Loredana Porcu

Klf1 Affects DNase II-Alpha Expression in the Central Macrophage of a Fetal Liver Erythroblastic Island: a Non-Cell-Autonomous Role in Definitive Erythropoiesis

ABSTRACT A key regulatory gene in definitive erythropoiesis is the erythroid Kruppel-like factor (Eklf or Klf1). Klf1 knockout (KO) mice die in utero due to severe anemia, while residual circulating red blood cells retain their nuclei. Dnase2a is another critical gene in definitive erythropoiesis. Dnase2a KO mice are also affected by severe anemia and die in utero. DNase II-alpha is expressed in the central macrophage of erythroblastic islands (CMEIs) of murine fetal liver. Its main role is to digest the DNA of the extruded nuclei of red blood cells during maturation. Circulating erythrocytes retain their nuclei in Dnase2a KO mice. Here, we show that Klf1 is expressed in CMEIs and that it binds and activates the promoter of Dnase2a. We further show that Dnase2a is severely downregulated in the Klf1 KO fetal liver. We propose that this downregulation of Dnase2a in the CMEI contributes to the Klf1 KO phenotype by a non-cell-autonomous mechanism.

Regulation of the human HBA genes by KLF4 in erythroid cell lines

KLF1/EKLF and related Krueppel‐like factors (KLFs) are variably implicated in the regulation of the HBB‐like globin genes. Prompted by the observation that four KLF sites are distributed in the human α‐globin gene (HBA) promoter, we investigated if KLFs could also act to modulate the expression of the HBA genes. Among the KLFs tested, only KLF4/GKLF bound specifically to three out of four α‐globin KLF sites. The occupancy of the same sites by KLF4 in vivo was confirmed by chromatin immunoprecipitation assays with KLF4‐specific antibodies. In luciferase reporter assays in MEL cells, high levels of the wild type HBA promoter, but not mutated promoters bearing point mutations that disrupted KLF4‐DNA binding, were transactivated by over‐expression of KLF4. In K562 cells, induced KLF4 expression with a Tet‐off regulated cassette stimulated the expression of the endogenous HBA genes. In a complementary assay in the same cell line, knocking down KLF4 with lentiviral delivered sh‐RNAs caused a parallel decrease in the transcription of the HBA genes. All experiments combined support a regulatory role of KLF4 in the control of HBA gene expression.

ΔN133p53 expression levels in relation to haplotypes of the TP53 internal promoter region

The transcription of the ΔN133p53 isoform of the TP53 gene is controlled by an internal promoter region (IPR) containing eight polymorphisms in 11 common haplotypes, following a resequencing of 47 Caucasians. We assayed the functional effects of the commonest six haplotypes on the promoter activity with a luciferase reporter system, in HeLa and 293T cells. These studies showed that different IPR haplotypes are associated with differences in the promoter activity resulting in marked variation in the baseline expression of ΔN133p53. In vivo quantitative‐polymerase chain reaction (PCR) on human tissues confirmed that the baseline levels of ΔN133p53 showed haplotype specific differences that paralleled those seen in vitro. When cell lines were treated with camptothecin, the fold‐increase in ΔN133p53 levels was dose‐dependent but haplotype‐independent (i.e., similar for all the haplotypes). Finally, we used an electrophoretic mobility shift assay to analyze the rs1794287 polymorphism and found changes in the pattern of protein binding. This partially confirmed our in silico analysis showing that the polymorphism rs1794287 can affect the function of the internal promoter by changing its affinity for several transcription factors. Thus, we showed that the expression of ΔN133p53 is under genetic control, and suggested the presence of interindividual differences underlying this mechanism. Hum Mutat 31:1–10, 2010.

A Novel Missense Mutation (C84R) in a Patient with Type II Vitamin D-dependent Rickets

A 7-year-old boy with severe rickets that by clinical analysis was diagnosed as affected by type II vitamin D-dependent rickets, was evaluated for mutations in the vitamin D receptor gene (VDR). The molecular analysis showed a homozygous state for a novel missense mutation (C84R) in a highly conserved nucleotide in the second Zn finger of the DNA binding domain.

The distal β-globin CACCC box is required for maximal stimulation of the β-globin gene by EKLF

The transcription factor erythroid Kruppel‐like factor (EKLF) specifically activates the β‐globin gene by interacting with the proximal β‐globin CACCC box, a known hot spot for thalassaemia mutations. This study investigated whether EKLF could also bind to, and activate from, the distal CACCC, which is a rare site of thalassaemia mutations. Using band shift and transient expression analysis with wild type, single and double CACCC mutants, we established that the distal CACCC box is weakly bound by EKLF, but, when mutated, significantly impairs EKLF‐dependent β‐globin stimulation. Thus, EKLF requires both CACCC boxes to maximally stimulate the β‐globin gene.

Regulation of the Human a-globin gene by KLF4 in erythroid cell lines M. British journal of Haematology. 2010. 149, 748-758

KLF1/EKLF and related Krueppel‐like factors (KLFs) are variably implicated in the regulation of the HBB‐like globin genes. Prompted by the observation that four KLF sites are distributed in the human α‐globin gene (HBA) promoter, we investigated if KLFs could also act to modulate the expression of the HBA genes. Among the KLFs tested, only KLF4/GKLF bound specifically to three out of four α‐globin KLF sites. The occupancy of the same sites by KLF4 in vivo was confirmed by chromatin immunoprecipitation assays with KLF4‐specific antibodies. In luciferase reporter assays in MEL cells, high levels of the wild type HBA promoter, but not mutated promoters bearing point mutations that disrupted KLF4‐DNA binding, were transactivated by over‐expression of KLF4. In K562 cells, induced KLF4 expression with a Tet‐off regulated cassette stimulated the expression of the endogenous HBA genes. In a complementary assay in the same cell line, knocking down KLF4 with lentiviral delivered sh‐RNAs caused a parallel decrease in the transcription of the HBA genes. All experiments combined support a regulatory role of KLF4 in the control of HBA gene expression.

Regulation of the human HBA genes by KLF4 in erythroid cell lines: Regulation of the Human HBA Genes

KLF1/EKLF and related Krueppel‐like factors (KLFs) are variably implicated in the regulation of the HBB‐like globin genes. Prompted by the observation that four KLF sites are distributed in the human α‐globin gene (HBA) promoter, we investigated if KLFs could also act to modulate the expression of the HBA genes. Among the KLFs tested, only KLF4/GKLF bound specifically to three out of four α‐globin KLF sites. The occupancy of the same sites by KLF4 in vivo was confirmed by chromatin immunoprecipitation assays with KLF4‐specific antibodies. In luciferase reporter assays in MEL cells, high levels of the wild type HBA promoter, but not mutated promoters bearing point mutations that disrupted KLF4‐DNA binding, were transactivated by over‐expression of KLF4. In K562 cells, induced KLF4 expression with a Tet‐off regulated cassette stimulated the expression of the endogenous HBA genes. In a complementary assay in the same cell line, knocking down KLF4 with lentiviral delivered sh‐RNAs caused a parallel decrease in the transcription of the HBA genes. All experiments combined support a regulatory role of KLF4 in the control of HBA gene expression.