Manorama C. John

Isolation of LUMINIDEPENDENS: A Gene Involved in the Control of Flowering Time in Arabidopsis

Plants have evolved the ability to regulate flowering in response to environmental signals such as temperature and photoperiod. The physiology and genetics of floral induction have been studied extensively, but the molecular mechanisms that underlie this process are poorly understood. To study this process, we isolated a gene, LUMINIDEPENDENS (LD), that is involved in the timing of flowering in Arabidopsis. Mutations in this gene render Arabidopsis late flowering and appear to affect light perception. The late-flowering phenotype of the ld mutation was partially suppressed by vernalization. Genomic and cDNA clones of the LD gene were characterized. The predicted amino acid sequence of the LD protein contains 953 residues and includes two putative bipartite nuclear localization signals and a glutamine-rich region.

Intact β-Adrenergic Response and Unmodified Progression Toward Heart Failure in Mice With Genetic Ablation of a Major Protein Kinase A Phosphorylation Site in the Cardiac Ryanodine Receptor

Increased phosphorylation of the cardiac ryanodine receptor (RyR)2 by protein kinase A (PKA) at the phosphoepitope encompassing Ser2808 has been advanced as a central mechanism in the pathogenesis of cardiac arrhythmias and heart failure. In this scheme, persistent activation of the sympathetic system during chronic stress leads to PKA “hyperphosphorylation” of RyR2-S2808, which increases Ca2+ release by augmenting the sensitivity of the RyR2 channel to diastolic Ca2+. This gain-of-function is postulated to occur with the unique participation of RyR2-S2808, and other potential PKA phosphorylation sites have been discarded. Although it is clear that RyR2 is among the first proteins in the heart to be phosphorylated by &bgr;-adrenergic stimulation, the functional impact of phosphorylation in excitation–contraction coupling and cardiac performance remains unclear. We used gene targeting to produce a mouse model with complete ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Whole-heart and isolated cardiomyocyte experiments were performed to test the role of &bgr;-adrenergic stimulation and PKA phosphorylation of Ser2808 in heart failure progression and cellular Ca2+ handling. We found that the RyR2-S2808A mutation does not alter the &bgr;-adrenergic response, leaves cellular function almost unchanged, and offers no significant protection in the maladaptive cardiac remodeling induced by chronic stress. Moreover, the RyR2-S2808A mutation appears to modify single-channel activity, although modestly and only at activating [Ca2+]. Taken together, these results reveal some of the most important effects of PKA phosphorylation of RyR2 but do not support a major role for RyR2-S2808 phosphorylation in the pathogenesis of cardiac dysfunction and failure.

Conditional forebrain deletion of the L-type calcium channel CaV1.2 disrupts remote spatial memories in mice

To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for remote memory consolidation, we generated conditional knockout mice in which the L-VGCC isoform Ca(V)1.2 was postnatally deleted in the hippocampus and cortex. In the Morris water maze, both Ca(V)1.2 conditional knockout mice (Ca(V)1.2(cKO)) and control littermates displayed a marked decrease in escape latencies and performed equally well on probe trials administered during training. In distinct contrast to their performance during training, Ca(V)1.2(cKO) mice exhibited significant impairments in spatial memory when examined 30 d after training, suggesting that Ca(V)1.2 plays a critical role in consolidation of remote spatial memories.

Rapid induction of genomic demethylation and T-DNA gene expression in plant cells by 5-azacytosine derivatives

We have optimized conditions for demethylation of the genome and induction of a silent, hypermethylated T-DNA gene (ipt) by 5-azacytosine (5-azaCyt) derivatives in a suspension culture of tobacco cells. In this system, 5-azacytidine (5-azaC) is more effective in causing genomic demethylation and ipt gene induction than 5-azaCyt or 5-azadeoxycytidine (5-azadC). A single treatment with 2.5 μM 5-azaC resulted in a maximal level of ipt gene induction without inhibiting cell growth. However, we could not reduce the level of genomic methylation below approximately 2/3 of that found in untreated controls, even after extensive 5-azaC treatment. Furthermore, remethylation of the genome occurred after removal of 5-azaC. The use of 5-azaC as an inducer of silent plant genes is discussed, along with differences in the response of plant and animal genomes to demethylating agents.

The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation

Genome-wide association studies have revealed that many low-penetrance breast cancer susceptibility loci are located in non-protein coding genomic regions; however, few have been characterized. In a comparative genetics approach to model such loci in a rat breast cancer model, we previously identified the mammary carcinoma susceptibility locus Mcs1a. We now localize Mcs1a to a critical interval (277 Kb) within a gene desert. Mcs1a reduces mammary carcinoma multiplicity by 50% and acts in a mammary cell-autonomous manner. We developed a megadeletion mouse model, which lacks 535 Kb of sequence containing the Mcs1a ortholog. Global gene expression analysis by RNA-seq revealed that in the mouse mammary gland, the orphan nuclear receptor gene Nr2f1/Coup-tf1 is regulated by Mcs1a. In resistant Mcs1a congenic rats, as compared with susceptible congenic control rats, we found Nr2f1 transcript levels to be elevated in mammary gland, epithelial cells, and carcinoma samples. Chromatin looping over ∼820 Kb of sequence from the Nr2f1 promoter to a strongly conserved element within the Mcs1a critical interval was identified. This element contains a 14 bp indel polymorphism that affects a human-rat-mouse conserved COUP-TF binding motif and is a functional Mcs1a candidate. In both the rat and mouse models, higher Nr2f1 transcript levels are associated with higher abundance of luminal mammary epithelial cells. In both the mouse mammary gland and a human breast cancer global gene expression data set, we found Nr2f1 transcript levels to be strongly anti-correlated to a gene cluster enriched in cell cycle-related genes. We queried 12 large publicly available human breast cancer gene expression studies and found that the median NR2F1 transcript level is consistently lower in ‘triple-negative’ (ER-PR-HER2-) breast cancers as compared with ‘receptor-positive’ breast cancers. Our data suggest that the non-protein coding locus Mcs1a regulates Nr2f1, which is a candidate modifier of differentiation, proliferation, and mammary cancer risk.

Gene Targeting in the Mouse

Establishment of methods to inactivate genes by homologous recombination in embryonic stem (ES) cells has provided great advantages to the field of mouse genetics. Using this technology, a number of null mutant mice, so-called knock-out mice, have been generated. The gene-targeting technology offers a strong tool that allows us to understand the function of a particular gene of interest in the whole animal and has contributed to studies in a wide variety of biological research areas. More recently, the original knock-out technology has been further refined to develop advanced strategies to generate conditional knock-out and knock-in mice. In this chapter, an overview of gene-targeting strategies is presented and procedures to generate these genetically engineered mice are discussed.

Irx4 Marks a Multipotent, Ventricular‐Specific Progenitor Cell

While much progress has been made in the resolution of the cellular hierarchy underlying cardiogenesis, our understanding of chamber‐specific myocardium differentiation remains incomplete. To better understand ventricular myocardium differentiation, we targeted the ventricle‐specific gene, Irx4, in mouse embryonic stem cells to generate a reporter cell line. Using an antibiotic‐selection approach, we purified Irx4+ cells in vitro from differentiating embryoid bodies. The isolated Irx4+ cells proved to be highly proliferative and presented Cxcr4, Pdgfr‐alpha, Flk1, and Flt1 on the cell surface. Single Irx4+ ventricular progenitor cells (VPCs) exhibited cardiovascular potency, generating endothelial cells, smooth muscle cells, and ventricular myocytes in vitro. The ventricular specificity of the Irx4+ population was further demonstrated in vivo as VPCs injected into the cardiac crescent subsequently produced Mlc2v+ myocytes that exclusively contributed to the nascent ventricle at E9.5. These findings support the existence of a newly identified ventricular myocardial progenitor. This is the first report of a multipotent cardiac progenitor that contributes progeny specific to the ventricular myocardium. Stem Cells 2016;34:2875–2888

Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer.

