Biology subjects

Mitra, S.

Publications and source records attributed to Mitra, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Competitive binding of STATs to receptor phospho-Tyr motifs accounts for altered cytokine responses in autoimmune disorders

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modelling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27R, as the main dynamical processes contributing to sustained pSTAT1 by IL-27. Mutation of Tyr613 on IL-27R decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with Systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

bioinformatics↗

bioRxiv2021

Role of extracellular mycobacteria in blood-retinal barrier invasion in a zebrafish model of ocular TB

Intraocular inflammation following mycobacterial dissemination to the eye is common in tuberculosis (TB)-endemic countries. However, the early host-pathogen interactions during ocular dissemination are unknown. In this study, we investigate the early events during mycobacterial invasion of the blood-retinal barriers (BRB) with fluorescent tagged Mycobacterium marinum (Mm), host macrophages and retinal vasculature in a zebrafish model of ocular TB. We found that Mm invaded the vascular endothelium either in extracellular or intracellular (inside phagocytes) state, typically 3-4 days post-injection (dpi). Extracellular Mm are phagocytosed in the retinal tissue, and progress to form a compact granuloma around 6 dpi. Intracellular Mm crossing the BRB are likely to be less virulent, and either persist inside solitary macrophages (most cases), or progress to loosely arranged granuloma (rare). The early interactions between mycobacteria and host immune cells can thus determine the course of disease during mycobacterial dissemination to the eye.

immunology↗

bioRxiv2020

Altered food habits? Understanding the feeding preference of free-ranging Grey langur (Semnopithecus entellus) within an urban settlement.

Urbanization affects concurrent human-animal movements as a result of altered resource availability and land use pattern, which leads to considerable ecological consequences. While some animals find themselves adrift, homeless with the uncertainty of resources resulting from the urban encroachment, few of them manage to survive by altering their natural behavioural patterns, and co-exist with humans. Folivorous colobines, such as grey langur, whose feeding repertoire largely consists of plant parts, tend to be more attuned to the urban high-calorie food sources to attain maximum fitness benefits within the concrete jungle having an insignificant green cover. However, such a mismatch between their generalized feeding behaviour and specialized gut physiology reminds us of the Liems paradox and demands considerable scientific attention which could tell us the story behind colobines successful co-existence within human settlements. Besides understanding their population dynamics, the effective management of these urbanized, free-ranging, non-human primate populations also depends on their altered feeding preferences, altogether which could lead us to the development of an ecologically sound urban ecosystem. Here, we have used a field-based experimental set up which allows langurs to choose between natural and urban food options, being independent of any inter-specific conflicts over resources due to food scarcity. The multinomial logit model reveals the choice-based decision making of these free-ranging grey langurs in an urban settlement of West Bengal, India, where they have not only learned to approach the human-provisioned urban food items but also shown a keen interest in it. While urbanization imposes tremendous survival challenges to these animals, it also opens up for various alternative options for human-animal co-existence which is reflected in this study, and could guide us for the establishment of a sustainable urban ecosystem in the future. HIGHLIGHTSO_LIThe feeding repertoire of free-ranging grey langurs at Dakshineswar largely consists of urban food items in contrast to the langurs of Nangi, and Nalpur who mostly depend on natural food sources. C_LIO_LIHigh human-langur interactions together with the scarcity of natural plant-based food sources could be considered as an intriguing driving force behind langurs altered feeding habits in Dakhineswar. C_LIO_LIThe field-based experimental set up allows free-ranging langurs to choose between natural and urban food options in an urban settlement like Dakhineswar. C_LIO_LIUrban food items outperformed natural food items as the most chosen one, indicating langurs altered feeding preferences which facilitate their successful co-existence within an urban ecosystem. C_LI

