Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

Telomere capture, a rare event that stabilizes chromosome breaks, is associated with certain genetic abnormalities in humans. Studies pertaining to the generation, maintenance, and biological effects of telomere formation are limited in metazoans. A mutation, mu2a, in Drosophila melanogaster decreases the rate of repair of double strand DNA breaks in oocytes, thus leading to chromosomes that have lost a natural telomere and gained a new telomere. Amino acid sequence, domain architecture, and protein interactions suggest that MU2 is an ortholog of human MDC1. The MU2 protein is a component of meiotic recombination foci and localizes to repair foci in S2 cells after irradiation in a manner similar to that of phosphorylated histone variant H2Av. Domain searches indicated that the protein contains an N-terminal FHA domain and a C-terminal tandem BRCT domain. Peptide pull-down studies showed that the BRCT domain interacts with phosphorylated H2Av, while the FHA domain interacts with the complex of MRE11, RAD50, and NBS. A frameshift mutation that eliminates the MU2 BRCT domain decreases the number and size of meiotic phospho-H2Av foci. MU2 is also required for the intra-S checkpoint in eye-antennal imaginal discs. MU2 participates at an early stage in the recognition of DNA damage at a step that is prerequisite for both DNA repair and cell cycle checkpoint control. We propose a model suggesting that neotelomeres may arise when radiation-induced chromosome breaks fail to be repaired, fail to arrest progression through meiosis, and are deposited in the zygote, where cell cycle control is absent and rapid rounds of replication and telomere formation ensue

MU2 and HP1a Regulate the Recognition of Double Strand Breaks in Drosophila melanogaster

Chromatin structure regulates the dynamics of the recognition and repair of DNA double strand breaks; open chromatin enhances the recruitment of DNA damage response factors, while compact chromatin is refractory to the assembly of radiation-induced repair foci. MU2, an orthologue of human MDC1, a scaffold for ionizing radiation-induced repair foci, is a widely distributed chromosomal protein in Drosophila melanogaster that moves to DNA repair foci after irradiation. Here we show using yeast 2 hybrid screens and co-immunoprecipitation that MU2 binds the chromoshadow domain of the heterochromatin protein HP1 in untreated cells. We asked what role HP1 plays in the formation of repair foci and cell cycle control in response to DNA damage. After irradiation repair foci form in heterochromatin but are shunted to the edge of heterochromatic regions an HP1-dependent manner, suggesting compartmentalized repair. Hydroxyurea-induced repair foci that form at collapsed replication forks, however, remain in the heterochromatic compartment. HP1a depletion in irradiated imaginal disc cells increases apoptosis and disrupts G2/M arrest. Further, cells irradiated in mitosis produced more and brighter repair foci than to cells irradiated during interphase. Thus, the interplay between MU2 and HP1a is dynamic and may be different in euchromatin and heterochromatin during DNA break recognition and repair

A feed forward circuit comprising Spt6, Ctk1 and PAF regulates Pol II CTD phosphorylation and transcription elongation

The C-terminal domain (CTD) of RNA polymerase II is sequentially modified for recruitment of numerous accessory factors during transcription. One such factor is Spt6, which couples transcription elongation with histone chaperone activity and the regulation of H3 lysine 36 methylation. Here, we show that CTD association of Spt6 is required for Ser2 CTD phosphorylation and for the protein stability of Ctk1 (the major Ser2 CTD kinase). We also find that Spt6 associates with Ctk1, and, unexpectedly, Ctk1 and Ser2 CTD phosphorylation are required for the stability of Spt6—thus revealing a Spt6–Ctk1 feed-forward loop that robustly maintains Ser2 phosphorylation during transcription. In addition, we find that the BUR kinase and the polymerase associated factor transcription complex function upstream of the Spt6–Ctk1 loop, most likely by recruiting Spt6 to the CTD at the onset of transcription. Consistent with requirement of Spt6 in histone gene expression and nucleosome deposition, mutation or deletion of members of the Spt6–Ctk1 loop leads to global loss of histone H3 and sensitivity to hydroxyurea. In sum, these results elucidate a new control mechanism for the regulation of RNAPII CTD phosphorylation during transcription elongation that is likely to be highly conserved

H3K36 Methylation Regulates Nutrient Stress Response in Saccharomyces cerevisiae by Enforcing Transcriptional Fidelity

Set2-mediated histone methylation at H3K36 regulates diverse activities, including DNA repair, mRNA splicing, and suppression of inappropriate (cryptic) transcription. Although failure of Set2 to suppress cryptic transcription has been linked to decreased lifespan, the extent to which cryptic transcription influences other cellular functions is poorly understood. Here, we uncover a role for H3K36 methylation in the regulation of the nutrient stress response pathway. We found that the transcriptional response to nutrient stress was dysregulated in SET2-deleted (set2Δ) cells and was correlated with genome-wide bi-directional cryptic transcription that originated from within gene bodies. Antisense transcripts arising from these cryptic events extended into the promoters of the genes from which they arose and were associated with decreased sense transcription under nutrient stress conditions. These results suggest that Set2-enforced transcriptional fidelity is critical to the proper regulation of inducible and highly regulated transcription programs

Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation

Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational modification analysis of affinity-isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C, although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro, whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1Δ cka2–8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2–8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation, a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNA polymerase II transcription through chromatin via phosphorylation of PAF-C

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

Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response

The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14

Redundant Functions for Nap1 and Chz1 in H2A.Z Deposition

Abstract H2A.Z is a histone H2A variant that contributes to transcriptional regulation, DNA damage response and limits heterochromatin spreading. In Saccharomyces cerevisiae, H2A.Z is deposited by the SWR-C complex, which relies on several histone chaperones including Nap1 and Chz1 to deliver H2A.Z-H2B dimers to SWR-C. However, the mechanisms by which Nap1 and Chz1 cooperate to bind H2A.Z and their contribution to H2A.Z deposition in chromatin is not well understood. Using structural modeling and molecular dynamics simulations, we identify a series of H2A.Z residues that form a chaperone-specific binding surface. Mutation of these residues revealed different surface requirements for Nap1 and Chz1 interaction with H2A.Z. Consistent with this result, we found that loss of Nap1 or Chz1 individually resulted in mild defects in H2A.Z deposition, but that deletion of both Nap1 and Chz1 resulted in a significant reduction of H2A.Z deposition at promoters and led to heterochromatin spreading. Together, our findings reveal unique H2A.Z surface dependences for Nap1 and Chz1 and a redundant role for these chaperones in H2A.Z deposition

Set2 methyltransferase facilitates cell cycle progression by maintaining transcriptional fidelity

Abstract Methylation of histone H3 lysine 36 (H3K36me) by yeast Set2 is critical for the maintenance of chromatin structure and transcriptional fidelity. However, we do not know the full range of Set2/H3K36me functions or the scope of mechanisms that regulate Set2-dependent H3K36 methylation. Here, we show that the APC/CCDC20 complex regulates Set2 protein abundance during the cell cycle. Significantly, absence of Set2-mediated H3K36me causes a loss of cell cycle control and pronounced defects in the transcriptional fidelity of cell cycle regulatory genes, a class of genes that are generally long, hence highly dependent on Set2/H3K36me for their transcriptional fidelity. Because APC/C also controls human SETD2, and SETD2 likewise regulates cell cycle progression, our data imply an evolutionarily conserved cell cycle function for Set2/SETD2 that may explain why recurrent mutations of SETD2 contribute to human disease

Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response

The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14