Repair of DNA interstrand cross-links is a complex process critical to which is the identification of sites of damage by specific proteins. We have recently identified the structural protein nonerythroid alpha spectrin (alphaSpIISigma) as a component of a nuclear protein complex in normal human cells which is involved in the repair of DNA interstrand cross-links and have shown that it forms a complex with the Fanconi anemia proteins FANCA, FANCC, and FANCG. Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well. That spectrin binds directly to the cross-linked DNA has been shown using purified bovine brain spectrin (alphaSpIISigma1/betaSpIISigma1)2. Binding of the Fanconi anemia (FA) proteins to the damaged DNA may be either direct or indirect via their association with alphaSpIISigma. These results demonstrate a role for alpha spectrin in the nucleus as well as a new function for this protein in the cell, an involvement in DNA repair. alphaSpIISigma may bind to cross-linked DNA and act as a scaffold to help in the recruitment of repair proteins to the site of damage and aid in their alignment and interaction with each other, thus enhancing the efficiency of the repair process.

Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf ؊/؊ ) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf ؊/؊ embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild-type and Eklf ؊/؊ early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf ؊/؊ early erythroid progenitor cells, which showed a delay in the G 1 -to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation.

In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.

The histone deacetylase inhibitors (HDACIs) butyrate and trichostatin A activate ␥-globin expression via a p38 mitogenactivating protein kinase (MAPK)-dependent mechanism. We hypothesized that downstream effectors of p38 MAPK, namely activating transcription factor-2 (ATF-2) and cyclic AMP response element (CRE) binding protein (CREB), are intimately involved in fetal hemoglobin induction by these agents. In this study, we observed increased ATF-2 and CREB1 phosphorylation mediated by the HDACIs in K562 cells, in conjunction with histone H4 hyperacetylation. Moreover, enhanced DNAprotein interactions occurred in the CRE in the G ␥-globin promoter (G-CRE) in vitro after drug treatments; subsequent chromatin immunoprecipitation assay confirmed ATF-2 and CREB1 binding to the G-CRE in vivo. Enforced expression of ATF-2 and CREB produced G ␥-promoter IntroductionThe growth factor erythropoietin (Epo) exerts its effects on commitment, proliferation, and differentiation of erythroid progenitors and globin chain synthesis through Janus kinase 2/Stat5 signaling and crosstalk with mitogen-activated protein kinase (MAPK) pathways. 1-3 p38 MAPK signaling is required for Epo mRNA stability and hemoglobin synthesis. 4,5 The reversible inhibition of p38 MAPK using SB203580 blocked Epo-dependent accumulation of mouse globin chains, 6 and studies in p38␣ Ϫ/Ϫ knockout mice showed a failure of definitive ␤ maj -globin gene expression. These studies confirm an Epo-p38 MAPK-dependent mechanism for hemoglobin synthesis. 7 The HDACI sodium butyrate (NaB) induces differentiation in erythroleukemia cells via Stat5 8,9 and p38 MAPK signaling. 10,11 Butyrate is a clinically useful fetal hemoglobin (HbF) inducer which has been used to treat individuals with sickle cell disease 12 and thalassemia 13 ; however, the molecular mechanism for NaB-mediated HbF induction is poorly understood. Recent data from Weinberg et al 14 showed that HbF induction by arginine butyrate is due in part to posttranslational mechanisms and increased ␥-globin mRNAtranslation.Several HDACIs, including trichostatin A (TSA), 10, and scriptaid,15,16 induce ␥-globin expression via p38 MAPK signaling. These studies suggest that different pharmacologic agents converge on the p38 MAPK pathway to activate ␥-globin expression. Four major MAPK pathways have been characterized: ERK1/2, ERK5/BMK1, cJun amino-terminal signal kinases (JNK), and p38. [17][18][19][20] Studies using erythroid progenitors, 21,22 knockout mice, 7 and K562 stable lines 10 suggest that p38␣ is the primary mediator of globin gene regulation.The downstream effector molecules of p38 MAPK signaling include MAPK-activated protein kinases 1 and 2, 23,24 PRAK, 25 ATF-1-4, CREB1, CREB2, and CREM. 26,27 Commonly, p38 phosphorylates ATF-2 and CREB to augment gene transcription. We recently demonstrated a p38 MAPK-dependent mechanism for NaB and TSA-induced ␥-globin expression. 10 Mechanistically, both agents bind a central zinc atom in HDAC to produce hyperacetylation of histone H3 (H3) and H4 28,29 to activate...

Mechanism(s) underlying activation of store-operated Ca2+ entry currents, I SOC, remain incompletely understood. F-actin configuration is an important determinant of channel function, although the nature of interaction between the cytoskeleton and I SOC channels is unknown. We examined whether the spectrin membrane skeleton couples Ca2+ store depletion to Ca2+ entry. Thapsigargin activated an endothelial cell I SOC (−45 pA at −80 mV) that reversed at +40 mV, was inwardly rectifying when Ca2+ was the charge carrier, and was inhibited by La3+ (50 μM). Disruption of the spectrin–protein 4.1 interaction at residues A207-V445 of βSpIIΣ1 decreased the thapsigargin-induced global cytosolic Ca2+ response by 50% and selectively abolished the endothelial cell I SOC, without altering activation of a nonselective current through cyclic nucleotide–gated channels. In contrast, disruption of the spectrin–actin interaction at residues A47-K186 of βSpIIΣ1 did not decrease the thapsigargin-induced global cytosolic Ca2+ response or inhibit I SOC. Results indicate that the spectrin–protein 4.1 interaction selectively controls I SOC, indicating that physical coupling between calcium release and calcium entry is reliant upon the spectrin membrane skeleton.

Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the ␤-globin promoter, where it establishes local chromatin structure permitting ␤-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the ␤-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and ␤-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

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