No abstract

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the gene loci. We discovered that the CTCF binding site located between and genes (CBS7/9) is critical for establishing and maintaining aberrant gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior genes. Consistent with the role of the CBS7/9 boundary in locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.

Accumulation of iron has been associated with the pathobiology of various disorders of the central nervous system. Our previous work has shown that hephaestin ( Heph ) and ceruloplasmin ( Cp ) double knockout (KO) mice induced iron accumulation in multiple brain regions and that this was paralleled by increased oxidative damage and deficits in cognition and memory. In this study, we enriched astrocytes and oligodendrocytes from the cerebral cortex of neonatal wild-type (WT), Heph KO and Cp KO mice. We demonstrated that Heph is highly expressed in oligodendrocytes, while Cp is mainly expressed in astrocytes. Iron efflux was impaired in Cp KO astrocytes and Heph KO oligodendrocytes and was associated with increased oxidative stress. The expression of Heph , Cp , and other iron-related genes was examined in astrocytes and oligodendrocytes both with and without iron treatment. Interestingly, we found that the expression of the mRNA encoding ferroportin 1, a transmembrane protein that cooperates with CP and HEPH to export iron from cells, was positively correlated with Cp expression in astrocytes, and with Heph expression in oligodendrocytes. Our findings collectively demonstrate that HEPH and CP are important for the prevention of glial iron accumulation and thus may be protective against oxidative damage.

We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens.