Locus-Specific Isolation of the Nanog Chromatin Identifies Regulators Relevant to Pluripotency of Mouse Embryonic Stem Cells and Reprogramming of Somatic Cells.

Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the Nanog promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells. Gene ontology analysis revealed that the identified proteins, including many RNA-binding proteins (RBPs), are enriched in RNA-related functions and gene expression. ChIP-qPCR experiments confirmed that BCLAF1, FUBP1, MSH6, PARK7, PSIP1, and THRAP3 occupy the Nanog promoter region in mESCs. Knockdown experiments of these factors show that they play varying roles in self-renewal, pluripotency gene expression, and differentiation of mESCs as well as in the reprogramming of somatic cells. Our results show the utility of unbiased identification of chromatin-associated proteins on a pluripotency gene in mESCs and reveal the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.

Polymorphisms in pattern recognition receptor genes of indigenous and White Leghorn breeds of chicken.

Functional polymorphisms in pattern recognition receptors (PRRs) modulate innate immunity and play a crucial role in resistance or susceptibility to diseases. The present study was carried out to explore polymorphic patterns in the coding sequences of PRR genes TLR3, TLR1LA (TLRs), MDA5, LGP2 (RLRs) and NOD1 (NLR) in chicken breeds of India, namely Ghagus (GH), Nicobari (NB) and the exotic White Leghorn (WLH) breed. Out of 209 SNPs observed in five genes among three breeds, 117 were synonymous (Syn) and 92 were non-synonymous (NS) SNPs. In TLR genes the highest polymorphism was observed in NB (16, 28) compared to GH (14, 16) and WLH (13, 19) breeds. In the MDA5 gene the highest polymorphism was observed in GH (12) compared to NB (eight) and WLH (four) breeds. However, an almost similar level of polymorphism was observed in the LGP2 gene among the three breeds. In the NOD1 gene, the highest polymorphism was observed in NB (27), followed by WLH (11) and GH (10) breeds. The overall highest number of SNPs was observed in NB (90), followed by GH (62) and the WLH (57) breed. With regard to variation in polymorphism among different classes of PRRs, the study revealed the highest polymorphism in TLRs compared to NOD1 and the RLR class of PRRs. Further, the domain locations of various Syn and NS SNPs in each PRR among the three breeds were identified. In silico analysis of NS SNPs revealed that most of them had a neutral effect on protein function. However, two each in TLR1LA and LGP2 and one in the MDA5 gene were predicted to be deleterious to protein function. The present study unravelled extensive polymorphism in the coding sequences of the TLR and NLR class of PRR genes, and the polymorphism was higher in indigenous chicken breeds.

Pattern recognition receptor genes expression profiling in indigenous chickens of India and White Leghorn.

Pattern recognition receptors (PRR) such as Toll-like receptors, NOD-like receptors, RIG-I helicase receptors, and C-type lectin receptors play a critical role in innate immunity as a first line of defense against invading pathogens through recognition of pathogen and/or damage-associated molecular patterns. Genetic makeup of birds is known to play a role in resistance or susceptibility to various infectious diseases. Therefore, the present study was carried out to elucidate the differential expression of PRR and some of the cytokine genes in peripheral blood mononuclear cells of indigenous chicken breeds such as Ghagus and Nicobari and an exotic chicken breed, White Leghorn (WLH). The stability of expression of reference genes in peripheral blood mononuclear cells of 3 breeds was first determined using NormFinder and BestKeeper programs. NormFinder determined B2M and G6PDH reference genes as the best combination with stability value of 0.38. Out of total 14 genes studied, expression of ten genes was found to be significantly different among 3 breeds after normalization with these reference genes. Ghagus breed showed higher level of expression of TLR1LB, TLR7, NOD1, NOD5, B-Lec, IFNß, IL1ß, and IL8 genes when compared to Nicobari breed. Further, Ghagus showed higher expression of TLR1LB, MDA5, LGP2, B-Lec, IL1ß, and IL8 genes as compared to WLH breed. Higher expression of LGP2 and MDA5 genes was observed in Nicobari compared to the WLH breed while higher expression of TLR7, NOD1, NOD5, and IFNß genes was observed in WLH as compared to Nicobari breed. No difference was observed in the expression of TLR1LA, TLR3, B-NK, and IFNα genes among 3 breeds. Study revealed significant breed effect in expression profile of PRR and some of the cytokine genes and Ghagus breed seems to have better expression profile of these genes linked to the innate immunity when compared to the WLH and Nicobar breeds.