Nynke Gillemans

Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12–13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

Erythroid Krüppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5′HS3 of the β-globin locus control region

Disruption of the gene for transcription factor EKLF (erythroid Krüppel‐like factor) results in fatal anaemia caused by severely reduced expression of the adult β‐globin gene, while other erythroid‐specific genes, including the embryonic ϵ‐ and fetal γ‐globin genes, are expressed normally. Thus, EKLF is thought to be a stage‐specific factor acting through the CACC box in the β‐gene promoter, even though it is already present in embryonic red cells. Here, we show that a β‐globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF−/− embryos. Thus, embryonic β‐globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR‐driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5′HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5′HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from γ‐ to β‐globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in β‐globin gene activation.

Fetal globin expression is regulated by Friend of Prmt1

An estimated 6% to 7% of the earths population carries a mutation affecting red blood cell function. The β-thalassemias and sickle cell disease are the most common monogenic disorders caused by these mutations. Increased levels of γ-globin ameliorate the severity of these diseases because fetal hemoglobin (HbF; α2γ2) can effectively replace adult hemoglobin (HbA; α2β2) and counteract polymerization of sickle hemoglobin (HbS; α2β(S)2). Therefore, understanding the molecular mechanism of globin switching is of biologic and clinical importance. Here, we show that the recently identified chromatin factor Friend of Prmt1 (FOP) is a critical modulator of γ-globin gene expression. Knockdown of FOP in adult erythroid progenitors strongly induces HbF. Importantly, γ-globin expression can be elevated in cells from β-thalassemic patients by reducing FOP levels. These observations identify FOP as a novel therapeutic target in β-hemoglobinopathies.

Friend of Prmt1, a Novel Chromatin Target of Protein Arginine Methyltransferases

ABSTRACT We describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified by both asymmetric and symmetric arginine methylation in vivo. Furthermore, Fop plays an important role in the ligand-dependent activation of estrogen receptor target genes, including TFF1 (pS2). Fop depletion results in an almost complete block of estradiol-induced promoter occupancy by the estrogen receptor. Our data indicate that Fop recruitment to the promoter is an early critical event in the activation of estradiol-dependent transcription.

Five Friends of Methylated Chromatin Target of Protein-Arginine-Methyltransferase[Prmt]-1 (Chtop), a Complex Linking Arginine Methylation to Desumoylation

Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity.

Erythropoiesis and globin switching in compound Klf1::Bcl11a mutant mice

B-cell lymphoma 11A (BCL11A) downregulation in human primary adult erythroid progenitors results in elevated expression of fetal γ-globin. Recent reports showed that BCL11A expression is activated by KLF1, leading to γ-globin repression. To study regulation of erythropoiesis and globin expression by KLF1 and BCL11A in an in vivo model, we used mice carrying a human β-globin locus transgene with combinations of Klf1 knockout, Bcl11a floxed, and EpoR(Cre) knockin alleles. We found a higher percentage of reticulocytes in adult Klf1(wt/ko) mice and a mild compensated anemia in Bcl11a(cko/cko) mice. These phenotypes were more pronounced in compound Klf1(wt/ko)::Bcl11a(cko/cko) mice. Analysis of Klf1(wt/ko), Bcl11a(cko/cko), and Klf1(wt/ko)::Bcl11a(cko/cko) mutant embryos demonstrated increased expression of mouse embryonic globins during fetal development. Expression of human γ-globin remained high in Bcl11a(cko/cko) embryos during fetal development, and this was further augmented in Klf1(wt/ko)::Bcl11a(cko/cko) embryos. After birth, expression of human γ-globin and mouse embryonic globins decreased in Bcl11a(cko/cko) and Klf1(wt/ko)::Bcl11a(cko/cko) mice, but the levels remained much higher than those observed in control animals. Collectively, our data support an important role for the KLF1-BCL11A axis in erythroid maturation and developmental regulation of globin expression.

Epigenetic silencing of spermatocyte-specific and neuronal genes by SUMO modification of the transcription factor sp3