animal behavior and cognition↗

bioRxiv2020

Functional Characterization of an Electromagnetic Perceptive Protein

Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in-vivo. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique to determine that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic activation at a single particle level. Trajectory analysis show notably different EPG protein kinetics before and after magnetic stimulation in both 2 (free vs bound particle) and 3 state (free vs intermediate vs bound particle) tracking models. This data provides additional information to support and understand the underlying biophysical mechanisms behind EPG activation by magnetic fields and provides evidence for the basis of magnetoreception in the EPG protein that will aid in future studies that seek to further understand this novel mechanism. This study is important for understanding magnetoreception as well as developing new technologies for magnetogenetics - the utilization of electromagnetic fields to remotely control cellular function. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/329946v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1ce4aborg.highwire.dtl.DTLVardef@e8bb89org.highwire.dtl.DTLVardef@1705113org.highwire.dtl.DTLVardef@dadf23_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Deubiquitinase UCHL1 Maintains Protein Homeostasis through PSMA7-APEH-Proteasome Axis in High-Grade Serous Ovarian Carcinoma

High-grade serous ovarian cancer (HGSOC) is characterized by chromosomal instability, DNA damage, oxidative stress, and high metabolic demand, which exacerbate misfolded, unfolded and damaged protein burden resulting in increased proteotoxicity. However, the underlying mechanisms that maintain protein homeostasis to promote HGSOC growth remain poorly understood. In this study, we report that the neuronal deubiquitinating enzyme, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is overexpressed in HGSOC and maintains protein homeostasis. UCHL1 expression was markedly increased in HGSOC patient tumors and serous tubal intraepithelial carcinoma (HGSOC precursor lesions). High UCHL1 levels correlated with higher tumor grade and poor patient survival. UCHL1 inhibition reduced HGSOC cell proliferation and invasion through the outer layers of omentum as well as significantly decreased the in vivo metastatic tumor growth in ovarian cancer xenografts. Transcriptional profiling of UCHL1 silenced HGSOC cells revealed the down-regulation of genes implicated with proteasome activity along with the upregulation of endoplasmic reticulum (ER) stress-induced genes. Reduced expression of proteasome subunit alpha 7 (PSMA7) and acylaminoacyl peptide hydrolase (APEH) resulted in a significant decrease in proteasome activity, impaired protein degradation, and abrogated HGSOC growth. Furthermore, the accumulation of polyubiquitinated proteins in the UCHL1 silenced cells led to attenuation of mTORC1 activity and protein synthesis, and induction of terminal unfolded protein response. Collectively, these results indicate that UCHL1 promotes HGSOC growth by mediating protein homeostasis through the PSMA7-APEH-proteasome axis. O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/316810v2_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@d38d5borg.highwire.dtl.DTLVardef@e58513org.highwire.dtl.DTLVardef@83e274org.highwire.dtl.DTLVardef@884ec3_HPS_FORMAT_FIGEXP M_FIG C_FIG ImplicationsThis study identifies the novel links in the proteostasis network to target protein homeostasis in HGSOC. It recognizes the potential of inhibiting UCHL1 and APEH to sensitize cancer cells to proteotoxic stress and as novel alternative therapeutic approaches.

cancer biology↗

bioRxiv2020

Neoplastic and Immune single cell transcriptomics define subgroup-specific intra-tumoral heterogeneity of childhood medulloblastoma.

Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. To better understand cellular heterogeneity in MB we use single-cell RNA sequencing, immunohistochemistry and deconvolution of transcriptomic data to profile neoplastic and immune populations in childhood MB samples. Neoplastic cells cluster primarily according to individual sample of origin which is in part due to the effect of chromosomal copy number gains and losses. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes and are associated with clinical outcomes, including discrete photoreceptor-like cells in MB subgroups GP3 and GP4 and nodule-associated neuronally-differentiated cells in subgroup SHH. We definitively chart the spectrum of MB immune cell infiltrates, which include subgroup/subtype-associated developmentally-related neuron-pruning and antigen presenting myeloid cells. MB cellular diversity is recapitulated in genetically engineered mouse subgroup-specific models of MB. These findings provide a clearer understanding of both the neoplastic and immune cell heterogeneity in MB.

