Epigenomic analysis of KLF1 haploinsufficiency in primary human erythroblasts

Heshusius, Steven; Grech, Laura; Gillemans, Nynke; Brouwer, Rutger W. W.; Dekker, Xander T. den; IJcken, Wilfred F. J. van; Nota, Benjamin; Felice, Alex E.; Dijk, Thamar B. van; Lindern, Marieke von; Borg, Joseph; Akker, Emile van den; Philipsen, Sjaak

doi:10.1038/s41598-021-04126-6

Cited by 10 publications

(12 citation statements)

References 54 publications

(95 reference statements)

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“…In K562 cells expressing the P173PfsX236 and C94X mutants, a dramatic reduction in the transcriptional activation of KLF1 target genes was observed as compared to the mock control, consistent with the haploinsufficiency effect predicted for these two variants [ 14 , 46 ]. This condition was accompanied by a slight increase in γ-globin gene expression levels that is to be considered of relevance given the presence of elevated background levels of fetal globin gene expression in K562 cells that may mask the effects of additional transactivation factors.…”

Section: Resultssupporting

confidence: 71%

“…The transcriptional activity of KLF1 was quantitatively determined by analyzing the mRNA levels of KLF1 target genes ( Figure 5 ). We thus examined the effects of these constructs on the expression levels of HBB, BCL11A, ZBTB7A, and HBG1/2 according to the role of KLF1 in promoting hemoglobin fetal-adult switching by directly up-regulating HBB and indirectly repressing fetal globin gene (HBG1/2) expression through the up-regulation of the transcription factor BCL11A and ZBTB7A [ 13 , 14 , 45 ].…”

Section: Resultsmentioning

confidence: 99%

“…In the last decades, several studies have contributed to identifying genetic factors located outside the β-globin locus, which are responsible for increased levels of HbF. Among these factors, KLF1 exerts a dual role in fetal-to-adult globin gene switching by directly activating the β-globin gene (HBB) and indirectly repressing fetal globin gene expression through the activation of BCL11A and ZBTB7A, a repressor of fetal globin gene (HBG1 and HBG2) expression ( Figure 1 ) [ 9 , 10 , 11 , 12 , 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

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Identification and Functional Analysis of Known and New Mutations in the Transcription Factor KLF1 Linked with β-Thalassemia-like Phenotypes

Catapano

Sessa

Trombetti

et al. 2023

Biology

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The erythroid transcriptional factor Krüppel-like factor 1 (KLF1) is a master regulator of erythropoiesis. Mutations that cause KLF1 haploinsufficiency have been linked to increased fetal hemoglobin (HbF) and hemoglobin A2 (HbA2) levels with ameliorative effects on the severity of β-thalassemia. With the aim of determining if KLF1 gene variations might play a role in the modulation of β-thalassemia, in this study we screened 17 subjects showing a β-thalassemia-like phenotype with a slight or marked increase in HbA2 and HbF levels. Overall, seven KLF1 gene variants were identified, of which two were novel. Functional studies were performed in K562 cells to clarify the pathogenic significance of these mutations. Our study confirmed the ameliorative effect on the thalassemia phenotype for some of these variants but also raised the notion that certain mutations may have deteriorating effects by increasing KLF1 expression levels or enhancing its transcriptional activity. Our results indicate that functional studies are required to evaluate the possible effects of KLF1 mutations, particularly in the case of the co-existence of two or more mutations that could differently contribute to KLF1 expression or transcriptional activity and consequently to the thalassemia phenotype.

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Section: Resultssupporting

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification and Functional Analysis of Known and New Mutations in the Transcription Factor KLF1 Linked with β-Thalassemia-like Phenotypes

Catapano

Sessa

Trombetti

et al. 2023

Biology

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“…: P1/P2 protamines, Pde8b , Trim28 , Tcfl5 ), and to the other diseases (i.e. : PIKFYVE in cataract, NFIA , SCN1A in neurological or epilepsy disturbances, KLF1 in hemoglobin persistenece, NR5A2 in pancreatic cancer), seem to confirm that the severity of the phenotype depends on the gene expression level [Jodar and Oliva, 2014; Mei et al , 2022; Ogura et al , 2022; Heshusius et al , 2022; Valassina et al , 2022; Sandhu et al , 2021; Leal et al , 2021; Tan et al , 2020; Xu et al , 2022]. We have documented variable phenotypes for a novel TCTE1 variant c.374T>G (p.Ile125Arg) that has been documented in seven males, including: azoospermia, crypto-, and severe oligoasthenozoospermics.…”

