ABSTRACT
Evidences indicate that locus control region (LCR) of beta-globin spatially closes to the downstream active gene promoter to mediate the transcriptional activation by looping. DNA binding proteins may play an important role in the looping formation. NF-E2 is one of the key transcription factors in beta-globin gene transcriptional activation. To shed light on whether NF-E2 is involved in this process, DS19MafKsiRNA cell pools were established by specifically knocked down the expression of MafK/NF-E2 p18, one subunit of NF-E2 heterodimer. In the above cell pools, it was observed that the occupancy efficiency of NF-E2 on beta-globin gene locus and the expression level of beta-globin genes were decreased. H3 acetylation, H3-K4 methylation and the deposition of RNA polymerase II, but not the recruitment of GATA-1, were also found reduced at the beta-globin gene cluster. Chromosome Conformation Capture (3C) assay showed that the cross-linking frequency between the main NF-E2 binding site HS2 and downstream structural genes was reduced compared to the normal cell. This result demonstrated that MafK/NF-E2 p18 recruitment was involved in the physical proximity of LCR and active beta-globin genes upon beta-globin gene transcriptional activation.
Subject(s)
Gene Expression Regulation/physiology , Globins/genetics , Locus Control Region/physiology , MafK Transcription Factor/physiology , Animals , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA Polymerase II/physiology , GATA1 Transcription Factor/physiology , Gene Silencing , Globins/biosynthesis , Histones/metabolism , Mice , RNA InterferenceABSTRACT
The alpha- and beta-globin gene clusters are subject to several levels of regulation leading to differentiation and developmental stage-specific expression. Not only the interaction of cis-acting elements and cell-specific trans-acting factors but also the chromosomal environment, nuclear compartmentalization of genome regions, and transcription factors play important roles in globin gene regulation research. FISH, with a refined technique and an incorporation of other molecular techniques, has become a powerful technique in gene regulation research, particular for the analysis of replication timing and primary transcripts as well as the research of the special organization of large-scale chromatin domains and nuclear compartmentalization at cellular and subcellular levels. This review will focus on the major impact of FISH on globin gene regulation research at different regulation levels, such as replicating timing, gene transcription, nuclear relocation, and chromosomal position effect.