cancer biology↗

bioRxiv2020

Comprehensive analysis of genomic diversity of SARS-CoV-2 in different geographic regions of India: An endeavour to classify Indian SARS-CoV-2 strains on the basis of co-existing mutations

Accumulation of mutations within the genome is the primary driving force for viral evolution within an endemic setting. This inherent feature often leads to altered virulence, infectivity and transmissibility as well as antigenic shift to escape host immunity, which might compromise the efficacy of vaccines and antiviral drugs. Therefore, we aimed at genome-wide analyses of circulating SARS-CoV-2 viruses for the emergence of novel co-existing mutations and trace their spatial distribution within India. Comprehensive analysis of whole genome sequences of 441 Indian SARS-CoV-2 strains revealed the occurrence of 33 different mutations, 21 being distinctive to India. Emergence of novel mutations were observed in S glycoprotein (7/33), NSP3 (6/33), RdRp/NSP12 (4/33), NSP2 (2/33) and N (2/33). Non-synonymous mutations were found to be 3.4 times more prevalent than synonymous mutations. We classified the Indian isolates into 22 groups based on the co-existing mutations. Phylogenetic analyses revealed that representative strain of each group divided themselves into various sub-clades within their respective clades, based on the presence of unique co-existing mutations. India was dominated by A2a clade (55.60%) followed by A3 (37.38%) and B (7%), but exhibited heterogeneous distribution among various geographical regions. The A2a clade mostly predominated in East India, Western India and Central India, whereas A3 clade prevailed in South and North India. In conclusion, this study highlights the divergent evolution of SARS-CoV-2 strains and co-circulation of multiple clades in India. Monitoring of the emerging mutations would pave ways for vaccine formulation and designing of antiviral drugs.

microbiology↗

bioRxiv2020

RoboCOP: Jointly computing chromatin occupancy profiles for numerous factors from chromatin accessibility data

Chromatin is the tightly packaged structure of DNA and protein within the nucleus of a cell. The arrangement of different protein complexes along the DNA modulates and is modulated by gene expression. Measuring the binding locations and level of occupancy of different transcription factors (TFs) and nucleosomes is therefore crucial to understanding gene regulation. Antibody-based methods for assaying chromatin occupancy are capable of identifying the binding sites of specific DNA binding factors, but only one factor at a time. On the other hand, epigenomic accessibility data like ATAC-seq, DNase-seq, and MNase-seq provide insight into the chromatin landscape of all factors bound along the genome, but with minimal insight into the identities of those factors. Here, we present RoboCOP, a multivariate state space model that integrates chromatin information from epigenomic accessibility data with nucleotide sequence to compute genome-wide probabilistic scores of nucleosome and TF occupancy, for hundreds of different factors at once. We apply RoboCOP to MNase-seq data to elucidate the protein-binding landscape of nucleosomes and 150 TFs across the yeast genome. Using available protein-binding datasets from the literature, we show that our model predicts the binding of these factors genome-wide more accurately than existing methods.

bioinformatics↗

bioRxiv2020

The novel Coronavirus enigma: Phylogeny and mutation analyses of SARS-CoV-2 viruses circulating in India during early 2020.

