An integrated structural model of the DNA damage-responsive H3K4me3 binding WDR76:SPIN1 complex with the nucleosome.

Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.

Histone acylation at a glance.

An important mechanism of gene expression regulation is the epigenetic modification of histones. The cofactors and substrates for these modifications are often intermediary metabolites, and it is becoming increasingly clear that the metabolic and nutritional state of cells can influence these marks. These connections between the balance of metabolites, histone modifications and downstream transcriptional changes comprise a metabolic signaling program that can enable cells to adapt to changes in nutrient availability. Beyond acetylation, there is evidence now that histones can be modified by other acyl groups. In this Cell Science at a Glance article and the accompanying poster, we focus on these histone acylation modifications and provide an overview of the players that govern these acylations and their connections with metabolism.

Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.

Paraspeckles are subnuclear RNA-protein structures that are implicated in important processes including cellular stress response, differentiation, and cancer progression. However, it is unclear how paraspeckles impart their physiological effect at the molecular level. Through biochemical analyses, we show that paraspeckles interact with the SWI/SNF chromatin-remodeling complex. This is specifically mediated by the direct interaction of the long-non-coding RNA NEAT1 of the paraspeckles with ARID1B of the cBAF-type SWI/SNF complex. Strikingly, ARID1B depletion, in addition to resulting in loss of interaction with the SWI/SNF complex, decreases the binding of paraspeckle proteins to chromatin modifiers, transcription factors, and histones. Functionally, the loss of ARID1B and NEAT1 influences the transcription and the alternative splicing of a common set of genes. Our findings reveal that dynamic granules such as the paraspeckles may leverage the specificity of epigenetic modifiers to impart their regulatory effect, thus providing a molecular basis for their function.

(mis)-Targeting of SWI/SNF complex(es) in cancer.

The ATP-dependent chromatin remodeling complex SWI/SNF (also called BAF) is critical for the regulation of gene expression. During the evolution from yeast to mammals, the BAF complex has evolved an enormous complexity that contains a high number of subunits encoded by various genes. Emerging studies highlight the frequent involvement of altered mammalian SWI/SNF chromatin-remodeling complexes in human cancers. Here, we discuss the recent advances in determining the structure of SWI/SNF complexes, highlight the mechanisms by which mutations affecting these complexes promote cancer, and describe the promising emerging opportunities for targeted therapies.

The disordered regions of the methyltransferase SETD2 govern its function by regulating its proteolysis and phase separation.

SETD2 is an important methyltransferase that methylates crucial substrates such as histone H3, tubulin, and STAT1 and also physically interacts with transcription and splicing regulators such as Pol II and various hnRNPs. Of note, SETD2 has a functionally uncharacterized extended N-terminal region, the removal of which leads to its stabilization. How this region regulates SETD2 half-life is unclear. Here we show that SETD2 consists of multiple long disordered regions across its length that cumulatively destabilize the protein by facilitating its proteasomal degradation. SETD2 disordered regions can reduce the half-life of the yeast homolog Set2 in mammalian cells as well as in yeast, demonstrating the importance of intrinsic structural features in regulating protein half-life. In addition to the shortened half-life, by performing fluorescence recovery after photobleaching assay we found that SETD2 forms liquid droplets in vivo, another property associated with proteins that contain disordered regions. The phase-separation behavior of SETD2 is exacerbated upon the removal of its N-terminal segment and results in activator-independent histone H3K36 methylation. Our findings reveal that disordered region-facilitated proteolysis is an important mechanism governing SETD2 function.

Effectiveness of preventive measures against COVID-19: A systematic review of In Silico modeling studies in indian context.

