Structural organization of chromosomal interactions during megakaryocytic differentiation
Date Issued
2008
Date
2008
Author(s)
Yen, Yin-Wen
Abstract
The Human Genome Project was initiated by the National Institute of Health and the Department of Energy of the United States in 1990 and completed its ultimate goal of decoding the genomic sequence of humans in 2003. A complete book of life enabled us to study evolution, growth and development, and diseases in a genome-wide scale. The human genome was conventionally considered as a contiguous, linear sequence entity. In fact, DNA is folded into multilayered, high-order structures that eventually packaged into chromosomes. The spatial organization of chromatin dramatically affects the function of the genome, including DNA replication, transcription, and repair. Furthermore, physiological processes at the organismal and cellular levels, such as differentiation, development, immune response and pathogenesis, are greatly influenced by the three-dimension (3D) chromosomal structures in the nucleus. For example, the transcription of mammalian α-globin gene is determined by the unique spatial arrangement of chromatin structure during erythroid differentiation. In the past, the analysis of 3D chromatin structures was limited by technical difficulties. Recently, a new methodology, termed chromosome conformation capture ( 3C ) has been developed to study chromosomes that are physically associated with nuclear protein complex. Such interactions require a prior knowledge of defined chromosomal regions. In order to examine physical associations in the unknown regions, a modified, high-throughput method of circular chromosome conformation capture ( 4C ) was developed to detect the intra- and interchromosomal interactions. The objective of my thesis research was to describe the structural organization of chromosomal interactions in human K562 cells during TPA-induced megakaryocytic differentiation. We adopted the 4C approach to investigate chromosomal interactions with the promoter sequence of the ITGA2B gene that is up-regulated in TPA-treated K562 cells. Altogether we identified 11 interchromosomal interactions. Eight of which were involved in the intragenic regions, whereas the remaining three were mapped to the intergenic regions. In this study, we also confirmed the interaction between ITGA2B and KSR2 only in the TPA-induced differentiation of K562 cells. By using the semi-quantitative RT-PCR assay to examine the transcripts of the ITGA2B-interacting genes, we found that the expression of both KSR2 and NAV2 was inducible after TPA treatment and that PLEKHA5, PCSK7, DACH1, LPHN3, SFRS15 and C1orf125 were constitutively expressed in K562 cells. These results suggest that actively transcribed genes are potentially clustered together in a nuclear space, as proposed to be in a transcription factory, which can be detected by 4C through the physical association with shared transcription complex. By using the bioinformatics approach, we also explored the transcription factor (TF)-binding motifs on the promoter sequences in the 1kb regions upstream of the ITGA2B-interacting genes. All but LPHN3, like ITGA2B, contain TATA-less promoters. It is likely that such a transcription factory, if indeed exists, may transcribe specifically the housekeeping and inducible genes without the TATA promoter with a transcription complex incorporating other TFs, such as SP1 and ETS. Further examination of the TF-binding motifs on the interactor sequences revealed that the potential binding sites, except SFRS15, were different from the ITGA2B promoter. These results likely reflect the dynamic nature of transcription machinery, in which during transcription the DNA sequence moves through immobilized protein complex.
Subjects
chromosome structure
megakaryocytic differentiation
circular chromosome conformation capture
SDGs
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