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BACKGROUND: Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug-targetable pathways. METHODS: Using case-control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus-specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence-based fine mapping, cross-population and cross-phenotype analyses, and gene-expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. RESULTS: A variant in TNFSF13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease-risk allele was also associated with up-regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion-deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up-regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. CONCLUSIONS: A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.) [ABSTRACT FROM AUTHOR]

Hemoglobin switching is a complex biological process not yet fully elucidated. The mechanism regulating the suppression of fetal hemoglobin (HbF) expression is of particular interest because of the positive impact of HbF on the course of diseases such as β-thalassemia and sickle cell disease, hereditary hemoglobin disorders that affect the health of countless individuals worldwide. Several transcription factors have been implicated in the control of HbF, of which BCL11A has emerged as a major player in HbF silencing. SOX6 has also been implicated in silencing HbF and is critical to the silencing of the mouse embryonic hemoglobins. BCL11A and SOX6 are co-expressed and physically interact in the erythroid compartment during differentiation. In this study, we observe that BCL11A knockout leads to post-transcriptional downregulation of SOX6 through activation of microRNA (miR)-365-3p. Downregulating SOX6 by transient ectopic expression of miR-365-3p or gene editing activates embryonic and fetal β-like globin gene expression in erythroid cells. The synchronized expression of BCL11A and SOX6 is crucial for hemoglobin switching. In this study, we identified a BCL11A/miR-365-3p/SOX6 evolutionarily conserved pathway, providing insights into the regulation of the embryonic and fetal globin genes suggesting new targets for treating β-hemoglobinopathies., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of β-thalassaemia. The homozygous mutation c.-196 C>T in the Aγ-globin (HBG1) promoter, which causes Sardinian δβ 0 -thalassaemia, is able to completely rescue the β-major thalassaemia phenotype caused by the β 0 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from β 0 -thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous β 0 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of β-thalassaemia irrespective of the β-globin gene (HBB) mutations., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

A key regulatory gene in definitive erythropoiesis is the transcription factor Krüppel-like factor 1 (Klf1). Klf1 null mice die in utero by day 15.5 (E15.5) due to impaired definitive erythropoiesis and severe anemia. Definitive erythropoiesis takes place in erythroblastic islands in mammals. Erythroblastic islands are formed by a central macrophage (Central Macrophage of Erythroblastic Island, CMEI) surrounded by maturating erythroblasts. Interferon-β (IFN-β) is activated in the fetal liver's CMEI of Klf1 null mice. The inhibitory effect of IFN-β on erythropoiesis is known and, therefore, we speculated that IFN-β could have contributed to the impairment of definitive erythropoiesis in Klf1 knockout (KO) mice fetal liver. To validate this hypothesis, in this work we determined whether the inactivation of type I interferon receptor (Ifnar1) would ameliorate the phenotype of Klf1 KO mice by improving the lethal anemia. Our results show a prolonged survival of Klf1/Ifnar1 double KO embryos, with an improvement of the definitive erythropoiesis and erythroblast enucleation, together with a longer lifespan of CMEI in the fetal liver and also a restoration of the apoptotic program. Our data indicate that the cytotoxic effect of IFN-β activation in CMEI contribute to the impairment of definitive erythropoiesis associated with Klf1 deprivation.

