Showing papers on "Chromosome conformation capture published in 2003"

The Dynamics of Chromosome Organization and Gene Regulation

Abstract: ▪ Abstract With the sequence of the human genome now complete, studies must focus on how the genome is functionally organized within the confines of the cell nucleus and the dynamic interplay between the genome and its regulatory factors to effectively control gene expression and silencing. In this review I describe our current state of knowledge with regard to the organization of chromosomes within the nucleus and the positioning of active versus inactive genes. In addition, I discuss studies on the dynamics of chromosomes and specific genetic loci within living cells and its relationship to gene activity and the cell cycle. Furthermore, our current understanding of the distribution and dynamics of RNA polymerase II transcription factors is discussed in relation to chromosomal loci and other nuclear domains.

3C technology: analyzing the spatial organization of genomic loci in vivo.

Abstract: Publisher Summary Technology has provided new insight into how chromosomes and chromosomal regions are organized in the context of the nucleus. Further intricate structural organizations are to be expected at great level of resolution when enhancers or other transcriptional regulatory elements communicate with distant promoters located in cis. Two novel, independently developed assays have allowed insight concerning the spatial organization of such genomic loci in vivo . One assay, called RNA-TRAP that involves targeting of horseradish peroxidase (HRP) to nascent RNA transcripts, followed by quantization of HRP-catalyzed biotin deposition on chromatin nearby. The other technique is 3C technology (chromosome conformation capture) and involves quantitation of cross-linking frequencies between two DNA restriction fragments as a measure of their frequency of interaction in the nuclear space. 3C technology is applied to analyze the conformation of a 200-kb region spanning the mouse β-globin gene cluster in its active and inactive transcriptional state. This chapter discusses a detailed protocol for 3C analysis in mammalian cells, evaluates interpretation of results, and illustrates the advantages and disadvantages over RNA-

Globins in space

Abstract: textThe aim of this thesis was to gain a detailed understanding of transcriptional regulation in the context of the living cell. The s-globin locus was used as a model system to study the question: how distal cisregulatory DNA elements communicate to activate tissue- and developmental stage-specific gene expression. This question was addressed through two different approaches. The first approach was based on the observation that globin gene transcription alternates between embryonic, fetal and adult genes in a flip-flop mechanism and the assumption that direct contacts between the Locus Control Region (LCR) and gene promoters is required for transcription (looping model). To monitor these putative interactions in the living cell we distinctly tagged the human s-globin locus on both the LCR and the s-globin gene. In a second approach, we adopted Chromosome Conformation Capture (3C) technology to measure the spatial conformation of the s-globin loci in man and mouse in vivo. This demonstrated that spatial interactions between the LCR, actively transcribed genes and distal DNase I hypersensitive regions occur in vivo. Furthermore, the data support the existence of an erythroid cellspecific nuclear compartment dedicated to the transcription of the globin genes by RNA polymerase (RNAP) II, called the active chromatin hub (ACH). The ACH model provides a mechanistic framework that may improve knowledge of transcription in the 3-dimensional space of the nucleus.