Biology subjects

Thakur, J.

Publications and source records attributed to Thakur, J..

8 recordsLinked to original sources

bioRxiv2024

Genomic and epigenomic maps of mouse centromeres and pericentromeres

Satellite DNA makes up [~]11% of the mouse genome, predominantly located in centromeric and pericentric regions, which are crucial for chromosome segregation. While comprehensive assemblies of these regions have been established in the human genome, they are still lacking in the mouse genome. In this study, we used PacBio long-read sequencing, CUT&RUN sequencing, DNA methylation analysis, and RNA sequencing to generate genomic and epigenomic maps of these regions. We find that centromeric regions are primarily occupied by 120-mer Minor satellites, with other Minor Satellite length variants, 112-mers and 112-64-dimers, localized at centromere-pericentric junctions. Pericentromeric regions are mainly composed of homogeneous Major satellites, while pericentric-chromosomal junctions contain a higher density of divergent satellites. Additionally, the density of non-satellite repeats increases progressively from centromeres to pericentromeres, and further toward chromosomal arm junctions. We found that 120-mer Minor satellites in the core centromere are highly enriched with CENP-A, while the 112-mers and 112-64-dimers show lower CENP-A levels. Homogeneous Major satellites are more enriched with H3K9me3 heterochromatin, whereas divergent Major satellites are preferentially associated with H3K27me3. Furthermore, DNA methylation levels are lower in centromeres compared to pericentric regions. We also observed that only a small subset of satellites is transcribed into RNA, particularly regions exhibiting lower DNA methylation density. Our comprehensive assembly and characterization of the genomic and epigenomic landscape of mouse centromeric and pericentric regions have major implications for satellite biology and ongoing mouse telomere-to-telomere (T2T) assembly efforts.

genomics↗

bioRxiv2023

DNA satellite and chromatin organization at house mouse centromeres and pericentromeres

Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of sequencing and assembling repetitive genomic regions. Using recently available PacBio long-read sequencing data from the C57BL/6 strain and chromatin profiling, we found that contrary to the previous reports of their highly homogeneous nature, centromeric and pericentromeric satellites display varied sequences and organization. We find that both centromeric minor satellites and pericentromeric major satellites exhibited sequence variations within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Additionally, we investigated CENP-A and H3K9me3 chromatin organization at centromeres and pericentromeres using Chromatin immunoprecipitation sequencing (ChIP-seq). We found that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence abundance and homogeneity at these regions. Furthermore, the transposable elements at centromeric regions are not part of functional centromeres as they lack CENP-A enrichment. Finally, we found that while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays lack phased organization. Interestingly, the homogeneous class of major satellites phase CENP-A and H3K27me3 nucleosomes as well, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. Overall, our findings reveal that house mouse centromeres and pericentromeres, which were previously thought to be highly homogenous, display significant diversity in satellite sequence, organization, and chromatin structure.

genomics↗

bioRxiv2023

CTCF-mediated insulation and chromatin environment modulate Car5b escape from X inactivation

BackgroundThe number and escape levels of genes that escape X chromosome inactivation (XCI) in female somatic cells vary among tissues and cell types, potentially contributing to specific sex differences. Here we investigate the role of CTCF, a master chromatin conformation regulator, in regulating escape from XCI. CTCF binding profiles and epigenetic features were systematically examined at constitutive and facultative escape genes using mouse allelic systems to distinguish the inactive X (Xi) and active X (Xa) chromosomes. ResultsWe found that escape genes are located inside domains flanked by convergent arrays of CTCF binding sites, consistent with the formation of loops. In addition, strong and divergent CTCF binding sites often located at the boundaries between escape genes and adjacent neighbors subject to XCI would help insulate domains. Facultative escapees show clear differences in CTCF binding dependent on their XCI status in specific cell types/tissues. Concordantly, deletion but not inversion of a CTCF binding site at the boundary between the facultative escape gene Car5b and its silent neighbor Siah1b resulted in loss of Car5b escape. Reduced CTCF binding and enrichment of a repressive mark over Car5b in cells with a boundary deletion indicated loss of looping and insulation. In mutant lines in which either the Xi-specific compact structure or its H3K27me3 enrichment was disrupted, escape genes showed an increase in gene expression and associated active marks, supporting the roles of the 3D Xi structure and heterochromatic marks in constraining levels of escape. ConclusionOur findings indicate that escape from XCI is modulated both by looping and insulation of chromatin via convergent arrays of CTCF binding sites and by compaction and epigenetic features of the surrounding heterochromatin.

genomics↗

bioRxiv2022

Integration of synthetic microbial consortia based bioprocessing with pyrolysis for efficient conversion of cellulose to valuables