BACKGROUND: In the absence of any approved treatment or vaccine against novel Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) infection, Non-Pharmaceutical Interventions (NPIs) are the cornerstone to prevent the disease, especially in a populous country like India. OBJECTIVES: To understand the effectiveness of NPIs reported in the contemporary literatures describing prediction models for prevention of the ongoing pandemic of SARS-CoV-2 specifically in Indian population. METHODS: Original research articles in English obtained through keyword search in PubMed, WHO Global Database for COVID19, and pre-print servers were included in the review. Thematic synthesis of extracted data from articles were done. RESULTS: Twenty-four articles were found eligible for the review - four published articles and twenty pre-print articles. Compartmental model was found to be the most commonly used mathematical model; along with exponential, time varying, neural network and cluster kinetic models. Social distancing, specifically lockdown, was the most commonly modelled intervention strategy. Additionally, contact tracing using smartphone application, international travel restriction, increasing hospital/ICU beds, changes in testing strategy were also dealt with. Social distancing along with increasing testing seemed to be effective in delaying the peak of the epidemic and reducing the peak prevalence. CONCLUSION: Although there is mathematical rationality behind implementation of social distancing measures including lockdown, this study also emphasised the importance of other associated measures like increasing tests and increasing the number of hospital and ICU beds. The later components are particularly important during the social mixing period to be observed after lifting of lockdown.

Quality of life of older people in an urban slum of India.

OBJECTIVES: The aim of this study was to examine different domains of quality of life (QOL) and their relationship to sociodemographic characteristics among older people in an urban slum in India. MATERIALS AND METHODS: A cross-sectional study was conducted (n = 120). Selected individuals were interviewed, and their QOL was assessed by the World Health Organization Quality of Life-BREF questionnaire. Statistical analysis was then performed. RESULTS: Of those included in the study population, 61.7% were men, all were Hindus, 46.7% were members of the general caste, 19.2% were illiterate, 35% were unemployed, and 72.5% lived with their joint family (i.e. extended family). Mean scores in each domain studied did not significantly differ between the sexes, age groups, castes, and family types. Subjects with more education, who were married, and with greater income had significantly better QOL scores. Mean scores were also better in certain domains among persons who had their own income and who resided with their children. CONCLUSION: Having low education, being single, lacking personal income, and not living with their children significantly reduced QOL in the elderly subjects. Attention should be given to these factors to help elderly individuals age in a healthy manner.

Red blood cell indices in different hemoglobinopathies: A cross-sectional study in Eastern India.

Introduction: Beta thalassemia and hemoglobin (HbE)-related hemoglobinopathies are common public health problems in developing countries. High-performance liquid chromatography (HPLC) is currently the diagnostic test of choice for carrier detection, but it is costly. Hence, some initial screening and complementary tests are required, which can be affordable. Aims: To find out the distribution of different red blood cell (RBC) indices in beta thalassemia trait (BTT) and HbE-related hemoglobinopathies and to determine their significance as screening tests to distinguish between these hemoglobinopathies. Study Settings and Design: This observational cross-sectional study has been carried out at an NABL (National Accreditation Board for Testing and Calibration Laboratories)-accredited Laboratory of Eastern India with approval from the concerned Institutional Ethics Committee from January 2021 to March 2021. Methods and Material: : HPLC tests and complete hemograms were performed on 2247 ethylenediaminetetraacetic acid anti-coagulated blood samples over 3 months. Patients <1 year of age or having a history of blood transfusion within the past 06 months were excluded. Statistical Analysis: : One-way analysis of variance along with Bonferroni post-hoc test was performed to find out significant differences of means of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hemoglobin%, red blood cell (RBC) count, and red cell distribution width (RDW-CV) among concerned hemoglobinopathies. Results: The results show a significant difference of total RBC count, RDW, MCV, MCH, and MCHC between BTT and E-trait. No significant difference of mean was found between HbE homozygous and E-beta. E-trait differs from both HbE homozygous and E-beta significantly in three parameters, namely, RDW, MCV and MCH. A value of MCV at ≤73.8 fl and MCH at ≤21.9 pg may be a clue of diagnosis for BTT rather than E-trait with >90% sensitivity and >80% specificity. Conclusion: RBC indices vary significantly between BTT and other HbE-related hemoglobinopathies. They can specially be utilized to differentiate BTT and E-trait as supportive tests in addition to the gold standard test of HPLC.

Serial Capture Affinity Purification and Integrated Structural Modeling of the H3K4me3 Binding and DNA Damage Related WDR76:SPIN1 Complex.

