ALBERT

Description: The zinc finger-encoding transacting factor EKLF binds key regulatory elements of many erythroid-specific genes, and is essential for definitive erythropoiesis. Mice lacking this factor (EKLF−/−) die of anemia by E15.5 of gestation, failing to activate β-globin gene transcription, and demonstrating a block in the erythroid differentiation program at the primitive erythroblast stage. In contrast, megakaryocytic progenitors are amplified in EKLFnull embryos, with increased Fli-1 gene expression (a marker of early megakaryocytic differentiation), consistent with the idea that EKLF modulates the megakaryocyticerythroid (M-E) differentiation switch. We have demonstrated that an amino terminal mutant of EKLF (Δ221EKLF), is required to induce chromatin remodeling at the β-globin promoter in an EKLF-null erythroid cell line, but additional amino terminal sequences are required for initiation of β-globin gene transcription (Brown et al., 2002). To evaluate the role of this chromatin remodeling (CR) domain in erythroid and megakaryocytic differentiation in vivo, we have generated a knock-in allele of EKLFCR allele. Similar to EKLF-null embryos, mice homozygous for this mutant allele die of anemia by E15.5 of gestation. In contrast to erythroid cells lacking EKLF, EKLFCR/CR progenitors demonstrate appropriate binding of the CR encoding domain to all EKLF-regulatory sequences; a block in erythropoiesis at a more a mature stage in differentiation a chromatin architecture and histone modification pattern at erythroid-specific genes that recapitulates the events observed in EKLF+/+ erythroblasts at a similar stage of erythroid ontogeny; a failure of terminal erythroid gene transcription. Examining the role of EKLFCR in megakaryopoiesis, we observed inhibition of megakaryocytic progenitor amplification in EKLFCR/CR fetal hematopoietic cell populations when compared to EKLF-null embryos; loss of Fli-1 gene expression in EKLFCR expressing cells; binding of the EKLFCR mutant protein to the Fli-1 promoter with inhibition of gene transcription; a repressed chromatin architecture at megakaryocytic gene loci. In contrast to these results, mice homozygous for a knockin allele encoding the zinc finger DNA binding domain alone (Δ253EKLF), a region shown previously to be sufficient for chromatin remodeling in vitro, demonstrate erythroid and megakaryocytic phenotypes that resemble those observed in EKLF-null hematopoietic progenitors. Taken together, our results suggest strongly that the unique EKLFCR domain is necessary and sufficient to modulate the chromatin-specific roles of EKLF at erythroid- and megakaryocytic-specific loci in definitive hematopoietic cells in vivo.

Description: Magnesium deprivation of pregnant rats produces in their offspring depression of intrauterine growth and severe anemia. The anemia is characterized by microcytosis, red cell fragmentation, increase in number of normoblasts and markedly decreased osmotic fragility.

Description: The administration of a Mg-deficient diet to adult rats for 4-5 wk causes anemia. The RBC are slightly smaller and flatter, and have a reduced hemoglobin component and a decreased osmotic fragility. 51Cr survival time is shortened when the affected cells are infused into normal adult rats. In vitro synthesis of heme and globin, as measured with 14C-glycine was the same as in control rats with "reticulocytosis-rich" anemia produced by bleeding or immunologically. The activity of a series of enzymes associated with energy production also failed to yield findings specific for Mg deficiency. It is suggested that the anemia of Mg deficiency is hemolytic and results from the combination of a reversible extrinsic defect and of an irreversible structural defect in the RBC.