BackgroundThis is a comprehensive analysis of 46 Indian SARS-CoV-2 genome sequences available from the NCBI and GISAID repository during early 2020. Evolutionary dynamics, gene-specific phylogeny and emergence of the novel co-evolving mutations in nine structural and non-structural genes among circulating SARS-CoV-2 strains in ten states of India have been assessed. Materials and methods46 SARS-CoV-2 nucleotide sequences submitted from India were downloaded from the GISAID (39/46) or from NCBI (7/46) database. Phylogenetic study and analyses of mutation were based on the nine structural and non-structural genes of SARS-CoV-2 strains. Secondary structure of RdRP/NSP12 protein was predicted with respect to the novel A97V mutation. ResultsPhylogenetic analyses revealed the evolution of "genome-type clusters" and adaptive selection of "L" type SARS-CoV-2 strains with genetic closeness to the bat SARS-like coronaviruses than pangolin or MERS-CoVs. With regards to the novel co-evolving mutations, 2 groups are seen to circulate in India at present: the "major group" (52.2%) and the "minor group" (30.4%), harboring four and five co-existing mutations, respectively. The "major group" mutations fall in the A2a clade. All the minor group mutations, except 11083G>T (L37F, NSP6) were unique to the Indian isolates. ConclusionThe study highlights rapidly evolving SARS-CoV-2 virus and co-circulation of multiple clades and sub-clades, driving this pandemic worldwide. This comprehensive study is a potential resource for monitoring the novel mutations in the viral genome, changes in viral pathogenesis, for designing vaccines and other therapeutics.

genomics↗

bioRxiv2020

CDK8 fine-tunes IL-6 transcriptional activities by limiting STAT3 resident time at the gene loci

Cytokines are highly pleiotropic ligands that critically contribute to a balanced immune response. We have an incomplete understanding of how cytokines elicit their functional pleiotropy, which has limited their therapeutic use. Here, using Interleukin-6 (IL-6) as a model system, we have performed detailed phosphoproteomic and transcriptomic studies in human Th-1 cells to address the molecular bases defining cytokine functional pleiotropy. We have identified CDK8 as a new negative regulator of STAT3 transcriptional activities that contributes to the diversification of IL-6 responses. We found that CDK8 is a major regulator of the IL-6 phosphoproteome and interacts with STAT3 in the nucleus upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecules inhibitors, reduced the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 Ser727 phosphorylation. This, in turn, resulted in retention of tyrosine phosphorylated STAT3 in the nucleus, which increased the binding of STAT3 to target DNA sites in the genome with a concomitant increase in STAT3 mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions resulted in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity via modulation of its gene loci resident time, critically contributing to tuning STAT3 mediated responses.

immunology↗

bioRxiv2019

Profiling the effects of rifaximin on the healthy human colonic microbiota using a chemostat model

Rifaximin is a low solubility antibiotic with activity against a wide range of bacterial pathogens. It accumulates in the intestine and is suitable for prolonged use. Three chemostat models (A, B and C) were used to investigate the effects of three rifaximin formulations (, {beta} and {kappa}, respectively) on the gut microbiome. Bacterial populations were monitored by bacterial culture and 16S rRNA gene amplicon (16S) sequencing. Limited disruption of bacterial populations was observed for rifaximin , {beta} and {kappa}. All formulations caused declines in total spores ([~]2 log10 cfu ml-1), Enterococcus spp. ([~]2 log10 cfu ml-1 in models A and C, and [~]1 log10 cfu ml-1 in model B), and Bacteroides spp. populations ([~]3 log10 cfu ml-1 in models A and C, and [~]4 log10 cfu ml-1 in model B). Bacterial populations fully recovered during antibiotic dosing in model C, and before the end of the experiment in models A and B. According to the taxonomic analysis, prior to rifaximin exposure, Bifidobacteriaceae, Ruminococcaceae, Acidaminococcaceae, Lachnospiraceae and Rikenellaceae families represented >92% of the total relative abundance, in all models. Within these families, 15 bacterial genera represented >99% of the overall relative abundance. Overall, the 16S sequencing and culture data showed similar variations in the bacterial populations studied. Among the three formulations, rifaximin {kappa} appeared to have the least disruptive effect on the colonic microbiota, with culture populations showing recovery in a shorter period and the taxonomic analysis revealing the least global variation in relative abundance of prevalent groups.

microbiology↗

bioRxiv2019

RNF144A shapes the hierarchy of cytokine signaling to provide protective immunity against influenza