FOXL2 belongs to the evolutionarily conserved forkhead box (FOX) superfamily and is a master transcription factor in a spectrum of developmental pathways, including ovarian and eyelid development and bone, cartilage and uterine maturation. To analyse its action, we searched for proteins that interact with FOXL2. We found that FOXL2 interacts with specific C-terminal propeptides of several fibrillary collagens. Because these propeptides can participate in feedback regulation of collagen biosynthesis, we inferred that FOXL2 could thereby affect the transcription of the cognate collagen genes. Focusing on COL1A2, we found that FOXL2 indeed affects collagen synthesis, by binding to a DNA response element located about 65Kb upstream of this gene. According to our hypothesis we found that in Foxl2(-/-) mouse ovaries, Col1a2 was elevated from birth to adulthood. The extracellular matrix (ECM) compartmentalizes the ovary during folliculogenesis, (with type I, type III and type IV collagens as primary components), and ECM composition changes during the reproductive lifespan. In Foxl2(-/-) mouse ovaries, in addition to up-regulation of Col1a2, Col3a1, Col4a1 and fibronectin were also upregulated, while laminin expression was reduced. Thus, by regulating levels of extracellular matrix components, FOXL2 may contribute to both ovarian histogenesis and the fibrosis attendant on depletion of the follicle reserve during reproductive aging and menopause., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Vitamin D response elements (VDREs) have been found in the promoter region of the MS-associated allele HLA-DRB1*15:01, suggesting that with low vitamin D availability VDREs are incapable of inducing *15:01 expression allowing in early life autoreactive T-cells to escape central thymic deletion. The Italian island of Sardinia exhibits a very high frequency of MS and high solar radiation exposure. We test the contribution of VDREs analysing the promoter region of the MS-associated DRB1 *04:05, *03:01, *13:01 and *15:01 and non-MS-associated *16:01, *01, *11, *07:01 alleles in a cohort of Sardinians (44 MS patients and 112 healthy subjects). Sequencing of the DRB1 promoter region revealed a homozygous canonical VDRE in all *15:01, *16:01, *11 and in 45/73 *03:01 and in heterozygous state in 28/73 *03:01 and all *01 alleles. A new mutated homozygous VDRE was found in all *13:03, *04:05 and *07:01 alleles. Functionality of mutated and canonical VDREs was assessed for its potential to modulate levels of DRB1 gene expression using an in vitro transactivation assay after stimulation with active vitamin D metabolite. Vitamin D failed to increase promoter activity of the *04:05 and *03:01 alleles carrying the new mutated VDRE, while the *16:01 and *03:01 alleles carrying the canonical VDRE sequence showed significantly increased transcriptional activity. The ability of VDR to bind the mutant VDRE in the DRB1 promoter was evaluated by EMSA. Efficient binding of VDR to the VDRE sequence found in the *16:01 and in the *15:01 allele reduced electrophoretic mobility when either an anti-VDR or an anti-RXR monoclonal antibody was added. Conversely, the Sardinian mutated VDRE sample showed very low affinity for the RXR/VDR heterodimer. These data seem to exclude a role of VDREs in the promoter region of the DRB1 gene in susceptibility to MS carried by DRB1* alleles in Sardinian patients.

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.

The persistence of high fetal hemoglobin level in adults may ameliorate the clinical phenotype of beta-thalassemia and sickle cell anemia. Several genetic variants responsible for hereditary persistence of fetal hemoglobin, linked and not linked to the beta globin gene cluster, have been identified in patients and in normal individuals. Monoallelic loss of KLF1, a gene with a key role in erythropoiesis, has been recently reported to be responsible for persistence of high levels of fetal hemoglobin. In a Sardinian family, high levels of HbF (22.1-30.9%) were present only in compound heterozygotes for the S270X nonsense and K332Q missense mutations, while the isolated S270X nonsense (haploinsufficiency) or K332Q missense mutation were associated with normal HbF levels (<1.5%). Functionally, the K332Q Klf1 mutation impairs binding to the BCl11A gene and activation of the γ- and β-globin promoters. Moreover, we report for the first time the association of KLF1 mutations with very high levels of zinc protoporphyrin.

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 alpha-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 alpha-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.

