Biomedical subjects

Shyam Prabhakar

Publications and source records attributed to Shyam Prabhakar.

13 recordsLinked to original sources

PubMed2025

Intrinsic changes in cell differentiation and identity drive impaired wound healing in aged female murine skin.

Cellular and molecular mechanisms that drive a perturbed wound microenvironment and impaired healing in aged skin have not been fully delineated. To obtain a comprehensive understanding of cell-intrinsic changes acquired during ageing that impact early responses to injury, we performed single-cell RNA sequencing in young and aged intact female murine skin and wounds 3 days post-injury. We observed that substantial changes in the mean proportional distribution and transcriptomic state of skin resident subpopulations in aged, but not young, tissues accompany a global increase in basal inflammation. This is driven by an altered signalling environment leading to impaired keratinocyte differentiation, loss of fibroblast identity and defective macrophage function. Further, we show that ageing-induced changes in skin resident cells persist after injury, resulting in increased expression of senescence-related genes in wound fibroblasts and aberrant monocyte-to-macrophage transitioning coupled to an enhanced inflammatory signature and defective intercellular signalling in comparison to wounds in young mice. In summary, our data highlights a contribution of both cell-intrinsic changes and an altered tissue microenvironment to poor wound healing responses in aged mice.

Animals↗

PubMed2025

Cell-type-specific DNA methylation dynamics in the prenatal and postnatal human cortex.

The human cortex undergoes extensive epigenetic remodeling during development, although the precise temporal and cell-type-specific dynamics of DNA methylation remain incompletely understood. In this study, we profiled genome-wide DNA methylation across human cortex tissue from donors aged 6 post-conception weeks to 108 years of age. We observed widespread, developmentally regulated changes in DNA methylation, with pronounced shifts occurring during early- and mid-gestation that were distinct from age-associated modifications in the postnatal cortex. Using fluorescence-activated nuclei sorting, we optimized a protocol for the isolation of SATB2-positive neuronal nuclei, enabling the identification of cell-type-specific DNA methylation trajectories in the developing cortex. Developmentally dynamic DNA methylation sites were significantly enriched near genes implicated in autism and schizophrenia, supporting a role for epigenetic dysregulation in neurodevelopmental conditions. Our findings underscore the prenatal period as a critical window of epigenomic plasticity in the brain with important implications for understanding the genetic basis of neurodevelopmental phenotypes.

Humans↗

PubMed2024

Quantification of escape from X chromosome inactivation with single-cell omics data reveals heterogeneity across cell types and tissues.

Several X-linked genes escape from X chromosome inactivation (XCI), while differences in escape across cell types and tissues are still poorly characterized. Here, we developed scLinaX for directly quantifying relative gene expression from the inactivated X chromosome with droplet-based single-cell RNA sequencing (scRNA-seq) data. The scLinaX and differentially expressed gene analyses with large-scale blood scRNA-seq datasets consistently identified the stronger escape in lymphocytes than in myeloid cells. An extension of scLinaX to a 10x multiome dataset (scLinaX-multi) suggested a stronger escape in lymphocytes than in myeloid cells at the chromatin-accessibility level. The scLinaX analysis of human multiple-organ scRNA-seq datasets also identified the relatively strong degree of escape from XCI in lymphoid tissues and lymphocytes. Finally, effect size comparisons of genome-wide association studies between sexes suggested the underlying impact of escape on the genotype-phenotype association. Overall, scLinaX and the quantified escape catalog identified the heterogeneity of escape across cell types and tissues.

X Chromosome Inactivation↗

PubMed2007

Detection of weakly conserved ancestral mammalian regulatory sequences by primate comparisons.

BACKGROUND: Genomic comparisons between human and distant, non-primate mammals are commonly used to identify cis-regulatory elements based on constrained sequence evolution. However, these methods fail to detect functional elements that are too weakly conserved among mammals to distinguish them from non-functional DNA. RESULTS: To evaluate a strategy for large scale genome annotation that is complementary to the commonly used distal species comparisons, we explored the potential of deep intra-primate sequence comparisons. We sequenced the orthologs of 558 kb of human genomic sequence, covering multiple loci involved in cholesterol homeostasis, in 6 non-human primates. Our analysis identified six non-coding DNA elements displaying significant conservation among primates but undetectable in more distant comparisons. In vitro and in vivo tests revealed that at least three of these six elements have regulatory function. Notably, the mouse orthologs of these three functional human sequences had regulatory activity despite their lack of significant sequence conservation, indicating that they are ancestral mammalian cis-regulatory elements. These regulatory elements could be detected even in a smaller set of three primate species including human, rhesus and marmoset. CONCLUSION: We have demonstrated that intra-primate sequence comparisons can be used to identify functional modules in large genomic regions, including cis-regulatory elements that are not detectable through comparison with non-mammalian genomes. With the available human and rhesus genomes and that of marmoset, which is being actively sequenced, this strategy can be extended to the whole genome in the near future.

Animals↗

PubMed2006

In vivo enhancer analysis of human conserved non-coding sequences.

Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultraconserved in human-mouse-rat. We tested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all approximately 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.

Animals↗

PubMed2006

Accelerated evolution of conserved noncoding sequences in humans.