WDR76 is a multifunctional protein involved in many cellular functions. With a diverse and complicated protein interaction network, dissecting the structure and function of specific WDR76 complexes is needed. We previously demonstrated the ability of the Serial Capture Affinity Purification (SCAP) method to isolate specific complexes by introducing two proteins of interest as baits at the same time. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone marker reader that specifically recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to the SCAP analysis of the SPIN1:SPINDOC complex, H3K4me3 was copurified with the WDR76:SPIN1 complex. In combination with crosslinking mass spectrometry, we built an integrated structural model of the complex which revealed that SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Lastly, interaction network analysis of copurifying proteins revealed the potential role of the WDR76:SPIN1 complex in the DNA damage response. Teaser: In contrast to the SPINDOC/SPIN1 complex, analyses reveal that the WDR76/SPIN1 complex interacts with core histones and is involved in DNA damage.

Elevated levels of the methyltransferase SETD2 causes transcription and alternative splicing changes resulting in oncogenic phenotypes.

The methyltransferase SETD2 regulates cryptic transcription, alternative splicing, and the DNA damage response. It is mutated in a variety of cancers and is believed to be a tumor suppressor. Counterintuitively, despite its important role, SETD2 is robustly degraded by the proteasome keeping its levels low. Here we show that SETD2 accumulation results in a non-canonical deposition of the functionally important H3K36me3 histone mark, which includes its reduced enrichment over gene bodies and exons. This perturbed epigenetic landscape is associated with widespread changes in transcription and alternative splicing. Strikingly, contrary to its role as a tumor suppressor, excessive SETD2 results in the upregulation of cell cycle-associated pathways. This is also reflected in phenotypes of increased cell proliferation and migration. Thus, the regulation of SETD2 levels through its proteolysis is important to maintain its appropriate function, which in turn regulates the fidelity of transcription and splicing-related processes.

DNA methylation mediated downregulation of histone H3 variant H3.3 affects cell proliferation contributing to the development of HCC.

Chromatin alterations brought by histone variants and modifications potentially regulate gene transcription from tumor initiation to progression. Histone H3.3 variant is one such epigenetic player important for disease progression and development. Though many studies have implicated H3.3 role in cancer progression and metastasis, its regulation, importance of specific modifications and chaperones have been not understood yet. We report DNA methylation mediated downregulation of histone H3 variant H3.3 in HCC and a concomitant increase in the level of the H3.2 variant. The loss of H3.3 in cancer tissues correlates with a decrease in the histone modifications associated with active transcription like H3K9/K14/K27Ac and H3K4Me3. The ectopic overexpression of H3.3 and H3.2 did not affect global PTMs and cell physiology, probably owing to the deregulation of specific histone chaperones CAF-1 (for H3.2) and HIRA (for H3.3) as observed in HCC tissues. Notably, knockdown of P150, a subunit of CAF-1 leads to a cell cycle arrest in S-phase in a neoplastic rat liver cell line, possibly due to the decrease in the histone levels necessary for DNA packaging. Remarkably, modulation of H3.3 in pre-neoplastic rat liver cells lead to an increase in cell proliferation and a decreased transcription of tumor suppressor genes, recapitulating the tumor cell phenotype. Our data suggests, inhibition of DNA methylation and histone deacetylation leads to the restoration of histone H3 variant expression in tumor cells.

Awareness Regarding Eye Donation and Effects of COVID-19 on Its Perception: A Community-Based Cross-Sectional Study From India.

OBJECTIVES: This study aimed to assess awareness toward eye donation and the effects of COVID-19 on perceptions about eye donation in an urban community of West Bengal, India. MATERIALS AND METHODS: Eligible adults residing in the area under study were chosen by simple random sampling and interviewed using a predesigned and pretested data collection schedule to assess awareness and perception. Based on their total awareness score, the participants were classified as having good awareness or poor awareness toward eye donation. RESULTS: Among 423 participants, 52.0% were male, 34.0% were 18 to 30 years old, 38.3% were educated up to secondary level, and 85.6% had eye disease in their family. Of total participants, 70.2% had heard about eye donation, with 59.9% of this group having knowledge of eye donation only after death and 57.9% having knowledge that donation must occur within 6 hours of death. In addition, 52.9% were aware about donation from home, 30.6% said only the cornea is transplantable, and 52.2% knew it can be preserved. Participants with higher education, higher socioeconomic status, and outside of the house occupational engagement had significantly higher chance of good eye donation awareness. Of total participants, 32.0% said that COVID-19 could be transmitted through eye donation, with 58.2% of participants opining that the pandemic was not a suitable time to consider donation of eyes. CONCLUSIONS: COVID-19 has had a negative impact on eye donation perceptions. Although most of our participants had heard about eye donation, their awareness regarding important aspects about eye donation were still lacking, thus calling for a more focused action plan and implementation of the same.