PKCε is a transforming oncogene and a predictive biomarker of various human cancers. However, a precise in vivo link of PKCε to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCε knockout mice (PKCε-CKO) using cre-lox technology. Homozygous PKCεLoxP/LoxP mice have normal body weight and phenotype. To determine what effect loss of PKCε would have on the prostate, the PKCεLoxP/LoxP mice were bred to probasin cre (PB-Cre4+) mice which express cre specifically in the prostate epithelium of postnatal mice. Western blot and immunohistochemical analyses showed reduced levels of PKCε specifically in the prostate of PKCε-CKO mice. Histopathological analyses of prostate from both PKCεLoxP/LoxP and prostate PKCε-CKO mice showed normal pathology. To determine the functional impact of prostate specific deletion of PKCε on prostate tumor growth, we performed an orthotopic xenograft study. Transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMPC1, 2×106) were implanted in the prostate of PKCε-CKO mice. Mice were sacrificed at 6th week post-implantation. Results demonstrated a significant (P<0.05) decrease in the growth of TRAMPC1 cells-derived xenograft tumors in PKCε-CKO mice compared to wild type. To determine a link of PKCε to ultraviolet radiation (UVR) exposure-induced epidermal Stat3 phosphorylation, PKCεLoxP/LoxP mice were bred to tamoxifen-inducible K14 Cre mice. PKCε deletion in the epidermis resulted in inhibition of UVR-induced Stat3 phosphorylation. In summary, our novel PKCεLoxP/LoxP mice will be useful for defining the link of PKCε to various cancers in specific organ, tissue, or cells.

Abstract LB-137: Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer

Evidence from our laboratory and others indicate that PKCe is a transforming oncogene and a predictive biomarker of various human cancers including prostate, breast, head and neck, lungs, brain, bladder and cutaneous squamous cell carcinoma (SCC). However, a precise in vivo link of PKCe and its downstream signaling components to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCe knockout mice using cre-lox technology. To do so, we generated a targeting vector in which Exon 4 of the PKCe gene was flanked by LoxP sites. This vector was used to generate mice carrying two floxed alleles of PKCe (PKCeLoxP/LoxP mice) by standard gene knockout methodology. Homozygous PKCeLoxP/LoxP mice have normal body weight and phenotype. To determine what effect loss of PKCe would have on the prostrate, the PKCeLoxP/LoxP mice were bred to prostate specific cre (PB-Cre4+). Western blot and immunohistochemical analyses showed inhibition of PKCe protein level in the prostate of PKCe-KO mice. However, no change in the PKCe protein level was observed in the spleen, liver and lungs of PKCe-KO mice. Also, PKCe deletion in prostate did not affect the levels of other PKC isoforms (PKCα, PKCβII, and PKCς). No significant difference was observed in the prostate weight of PKCeLoxP/LoxP and PKCe-KO mice. Histopathological analyses of prostate from both PKCeLoxP/LoxP and prostate PKCe-KO mice showed normal pathology in the PKCe-KO prostate. To determine the functional impact of prostate specific deletion of PKCe on prostate tumor growth, we performed an orthotopic xenograft study. In this experiment, TRAMP mouse tumor cells (TRAMPC1, 2×106) were implanted in the prostate. Mice were sacrificed at 6 weeks post-implantation. Results demonstrated a significant (P Citation Format: Bilal B. Hafeez, Louise Meske, Anupama Singh, Ashok Singh, Weixiong Zhong, Patricia Powers, Manorama John, Anne Griep, Ajit Verma. Tissue-specific conditional PKCe knockout mice: a model to precisely reveal PKCe functional role in initiation, promotion and progression of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-137. doi:10.1158/1538-7445.AM2015-LB-137