miRNA-mRNA integrative expression mapping during mouse embryonic stem cell to Neuron progenitor differentiation

Genome-wide transcriptome profiling of mammalian cells has given more insight into complexity of RNA world. Vast majority of RNAs are falling under non-coding category, among which ~22 nucleotides RNA is known as microRNA. It regulates gene expression by repressing translationally or cleaving mRNAs. 6-8 nucleotides region at 5’ end of miRNAs bind to 3’ UTR region of mRNA called ‘seed region’. This binding is either completely or incompletely complementary. Complete complementarity associates with RNA Induced Silencing Complex (RISC) that able to degrade mRNA with the help of Argonaute protein (Ago). But, incomplete complementarity is believed to be in a type of poised state that can be removed during requirement of that particular protein synthesis. mRNA translational repression by miRNA leads to subsequent mRNA destabilization. These dynamics of miRNA and mRNA integrative expression was further studied in embryonic stem cell to Neuron Progenitor differentiation system. Upon retinoic acid treatment to mouse embryonic stem cell line (R1), without LIF and 2i inhibitors that maintain undifferentiating state in ESC, cells were differentiated to neuronal cell lineage. To study, the dynamic changes in expression during differentiation we performed both microRNA and mRNA sequencing separately in these two states with biologically duplicated samples. Interestingly, we came up with 82 differentially regulated miRNAs, 9 lncRNAs and 4336 mRNA genes during this phage. Along with these 31 novel miRNAs were identified, among them 17 were identified that specific to ESC and 14 were in NP. Interaction maps of miRNA-mRNA was confirmed the down regulation of targeted mRNAs due to upregulated miRNAs during differentiation. KEGG pathways analysis of these genes showed downregulation of signaling pathways regulating pluripotency of stem cells (by miR-466m-3p, miR-466k, miR-1198 and miR344e) and upregulation of axon guidance and Neurotrophin signaling pathways that involves in differentiation of neuronal cells. These studies reveal genome wide miRNAs and mRNAs interaction map in ESC to NP differentiation. That will help in understanding of controlled gene expression patterns during neuronal differentiation due to miRNAs and lncRNAs parallel expressions

Involvement of androgen receptor gene in male gonad differentiation in Indian garden lizard, Calotes versicolor

The Indian garden lizard, Calotes versicolor, shows neither cytologically distinguishable sex chromosomes nor temperature dependent sex determination. However, previous studies on the administration of androgens to embryos during early development have shown reversal towards male sex. We have cloned and sequenced a cDNA fragment of the androgen receptor (AR) gene (CvAR) and characterized its expression. CvAR shares much homology with AR gene of other vertebrates at the nucleotide and amino acid levels indicating their evolutionary conservation. Whole mount RNA in situ hybridization (WRISH) and semi-quantitative RT-PCR analyses of its expression in the genital ridge of individual embryos from different stages of development, demonstrate its initiation by day 10 which is later than CvSox9 but occurs dimorphically only in those embryonic gonads which express CvSox9, a male specific marker. It suggests that AR expression may be crucial in the male sex determination pathway in C. versicolor

Spatiotemporal Dynamics of 3D Genome Architecture and Gene Expression during Lactogenic Differentiation of Murine Mammary Epithelial Cells

Orchestration of differential gene expression program during cellular development and differentiation is tightly coordinated in each and every cell type. Cell-type specific gene expression is primarily regulated by the spatial arrangement of genome within the 3-dimensional space of the cell nucleus. Recent evidences suggest that interphase chromosome territories are non-randomly organized in a cell-type specific manner and their neighbourhood are interlinked with cell-type specific gene expression patterns. In this study we made an attempt to study chromosomal dynamics and gene expression in the context of mammary epithelial cells lactogenic differentiation under the influence of lactogenic hormones. We derived genome-wide chromosome conformation capture HiC based chromosome interactions and gene expression by RNA-seq in undifferentiated murine mammary epithelial cells and hydrocortisone and Prolactin hormone treated cells.  We found that chromosome territories are non-randomly organized in HC11 cells undifferentiated as well as differentiated cells-types. There seems to be increased and decreased interactions within and in between chromosomes upon differentiation. We derived A & B compartments within each chromosome and their relationship with gene expression. We also studied topologically associated domain reorganization during differentiation which indicates that majority of the TADs are highly conserved and minor or less TADs are highly reorganized during signalling. From these studies, we concluded that differentiation signals promote subtle shift of chromosomal territories neighbourhood but promotes extensive reorganization within or between chromosomes

RNA sequencing of murine mammary epithelial stem-like cells (HC11) undergoing lactogenic differentiation and its comparison with embryonic stem cells

Abstract Objectives Understanding of transcriptional networks specifying HC11 murine mammary epithelial stem cell-like cells (MEC) in comparison with embryonic stem cells (ESCs) and their rewiring, under the influence of glucocorticoids (GC) and prolactin (PRL) hormones, is critical for elucidating the mechanism of lactogenesis. In this data note, we provide RNA sequencing data from murine MECs and ESCs, MECs treated with steroid hormone alone and in combination with PRL. This data could help in understanding temporal dynamics of mRNA transcription that impact the process of lactogenesis associated with mammary gland development. Further integration of these data sets with existing datasets of cells derived from various stages of mammary gland development and different types of breast tumors, should pave the way for effective prognosis and to develop therapies for breast cancer. Data description We have generated RNA-sequencing data representing steady-state levels of mRNAs from murine ESCs, normal MECs (N), MECs primed (P) with hydrocortisone (HC) alone and in combination with PRL hormone by using Illumina sequencing platform. We have generated ~ 58 million reads for ESCs with an average length of ~ 100 nt and an average 115 million good quality mapped reads with an average length of ~ 150 nt for different stages of MECs differentiation

Inhibitors of Apoptosis Protein Antagonists (Smac Mimetic Compounds) Control Polarization of Macrophages during Microbial Challenge and Sterile Inflammatory Responses

Apoptosis is a physiological cell death process essential for development, tissue homeostasis, and for immune defense of multicellular animals. Inhibitors of apoptosis proteins (IAPs) regulate apoptosis in response to various cellular assaults. Using both genetic and pharmacological approaches we demonstrate here that the IAPs not only support opportunistic survival of intracellular human pathogens like Chlamydia pneumoniae but also control plasticity of iNOS+ M1 macrophage during the course of infection and render them refractory for immune stimulation. Treatment of Th1 primed macrophages with birinapant (IAP-specific antagonist) inhibited NO generation and relevant proteins involved in innate immune signaling. Accordingly, birinapant promoted hypoxia, angiogenesis, and tumor-induced M2 polarization of iNOS+ M1 macrophages. Interestingly, birinapant-driven changes in immune signaling were accompanied with changes in the expression of various proteins involved in the metabolism, and thus revealing the new role of IAPs in immune metabolic reprogramming in committed macrophages. Taken together, our study reveals the significance of IAP targeting approaches (Smac mimetic compounds) for the management of infectious and inflammatory diseases relying on macrophage plasticity