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Isolation of copy number variations and chromosomal duplications at high frequency in the laboratory suggested that Caenorhabditis elegans tolerates increased gene dosage. Here, we addressed if a general dosage compensation mechanism acts at the level of mRNA expression in C. elegans. We characterized gene dosage and mRNA expression in 3 chromosomal duplications and a fosmid integration strain using DNA-seq and mRNA-seq. Our results show that on average, increased gene dosage leads to increased mRNA expression, pointing to a lack of genome-wide dosage compensation. Different genes within the same chromosomal duplication show variable levels of mRNA increase, suggesting feedback regulation of individual genes. Somatic dosage compensation and germline repression reduce the level of mRNA increase from X chromosomal duplications. Together, our results show a lack of genome-wide dosage compensation mechanism acting at the mRNA level in C. elegans and highlight the role of epigenetic and individual gene regulation contributing to the varied consequences of increased gene dosage., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Progression of a cell along a differentiation path is characterized by changes in gene expression profiles. Alterations of these transcriptional programs result from cell type-specific transcription factors that act in a dynamic chromatin environment. Understanding the precise contribution of these molecular factors during the differentiation process requires accessing specific cell types within a developing organ. This chapter describes a streamlined and alternative version of INTACT, a method enabling the isolation of specific cell populations by affinity-purification of tagged nuclei and the subsequent analysis of gene expression, transcription factor binding profiles, as well as chromatin state at a genome-wide scale. In particular, modifications of the nuclei isolation, capture, and purification procedures are proposed that improve time scale, yield, and purity. In addition, the combination of different tags enables the analysis of distinct cell populations from a single transgenic line and the subtractive purification of subpopulations of cells, including those for which no specific promoter is available. Finally, we describe a chromatin immunoprecipitation protocol that has been successfully used to profile histone modifications and other chromatin-associated proteins such as RNA Polymerase II in different cell populations of the Arabidopsis root, including the quiescent center of the stem cell niche.

Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)