Improved technologies are needed for sustainable conversion of cellulosic waste to valuable products. Here we demonstrate the successful integration of a synthetic microbial consortium (SynCONS) based consolidated bioprocessing with pyrolysis to produce commodity chemicals from cellulose. Promising microbial partners were rationally identified from 7626 organisms via comparative metabolic mapping which led to establishing two promising SynCONS with abilities to convert cellulose to ethanol and lactate in bioreactors. The partners in the two SynCONS were a) the mesophilic fungus Trichoderma reesei grown sequentially with the thermophilic bacterium Parageobacillus thermoglucosidasius NCIMB 11955 (TrPt) and b) a thermophilic bacterium Thermobifida fusca grown together with Parageobacillus thermoglucosidasius NCIMB 11955 (TfPt). TrPt sequential bioprocessing resulted in 39% (g/g) cellulose consumption with product yields up to 9.3% g/g (ethanol + lactate). The TfPt co-cultures demonstrated a cellulose consumption of 30% (g/g) and combined yields of ethanol and lactic acid up to 23.7% g/g of consumed cellulose. The total product yields were further enhanced (51% g/g cellulose) when commercially available cellulases were used in place of T. fusca. Furthermore, when the metabolically engineered ethanol-producing strain of P. thermoglucosidasius TM242 (TfPt242) was substituted in the thermophilic TfPt co-culture consortium, ethanol yields were substantially higher (32.7% g/g of consumed cellulose). Finally, subjecting the residual cellulose and microbial biomass to pyrolysis resulted in carbon material with physicochemical properties similar to commercially available activated carbon as analysed using Scanning Electron Microscopy, X-Ray Diffraction and Raman spectroscopy. Overall, the integration of this synthetic microbial consortia-based bioprocessing strategy with pyrolysis demonstrated a promising strategy for conversion of waste cellulose to chemicals, biofuels, and industrial carbon potentially suitable for several industrial applications.

bioengineering↗

bioRxiv2021

Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries

Vertebrate genomes are partitioned into Topologically Associating Domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at TAD boundaries correlates with better insulation, however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer:promoter interactions and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters we show that active TAD enhancers but not promoters induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

genomics↗

bioRxiv2020

Kinetic isotope tracing of glycerol and de novo proteogenic amino acids in Human Lung Carcinoma cells using glucose

13C based Isotopic tracers of the media components can be used to kinetically track their contribution in the cell systems. A tracer ([U-13C6] glucose) was used to monitor its contribution into the central carbon metabolic pathways of human lung carcinoma (A549) cells by Gas chromatography-mass spectrometry (GC-MS) based mass isotopomer analysis. Calculated average 13C of methanolic extracts (glycerol: 5.46{+/-}3.53 % and lactate: 74.4{+/-}2.65 %), protein acid hydrolysates (serine: 4.51{+/-}0.21 %, glycine: 2.44{+/-}0.31 %, alanine: 24.56{+/-}0.59 %, glutamate: 8.81{+/-}0.85 %, proline: 6.96{+/-}0.53 % and aspartate: 10.72{+/-}0.95 %) and the culture filtrate (glycerol: 43.14{+/-}1.45 % and lactate: 81.67{+/-}0.91 %), showed significant contribution of 13C glucose. We observed the Warburg effect with higher levels of 13C lactate in the culture filtrate. 13C glycerol levels in culture supernatant showed significant increase with time and amino acids of glucogenic origin also contributed cellular protein biomass. The workflow adopted in this study for 13C analysis could be useful for the metabolic phenotyping of other mammalian cell systems under normal and perturbed (cancer and infection) conditions.

biochemistry↗

bioRxiv2019

Architectural RNA is required for heterochromatin organization

In addition to its known roles in protein synthesis and enzyme catalysis, RNA has been proposed to stabilize higher-order chromatin structure. To distinguish presumed architectural roles of RNA from other functions, we applied a ribonuclease digestion strategy to our CUT&RUN in situ chromatin profiling method (CUT&RUN.RNase). We find that depletion of RNA compromises association of the murine nucleolar protein Nucleophosmin with pericentric heterochromatin and alters the chromatin environment of CCCTC-binding factor (CTCF) bound regions. Strikingly, we find that RNA maintains the integrity of both constitutive (H3K9me3 marked) and facultative (H3K27me3 marked) heterochromatic regions as compact domains, but only moderately stabilizes euchromatin. To establish the specificity of heterochromatin stabilization by RNA, we performed CUT&RUN on cells deleted for the Firre long non-coding RNA and observed disruption of H3K27me3 domains on several chromosomes. We conclude that RNA maintains local and global chromatin organization by acting as a structural scaffold for heterochromatic domains.

genomics↗

bioRxiv2019

Trans- and cis-acting effects of the lncRNA Firre on epigenetic and structural features of the inactive X chromosome

Firre encodes a lncRNA involved in nuclear organization in mammals. Here we find that Firre RNA is transcribed from the active X chromosome (Xa) and exerts trans-acting effects on the inactive X chromosome (Xi). Allelic deletion of Firre on the Xa in a mouse hybrid fibroblast cell line results in a dramatic loss of the histone modification H3K27me3 and of components of the PRC2 complex on the Xi as well as the disruption of the perinucleolar location of the Xi. These features are measurably rescued by ectopic expression of a mouse or human Firre/FIRRE cDNA transgene, strongly supporting a conserved trans-acting role of the Firre transcript in maintaining the Xi heterochromatin environment. Surprisingly, CTCF occupancy is decreased on the Xi upon loss of Firre RNA, but is partially recovered by ectopic transgene expression, suggesting a functional link between Firre RNA and CTCF in maintenance of epigenetic features and/or location of the Xi. Loss of Firre RNA results in dysregulation of genes implicated in cell division and development, but not in reactivation of genes on the Xi, which retains its bipartite structure despite some changes in chromatin contact distribution. Allelic deletion or inversion of Firre on the Xi causes localized redistribution of chromatin contacts, apparently dependent on the orientation of CTCF binding sites clustered at the locus. Thus, the Firre locus and its RNA have roles in the maintenance of epigenetic features and structure of the Xi.

genomics↗