Russell C. DeKelver

SON Controls Cell-Cycle Progression by Coordinated Regulation of RNA Splicing

It has been suspected that cell-cycle progression might be functionally coupled with RNA processing. However, little is known about the role of the precise splicing control in cell-cycle progression. Here, we report that SON, a large Ser/Arg (SR)-related protein, is a splicing cofactor contributing to efficient splicing of cell-cycle regulators. Downregulation of SON leads to severe impairment of spindle pole separation, microtubule dynamics, and genome integrity. These molecular defects result from inadequate RNA splicing of a specific set of cell-cycle-related genes that possess weak splice sites. Furthermore, we show that SON facilitates the interaction of SR proteins with RNA polymerase II and other key spliceosome components, suggesting its function in efficient cotranscriptional RNA processing. These results reveal a mechanism for controlling cell-cycle progression through SON-dependent constitutive splicing at suboptimal splice sites, with strong implications for its role in cancer and other human diseases.

SRSF2 Is Essential for Hematopoiesis, and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing

ABSTRACT Myelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2flox/flox mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage− Sca+ c-Kit+ cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes.

SON Protein Regulates GATA-2 through Transcriptional Control of the MicroRNA 23a∼27a∼24-2 Cluster

Background: SON is a recently characterized splicing factor with potential functions in transcription. Results: SON knockdown increases miR-27a through transcriptional activation, which in turn down-regulates GATA-2. Conclusion: Transcriptional repression by SON on the miR-23a∼27a∼24-2 promoter controls the GATA-2 protein level. Significance: Understanding SON regulation of microRNA transcription provides a mechanism of SON-mediated gene expression and its effect on hematopoiesis. SON is a DNA- and RNA-binding protein localized in nuclear speckles. Although its function in RNA splicing for effective cell cycle progression and genome stability was recently unveiled, other mechanisms of SON functions remain unexplored. Here, we report that SON regulates GATA-2, a key transcription factor involved in hematopoietic stem cell maintenance and differentiation. SON is highly expressed in undifferentiated hematopoietic stem/progenitor cells and leukemic blasts. SON knockdown leads to significant depletion of GATA-2 protein with marginal down-regulation of GATA-2 mRNA. We show that miR-27a is up-regulated upon SON knockdown and targets the 3′-UTR of GATA-2 mRNA in hematopoietic cells. Up-regulation of miR-27a was due to activation of the promoter of the miR-23a∼27a∼24-2 cluster, suggesting that SON suppresses this promoter to lower the microRNAs from this cluster. Our data revealed a previously unidentified role of SON in microRNA production via regulating the transcription process, thereby modulating GATA-2 at the protein level during hematopoietic differentiation.

Attenuation of AML1-ETO cellular dysregulation correlates with increased leukemogenic potential

AML1-ETO (RUNX1-ETO) fusion proteins are generated by the 8;21 translocation, commonly found in acute myeloid leukemia, which fuses the AML1 (RUNX1) and ETO (MTG8, RUNX1T1) genes. Previous studies have shown that AML1-ETO interferes with AML1 function but requires additional cooperating mutations to induce leukemia development. In mouse models, AML1-ETO forms lacking the C-terminus have been shown to have greatly enhanced leukemogenic potential. Here, we investigate the role of 2 AML1-ETO C-terminal-interacting proteins, N-CoR, a transcriptional corepressor, and SON, a splicing/transcription factor required for cell cycle progression, in AML1-ETO-induced leukemia development. AML1-ETO-W692A loses N-CoR binding at NHR4, displays attenuated transcriptional repression ability and decreased cellular dysregulation, and promotes leukemia in vivo. These results support the importance of the degree of dysregulation by AML1-ETO in cellular transformation and demonstrate that AML1-ETO-W692A can be used as an effective experimental model for determining which factors compromise the leukemogenic potential of AML1-ETO.

SON regulates GATA-2 through transcriptional control of the miR-23a~27a~24-2 cluster

Background: SON is a recently characterized splicing factor with potential functions in transcription. Results: SON knockdown increases miR-27a through transcriptional activation, which in turn down-regulates GATA-2. Conclusion: Transcriptional repression by SON on the miR-23a∼27a∼24-2 promoter controls the GATA-2 protein level. Significance: Understanding SON regulation of microRNA transcription provides a mechanism of SON-mediated gene expression and its effect on hematopoiesis. SON is a DNA- and RNA-binding protein localized in nuclear speckles. Although its function in RNA splicing for effective cell cycle progression and genome stability was recently unveiled, other mechanisms of SON functions remain unexplored. Here, we report that SON regulates GATA-2, a key transcription factor involved in hematopoietic stem cell maintenance and differentiation. SON is highly expressed in undifferentiated hematopoietic stem/progenitor cells and leukemic blasts. SON knockdown leads to significant depletion of GATA-2 protein with marginal down-regulation of GATA-2 mRNA. We show that miR-27a is up-regulated upon SON knockdown and targets the 3′-UTR of GATA-2 mRNA in hematopoietic cells. Up-regulation of miR-27a was due to activation of the promoter of the miR-23a∼27a∼24-2 cluster, suggesting that SON suppresses this promoter to lower the microRNAs from this cluster. Our data revealed a previously unidentified role of SON in microRNA production via regulating the transcription process, thereby modulating GATA-2 at the protein level during hematopoietic differentiation.

Cooperation between RUNX1-ETO9a and Novel Transcriptional Partner KLF6 in Upregulation of Alox5 in Acute Myeloid Leukemia

Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene Alox5, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of Alox5 expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα in vitro. However, Alox5 is not essential for the induction of leukemia by RUNX1-ETO9a in vivo. Finally, we demonstrate that the upregulation of Alox5 by RUNX1-ETO9a occurs via the C2H2 zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, KLF6 is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.

RUNX1–ETO induces a type I interferon response which negatively effects t(8;21)-induced increased self-renewal and leukemia development

Abstract The 8;21 translocation is the most common chromosomal aberration occurring in acute myeloid leukemia (AML). This translocation causes expression of the RUNX1–ETO (AML1–ETO) fusion protein, which cooperates with additional mutations in leukemia development. We report here that interferons (IFNs) and IFN-stimulated genes are a group of genes consistently up-regulated by RUNX1–ETO in both human and murine models. RUNX1–ETO-induced up-regulation of IFN-stimulated genes occurs primarily via type I IFN signaling with a requirement for the IFNAR complex. Addition of exogenous IFN in vitro significantly reduces the increase in self-renewal potential induced by both RUNX1–ETO and its leukemogenic splicing isoform RUNX1–ETO9a. Finally, loss of type I IFN signaling via knockout of Ifnar1 significantly accelerates leukemogenesis in a t(8;21) murine model. This demonstrates the role of increased IFN signaling as an important factor inhibiting t(8;21) fusion protein function and leukemia development and supports the use of type I IFNs in the treatment of AML.