Cytokine-induced signaling pathways are tightly regulated and self-limiting, as their dysregulation causes immune disorders and cancer. The precise mechanisms that fine-tune these responses are incompletely understood. We show that the E3 ubiquitin ligase RNF144A is an IL-2/STAT5-induced gene in T cells and critically orchestrates the hierarchy of IL-2R signaling to promote STAT5 activation and limit RAF-ERK-MAPK output from the IL-2R. Mechanistically, RNF144A increased the interaction between IL-2R{beta} and STAT5 and polyubiquitinated RAF1, enhancing its proteasomal degradation and preventing the formation of the potent RAF1/BRAF kinase complex. CD8+ T cells from Rnf144a-/- mice had impaired IL-2-induction of effector genes, including Tnf and granzymes, and these mice demonstrated increased susceptibility to influenza. Reduced RNF144A expression was associated with more severe influenza in humans and its expression in patients was a biomarker distinguishing moderate from severe disease. These studies reveal a vital physiological role for RNF144A in maintaining the fidelity of IL-2R signaling in CTLs to prevent severe inflammation in response to infection.\n\nOne Sentence SummaryRNF144A promotes anti-viral immunity by regulating the hierarchy of cytokine signal output

immunology↗

bioRxiv2019

Adverse childhood experiences in families with multiple members diagnosed to have psychiatric illnesses

ObjectiveAdverse Childhood Experiences (ACEs) are linked to the development of a number of psychiatric illnesses in adulthood. Our study examined the pattern of ACEs and their relation to the age of onset (AAO) of major psychiatric conditions in individuals from families that had [≥] 2 first degree relatives with major psychiatric conditions (multiplex families) identified as part of an ongoing longitudinal study.\n\nMethodsOur sample consisted of 509 individuals from 215 families. Of these, 268 were affected i.e diagnosed with bipolar disorder (BPAD) (n=61), obsessive-compulsive disorder (OCD) (n=58), schizophrenia (n=52), substance dependence (SUD) (n=59), or co-occurring diagnoses (n=38); while 241 were at-risk first degree relatives (FDRs) who were either unaffected (n=210) or had other depressive or anxiety disorders (n=31). All individuals were evaluated using the Adverse Childhood Experiences - International Questionnaire (ACE-IQ) and ACE binary and frequency scores were calculated.\n\nResultsIt was seen that affected males, as a group, had the greatest ACE scores in our sample. A cox mixed-effects model fit by gender revealed that higher ACE binary and frequency scores were associated with significantly increased risk for an earlier AAO of psychiatric diagnoses in males. A similar model that evaluated the effect of diagnosis revealed an earlier AAO in OCD and SUD, but not in schizophrenia and BPAD.\n\nConclusionsOur study indicates that ACEs brought forward the onset of major psychiatric conditions in men and in individuals diagnosed with OCD and SUD. Ongoing longitudinal assessments in FDRs from these families are expected to identify mechanisms underlying this relationship.

neuroscience↗

bioRxiv2019

Molecular profiling of driver events and tumor-infiltrating lymphocytes in metastatic uveal melanoma

Uveal melanoma (UM) is a rare form of melanoma with a genetics and immunology that is different from skin melanoma. Previous studies have identified genetic driver events of early stage disease when the tumor is confined to the eye. However due to lack of a clinical rationale to biopsy metastatic disease, access to tumor material to perform molecular profiling of metastases has been limited. In this study, we have characterized genomic events in UM metastases using whole-genome sequencing of fresh frozen biopsies from thirty-two patients and profiled the transcriptomes of individual tumor infiltrating lymphocytes in eight patients by single-cell sequencing. We find that 91% of the patients have metastases carrying inactivating events in the tumor suppressor BAP1 and this coincided with somatic alterations in GNAQ, GNA11, CYSLTR2, PLCB4, SF3B1 and/or CDKN2A. Mutational signature analysis revealed a rare subset of tumors with prominent signs of UV damage, associated with outlier mutational burden. We study copy number variations (CNV) and find overrepresented events, some of which were not altered in matched primary eye tumors. A focused siRNA screen identified functionally significant genes of some of the segments recurrently gained. We reintroduced a functional copy of BAP1 into a patient-derived BAP1 deficient tumor cell line and found broad transcriptomic changes of genes associated with subtype distinction and prognosis in primary UM. Lastly, our analysis of the immune microenvironments of metastases revealed a presence of tumor-reactive T cells. However, a majority expressed the immune checkpoint receptors TIM-3, LAG3 and TIGIT, and to a lesser extent PD-1. These results provide an updated view of genomic events represented in metastatic UM and immune interactions in advanced lesions.