Changes in gene regulation likely influenced the profound phenotypic divergence of humans from other mammals, but the extent of adaptive substitution in human regulatory sequences remains unknown. We identified 992 conserved noncoding sequences (CNSs) with a significant excess of human-specific substitutions. These accelerated elements were disproportionately found near genes involved in neuronal cell adhesion. To assess the uniqueness of human noncoding evolution, we examined CNSs accelerated in chimpanzee and mouse. Although we observed a similar enrichment near neuronal adhesion genes in chimpanzee, the accelerated CNSs themselves exhibited almost no overlap with those in human, suggesting independent evolution toward different neuronal phenotypes in each species. CNSs accelerated in mouse showed no bias toward neuronal cell adhesion. Our results indicate that widespread cis-regulatory changes in human evolution may have contributed to uniquely human features of brain development and function.

Animals↗

PubMed2006

Primate-specific evolution of an LDLR enhancer.

BACKGROUND: Sequence changes in regulatory regions have often been invoked to explain phenotypic divergence among species, but molecular examples of this have been difficult to obtain. RESULTS: In this study we identified an anthropoid primate-specific sequence element that contributed to the regulatory evolution of the low-density lipoprotein receptor. Using a combination of close and distant species genomic sequence comparisons coupled with in vivo and in vitro studies, we found that a functional cholesterol-sensing sequence motif arose and was fixed within a pre-existing enhancer in the common ancestor of anthropoid primates. CONCLUSION: Our study demonstrates one molecular mechanism by which ancestral mammalian regulatory elements can evolve to perform new functions in the primate lineage leading to human.

Amino Acid Motifs↗

PubMed2006

Close sequence comparisons are sufficient to identify human cis-regulatory elements.

Cross-species DNA sequence comparison is the primary method used to identify functional noncoding elements in human and other large genomes. However, little is known about the relative merits of evolutionarily close and distant sequence comparisons. To address this problem, we identified evolutionarily conserved noncoding regions in primate, mammalian, and more distant comparisons using a uniform approach (Gumby) that facilitates unbiased assessment of the impact of evolutionary distance on predictive power. We benchmarked computational predictions against previously identified cis-regulatory elements at diverse genomic loci and also tested numerous extremely conserved human-rodent sequences for transcriptional enhancer activity using an in vivo enhancer assay in transgenic mice. Human regulatory elements were identified with acceptable sensitivity (53%-80%) and true-positive rate (27%-67%) by comparison with one to five other eutherian mammals or six other simian primates. More distant comparisons (marsupial, avian, amphibian, and fish) failed to identify many of the empirically defined functional noncoding elements. Our results highlight the practical utility of close sequence comparisons, and the loss of sensitivity entailed by more distant comparisons. We derived an intuitive relationship between ancient and recent noncoding sequence conservation from whole-genome comparative analysis that explains most of the observations from empirical benchmarking. Lastly, we determined that, in addition to strength of conservation, genomic location and/or density of surrounding conserved elements must also be considered in selecting candidate enhancers for in vivo testing at embryonic time points.

Animals↗

PubMed2005

Mapping cis-regulatory domains in the human genome using multi-species conservation of synteny.

Our inability to associate distant regulatory elements with the genes they regulate has largely precluded their examination for sequence alterations contributing to human disease. One major obstacle is the large genomic space surrounding targeted genes in which such elements could potentially reside. In order to delineate gene regulatory boundaries, we used whole-genome human-mouse-chicken (HMC) and human-mouse-frog (HMF) multiple alignments to compile conserved blocks of synteny (CBSs), under the hypothesis that these blocks have been kept intact throughout evolution at least in part by the requirement of regulatory elements to stay linked to the genes they regulate. A total of 2116 and 1942 CBSs >200 kb were assembled for HMC and HMF, respectively, encompassing 1.53 and 0.86 Gb of human sequence. To support the existence of complex long-range regulatory domains within these CBSs, we analyzed the prevalence and distribution of chromosomal aberrations leading to position effects (disruption of a gene's regulatory environment), observing a clear bias not only for mapping onto CBS but also for longer CBS size. Our results provide an extensive data set characterizing the regulatory domains of genes and the conserved regulatory elements within them.

Animals↗

PubMed2005

Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences.

An important step toward improving the annotation of the human genome is to identify cis-acting regulatory elements from primary DNA sequence. One approach is to compare sequences from multiple, divergent species. This approach distinguishes multispecies conserved sequences (MCS) in noncoding regions from more rapidly evolving neutral DNA. Here, we have analyzed a region of approximately 238kb containing the human alpha globin cluster that was sequenced and/or annotated across the syntenic region in 22 species spanning 500 million years of evolution. Using a variety of bioinformatic approaches and correlating the results with many aspects of chromosome structure and function in this region, we were able to identify and evaluate the importance of 24 individual MCSs. This approach sensitively and accurately identified previously characterized regulatory elements but also discovered unidentified promoters, exons, splicing, and transcriptional regulatory elements. Together, these studies demonstrate an integrated approach by which to identify, subclassify, and predict the potential importance of MCSs.

Animals↗

PubMed2005

Gene expression patterns define key transcriptional events in cell-cycle regulation by cAMP and protein kinase A.

Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), a PKA-selective cAMP analog, alters the expression of approximately 4,500 of approximately 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPT-cAMP. Changes in mRNA and protein expression of several cell-cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2 h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.

Animals↗

PubMed2004

The sequence and analysis of duplication-rich human chromosome 16.

Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.

Animals↗

PubMed2004

The DNA sequence and comparative analysis of human chromosome 5.

Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.

Animals↗