Differential regulation of the alpha-globin locus by Kruppel-like factor 3 in erythroid and non-erythroid cells

Background: Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription. Results: We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3−/− mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3−/− erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions. Conclusions: These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression. </p

Transcriptional regulators of haematopoiesis

The gene encoding the transcriptional regulator Krüppel-like Factor 3 (Klf3) is expressed highly during erythroid development. To investigate its biological role we have been studying a Klf3 knockout mouse model, where loss of Klf3 leads to numerous erythroid defects and mild anaemia. However, this has been complicated by upregulation of the related transcription factor Klf8 in Klf3 null tissue. These two factors are highly homologous and are able to regulate an overlapping set of target genes, suggesting that Klf8 may functionally compensate for the loss of Klf3. In this study, we have addressed this issue by comparing normal, Klf3, Klf8 and Klf3-Klf8 double mutant mouse lines. This approach has allowed us to determine that Klf3 and Klf8 can both contribute to the repression of embryonic globin gene expression during definitive erythropoiesis. Additionally, we have identified an intriguing new Klf3 target gene, a novel short isoform of the master myeloid regulator Pu.1. This isoform, which we have named Pu.2, is expressed via an internal promoter mapping to a retroviral long terminal repeat present in the mouse Pu.1 locus. We have determined that this and other related promoters are regulated by Klf3. Furthermore, we have found that Pu.2 negatively regulates Pu.1 activity and overexpression of Pu.2 in human myelogenous leukaemia cells induces erythroid differentiation. This study concludes that Klf3 plays a key role in erythroid biology that is achieved in part by regulating expression of factors such as Klf8 and Pu.2

PU.1 and Haematopoietic Cell Fate: Dosage Matters

The ETS family transcription factor PU.1 is a key regulator of haematopoietic differentiation. Its expression is dynamically controlled throughout haematopoiesis in order to direct appropriate lineage specification. Elucidating the biological role of PU.1 has proved challenging. This paper will discuss how a range of experiments in cell lines and mutant and transgenic mouse models have enhanced our knowledge of the mechanisms by which PU.1 drives lineage-specific differentiation during haematopoiesis

High prevalence of de novo metabolic dysfunction-associated fatty liver disease after liver transplantation and the role of controlled attenuation parameter

Abstract Background & Aims Although non-alcoholic fatty liver disease (NAFLD) remains an uncommon indication for liver transplantation (LT) in the Chinese, the prevalence of NAFLD is increasing. We aimed to determine the prevalence of de novo steatosis and metabolic dysfunction-associated fatty liver disease (MAFLD) after LT. Methods Transient elastography assessment for liver stiffness and controlled attenuation parameter (CAP) were performed after LT in 549 patients at median time of 77 months from LT. CAP was compared with implant liver biopsy, and also validated in 42 patients with post-LT liver biopsy. Longitudinal history including diabetes mellitus (DM), dyslipidemia, hypertension, and immunosuppressive regimen were recorded. Results The optimal cut-off level of CAP for diagnosing at least mild (≥ S1) and moderate-to-severe steatosis (≥ S2/3) was 266 and 293 dB/m respectively, with AUROC of 0.740 and 0.954 respectively. Using this newly derived cut-off, 28.9% patients have de novo NAFLD, of which 95.6% fulfilled the criteria for MAFLD. After multivariate analysis, BMI (HR 1.34), DM (HR 2.01), hypertension (HR 2.03), HDL-cholesterol (HR 0.25), LDL-cholesterol (HR 1.5) and cryptogenic cirrhosis (HR 4.85) were associated with the development of S2/3 graft steatosis. de novo NAFLD was associated with higher incidence of new-onset hypertension (p  40 U/L; p = 0.008), but not associated with graft fibrosis (defined as liver stiffness > 12 kPa; p = 0.761). Conclusion Although NAFLD remains an uncommon primary liver disease indication for LT in Chinese patients, post-transplant de novo graft steatosis is common and the majority is classified as MAFLD. Development of graft steatosis is not associated with an increase in graft fibrosis but was associated with worse metabolic control and graft dysfunction. Routine CAP measurement to detect de novo graft steatosis should be considered after LT regardless of the primary indication of LT

Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection

Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death in replication stressed cells results in distinct cellular outcomes depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and cancer genome evolution

Recurrent hungry bone syndrome in a kidney transplant recipient with a history of parathyroidectomy: A case report

Background: The hungry bone syndrome (HBS) is a well described phenomenon occurring shortly after parathyroidectomy characterized by rapid bone formation with concomitant hypocalcemia, hypophosphatemia and hypomagnesemia requiring intensive management. Recurrent HBS occurring in isolation from parathyroidectomy has not been reported.Case presentation: We describe a case of recurrent HBS in a kidney transplant recipient (KTR) developing years after parathyroidectomy. The KTR was a 49 year-old lady who had undergone successful total parathyroidectomy without re-implantation 14 years prior and cadaveric kidney transplantation 12 years prior. She had a stable creatinine level of 220μmol/L and an estimated glomerular filtration rate (eGFR) of 20 mL/min-1.73m2. She presented to us with severe hypercalcemia, likely a result of excessive calcium and vitamin D supplementation, and acute kidney injury. Serum creatinine, calcium, phosphate, magnesium, alkaline phosphatase (ALP), and intact parathyroid hormone (iPTH) levels on admission were 743μmol/L, 4.8 mmol/L, 1.8 mmol/L, 0.75 mmol/L, 48IU/L and <0.1 pmol/L, respectively. Vigorous intravenous fluids were given in addition to withdrawal of calcium carbonate and calcitriol. Clinical improvement was evident with falling serum creatinine and calcium levels. However, this was followed 2–3 days after admission by an unexplained rise in ALP from a baseline of 48IU/L to a peak level of 1150IU/L over the next week, accompanied by the development of severe hypocalcemia, hypomagnesemia and a persistent drop in phosphate levels. The patient required large doses of calcium carbonate, calcitriol and magnesium lactate to maintain blood mineral levels. The ALP progressively decreased subsequently and the serum levels of calcium, phosphate and magnesium began to stabilize in the next 2–3 weeks.Conclusion: Recurrent HBS can occur years after parathyroidectomy in KTRs. We hypothesize that hypercalcemia and its rapid correction might have been the trigger in this particular patient. The mechanism is not well understood but might involve bone remodeling pathways that are independent of parathyroid hormone

A CpG mutational hotspot in a ONECUT binding site accounts for the prevalent variant of hemophilia B Leyden.

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