ABSTRACT
The plant cortical microtubule array is a dynamic structure that confers cell shape and enables plants to alter their growth and development in response to internal and external cues. Cells use a variety of microtubule regulatory proteins to spatially and temporally modulate the intrinsic polymerization dynamics of cortical microtubules to arrange them into specific configurations and to reshape arrays to adapt to changing conditions. To obtain mechanistic insight into how particular microtubule regulatory proteins mediate the dynamic (re)structuring of cortical microtubule arrays, we need to measure their effect on the dynamics of cortical microtubules. In this chapter, we describe new ImageJ plugins to generate kymographs from time-lapse images and to analyze them to measure the parameters that quantitatively describe cortical microtubule dynamics.
Subject(s)
Kymography/methods , Microtubules/metabolism , Plants/metabolism , Polymerization , User-Computer InterfaceABSTRACT
Prenatal stress defines long-term phenotypes through epigenetic programming of the offspring. These effects are potentially mediated by glucocorticoid release and by sex. We hypothesized that the glucocorticoid receptor (Gr, Nr3c1) fashions the DNA methylation profile of offspring. Consistent with this hypothesis, fetal Nr3c1 heterozygosity leads to altered DNA methylation landscape in fetal placenta in a sex-specific manner. There was a significant overlap of differentially methylated genes in fetal placenta and adult frontal cortex in Nr3c1 heterozygotes. Phenotypically, Nr3c1 heterozygotes show significantly more anxiety-like behavior than wildtype. DNA methylation status of fetal placental tissue is significantly correlated with anxiety-like behavior of the same animals in adulthood. Thus, placental DNA methylation might predict behavioral phenotypes in adulthood. Our data supports the hypothesis that Nr3c1 influences DNA methylation at birth and that DNA methylation in placenta correlates with adult frontal cortex DNA methylation and anxiety-like phenotypes.
Subject(s)
Anxiety Disorders/genetics , Behavior, Animal , DNA Methylation , Placenta , Receptors, Glucocorticoid/deficiency , Sex Factors , Animals , CpG Islands , Disease Models, Animal , Epigenesis, Genetic , Female , Fetus , Male , Mice , Mice, Knockout , Pregnancy , Prenatal Exposure Delayed Effects/geneticsABSTRACT
Background: The idea that changes to the host immune system are critical for cancer progression was proposed a century ago and recently regained experimental support. Results: Herein, the hypothesis that hepatocellular carcinoma (HCC) leaves a molecular signature in the host peripheral immune system was tested by profiling DNA methylation in peripheral blood mononuclear cells (PBMC) and T cells from a discovery cohort (n = 69) of healthy controls, chronic hepatitis, and HCC using Illumina 450K platform and was validated in two validation sets (n = 80 and n = 48) using pyrosequencing. Conclusions: The study reveals a broad signature of hepatocellular carcinoma in PBMC and T cells DNA methylation which discriminates early HCC stage from chronic hepatitis B and C and healthy controls, intensifies with progression of HCC, and is highly enriched in immune function-related genes such as PD-1, a current cancer immunotherapy target. These data also support the feasibility of using these profiles for early detection of HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , DNA Methylation , Epigenomics/methods , Hepatitis, Chronic/genetics , Liver Neoplasms/genetics , Adult , Aged , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/immunology , Case-Control Studies , Disease Progression , Early Detection of Cancer , Female , Hepatitis, Chronic/immunology , Humans , Leukocytes, Mononuclear/chemistry , Liver Neoplasms/diagnosis , Liver Neoplasms/immunology , Male , Middle Aged , Neoplasm Staging , Programmed Cell Death 1 Receptor/genetics , Sequence Analysis, DNA , T-Lymphocytes/chemistryABSTRACT
AIM: DNA methylation downregulates transcription. However, a large number of genes, which are unmethylated in the promoter region, are inactive. We tested the hypothesis that these genes are regulated by DNA methylation of upstream regulators. METHODS: We inhibited DNMT1 with 5-aza-2'-deoxycytidine or depleted it with shRNA to map the transcription initiation positions controlled by DNMT1 using ChIPseq with RNApolIIser5 antibody. Ingenuity pathway analysis identified potential methylated upstream regulators. Their functional role in controlling unmethylated promoters was determined by CRISPR/Cas9 gene editing. RESULTS: We show that a large group of unmethylated promoters is regulated by DNMT1 through DNA methylation dependent silencing of upstream regulators such as transcription factor HNF4A. CONCLUSION: The landscape of genes regulated by DNA methylation is more wide-ranging than genes downregulated by methylation of their own cis-regulatory sequences; regulation of unmethylated promoters is dependent on the methylation state of upstream trans regulators.