome binding.

Eukaryotic transcription and mRNA processing depend upon the coordinated interactions of many proteins, including Spn1 and Spt6, which are conserved across eukaryotes, are essential for viability, and associate with each other in some of their biologically important contexts. Here we report crystal structures of the Spn1 core alone and in complex with the binding determinant of Spt6. Mutating interface residues greatly diminishes binding in vitro and causes strong phenotypes in vivo, including a defect in maintaining repressive chromatin. Overexpression of Spn1 partially suppresses the defects caused by an spt6 mutation affecting the Spn1 interface, indicating that the Spn1-Spt6 interaction is important for managing chromatin. Spt6 binds nucleosomes directly in vitro, and this interaction is blocked by Spn1, providing further mechanistic insight into the function of the interaction. These data thereby reveal the structural and biochemical bases of molecular interactions that function in the maintenance of chromatin structure.

McDonald SM ，Close D ，Xin H ，Formosa T ，Hill CP ... -  《-》

Spn1 and Its Dynamic Interactions with Spt6, Histones and Nucleosomes.

Histone chaperones facilitate the assembly and disassembly of nucleosomes and regulate DNA accessibility for critical cellular processes. Spn1 is an essential, highly conserved histone chaperone that functions in transcription initiation and elongation in a chromatin context. Here we demonstrate that Spn1 binds H3-H4 with low nanomolar affinity, residues 85-99 within the acidic N-terminal region of Spn1 are required for H3-H4 binding, and Spn1 binding to H3-H4 dimers does not impede (H3-H4)2 tetramer formation. Previous work has shown the central region of Spn1 (residues 141-305) is important for interaction with Spt6, another conserved and essential histone chaperone. We show that the C-terminal region of Spn1 also contributes to Spt6 binding and is critical for Spn1 binding to nucleosomes. We also show Spt6 preferentially binds H3-H4 tetramers and Spt6 competes with nucleosomes for Spn1 binding. Combined with previous results, this indicates the Spn1-Spt6 complex does not bind nucleosomes. In contrast to nucleosome binding, we found that the Spn1-Spt6 complex can bind H3-H4 dimers and tetramers and H2A-H2B to form ternary complexes. These important results provide new information about the functions of Spn1, Spt6, and the Spn1-Spt6 complex, two essential and highly conserved histone chaperones.

Li S ，Edwards G ，Radebaugh CA ，Luger K ，Stargell LA ... -  《-》

The Abundant Histone Chaperones Spt6 and FACT Collaborate to Assemble, Inspect, and Maintain Chromatin Structure in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Spt6 protein is a conserved chromatin factor with several distinct functional domains, including a natively unstructured 30-residue N-terminal region that binds competitively with Spn1 or nucleosomes. To uncover physiological roles of these interactions, we isolated histone mutations that suppress defects caused by weakening Spt6:Spn1 binding with the spt6-F249K mutation. The strongest suppressor was H2A-N39K, which perturbs the point of contact between the two H2A-H2B dimers in an assembled nucleosome. Substantial suppression also was observed when the H2A-H2B interface with H3-H4 was altered, and many members of this class of mutations also suppressed a defect in another essential histone chaperone, FACT. Spt6 is best known as an H3-H4 chaperone, but we found that it binds with similar affinity to H2A-H2B or H3-H4. Like FACT, Spt6 is therefore capable of binding each of the individual components of a nucleosome, but unlike FACT, Spt6 did not produce endonuclease-sensitive reorganized nucleosomes and did not displace H2A-H2B dimers from nucleosomes. Spt6 and FACT therefore have distinct activities, but defects can be suppressed by overlapping histone mutations. We also found that Spt6 and FACT together are nearly as abundant as nucleosomes, with ∼24,000 Spt6 molecules, ∼42,000 FACT molecules, and ∼75,000 nucleosomes per cell. Histone mutations that destabilize interfaces within nucleosomes therefore reveal multiple spatial regions that have both common and distinct roles in the functions of these two essential and abundant histone chaperones. We discuss these observations in terms of different potential roles for chaperones in both promoting the assembly of nucleosomes and monitoring their quality.

McCullough L ，Connell Z ，Petersen C ，Formosa T ... -  《-》

Casein Kinase II Phosphorylation of Spt6 Enforces Transcriptional Fidelity by Maintaining Spn1-Spt6 Interaction.

Spt6 is a histone chaperone that associates with RNA polymerase II and deposits nucleosomes in the wake of transcription. Although Spt6 has an essential function in nucleosome deposition, it is not known whether this function is influenced by post-translational modification. Here, we report that casein kinase II (CKII) phosphorylation of Spt6 is required for nucleosome occupancy at the 5' ends of genes to prevent aberrant antisense transcription and enforce transcriptional directionality. Mechanistically, we show that CKII phosphorylation of Spt6 promotes the interaction of Spt6 with Spn1, a binding partner required for chromatin reassembly and full recruitment of Spt6 to genes. Our study defines a function for CKII phosphorylation in transcription and highlights the importance of post-translational modification in histone chaperone function.

Dronamraju R ，Kerschner JL ，Peck SA ，Hepperla AJ ，Adams AT ，Hughes KD ，Aslam S ，Yoblinski AR ，Davis IJ ，Mosley AL ，Strahl BD ... -  《Cell Reports》

Essential histone chaperones collaborate to regulate transcription and chromatin integrity.

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.

Viktorovskaya O ，Chuang J ，Jain D ，Reim NI ，López-Rivera F ，Murawska M ，Spatt D ，Churchman LS ，Park PJ ，Winston F ... -  《-》