Physiological and Aberrant γ-Globin Transcription During Development.

The expression of the fetal Gγ- and Aγ-globin genes in normal development is confined to the fetal period, where two γ-globin chains assemble with two α-globin chains to form α2γ2 tetramers (HbF). HbF sustains oxygen delivery to tissues until birth, when ß-globin replaces γ-globin, leading to the formation of α2ß2 tetramers (HbA). However, in different benign and pathological conditions, HbF is expressed in adult cells, as it happens in the hereditary persistence of fetal hemoglobin, in anemias and in some leukemias. The molecular basis of γ-globin differential expression in the fetus and of its inappropriate activation in adult cells is largely unknown, although in recent years, a few transcription factors involved in this process have been identified. The recent discovery that fetal cells can persist to adulthood and contribute to disease raises the possibility that postnatal γ-globin expression could, in some cases, represent the signature of the fetal cellular origin.

FUS-dependent liquid-liquid phase separation is important for DNA repair initiation.

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

miR-129-5p: A key factor and therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a relentless and fatal neurological disease characterized by the selective degeneration of motor neurons. No effective therapy is available for this disease. Several lines of evidence indicate that alteration of RNA metabolism, including microRNA (miRNA) processing, is a relevant pathogenetic factor and a possible therapeutic target for ALS. Here, we showed that the abundance of components in the miRNA processing machinery is altered in a SOD1-linked cellular model, suggesting consequent dysregulation of miRNA biogenesis. Indeed, high-throughput sequencing of the small RNA fraction showed that among the altered miRNAs, miR-129-5p was increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients. We demonstrated that miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD. ELAVL4/HuD is predominantly expressed in neurons, where it controls several key neuronal mRNAs. Overexpression of pre-miR-129-1 inhibited neurite outgrowth and differentiation via HuD silencing in vitro, while its inhibition with an antagomir rescued the phenotype. Remarkably, we showed that administration of an antisense oligonucleotide (ASO) inhibitor of miR-129-5p to an ALS animal model, SOD1 (G93A) mice, result in a significant increase in survival and improved the neuromuscular phenotype in treated mice. These results identify miR-129-5p as a therapeutic target that is amenable to ASO modulation for the treatment of ALS patients.

SOX6 blocks the proliferation of BCR-ABL1+ and JAK2V617F+ leukemic cells.

SOX6 is a HMG-box transcription factor expressed in a wide range of tissues. Recent data show that SOX6 expression is altered in different cancers, in the majority of cases being downregulated. To date, no data are available about SOX6 role in hematological malignancies. Here we demonstrate that SOX6 overexpressing BCR-ABL1+ B-ALL cells are unable to promote leukemia in a mouse model. Starting from this observation, we extended our study to a panel of human leukemic cells carrying genetic lesions distinctive of different types of leukemias and myeloproliferative disorders (the BCR-ABL1 translocation and the JAK2V617F amino acid substitution) to dissect the cellular events induced by SOX6. The inhibition of proliferation is the invariant outcome of SOX6 overexpression but it is achieved via two different cellular responses: terminal differentiation in erythroid-biased cells, irrespectively of their mutation, and apoptosis in megakaryocytic-primed and lymphoid cells. Within this context, cells carrying the highest copy number of the JAK2V617F allele better counteract the SOX6-imposed growth arrest. The interrogation of the GEPIA (Gene Expression Profiling Interactive Analysis) human dataset reveals that SOX6 is downregulated in a cohort of AML patients, uncovering a wide anti-proliferative role of SOX6 in a variety of mutant backgrounds.

The Pleiotropic Effects of GATA1 and KLF1 in Physiological Erythropoiesis and in Dyserythropoietic Disorders.

In the last few years, the advent of new technological approaches has led to a better knowledge of the ontogeny of erythropoiesis during development and of the journey leading from hematopoietic stem cells (HSCs) to mature red blood cells (RBCs). Our view of a well-defined hierarchical model of hematopoiesis with a near-homogeneous HSC population residing at the apex has been progressively challenged in favor of a landscape where HSCs themselves are highly heterogeneous and lineages separate earlier than previously thought. The coordination of these events is orchestrated by transcription factors (TFs) that work in a combinatorial manner to activate and/or repress their target genes. The development of next generation sequencing (NGS) has facilitated the identification of pathological mutations involving TFs underlying hematological defects. The examples of GATA1 and KLF1 presented in this review suggest that in the next few years the number of TF mutations associated with dyserythropoietic disorders will further increase.