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

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

No Rise in Incidence but Geographical Heterogeneity in the Occurrence of Primary Biliary Cirrhosis in North East England

In this study, we examined temporal changes in the incidence of primary biliary cirrhosis (PBC) and investigated associations between PBC incidence and sociodemographic factors and spatial clustering. We included 982 patients aged ≥40 years from North East England with incident PBC diagnosed during 1987–2003. Age-standardized incidence rates with 95% confidence intervals were calculated. Negative binomial regression was used to analyze incidence and socioeconomic deprivation. Clustering analysis was performed using point process methods, testing the null hypothesis that disease risk does not vary spatially and that PBC cases occur independently. The age-standardized incidence rate was 53.50 per million persons per year (95% confidence interval: 48.65, 58.35) in 1987–1994 and 45.09 per million persons per year (95% confidence interval: 41.10, 49.07) in 1995–2003. Risk of PBC increased in areas with higher levels of socioeconomic deprivation (P = 0.035). More specifically, risk increased in areas with higher levels of overcrowded homes (P = 0.040), higher levels of households without cars (P < 0.001), and higher levels of non-owner-occupied homes (P < 0.001). Overall, there was evidence of spatial clustering (P = 0.001). The findings confirm that overall incidence of PBC did not rise over time, but sociodemographic variations suggest that certain aspects of deprivation are involved in its etiology

Study of depression among patients with type 2 diabetes mellitus at a tertiary care hospital

Background: Diabetes and depression are two major issues related to community health. Diabetes patients frequently co-occur with depression. Diabetes patients frequently co-occur with depression, which calls for serious attention because delayed diagnosis and treatment can worsen the patients' complications. Assessing the prevalence of depression in diabetic patients and identifying the various factors associated with it were the objectives of this research study. Methods: In this study 70 adult patients suffering from type 2 DM participated in this 6-month prospective study. Sociodemographic data and clinical features of the participants were collected. The presence and severity of depressive symptoms in patients have been assessed by a PHQ9 questionnaire. Ethical approval was taken before the commencement of the study. SPSS (Version 20) was used for data analysis. Results: The majority of the patients were from 41-50 years of age group (32.9%) with a female predominance (58.6%), with no symptoms of depression before type 2 DM (92.9%). Most of them had primary education (32.9%) and majority were homemakers (44.3%) residing in urban are (65.7%) living joint family setup (68.6%). Most of them had 11 to 20 years of type 2 DM duration (745.8%) with a high family history of type 2 DM (75.7%). Majority of them were on Oral therapy (47.1%) with ophthalmic complications (32.9%). The majority of them had mild depression (5-9) i.e. 67.1%. Conclusions: Due to patient-specific diabetes management and inappropriate diabetes treatment, the majority of cases were found to have depressive disorders. This article focused on a few common factors and their relationships that lead to depression in people with diabetes

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

Evaluation of EED Inhibitors as a Class of PRC2-Targeted Small Molecules for HIV Latency Reversal

A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development

Genetic interaction mapping informs integrative structure determination of protein complexes

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on similar to 500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations

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