cancer biology↗

bioRxiv2019

Microbiota supplementation with Bifidobacterium and Lactobacillus modifies the preterm infant gut microbiota and metabolome

Supplementation with members of the early-life microbiota or probiotics is becoming increasingly popular to attempt to beneficially manipulate the preterm gut microbiota. We performed a large longitudinal study comprising two preterm groups; 101 orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 non-supplemented (Control) matched by age, sex, birth-mode, and diet. 16S rRNA metataxonomic profiling on stool samples (n = 592) indicated a predominance of Bifidobacterium, and a reduction of pathobionts in the Bif/Lacto group. Metabolic phenotyping found a parallel increase in fecal acetate and lactate in the Bif/Lacto group compared to the Control group, which positively correlated with Bifidobacterium abundance consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides and reduced gut pH. This study demonstrates that microbiota supplementation can modify the preterm microbiome and the gastrointestinal environment to more closely resemble that of a full-term infant.

microbiology↗

bioRxiv2019

Kinetics of cytokine receptor trafficking determine signaling and functional selectivity

Cytokines activate downstream signaling networks via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered variants of IL-6 with different affinities to the gp130 receptor chain to investigate how cytokine receptor binding kinetics influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes, from minimal to full agonist, and induced biased signaling, with changes in receptor binding kinetics affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This, in turn, leads to a graded gene expression response and substantial differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

immunology↗

bioRxiv2019

Genetic screening identifies a SUMO protease dynamically maintaining centromeric chromatin and the associated centromere complex

Centromeres are defined by a unique self-propagating chromatin structure featuring nucleosomes containing the histone H3 variant CENP-A. CENP-A turns over slower than general chromatin and a key question is whether this unusual stability is intrinsic to CENP-A nucleosomes or rather imposed by external factors. We designed a specific genetic screen to identify proteins involved in CENP-A stability based on SNAP-tag pulse chase labeling. Using a double pulse-labeling approach we simultaneously assay for factors with selective roles in CENP-A chromatin assembly. We discover a series of new proteins involved in CENP-A propagation, including proteins with known roles in DNA replication, repair and chromatin modification and transcription, revealing that a broad set of chromatin regulators impacts in CENP-A transmission through the cell cycle. The key factor we find to strongly affect CENP-A stability is SENP6. This SUMO-protease controls not only the levels of chromatin bound CENP-A but is required for the maintenance of virtually the entire centromere and kinetochore, with the exception of CENP-B. Acute depletion of SENP6 protein reveals its requirement for maintaining centromeric CENP-A levels throughout the cell cycle, suggesting that a dynamic SUMO cycle underlies a continuous surveillance of the centromere complex.

cell biology↗

bioRxiv2019

Spt6 is a maintenance factor for centromeric CENP-A

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This constitutes both an opportunity to remodel the underlying chromatin as well as the potential danger of losing epigenetic information. Centromeric transcription has been shown to be required for stable incorporation of the centromere-specific histone dCENP-A in M/G1-phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and shows enhanced association with non-phosphorylatable dCENP-A mutants compared to histone H3, while phosphomimetic residues alleviate association with Spt6. We conclude that Spt6 acts as a conserved CENP-A maintenance factor, which is required during transcription-mediated chromatin remodelling at the centromere to ensure long-term stability of epigenetic centromere identity.

cell biology↗