Subcellular Transport of EKLF and Switch-On of Murine Adult β_maj Globin Gene Transcription

Abstract

Erythroid Krüppel-like factor (EKLF) is an essential transcription factor for mammalian ␤-like globin gene switching, and it specifically activates transcription of the adult ␤ globin gene through binding of its zinc fingers to the promoter. It has been a puzzle that in the mouse, despite its expression throughout the erythroid development, EKLF activates the adult ␤ maj globin promoter only in erythroid cells beyond the stage of embryonic day 10.5 (E10.5) but not before. We show here that expression of the mouse ␤ maj globin gene in the aorta-gonad-mesonephros region of E10.5 embryos and in the E14.5 fetal liver is accompanied by predominantly nuclear localization of EKLF. In contrast, EKLF is mainly cytoplasmic in the erythroid cells of E9.5 blood islands in which ␤ maj is silenced. Remarkably, in a cultured mouse adult erythroleukemic (MEL) cell line, the activation of the ␤ maj globin gene by dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) induction is also paralleled by a shift of the subcellular location of EKLF from the cytoplasm to the nucleus. Blockage of the nuclear import of EKLF in DMSO-induced MEL cells with a nuclear export inhibitor repressed the transcription of the ␤ maj globin gene. Transient transfection experiments further indicated that the full-sequence context of EKLF was required for the regulation of its subcellular locations in MEL cells during DMSO induction. Finally, in both the E14.5 fetal liver cells and induced MEL cells, the ␤-like globin locus is colocalized the PML oncogene domain nuclear body, and concentrated with EKLF, RNA polymerase II, and the splicing factor SC35. These data together provide the first evidence that developmental stage-and differentiation state-specific regulation of the nuclear transport of EKLF might be one of the steps necessary for the switch-on of the mammalian adult ␤ globin gene transcription.In mammals, the ␤-like and ␣-like families are individually clustered on different chromosomes. Functional members of both gene clusters are arranged in the order of their expression during erythroid development (globin switch). In the mouse, the ␤-like globin locus is arranged as follows: 5Ј-ε y (embryonic)-␤h1 (embryonic)-␤ maj (fetal and adult)-␤ min (fetal and adult, minor species)-3Ј (21, 2a) (top diagram in Fig. 1B). The hematopoietic cells, including the erythroid ones in the embryo, initially arise in the blood island that forms at embryonic day 7.5 (E7.5) (17). Within the primitive erythroblasts derived from the yolk sac are the embryonic genes, including ε y and ␤h1, as transcribed previously (26). The adult ␤ maj globin gene, on the other hand, is silenced in E9.5 yolk sac, but it starts to be expressed on E10.5 in the erythroblasts in the aorta-gonadmesonephros (AGM) region and the fetal liver of the embryo (26,38,54,56). During development, the major site of the erythropoiesis shifts to the fetal liver and then again to the bone marrow at the time of birth. The erythroid cells generated in the latter-named two tissues represent a def...

Non-random subcellular distribution of variant EKLF in erythroid cells

“…During the course of these studies, Shyu et al also demonstrated that EKLF can be found in the cytoplasm [55]. Although this conclusion is similar to ours, there are large inconsistencies in the details of the two studies that are hard to resolve but may be due to differences in experimental conditions and antibodies used.…”

Induction of adult levels of -globin in human erythroid cells that intrinsically express embryonic or fetal globin by transduction with KLF1 and BCL11A-XL

“…2,36 As shown above, HiDEP-1 cells express a similar level of endogenous KLF1 to adult erythroid cells on differentiation in erythroid culture. However, in mouse erythroid cells, KLF1 is detected in both the cytoplasm and nucleus 37,38 with developmental stage-specific nuclear import potentially involved in the switch to adult globin expression. 38,39 We, therefore, compared the sub-cellular localization of KLF1 in HiDEP-1 cells with adult PB CD34 + derived erythroid cells at the same developmental stage ( Figure 3A).…”

“…However, in mouse erythroid cells, KLF1 is detected in both the cytoplasm and nucleus 37,38 with developmental stage-specific nuclear import potentially involved in the switch to adult globin expression. 38,39 We, therefore, compared the sub-cellular localization of KLF1 in HiDEP-1 cells with adult PB CD34 + derived erythroid cells at the same developmental stage ( Figure 3A). In both, KLF1 was detected in the cytoplasmic and nuclear fractions ( Figure 3B), with at least as much KLF1 detected in the nuclear fraction of HiDEP-1 as in adult cells.…”

EKLF/KLF1, a Tissue-Restricted Integrator of Transcriptional Control, Chromatin Remodeling, and Lineage Determination

“…In spite of the existence of two domains that behave as strong nuclear localization signals, a significant proportion of KLF1 quite unexpectedly resides in the cytoplasm (14,73,75,122). Immunofluorescent and biochemical assays show that a substantial proportion of endogenous KLF1 is cytoplasmic at steady state in all erythroid cells examined (primary and MEL), that this pattern does not appear to change during either erythroid development or terminal differentiation, and that its nuclear/cytoplasmic ratio is not altered by a variety of treatments (122).…”

“…KLF1-associated transcription factories tend to colocalize with loci that are highly transcribed in erythroid cells. For example, the globin genes associate with other active genes, revealing extensive and preferential intra-and interchromosomal transcription interactomes that are larger than transcription factories without KLF1 (74) and that may include PML bodies and Sc35 (75). Such interactions likely go hand in hand with the KLF1-, GATA1-and FOG1-dependent extrusion of the ␤-globin locus away from the nuclear periphery (76), an event that is necessary for its transcriptional activation (77).…”