SUMO modification of transcription factors is linked to repression of transcription. The physiological significance of SUMO attachment to a particular transcriptional regulator, however, is largely unknown. We have employed the ubiquitously expressed murine transcription factor Sp3 to analyze the role of SUMOylation in vivo. We generated mice and mouse embryonic fibroblasts (MEFs) carrying a subtle point mutation in the SUMO attachment sequence of Sp3 (IKEE553D mutation). The E553D mutation impedes SUMOylation of Sp3 at K551 in vivo, without affecting Sp3 protein levels. Expression profiling revealed that spermatocyte-specific genes, such as Dmc1 and Dnahc8, and neuronal genes, including Paqr6, Rims3, and Robo3, are de-repressed in non-testicular and extra-neuronal mouse tissues and in mouse embryonic fibroblasts expressing the SUMOylation-deficient Sp3E553D mutant protein. Chromatin immunoprecipitation experiments show that transcriptional de-repression of these genes is accompanied by the loss of repressive heterochromatic marks such as H3K9 and H4K20 tri-methylation and impaired recruitment of repressive chromatin-modifying enzymes. Finally, analysis of the DNA methylation state of the Dmc1, Paqr6, and Rims3 promoters by bisulfite sequencing revealed that these genes are highly methylated in Sp3wt MEFs but are unmethylated in Sp3E553D MEFs linking SUMOylation of Sp3 to tissue-specific CpG methylation. Our results establish SUMO conjugation to Sp3 as a molecular beacon for the assembly of repression machineries to maintain tissue-specific transcriptional gene silencing.

HS5 of the human β-globin Locus Control Region: a developmental stage-specific border in erythroid cells.

Elements with insulator/border activity have been characterized most extensively in Drosophila melanogaster. In vertebrates, the first example of such an element was provided by a hypersensitive site of the chicken β‐globin locus, cHS4. It has been proposed that the homologous site in humans, HS5, functions as a border of the human β‐globin locus. Here, we have characterized HS5 of the human β‐globin locus control region. We have examined its tissue‐specificity and assessed its insulating properties in transgenic mice using a lacZ reporter assay. Most importantly, we have tested its enhancer blocking activity in the context of the full β‐globin locus. Our results show that HS5 is erythroid‐specific rather than ubiquitous in human tissues. Furthermore, HS5 does not fulfil the criteria of a general in vivo insulator in the transgene protection assay. Finally, a HS5 conditional deletion from the complete locus demonstrates that HS5 has no discernable activity in adult erythroid cells. Sur prisingly, HS5 functions as an enhancer blocker in embryonic erythroid cells. We conclude that HS5 is a developmental stage‐specific border in erythroid cells.

Specificity Protein 2 (Sp2) Is Essential for Mouse Development and Autonomous Proliferation of Mouse Embryonic Fibroblasts

Background The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. Methodology/Principal Findings Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. Conclusions/Significance Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with the phenotypes of Sp1, Sp3 and Sp4 knockout strains shows that, despite their structural similarity and evolutionary relationship, all four glutamine-rich members of the Sp family of transcription factors have distinct non-redundant functions in vivo.

Hereditary Persistence of Fetal Hemoglobin in two patients with KLF1 haploinsufficiency due to 19p13.2–p13.12/13 deletion

FRANCESCO ZAJA,* VALENTINA TABANELLI, CLAUDIO AGOSTINELLI, ANGELICA CALLERI, ANNALISA CHIAPPELLA, MARZIA VARETTONI, PIER LUIGI ZINZANI, STEFANO VOLPETTI, ELENA SABATTINI, RENATO FANIN, AND STEFANO A. PILERI Clinica Ematologica, DISM, Azienda Sanitaria Universitaria Integrata, Udine, Italy; Unit of Haematopathology, European Institute of Oncology, Milan, Italyi; Unit a di Emolinfopatologia, Dipartimento di Ematologia & Oncologia Azienda Ospedaliero – Universitaria, Bologna, Italy; Department of Experimental, Diagnostic and Specialty Medicine, Bologna University School of Medicine, Bologna, Italy; S.C. Ematologia A.O.U. Citt a della Salute e della Scienza, Torino, Italy; Dipartimento di Ematologia e Oncologia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Institute of Hematology “L. e A. Ser agnoli”, University of Bologna, Bologna, Italy Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: AIRC 5x1000 n.10007 (to S.A.P). Conflict of interest: FZ received research funding and honoraria from Roche, Celgene, GSK, Novartis, Takeda, Janssen, Gilead, Mundipharma. SAP and PLZ are members of Takeda/ Millennium Advisory Board. MV received honoraria from Janssen. AC received honoraria from Celgene, Roche, Jannsen, Teva, Pfizer, Amgen. ES received honoraria from Novartis. F.Z. and V.T. equally contributed to the study *Correspondence to: Francesco Zaja, MD, Clinica Ematologica, DISM, Azienda Sanitaria Universitaria Integrata di Udine, P. le S. Maria della Misericordia 15 33100 Udine, Italy. Tel.:1 39 0432 559604. Fax:1 39 0432 559661. E-mail francesco.zaja@asuiud.sanita.fvg.it AIRC 5x1000 n.10007 (to S.A.P). Received for publication: 24 September 2016; Revised: 28 September 2016; Accepted: 29 September 2016 Published online: 3 October 2016 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.24571