Description: Abstract 3189 Binding of the Krüppel-like Factor 1 (KLF1) to the β-globin gene promoter is required for developmental stage-specific chromatin remodeling and transcriptional initiation. Interaction with a long-range enhancer, the Locus Control Region (LCR) is also required for maximal gene expression. KLF1 binds the LCR, and has been described recently as a facilitator of the proximal clustering of the LCR with the β-globin gene promoter region. To elucidate the role(s) of KLF1 at the LCR and their relationship to β-globin gene activation, we evaluated KLF1-directed events across the β-globin locus using a 4-OH-Tamoxifen KLF-1 inducible erythroid cell line. KLF1 binding was maximal 1 hour post-induction at the LCR, whereas 2 hours elapsed prior to maximal occupancy at the β-globin promoter. The site-specific differential in factor occupancy is consistent with the notion that the LCR serves as a nucleating docking element for sequence-specific transcription factors as well as the RNA Pol-II complex. LCR occupancy by Pol-II, p45 NF-E2, GATA-1 and the SCL/LMO2/Ldb1 complex was KLF1-independent. In contrast, the occupancy of the β-globin promoter by Pol-II and erythroid-restricted factors was dependent on maximal KLF-1 binding. Interestingly, the SCL/LMO2/Ldb1 complex was recruited first, consistent with the idea that this complex is required for LCR/promoter interaction. Interestingly, we have identified a direct protein-protein interaction between the carboxy terminal (aa221–396) domain of KLF1 (Δ221KLF1) and Ldb1, the factor implicated as critical for distal regulatory loci interactions at the β-globin locus. To dissect these events in vivo, we took advantage of a novel KLF1 knock-in strain (Δ221KLF1). In these animals, the endogenous KLF1 gene is replaced with a carboxy-terminal domain (aa221–396) expression cassette, resulting in DNA binding of the mutant factor, with β-globin chromatin remodeling. However, Δ221KLF1 fetal liver erythroblasts fail to activate the KLF1-dependent gene network, and have the identical differentiation defect characteristic of KLF1-null erythroblasts. Chromatin remodeling at the β-globin failed to result in recruitment of GATA-1, NF-E2, or the SCL/LMO2/Ldb1 complex. In contrast, ChIP analyses of the LCR in Δ221KLF1 erythroblasts revealed rescue of RNA Pol-II, GATA-1 and the SCL/LMO2/Ldb1 complex occupancy to wild type levels. Structural studies, utilizing the chromosome conformation capture (3C) assay, revealed that an incomplete re-configuration of the locus in Δ221 KLF1 mice. In conclusion, the chromatin remodeling domain of KLF1 is sufficient to reconfigure the LCR. However, KLF1-dependent Ldb1 recruitment at the LCR is insufficient to promote adequate communication between the two regulatory elements of the β-globin gene. Preliminary data suggest that an adjacent domain (aa163–221) is sufficient to rescue β-globin transcription in vivo. Together, these domains (aa163–396) are sufficient to promote the appropriate loading of transcription factors at the β-globin promoter in conjunction with locus structural re-configuration. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 461 Krüppel-like factor 1 (KLF1) is essential for erythroid gene expression. Key molecular mechanisms modulated by this transacting factor have been elucidated at the b-globin locus. KLF1 has been associated with recruitment of SWI/SNF and RNA polymerase (PolII) complexes necessary for chromatin remodeling and gene transcription respectively, and for facilitating the apposition of the promoter with the far-upstream locus control region. More recently, KLF1 has been implicated in the regulation of an erythroid-specific gene program unlinked to the b-globin locus. Coordinated expression of these genes, including Alpha Hemoglobin-Stabilizing Protein (AHSP), a factor required for globin tetramer stability, and the red cell membrane protein Dematin, are critical for erythroid ontogeny. To compare the role(s) of KLF1 at these loci, we have used a unique 4-OH-Tamoxifen (4-OHT) inducible erythroid cell line, which facilitates the characterization of the temporal kinetics of KLF1-dependent erythroid gene activation. In preliminary experiments, we observed that KLF1 binding was maximal at the three loci within 60 minutes of 4-OHT induction. AHSP and dematin primary RNA transcripts followed similar kinetics, being maximal at 60-90 minutes post-induction. In contrast, b-globin gene transcription reached a plateau 4-6 hours post-induction. From these observations, we hypothesized that transcriptional activation at AHSP and dematin differs from that observed at the b-globin cluster. Consistent with this hypothesis, we observed significant differences in chromatin remodeling at the three loci. At the b-globin promoter, we observed a small but statistically significant increase in DNaseI sensitivity, a measure of chromatin remodeling, with KLF1 binding. In contrast, we observed a complete loss of DNaseI resistance after KLF1 binding at the AHSP and dematin promoters. Consistent with these findings, we observed a five-fold reduction in histone H3 occupancy at the AHSP and dematin promoters, contrasting with no significant change in occupancy at the b-promoter. Importantly, these differences were not observed in regions 1-5 kb upstream of the promoters. These observations, coupled with similar differences in DNaseI hypersensitivity and histone occupancy in fetal liver erythroblasts from wild type and KLF1-null mice, suggest a profound difference in the mechanisms of chromatin remodeling at KLF1-dependent erythroid gene loci. To explore the potential mechanisms underlying these differences in chromatin accessibility, we examined the kinetics of recruitment of other transacting factors and co-activators to the three loci. We observed similar increases in binding of serine-5 phosphorylated PolII, GATA-1, and p45NF-E2 at the promoters. In contrast, binding of BRG1, the core ATPase component of the SWI/SNF complex differed between the b–promoter and the other erythroid genes. Although BRG1 binding was co-incident with KLF1 binding to the b-gene, we observed significant albeit weak binding of this complex to the AHSP and Dematin promoters only after maximal gene transcription had occurred. Our results suggest that different KLF1 multiprotein complexes are recruited to remodel target gene promoters in vivo. Furthermore, we propose that KLF1's chromatin remodeling capabilities are not limited to the recruitment of the SWI/SNF complexes Disclosures: No relevant conflicts of interest to declare.

Description: The 17q23 amplicon is associated with poor outcome in ER+ breast cancers, but the causal genes to endocrine resistance in this amplicon are unclear. Here, we interrogate transcriptome data from primary breast tumors and find that among genes in 17q23, PRR11 is a key gene associated with a poor response to therapeutic estrogen suppression. PRR11 promotes estrogen-independent proliferation and confers endocrine resistance in ER+ breast cancers. Mechanistically, the proline-rich motif-mediated interaction of PRR11 with the p85α regulatory subunit of PI3K suppresses p85 homodimerization, thus enhancing insulin-stimulated binding of p110-p85α heterodimers to IRS1 and activation of PI3K. PRR11-amplified breast cancer cells rely on PIK3CA and are highly sensitive to PI3K inhibitors, suggesting that PRR11 amplification confers PI3K dependence. Finally, genetic and pharmacological inhibition of PI3K suppresses PRR11-mediated, estrogen-independent growth. These data suggest ER+/PRR11-amplified breast cancers as a novel subgroup of tumors that may benefit from treatment with PI3K inhibitors and antiestrogens.