Section: Discussionmentioning

confidence: 93%

Tcte1knockout influence on energy chain transportation, apoptosis and spermatogenesis – implications for male infertility

Olszewska

Malcher

Stokowy

et al. 2022

Preprint

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STUDY QUESTION: Is Tcte1 mutation causative for male infertility? SUMMARY ANSWER: Collected data underline the complex and devastating effect of the single-gene mutation on testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY: Latest data revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity, and is built from DRC1-DRC7 proteins. DRC5 (TCTE1) - one of N-DRC element, has been already reported as a candidate for abnormal sperm flagella beating, however, only in restricted manner with no clear explanation of respective observations. STUDY DESIGN, SIZE, DURATION: Using CRISPR/Cas9 genome editing technique, mouse knockout line of Tcte1 gene was created on the basis of C57Bl/6J strain. Then, the mouse reproductive potential, semen characteristics, testicular gene expression level, sperm ATP and testis apoptosis level measurements have been performed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n=248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild type (WT - control group), heterozygous Tcte1+/-, and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) has been done for mice testis tissues. STRING interactions have been checked for protein-protein interactions, based on changed expression level of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the ATP amount in spermatozoa, the luminescence level was measured. Also, immunofluorescent in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n=137 non-obstructive azoospermia or cryptozoospermia, n=111 samples with spectrum of oligoasthenoteratozoospermia, including n=47 with asthenozoospermia) has been extracted to perform genomic sequencing (WGS, WES or Sanger). Protein prediction modeling of human identified variants and the exon 3 structure deleted in mouse knockout has been also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for homozygous males with revealed oligoasthenoteratozoospermia, while heterozygous animals (fertile) manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing, linked with apoptosis, or spermatogenesis. In Tcte1-/- males the protein revealed only residual amounts in sperm head nucleus, and was not transported to sperm flagella, as other N-DRC components. Decreased ATP level (2.4-fold lower) was found in spermatozoa of homozygous mice, together with disturbed tail:midpiece ratio, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at similar level in all three mouse genotypes. Mutation screening of human infertile males revealed 1 novel and 5 ultrarare heterogeneous variants (predicted as disease causing) in 6.05% of patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus, suggesting the disrupted TCTE1 interaction with its binding partners located within the axoneme.

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“…During in vitro erythroblast differentiation, cells massively upregulate globin expression to produce hemoglobin [ 32 ]. Total hemoglobin expression was followed during the first days of differentiation.…”

Section: Resultsmentioning

confidence: 99%

In Vitro Erythropoiesis at Different pO2 Induces Adaptations That Are Independent of Prior Systemic Exposure to Hypoxia

Simionato

Rabe

Gallego-Murillo

et al. 2022

Cells

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Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. At high altitude, EPO levels first increase and then decrease, although erythropoiesis remains elevated at a stable level. The roles of hypoxia and related EPO adjustments are not fully understood, which has contributed to the formulation of the theory of neocytolysis. We aimed to evaluate the role of oxygen exclusively on erythropoiesis, comparing in vitro erythroid differentiation performed at atmospheric oxygen, a lower oxygen concentration (three percent oxygen) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in different erythroid development, suggesting no retention of a high-altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at three percent oxygen by a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation of the concept of neocytolysis.

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Epigenomic analysis of KLF1 haploinsufficiency in primary human erythroblasts

Cited by 10 publications

References 54 publications

Identification and Functional Analysis of Known and New Mutations in the Transcription Factor KLF1 Linked with β-Thalassemia-like Phenotypes

Identification and Functional Analysis of Known and New Mutations in the Transcription Factor KLF1 Linked with β-Thalassemia-like Phenotypes

Tcte1knockout influence on energy chain transportation, apoptosis and spermatogenesis – implications for male infertility

In Vitro Erythropoiesis at Different pO2 Induces Adaptations That Are Independent of Prior Systemic Exposure to Hypoxia

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