Understanding social problems during lockdown and its relationship to perceived stress - An online survey among adult residents of India.

CONTEXT: Nationwide lock down imposed due to Covid 19 might bring about various social problems, constraints in availing essential services, difficulty in taking care of morbid elderly persons and inaccessibility to health care which can lead to stress. AIMS: To identify social problems among adult Indian residents during lockdown period, to assess perceived stress among them and to identify the factors associated with it. SETTINGS AND DESIGN: The study was conducted through the online platform using a google form among adult residents of India. Perceived stress was assessed by Perceived Stress Scale 10. STATISTICAL ANALYSIS USED: Data were analysed using SPSS version 16.0., using chi square, Odd's Ratio and binary logistic regression. RESULTS: One fourth of the respondents faced economic problem, 11.7% of them had experienced some sort of violence and worsening of familial relationship. The problems encountered in seeking care were chi square test due to non availability of transportation facilities and health services. Overall 80.78% of the respondents were observed to have moderate and high levels of perceived stress. Multivariate analysis revealed single marital status, owing a debt and experiencing violence had 1.62, 1.8 and 9 times higher odds of having moderate to high perceived stress. Violence was identified as its chief correlate. CONCLUSIONS: The present study found negative economic and social consequences on the Indian population and also posed an enormous psychological stress due to sudden lock down in pandemic situation.

The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain.

Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing.

The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

Regulation of SETD2 stability is important for the fidelity of H3K36me3 deposition.

BACKGROUND: The histone H3K36me3 mark regulates transcription elongation, pre-mRNA splicing, DNA methylation, and DNA damage repair. However, knowledge of the regulation of the enzyme SETD2, which deposits this functionally important mark, is very limited. RESULTS: Here, we show that the poorly characterized N-terminal region of SETD2 plays a determining role in regulating the stability of SETD2. This stretch of 1-1403 amino acids contributes to the robust degradation of SETD2 by the proteasome. Besides, the SETD2 protein is aggregate prone and forms insoluble bodies in nuclei especially upon proteasome inhibition. Removal of the N-terminal segment results in the stabilization of SETD2 and leads to a marked increase in global H3K36me3 which, uncharacteristically, happens in a Pol II-independent manner. CONCLUSION: The functionally uncharacterized N-terminal segment of SETD2 regulates its half-life to maintain the requisite cellular amount of the protein. The absence of SETD2 proteolysis results in a Pol II-independent H3K36me3 deposition and protein aggregation.

Perception and Willingness towards Eye Donation in an urban community of West Bengal.

INTRODUCTION: Corneal blindness, though treatable, contributes largely to the burden of blindness. With 1.2 million existing cases of corneal blindness in India, the demand for cornea collection is increasing. 69349 corneas were collected in 2018-19 but it is much less than what is needed. Faulty perceptions and unwillingness are two major hurdles against a scenario favourable towards eye donation. The objectives of the study was to assess the perception and willingness towards eye donation and the socio-demographic variables that affect such perception and willingness, in an urban community of West Bengal. MATERIALS AND METHODS: In this cross-sectional study conducted among adult residents of the urban field practice area of Medical College Kolkata, data was collected from randomly selected eligible members, one each from all the available families by interviewing them using a validated pre tested schedule. RESULTS: Eighty nine (52.3%) among 170 interviewed participants had Secondary level education or higher, 49.4% had outdoor engagement. 128 participants reported ocular morbidity in the family but only 126 had heard about eye donation. 98 (77.8%) of these 126, had favourable perception towards eye donation. Among these 98 participants, 40 were willing to pledge for eye donation. Higher education and outdoor engagement significantly predicted favourable perception, while willingness significantly increased with participants belonging to higher socio-economic class and with higher education. CONCLUSIONS: Compared to favourable perception, willingness of pledging eyes was lesser due to various false notions. Proper education through usage of means of mass communication and stress upon school education is needed to address these hurdles.

The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity.

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.