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., (J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart * New York.)

beta-Thalassemia and sickle cell disease both display a great deal of phenotypic heterogeneity, despite being generally thought of as simple Mendelian diseases. The reasons for this are not well understood, although the level of fetal hemoglobin (HbF) is one well characterized ameliorating factor in both of these conditions. To better understand the genetic basis of this heterogeneity, we carried out genome-wide scans with 362,129 common SNPs on 4,305 Sardinians to look for genetic linkage and association with HbF levels, as well as other red blood cell-related traits. Among major variants affecting HbF levels, SNP rs11886868 in the BCL11A gene was strongly associated with this trait (P < 10(-35)). The C allele frequency was significantly higher in Sardinian individuals with elevated HbF levels, detected by screening for beta-thalassemia, and patients with attenuated forms of beta-thalassemia vs. those with thalassemia major. We also show that the same BCL11A variant is strongly associated with HbF levels in a large cohort of sickle cell patients. These results indicate that BCL11A variants, by modulating HbF levels, act as an important ameliorating factor of the beta-thalassemia phenotype, and it is likely they could help ameliorate other hemoglobin disorders. We expect our findings will help to characterize the molecular mechanisms of fetal globin regulation and could eventually contribute to the development of new therapeutic approaches for beta-thalassemia and sickle cell anemia.

The transcription factor erythroid Kruppel-like factor (EKLF) specifically activates the beta-globin gene by interacting with the proximal beta-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 beta-globin stimulation. Thus, EKLF requires both CACCC boxes to maximally stimulate the beta-globin gene., (Copyright 2004 Blackwell Publishing Ltd)

The silent beta-thalassemia mutation, beta(+)-101C-->T, is the only mutation currently described in the distal beta-globin CACCC box. We present a novel mutation, a C-->G transversion, in the same position. Expression analysis in heterozygous subjects demonstrated that the mutation determines a 20% reduction in the output of the beta-globin gene. DNA-protein interaction and transactivation analysis correlated the decrease in the beta-globin synthesis with the reduced binding and transactivation of EKLF to the mutant promoter. These data predict that the beta-101C-->G mutation will display a silent thalassemia phenotype similar to that of the beta-101C-->T mutation.

The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human beta-globin locus control region is required for high level globin gene expression. We used an oligonucleotide of the NF-E2 tandem repeat, within HS2, as recognition site probe to screen a K562 cDNA library for interacting transcription factors. A 2.3 kb full length cDNA encoding the b-zip transcription factor MafF was isolated. MafF can form both homodimers and high affinity heterodimers with Nrf1, Nrf2 and Nf-E2, three members of the CNC-bZip family. Despite obvious structural similarities with the other small Maf proteins, MafF differs in its tissue distribution and its inability to repress transcription when overexpressed as homodimer. In fact, in different cell lines and on different promoters (gamma-globin, beta-globin and glutamylcysteine synthetase genes) the MafF homodimers do not appreciably affect transcription of target promoters, whereas MafF/CNC member heterodimers act as weak transcriptional activators. Even though MafF was cloned using probes derived from the globin LCR, it is in the context of the GCSl promoter and in combination with Jun that MafF shows a rather distinct and specific regulatory role. These observations suggest that a complex network of small Maf and CNC-AP1 protein interactions might be involved in regulating transcription in diverse tissues or developmental stages.

Hypersensitive site 2 located in the beta-globin locus control region confers high levels of expression to the genes of the beta-globin cluster. A tandem repeat of the consensus sequence for the transcription factors AP1 and NF-E2 (activating protein 1 and nuclear factor erythroid 2, respectively) is present within hypersensitive site 2 and is absolutely required for strong enhancer activity. This sequence binds, in vitro and in vivo, to ubiquitous proteins of the AP1 family and to the recently cloned erythroid-specific transcription factor NF-E2. Using the tandem repeat as a recognition site probe to screen a lambda gt11 cDNA expression library from K562 cells, we isolated several DNA binding proteins. Here, we report the characterization of one of the clones isolated. The gene, which we named Nrf2 (NF-E2-related factor 2), is encoded within a 2.2-kb transcript and predicts a 66-kDa protein with a basic leucine zipper DNA binding domain highly homologous to that of NF-E2. Although Nrf2 is expressed ubiquitously, a role of this protein in mediating enhancer activity of hypersensitive site 2 in erythroid cells cannot be excluded. In this respect, Nrf2 contains a powerful acidic activation domain that may participate in the transcriptional stimulation of beta-globin genes.